Quantitative determination of insulin by mass spectrometry

Abstract

To provide a method for determining an amount of insulin in a sample by mass spectroscopy.SOLUTION: Provided is a mass spectrometry for detecting and quantifying insulin in a biological sample using a purification method in conjunction with tandem mass spectroscopy or high-resolution / high-precision mass spectroscopy technique. More specifically, a method for determining an amount of insulin in a biological sample with the tandem mass spectroscopy, comprises: (a) subjecting a sample to a condition suitable for obtaining an insulin B chain from insulin; (b) processing the sample from the step (a) to obtain an insulin B chain enriched fraction; (c) subjecting the concentrated insulin B chain to an ionization source under a condition suitable for generating one or more insulin B chain ions detectable with the mass spectroscopy; and (d) determining an amount of one or more insulin B chain ions with the tandem mass spectroscopy, where an amount of ions determined in the step (d) is associated with the amount of insulin in the sample.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related patent applications This application, in its entirety and for all purposes, is incorporated herein by reference. The main interest of U.S. Provisional Application No. 61 / 427,749, filed on December 28, 2010, is It is something that stretches.

[0002] Field of Invention This invention relates to the quantitative measurement of insulin. In a particular embodiment, this invention relates to the quantitative measurement of insulin by mass This paper describes a method for quantitatively measuring insulin by analysis. [Background technology]

[0003] Background of the Invention The following description of the background of the present invention is provided merely to aid in understanding the present invention. This does not constitute a description or representation of the prior art of the present invention.

[0004] Insulin consists of A and B chains linked by disulfide crosslinks between cysteine ​​residues. It is a small peptide consisting of 51 amino acids in two chains, represented as [expression]. Human insulin The molecule has a molecular weight of approximately 5607.4 amu. Chain A has 21 amino acids, and chain B The chain has 30 amino acids.

[0005] Insulin is a central hormone that regulates fat and steroid metabolism in the body. When blood sugar levels rise after a meal, insulin is released into the bloodstream, distributing glucose from the circulation to the cells. To enable transportation inside.

[0006] Diabetes is caused by a lack of insulin production or utilization. Insulin is used in diabetes It is often administered to treat illness. Diabetes and its complications are a serious public health problem. The question is: Therefore, insulin in samples from patients with diabetes and pre-diabetes. Quantitative measurement of ions is important both as a diagnostic tool and for monitoring patient treatment. .

[0007] Immunological techniques are widely used for insulin quantification (e.g., Manley et al., Clin Chem., 20 (See 2007, Vol. 53, pp. 922-932), several qualities for the detection and / or quantification of insulin. Quantitative analysis methods have been reported. For example, Stocklin R. et al., Diabetes, 1997, Vol. 46, pp. 44-50. Immunoaffinity chromatography - solid-phase extraction - HPLC - single mass spectrometry (Reporting the quantitative determination of insulin in serum samples), Darby SM et al., J. Anal Toxicol, 20 2001, Vol. 25, pp. 8-14 (SPE-HPLC-MS determination of plasma insulin levels above physiological levels) (Reporting the quantity), Fierens C. et al., Rapid Commun. Mass Spectrom., 2001, Vol. 15, 14 Pages 33-41 (Reporting the detection of insulin in aqueous solution by HPLC-(ESI)MS / MS) (Magnes, C. et al., 52) nd ASMS Conference, May 2004 (High resolution / high precision mass fraction (Reports on the quantitative determination of insulin in serum by analysis), Thevis, M. et al., Anal. Chem., 200 Year 5, Volume 77, pp. 3579-3585 (For quantitative analysis of insulin from plasma and detection of insulin B chain) Immunoaffinity chromatography-solid-phase extraction-HPLC-tandem mass spectrometry (Reported), Thevis, M. et al., Anal. Chem., 2006, Vol. 77, pp. 3579-3585 and Thomas, A . et al., Analyst. Chem., 2007, Vol. 79, pp. 2518-2524 (Quantitative determination of insulin from plasma and insulin Solid-phase extraction for detection of phosphorus B chain - immunoaffinity chromatography - solid-phase extraction - Uytfanghe, K. et al., who have reported on HPLC-tandem mass spectrometry, present the Rapid Comm Mass S pectrom., 2007, Vol. 21, pp. 819-821 (Immuno-affective quantification of insulin from serum) This report describes a method involving nitty chromatography, solid-phase extraction, HPLC, and tandem mass spectrometry. ), Rodriguez-Cabaleiro D. et al., Clin Chem., 2007, Vol. 53, pp. 1462-1469 (from plasma) Immunoaffinity chromatography for insulin quantification and detection of insulin B chains --Solid-phase extraction-HPLC-tandem mass spectrometry method reported), Thevis, M. et al., Mass S pectrom. Reviews, 2008, Vol. 27, pp. 35-50 (Quantification of insulin from plasma and insulin Immunoaffinity chromatography-solid-phase extraction-HPLC-tank for detection of B chain (Reports on the Ndem Mass Spectrometry method) and Guedes, S., J. Am Soc Mass Spectrom, 2009 See Volume 20, pages 1319-1326. [Overview of the Initiative] [Means for solving the problem]

[0008] Summary of the Invention This invention provides a method for determining the amount of insulin in a sample by mass spectrometry.

[0009] In one embodiment, this method uses tandem mass spectrometry. Several tandem mass In the analytical embodiment, this method analyzes insulin in a biological sample taken from a human. for determining the amount of. In some embodiments, the method comprises: (a) subjecting a sample to solid phase extraction (SPE) and high performance liquid chromatography (HPLC) to obtain an insulin enriched fraction from the sample; (b) subjecting the enriched insulin (enriched insul ine) to an ionization source under conditions suitable for generating one or more insulin ions detectable by mass spectrometry; (c) determining the amount of one or more insulin ions by tandem mass spectrometry, wherein the sample has not been subjected to immunopurification prior to ionization. In these embodiments, the amount of insulin in the

[0010] sample is determined using the amount of one or more ions determined in step (c). In some embodiments, the sample is subjected to acidic conditions prior to ionization in positive ion mode. In some related embodiments, subjecting the sample to acidic conditions comprises subjecting the enriched insulin to formic acid. In some related embodiments, one or more ions determined in step (c) comprise insulin precursor ions selected from the group of ions having a mass to charge ratio (m / z) of 1162.5 ± 0.5 and 968 .9 ± 0.5. In further related embodiments, one or more ions determined in step (c) comprise one or more fragment ions selected from the group of ions having an m / z of 226.2 ± 0.5 and 135.9 ± 0 One or more fragments from an insulin precursor ion having a m / z of 0.5 ± 0.5 One or more insulin precursor ions having an ion and a m / z of 968.9±0.5 It contains several fragment ions. In related embodiments, one or more fragment ions from each precursor ion. The fragment ions have m / z values ​​of 226.2±0.5 and 135.9±0.5. It contains one or more fragment ions selected from the group consisting of ions.

[0011] In some embodiments, the sample is salted before ionization in positive ion mode. It is subjected to basic conditions. In some related embodiments, the sample is subjected to basic conditions. The step includes the step of subjecting the sample to ammonia. In some related embodiments, The one or more ions determined in step (c) are 1453.8±0.5 and 116 Insulin precursor ions selected from the group consisting of ions with a m / z of 3.0 ± 0.5 This includes one or more i determined in step (c). In further related embodiments, one or more i ON is a group of ions having m / z values ​​of 226.2±0.5 and 135.9±0.5. It comprises one or more fragment ions selected from the following. In other related embodiments, One or more fragment ions are insulin precursors with a frequency of 1453.8 m / z. One or more fragment ions from ON and a mass-to-charge ratio of 1163.0 ± 0.5 It contains one or more fragment ions from the insulin precursor ion.

[0012] Several implementations of using tandem mass spectrometry to determine the amount of insulin in a sample In this case, the method is (a) a step of concentrating insulin in the sample by extraction technique; ( b) Steps to obtain an insulin-enriched fraction from the sample The purified insulin from (a) is subjected to high-performance liquid chromatography (HPLC). (c) Conditions suitable for generating insulin precursor ions detectable by mass spectrometry The step below involves providing concentrated insulin as an ionization source, wherein the insulin precursor The ON has a m / z of 1162.5 ± 0.5, step; (d) detectable by mass spectrometry. Approximately 4 insulin precursor ions are used to generate one or more fragment ions. Steps to provide collision-induced dissociation at collision energies in the range of 0 to 70 eV; (e) mass This includes the step of determining the amount of one or more of the aforementioned fragment ions by analysis. In these embodiments, the amount of ions determined in step (e) is added to the sample It relates to the amount of n. In some embodiments, the extraction technique is solid-phase extraction (SPE). be.

[0013] In some embodiments, the sample is acidified before ionization in positive ion mode. To be subjected to conditions. In some related embodiments, the step of subjecting the sample to acidic conditions is, The process includes the step of subjecting the sample to formic acid. In an alternative embodiment, the sample is based before ionization. Subject to basic conditions. In some related embodiments, the step of subjecting the sample to basic conditions. It contains ammonia.

[0014] In some embodiments, the collision energy is, for example, in the range of about 40-50 eV. These are in the range of approximately 40-60 eV. In some embodiments, step (d) The one or more fragment ions generated are 226.2±0.5 and 135.9± It contains one or more ions selected from the group consisting of ions having an m / z of 0.5.

[0015] To determine the amount of insulin in a biological sample taken from a human, a tandem solution is used. In some embodiments using quantitative analysis, the method involves (a) separating the sample from insulin. (b) A step of subjecting the insulin A chain to conditions suitable for obtaining insulin A chains; (b) a step of providing the insulin A chain concentrated fraction To obtain the sample from step (a), solid-phase extraction (SPE) and high-performance liquid chromatography are performed. (c) A step of subjecting to HPLC; (c) One or more steps that can be detected by mass spectrometry Concentrated insulin A chains as an ionization source under conditions suitable for generating insulin A chain ions. (d) A step of providing to the following: (d) Tandem mass spectrometry to determine one or more insulin A chain ions The step includes determining the amount of The amount of ions is correlated with the amount of insulin in the sample.

[0016] In some related embodiments, the sample is placed before ionization in positive ion mode. Expose to acidic conditions. In some related embodiments, the step of exposing the sample to acidic conditions. This includes the step of subjecting the sample to formic acid. In some embodiments, step ( a) The insulin A chain obtained in this case is not chemically modified before ionization. In a connected embodiment, one or more ions determined in step (d) are 1192. Selected from the group consisting of ions having m / z values ​​of 9±0.5 and 795.4±0.5. Contains nsrin A chain precursor ions. In some related embodiments, step (d) The one or more ions defined are 513.0±0.5, 399.0±0.5, and 236. One selected from the group of ions having m / z values ​​of 0±0.5 and 133.0±0.5 or It contains multiple fragment ions. In some related embodiments, in step (d) The one or more ions determined are insulin with a m / z of 1192.9 ± 0.5. One or more fragment ions from the A chain precursor ion and 795.4±0.5 m / It contains one or more fragment ions from an insulin A chain precursor ion having z. In some related embodiments, one or more fragment ions from each precursor ion The values ​​are 513.0±0.5, 399.0±0.5, 236.0±0.5, and 133.0± One or more fragments selected from the group consisting of ions having an m / z of 0.5 Includes ON.

[0017] In an alternative embodiment, the method involves the insulin obtained in step (a) before ionization. The step further includes chemically modifying chain A. In some embodiments, the chemical modification The modification includes the step of alkylating the insulin A chain. In further related embodiments, The one or more ions determined in step (d) are 1306.0±0.5 and 87 Alkylated insulin selected from the group consisting of ions having an m / z of 1.0 ± 0.5 Contains A chain precursor ions. In other related embodiments, one or The multiple ions are 570.0±0.5, 456.0±0.5, 293.0±0.5 and One or more fragments selected from the group of ions having an m / z of 133.0 ± 0.5 Contains ion ions. In other related embodiments, one or more determined in step (d) The number of ions is alkylated insulin A chain precursor with m / z of 1306.0±0.5. One or more fragment ions from ON and having a m / z of 871.0 ± 0.5 It contains one or more fragment ions from alkylated insulin A chain precursor ions. In some related embodiments, one or more flags from each alkylated precursor ion Menthion ions were 570.0±0.5, 456.0±0.5, 293.0±0.5 and 1 One or more fractions selected from the group consisting of ions having a m / z of 33.0 ± 0.5 Contains ions.

[0018] In some embodiments, to determine the amount of insulin in a biological sample, Dem mass spectrometry is used. In these embodiments, the method is to (a) measure the sample and insulin (b) A step of subjecting the insulin B chain to conditions suitable for obtaining insulin B chains; (b) Concentration of insulin B chains Step (a) processing the sample from step (c) mass spectrometry to obtain a fraction; Concentrated under conditions suitable for generating one or more detectable insulin B chain ions. (d) providing the nsrin B chain as an ionization source; (f) tandem mass spectrometry to obtain one or more ions. The step includes determining the amount of a certain number of insulin B chain ions. In these embodiments, The amount of ions determined in step (d) is correlated with the amount of insulin in the sample.

[0019] In some embodiments, the process in step (b) is solid-phase extraction (SPE), high-performance liquid The step of enriching insulin B chains by chromatography (HPLC) or both. Includes. In some relevant embodiments using both SPE and HPLC, two concentrations The technology can be implemented online.

[0020] In some embodiments, the biological sample includes human plasma or serum sample. In that related embodiment, the amount of insulin determined is the amount obtained from a sample taken from a human. This is the amount of insulin present.

[0021] In some embodiments, the ionizer is an electrospray source such as a heated ESI source. It is an ionization (ESI) source.

[0022] In some embodiments, the sample is acidified before ionization in positive ion mode. To be subjected to conditions. In some related embodiments, the step of subjecting the sample to acidic conditions is, The procedure includes the step of subjecting the sample to formic acid.

[0023] In some embodiments, the insulin B chain is not chemically modified before ionization. In some related embodiments, one or more ions determined in step (d) are It has m / z values ​​of 1144.2±0.5, 858.3±0.5, and 686.8±0.5. It contains an insulin B chain precursor ion selected from the group consisting of ions. Several related practices In terms of morphology, one or more ions determined in step (d) are 906.0 ± 0. 5, 825.0±0.5, 768.5±0.5, 753.0±0.5, 703.0±0. 5. A group consisting of ions having m / z values ​​of 345.0±0.5 and 226.2±0.5, e.g. For example, 768.5±0.5, 753.0±0.5, 345.0±0.5 and 226.2± For example, a group consisting of ions with an m / z of 0.5, such as 768.5±0.5 and 753 One or more fractions selected from the group consisting of ions having an m / z of 0.0 ± 0.5 Contains ions. In some embodiments, one determined in step (d) Alternatively, multiple ions are insulin B chain precursor ions with a m / z of 1144.2 ± 0.5. Fragment ions from insulin B chain precursor with a m / z of 858.3±0.5 Fragment ions from ON and insulin B chains with a m / z of 686.8±0.5 Two or more fragments selected from the group consisting of fragment ions from precursor ions Contains ions.

[0024] In some embodiments, tandem mass spectrometry is performed with a mass-to-charge ratio of 686.8 ± 0.5. The steps include generating a human insulin B chain precursor ion having (m / z) and the precursor ion to 906.0±0.5, 825.0±0.5, 768.5±0.5, 753.0±0.5 , having m / z values ​​of 703.0±0.5, 345.0±0.5 and 226.2±0.5 One or more fragment ions selected from the group consisting of ONs, e.g., 768.5 One or more fragments selected from the group consisting of ±0.5 and 753.0±0.5 This includes a step of fragmenting the material into ions or other forms.

[0025] In some embodiments, tandem mass spectrometry is performed in the range of 10 to 25 V (including endpoints). The process includes the step of fragmenting the precursor ions using the collision energy within the enclosure.

[0026] In alternative embodiments, the insulin B chain is chemically modified before ionization. In one embodiment, the chemical modification includes the step of alkylating the insulin B chain. In some embodiments, one or more ions determined in step (d) are It has m / z values ​​of 1181.9±0.5, 886.9±0.5, and 709.8±0.5. It contains alkylated insulin B chain precursor ions selected from the group consisting of several ions. In the embodiment, one or more ions determined in step (d) are 345.0 One or more ions selected from the group of ions having m / z values ​​of ±0.5 and 226.2±0.5. It contains a number of fragment ions. In some embodiments, determined in step (d) One or more ions have a mass-to-charge ratio (m / z) of 1181.9 ± 0.5. Fragment ions from lysyllated insulin B-chain precursor ions, 886.9±0.5 m Fragment ions from alkylated insulin B chain precursor ions having / z and 709 Fragments from alkylated insulin B chain precursor ions with a m / z of 0.8±0.5 It contains two or more fragment ions selected from the group consisting of ions. In this embodiment, the fragment ions from each precursor ion are 345.0 ± 0.5 and It contains ions selected from the group consisting of ions having an m / z of 226.2 ± 0.5.

[0027] In a second embodiment, the specific method described herein determines the amount of insulin in a sample. To do this, we will use high-resolution / high-precision mass spectrometry. In some embodiments, the method is suitable for (a) generating polyvalent insulin ions The step involves providing insulin from a sample to an ionization source under certain conditions, wherein the insulin Phosphate ions can be detected by mass spectrometry; and (b) high-resolution / high-precision mass spectrometry. The process includes the step of determining the amount of one or more polyvalent insulin ions by analysis. In this embodiment, the amount of one or more ions determined in step (b) is added to the sample It relates to the amount of insulin inside. In some embodiments, high-resolution / high-precision mass The analysis will be performed with a FWHM of 10,000 and a mass accuracy of 50 ppm. In some embodiments, high-resolution / high-precision mass spectrometry is performed using high-resolution / high-precision time-of-flight (T) spectroscopy. The procedure is performed using an OF-type mass spectrometer. In some embodiments, the ionization conditions are acidic. This includes the ionization of insulin under certain conditions. In some related embodiments, acidic conditions are This includes treatment of the sample with formic acid before ionization. In some embodiments, polyvalent Insulin ions are 4+, 5+, and 6+ valent insulin ions. The selection is made from a group consisting of insulin ions.

[0028] In some embodiments, one or more insulin ions in the 6+ charged state are approximately It contains one or more ions having an m / z within the range of 968.8 ± 1.5. In the embodiment, one or more insulin ions in the 6+ charged state are 968.28± 0.1, 968.45±0.1, 968.62±0.1, 968.79±0.1, 968 0.95±0.1, 969.12±0.1, 969.28±0.1, 969.45±0.1 , one or more selected from the group consisting of ions having an m / z of 969.61±0.1 This includes ions such as those having an m / z of 968.95±0.1.

[0029] In some embodiments, one or more insulin ions in a 5+ charged state are approximately It contains one or more ions having an m / z within the range of 1162.5 ± 1.0. In this embodiment, one or more insulin ions in a 5+ charged state are 1161.7 2±0.1, 1161.92±0.1, 1162.12±0.1, 1162.32±0. 1, 1162.52±0.1, 1162.72±0.1, 1162.92±0.1, 11 Select from the group consisting of ions having m / z values ​​of 63.12±0.1 and 1163.32±0.1. One or more ions are selected, for example, ions having a m / z of 1162.54±0.1. Includes things like n.

[0030] In some embodiments, one or more insulin ions in a 4+ charged state are approximately It contains one or more ions having an m / z within the range of 1452.9 ± 0.8.

[0031] In any of the methods described herein, the sample may include a biological sample. In that embodiment, the biological sample may include biological fluids such as urine, plasma, or serum. In some embodiments, the biological sample is a human, for example, an adult male or female, This may include samples from young men or women. Here, "young" refers to those under 18 years of age or under 15 years of age. The person must be under 12 years old or under 10 years old. Human samples are used to diagnose or monitor a disease or condition. To analyze for the purpose of monitoring, or to monitor the effectiveness of treatment for a disease or condition. This is possible. In some related embodiments, the methods described herein involve taking from humans It can be used to determine the amount of insulin in a biological sample in such a case.

[0032] In embodiments utilizing tandem mass spectrometry, tandem mass spectrometry is used, for example, for multiple inverse This includes response monitoring, precursor ion scanning, or product ion scanning. This can be carried out by methods known in the technical field.

[0033] In some embodiments, tandem mass spectrometry uses one or more fractional precursor ions. The process includes the step of fragmenting into fragment ions. In an embodiment for determining the quantity, the measured ion quantity is related to the amount of insulin in the sample. To achieve this, the quantity can be subjected to any mathematical operation known in the art. For example, As part of the step of determining the amount of insulin in the sample, two or more fragments The amount of ON can be totaled.

[0034] In any of the methods described herein, the analyte (for example, Insulin, chemically modified or unmodified insulin A chain, or chemically modified (Modified or unmodified insulin B chains) are subjected to high-performance liquid chromatography before ionization. The sample can be purified by HPLC. The method described herein In either of these methods, the analyte is subjected to, for example, solid-phase extraction (SPE) on a column. The sample can be purified by extraction techniques such as the following. In some embodiments However, the extraction technique is not an immunopurification technique. Specifically, in some embodiments, S PE columns are not immunoaffinity columns. In some embodiments, immunosinophils The manufacturing process is not used at any stage of the method. In some embodiments, extraction techniques HPLC also enables automated sample processing and analysis through online methods. It can be done.

[0035] In some embodiments, high-resolution / high-precision mass spectrometry is performed at approximately 10,000 or more, for example. For example, over 15,000, for instance, over 20,000, for instance, over 25,000 The process is carried out with a resolution (FWHM) of 00 or higher. In some embodiments, high resolution High-precision mass spectrometry can detect levels of approximately 50 ppm or less, for example, approximately 20 ppm or less, or even around 10 ppm. The following will be performed with an accuracy of approximately 5 ppm or less, or approximately 3 ppm or less. Several implementations In terms of morphology, high-resolution / high-precision mass spectrometry offers a resolution of approximately 10,000 or higher (FWHM). And it is carried out with an accuracy of approximately 50 ppm or less. In some embodiments, the resolution is approximately 1 The number is over 5,000, and the accuracy is approximately 20 ppm or less. In some embodiments, The resolution is approximately 20,000 or higher, and the accuracy is approximately 10 ppm or less, preferably. The resolution is approximately 20,000 or higher, and the accuracy is approximately 5 ppm or less, for example, approximately 3 ppm. The following are some examples.

[0036] In some embodiments, high-resolution / high-precision mass spectrometry is performed using an orbit trap type mass spectrometry. Mass spectrometer, time-of-flight (TOF) type mass spectrometer, or Fourier transform ion cyclotron resonance type mass spectrometer This can be done using a quantitative analyzer (sometimes known as a Fourier transform mass spectrometer). can.

[0037] In some embodiments, one or more detectable by high-resolution / high-precision mass spectrometry can be detected. The number of insulin ions is approximately 1452.9±0.8, 1162.5±1, and 968.8± One or more ions selected from the group consisting of ions having m / z in the range of 1.5 These ions have charges of 4+, 5+, and 6+, respectively. This corresponds to the phosphorus ion. These single isotope ions with these charges are within the range listed m It mainly resides in / z. However, naturally occurring isotopic variants with lower abundances... It may exist outside the range. Insulin ions within the range of 1162.5±1 are preferably about 1161.72±0.1, 1161.92±0.1, 1162.12±0.1, 1162 0.32±0.1, 1162.52±0.1, 1162.72±0.1, 1162.92± Insulin with m / z values ​​of 0.1, 1163.12±0.1, and 1163.32±0.1 This includes ions, such as those with a m / z of 1162.54 ± 0.1. 968. Insulin ions within the range of 8±1.5 are preferably about 968.28±0.1, 968 0.45±0.1, 968.62±0.1, 968.79±0.1, 968.95±0.1 , 969.12±0.1, 969.28±0.1, 969.45±0.1, 969.61 Insulin ions with a m / z of ±0.1, for example, 968.95 ± 0.1 m / z It contains ions, etc. In some embodiments, one is detected by mass spectrometry. Alternatively, a method that correlates the amount of multiple insulin ions with the amount of insulin protein in the sample. The comparison includes comparison with internal standards such as human or non-human insulin protein. It can be isotope-labeled in some cases.

[0038] In any of the methods described herein, the sample is a biological sample, preferably, for example It may include a body fluid sample containing plasma or serum.

[0039] Mass spectrometry (tandem or high-resolution / high-precision) is performed in positive ion mode. This is possible. Alternatively, mass spectrometry can be performed in negative ion mode. Example For example, including atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). Insulin can be ionized using various ionization sources. Several implementations In terms of form, insulin and / or chemically modified or unmodified insulin Insulin A chain or insulin B chain is ionized in positive ion mode by ESI. do.

[0040] In any of the methods described herein, an internal standard that can be detected independently is added to the sample. This can be done, and the amount is also determined in the sample. Implementation using an internal standard that can be detected independently. In this state, all or part of both the analyte and the internal standard present in the sample are When activated, it generates multiple ions that can be detected by a mass spectrometer, and from each of them the following is generated: One or more ions are detected by mass spectrometry. In these embodiments, the target The presence or amount of ions generated from the analyte is determined by comparison with the amount of the detected internal standard ion. This can be correlated with the presence of the target analyte in the sample.

[0041] Alternatively, the amount of insulin in the sample is determined by comparison with one or more external reference standards. This is possible. Specific examples of external reference standards include human or non-human insulin, and synthetic insulin. Blank plasma or serum to which an insulin analog or its isotope-labeled variant has been added. ru.

[0042] In some embodiments, the method is approximately 10 μIU / mL to 500 μIU / mL (approximately 60 pmol / L to 3000 pmol / L or approximately 0.35 ng / mL to 17.4 ng / m³ To determine the amount of insulin in a sample within the range of L (including the endpoints) It is possible.

[0043] In this specification, unless otherwise specified, the singular forms "a," "an," and "the" are used. , and include multiple referents. Therefore, for example, a reference to "a protein" includes multiple referents. It contains a number of protein molecules.

[0044] As used herein, the terms “purify,” “purify,” and “concentrate” are used in relation to the subject matter. This does not mean removing all substances other than the analyte(s) More importantly, these terms refer to other substances in the sample that may interfere with the detection of the target analyte. This refers to a procedure that increases the amount of one or more of the target analytes compared to the components of the other components. The purification of the sample by means of one or more interfering substances, for example, selected by mass spectrometry. One or more factors that may or may not interfere with the detection of parent or daughter ions. It allows for a relative decrease in the substance. When using this term, the relative decrease refers to the amount of the purified substance. It is essential that any substances present in the material along with the analyte be completely removed by purification. It is not necessary.

[0045] As used herein, the terms "immunopurification" or "to immunopurify" refer to one of the subjects. Alternatively, an antibody containing polyclonal or monoclonal antibodies to concentrate multiple analytes. This refers to a purification procedure that utilizes immunopurification. Immunopurification is one of the immunopurification methods known in the art. This can be carried out using a solid support, for example, a column. Conjugated in a well, tube, gel, capsule, particle or similar form. This method utilizes antibodies that are bound in one or another state. Immunopurification is used in the context described herein. In addition, the procedure often referred to in this art as immunoprecipitation, and also the procedure often referred to in this art as affiliate A procedure called immunochromatography or immunoaffinity chromatography. Includes without restriction.

[0046] As used herein, the term "immune particle" refers to its surface (on and / or within the particle). Capsules and beads containing antibodies that are conjugated, bound, or otherwise bound. , means gel particles or similar. In certain preferred embodiments, immune particles are These are Sepharose or agarose beads. In a preferred alternative embodiment, the immune particles are , including glass, plastic, or silica beads or silica gel.

[0047] As used herein, the term "anti-insulin antibody" means an affinity for insulin. This refers to any polyclonal or monoclonal antibody having sexual properties. In various embodiments, In this regard, the specificity of insulin antibodies against chemical species other than insulin may differ. For example, in a particular preferred embodiment, the anti-insulin antibody against insulin It is specific and therefore has little to no affinity for chemical species other than insulin. Although not possessing, in other preferred embodiments, the anti-insulin antibody is nonspecific. It binds to specific chemical species other than insulin.

[0048] As used herein, the term "sample" means any sample that may contain the analyte. This means. As used herein, the term "body fluids" refers to fluids that are separated from an individual's body. It refers to any fluid that can be used for this purpose. For example, "body fluids" include blood, plasma, serum, bile, and saliva. It may include urine, tears, sweat and similar substances. In a preferred embodiment, the sample is human or This includes a sample of their bodily fluids, preferably plasma or serum.

[0049] As used herein, the terms "solid-phase extraction" or "SPE" refer to the process by which a solution passes or circumferentially... Dissolution or suspension in a solution (i.e., mobile phase) relative to a flowing solid (i.e., solid phase). This refers to a method of separating a mixture of chemical substances into its components as a result of the affinity of those components. Depending on the circumstances, when the mobile phase flows through or around the solid phase, undesirable components of the mobile phase may be lost. The solid phase retains the analyte, potentially leading to the purification of the analyte in the mobile phase. In some cases, the analyte is held by the solid phase, and undesirable components of the mobile phase pass through the solid phase. It may be possible for it to flow around it. In these cases, for further processing or analysis Next, a second mobile phase is used to elute the retained analyte from the solid phase. (Containing TFLC) SPE can function with a single-mode or mixed-mode mechanism. The mixed-mode mechanism is used in the same column. This method utilizes ion exchange and hydrophobicity retention, for example, in a mixed-mode SPE column. The solid phase may exhibit strong anion exchange and hydrophobicity retention, or strong cation exchange and hydrophobicity. It may indicate the retention of sexual characteristics.

[0050] Generally, the affinity of an SPE column packing material to the analyte is determined by one or more chemical factors. This can be due to one of several mechanisms, such as interactions or immunoaffinity interactions. In this embodiment, insulin SPE is performed without using an immunoaffinity column packing material. In other words, in some embodiments, insulin is not an immunoaffinity column. The sample is purified using a PE column.

[0051] As used herein, the term "chromatography" refers to the process of transporting liquids or gases. When a mixture of chemicals that can be detected flows around or over a stationary liquid or solid phase, the difference in chemicals occurs. This refers to a method of separating components as a result of alternative distribution.

[0052] As used herein, the terms "liquid chromatography" or "LC" refer to fluid chromatography. When a fine substance permeates uniformly into a column or capillary passage, one or more fluid solutions This refers to a method in which components are selectively delayed. The delay involves one or more stationary phases and bulk fluids. That is, when this fluid moves between the mobile phase and the stationary phase (one or more) This is due to the distribution of components in a mixture. An example of "liquid chromatography" is reversed-phase liquid chromatography. Chromatography (RPLC), high-performance liquid chromatography (HPLC), and turbulent liquid chromatography. Chromatography (TFLC) (sometimes high-turbulence liquid chromatography (HTLC) or Examples include (known as high-processing liquid chromatography).

[0053] In this specification, "high-performance liquid chromatography" or "HPLC" (sometimes referred to as "high-performance liquid chromatography") is used. The term "pressure liquid chromatography" (known as "pressure liquid chromatography") refers to the process of controlling the mobile phase under pressure while stationary phase is controlled. Generally, a liquid is forced through a tightly packed column to increase the degree of separation. This refers to chromatography.

[0054] As used herein, "turbulent liquid chromatography" or "TFLC" (sometimes highly turbulent) (known as fluid chromatography or high-rate liquid chromatography) The term refers to using the turbulent flow of the substance being assayed through a column packing material as the basis for separation. TFLC refers to a type of chromatography that involves two steps before analysis by mass spectrometry. It was applied to the preparation of samples containing unnamed drugs. For example, Zimmer et al., J Chromatogr, A854, See pages 23-35 (1999). TFLC is further explained in U.S. Patent No. 5,966. Nos. 8,367, 5,919,368, 5,795,469 and 5,772,8 See also issue 74. Those skilled in the art understand "turbulence." A fluid flows slowly and smoothly. When this occurs, the flow is called "laminar flow." For example, when flowing through an HPLC column at a low flow rate... The moving fluid is laminar. In laminar flow, the motion of fluid particles is regular, and the particles The water generally moves in a substantially linear fashion. At higher speeds, the inertia of the water acts as a frictional force against the fluid. The current is overcome, and turbulence is generated. Fluids that are not in contact with the irregular boundary "overtake" it, It is slowed down by friction or deflected by an uneven surface. The fluid flows turbulently. If so, it will swirl in a vortex due to greater "resistance" than when the flow is laminar (and It flows (in a vortex). This helps in determining whether a fluid flow is laminar or turbulent. Many references are available for this purpose (for example, Turbulent Flow Analysis: Measure ment and Prediction, PS Bernard & JM Wallace, John Wiley & Sons, Inc. (2000) ), An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge Uni versity Press (2001)).

[0055] As used herein, the terms "gas chromatography" or "GC" refer to sample The mixture is evaporated and moves through a column containing a stationary phase consisting of a liquid or particulate solid. Inject into the flow of carrier gas (as nitrogen or helium) and compound against the stationary phase This refers to chromatography, which separates component compounds according to their affinity.

[0056] As used herein, the terms "large particle column" or "extraction column" refer to approximately 50 μm. This refers to a chromatography column containing average particle diameters greater than m. When used in this context, the term "approximately" means ±10%.

[0057] As used herein, the term "analytical column" refers to the determination of the presence or quantity of the analyte. Sufficient chloroform to enable separation of substances in the sample eluted from the column. This refers to a chromatography column having a matrix plate. M is a substance that is not retained in order to obtain a purified sample for further analysis. This is often distinguished from "extraction columns," which have the general purpose of separating or extracting substances. In this context, the term "approximately" means ±10%. Preferred practice Morphologically, the analytical column contains particles with a diameter of approximately 5 μm.

[0058] As used herein, the terms “online” and “inline” mean, for example, Used in "online automated fashion" or "online extraction". As indicated, this means a procedure that is performed without the need for operator intervention. In contrast, "off As used herein, the term "line" refers to a situation requiring manual intervention by an operator. This refers to the procedure. Therefore, the sample is subjected to precipitation, and then the supernatant is manually autosampled. When loading into a ra, the settling and loading steps are offline from the subsequent steps. In various embodiments of the method, one or more steps are performed online automatically. It is possible.

[0059] As used herein, the terms "mass spectrometry" or "MS" refer to the analysis of compounds by their quality. MS refers to analytical techniques for identification based on quantity. MS stands for mass-to-charge ratio of ions or "m / z". MS technology is a method of filtering, detecting, and measuring ions based on [the following criteria]. Generally, (1) the step of ionizing the compound to produce a charged compound, and (2) the step of charging The process includes the steps of detecting the molecular weight of a compound and calculating its mass-to-charge ratio. It can be ionized and detected in stages. A "mass spectrometer" is generally an ionization device. This includes a mass spectrometer and an ion detector. Generally, when one or more molecules of the target are ionized... Then, the ions are introduced into a mass spectrometer, where, due to a combination of magnetic and electric fields... Ions then follow paths in space that depend on their mass ("m") and charge ("z"). The US has published a paper titled "Mass Spectrometry From Surfaces." No. 6,204,500, "Methods and Apparatus for Tandem Mass Spectrometry" Publication No. 6,107,623, titled "r Tandem Mass Spectrometry)," The 6,268th edition, entitled "DNA Diagnostics Based on Mass Spectrometry," No. 144, "Surface-enhanced photosensitive coupling and emission for desorption and detection of analytes (Surface- Enhanced Photolabile Attachment And Release For Deoption And Detection Of Analyzes Issues 6, 124, and 137 of the Prostate Cancer and Prostatic Diseases, titled "tes)", by Wright et al. iseases, 1999, Vol. 2, pp. 264-2676, and Merchant and Weinberger, Electrophoresis, 20 See Volume 21, pp. 1164-1167, 2000.

[0060] As used herein, "high-resolution / high-precision mass spectrometry" refers to a unique chemical ion. The load is loaded with sufficient precision and accuracy to confirm chemical ions. This refers to mass spectrometry performed using a mass spectrometer capable of measuring the mass-to-charge ratio of different types of electricity. To confirm a specific chemical ion, the individual isotope peaks of that ion can be easily identified. It is possible for the ions in a given case. Specific decomposition is necessary to identify the unique chemical ions. The accuracy and mass precision vary depending on the mass and charge state of the ions.

[0061] As used herein, "resolution" or "resolution (FWHM)" (in the art, "m / ") Δm 50% The term (also known as "mass peak at 50% of maximum height") refers to the mass peak at 50% of maximum height. This refers to the observed mass-to-charge ratio divided by the width (full width at half maximum, "FWHM"). It is the effect of differences in resolution. This is shown in Figures 1A-C, which display the theoretical mass spectrum of an ion with a frequency of approximately 1093 m / z. Figure 1A shows a resolution of approximately 3000 (typical operating conditions for a standard quadrupole mass spectrometer). The theoretical mass spectra obtained by the mass spectrometer are shown. As can be seen in Figure 1A, each The isotope peaks cannot be identified. In comparison, Figure 1B has a resolution of approximately 10,000. The theoretical mass spectrum obtained by a mass spectrometer is shown, but individual isotope peaks cannot be clearly identified. Figure 1C shows the theoretical mass spectrum obtained by a mass spectrometer with a resolution of approximately 12,000. This demonstrates that, at this highest resolution, individual isotope peaks are less than 1% from baseline. Includes the given.

[0062] As used herein, "specific chemical ions" in relation to mass spectrometry refer to single-atom constituents. This refers to a single ion having a specific charge. A single ion can be monovalent or polyvalent.

[0063] As used herein, the term "accuracy" (or "mass accuracy") in relation to mass spectrometry is used in this specification. The term refers to the possible bias that can occur in the instrument's response from the true m / z of the ion being considered. Degrees are generally expressed in parts per million (ppm). The effect of differences in mass accuracy is expressed as 1093 For the theoretical peak at 0.52094 m / z, between the detected m / z and the actual m / z Figures 2A-D show the boundaries of possible differences. Figure 2A shows the detection with an accuracy of 120 ppm. This shows the range in which m / z can occur. In contrast, Figure 2B shows the m detected with an accuracy of 50 ppm. The range in which / z can occur is shown. Figures 2C and 2D show the results with accuracy of 20 ppm and 10 ppm. This indicates a narrower range of possible m / z values.

[0064] The high-resolution / high-precision mass spectrometry method of the present invention is available in 10,000, 15,000, and 20,000 resolutions. 25,000, 50,000, 100,000 or even greater values ​​than FW This can be carried out using an instrument capable of performing mass spectrometry with HM. Similarly, the method of the present invention This refers to concentrations of 50 ppm, 20 ppm, 15 ppm, 10 ppm, 5 ppm, less than 3 ppm, or Therefore, it can be performed using instruments that can perform mass spectrometry with a low level of accuracy. Instruments capable of these performance characteristics include certain orbital mass spectrometers, flight time (" A TOF (Time-of-Flight) type mass spectrometer or a Fourier transform ion cyclotron resonance mass spectrometer is incorporated. It can be seen. In a preferred embodiment, the method is an orbitrap mass spectrometer or a TOF type mass spectrometer. This is performed using instruments including quantitative analyzers.

[0065] The term "orbitrap" refers to an ion trap consisting of a barrel-shaped outer electrode and a coaxial inner electrode. Describe the trap. Ions are injected tangentially into the electric field between the electrodes, and the ions and electrodes The electrostatic interaction between them balances the centrifugal force as the ions orbit the coaxial inner electrode. When ions orbit the coaxial inner electrode, the orbit of the trapped ions becomes The central electrode vibrates along its axis at a harmonic frequency corresponding to the mass-to-charge ratio of the element. Detection of orbital frequency. This allows for high-precision (low 1-2 ppm) and high-resolution (FWHM) orbit trapping. It can be used as a mass spectrometer with a capacity of approximately 200,000. A mass spectrometer based on the P is incorporated herein by reference as a whole, U.S. Patent Details are provided in publication No. 6,995,364. The use of the Orbitrap analyzer is varied. Qualitative and quantitative analysis of the analyte has been reported. For example, U.S. Patent Application Publication No. 200 Application No. 8 / 0118932 (filed November 9, 2007), Bredehoft et al., Rapid Commun. Mas s Spectrom., 2008, Vol. 22, pp. 477-485, Le Breton et al., Rapid Commun. Mass Spectrom. Thevis et al., 2008, Vol. 22, pp. 3130-3136, Mass Spectrom. Reviews, 2008, Vol. 27, pp. 35-3136. Page 50, Thomas et al., J. Mass Spectrom., 2008, Vol. 43, pp. 908-9015, Schenk et al., BMC Medica l Genomics, 2008, Vol. 1, p. 41 and Olsen et al., Nature Methods, 2007, Vol. 4, pp. 709-7012. See below.

[0066] As used herein, the term "operating in negative ion mode" means operating in negative ion mode. This refers to a mass spectrometry method that generates and detects ions. It operates in positive ion mode. As used herein, the term refers to a mass spectrometry method that generates and detects positive ions. In a preferred embodiment, mass spectrometry is performed in positive ion mode.

[0067] As used herein, the term "ionization" or "to ionize" means one or more electrons This refers to a method of generating analyte ions with a net charge equal to that of the individual units. (Negative ions) An ion has a net negative charge of one or more electron units, while a positive ion has one or more electron units. It has a net positive charge in units of electrons.

[0068] As used herein, the terms "electron ionization" or "EI" refer to the gas phase or vapor phase. This refers to the way in which the object being analyzed interacts with the flow of electrons. The suction then generates analyte ions that can be subjected to mass spectrometry.

[0069] As used herein, the terms "chemical ionization" or "Cl" refer to reagent gases (for example) For example, ammonia is subjected to electron bombardment, and the interaction between the reagent gas ions and the analyte molecules results in This refers to a method by which analyte ions are generated.

[0070] As used herein, the terms “fast atomic impact” or “FAB” mean “high energy A beam of atoms (often Xe or Ar) collides with a non-volatile sample, and the components contained in the sample This refers to a method of detaching a molecule and ionizing it. The test sample is glycerol, thioglycerol L, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitro Phenyloctyl ether, sulfolane, diethanolamine, and triethanolamine It dissolves in viscous liquid matrices such as the following. A suitable matrix for the compound or sample. Choice is an empirical process.

[0071] As used herein, "matrix-assisted laser desorption / ionization" or "MALDI" The term refers to the photoionization, protonation, deprotonation, and clustering of non-volatile samples. Lasers desorb and ionize the analyte in a sample through various ionization pathways, including decay. - This refers to a method of exposure to irradiation. For MALDI, the sample is subjected to irradiation to promote the desorption of the analyte molecules. It mixes with the advancing energy absorption matrix.

[0072] As used herein, "surface-enhancing laser desorption ionization" or "SELDI" The term refers to the photoionization, protonation, deprotonation, and clustering of non-volatile samples. Lasers desorb and ionize the analyte in a sample through various ionization pathways, including decay. - This refers to other methods of exposure to irradiation. For SELDI, the sample is generally one of the subjects. Alternatively, it can be bonded to a surface that preferentially holds multiple analytes. Similar to MALDI, this method The method may also utilize energy-absorbing materials that promote ionization.

[0073] As used herein, the term "electrospray ionization" or "ESI" is used in this specification. This refers to a method of passing a solution through a short capillary tube to which a high positive or negative potential is applied at the end. The solution that reaches the surface evaporates (atoms), forming a jet of very small droplets of solution in the solvent vapor. It becomes a spray. This mist of droplets flows through the evaporation chamber. As the droplet becomes smaller, ions and neutral molecules are released due to the natural repulsion between the charges. The surface charge density increases until the point in time when this occurs.

[0074] As used herein, the terms "atmospheric pressure chemical ionization" or "APCI" are defined as ES It refers to a mass spectrometry method similar to I, but APCI involves ions occurring in a plasma at atmospheric pressure. Ions are generated through molecular reactions. The plasma is maintained by a discharge between the spray capillary and the counter electrode. It is then maintained. Next, the ion generally uses a set of differential exhaust skimmer stages. Further extraction into the mass spectrometer. Using countercurrent, dry, and preheated N2 gas, the solvent Removal can be improved. Gas-phase ionization in APCI separates less polar species. It may be more effective than ESI for analysis.

[0075] The terms "atmospheric pressure photoionization" or "APPI" are used herein to refer to molecular M The ionization mechanism is mass spectrometry, which involves the absorption of photons and the emission of electrons to produce molecular ions M+. This refers to the form of the photon. Since the photon energy is generally just above the ionization potential, the molecular ion It is less susceptible to dissociation. In many cases, it can be used to analyze samples without the need for chromatography. This is possible, and can result in considerable time and cost savings. (Water vapor or proto) In the presence of a volatile solvent, molecular ions can abstract H to form MH+. This tends to occur when there is rotonic affinity. The sum of M+ and MH+ is constant. Therefore, this does not affect the accuracy of the quantification. Drug compounds in protic solvents are usually Although observed as MH+, nonpolar compounds such as naphthalene or testosterone are usually Forms M+. For example, see Robb et al., Anal. Chem., 2000, Vol. 72 (No. 15), pp. 3653-3659. See reference.

[0076] As used herein, the terms "inductively coupled plasma" or "ICP" are used in most contexts. The sample is partially ionized at a sufficiently high temperature to atomize and ionize the elements. It means the way in which it interacts with gas.

[0077] As used herein, the term "field desorption" refers to the process of ionizing a non-volatile test specimen onto an ionized surface. This method involves placing the analyte on top of the sample and using a strong electric field to generate ions of the analyte to be analyzed.

[0078] As used herein, the term "desorption" means the removal of the analyte from the surface and / or This refers to the penetration of the analyte into the gas phase. Laser desorption and thermal desorption involve laser desorption of a sample containing the analyte. This is a technique that uses a laser pulse to thermally desorb substances into the gas phase. The laser has a metal base. The back surface of a specially made 96-well plate is irradiated. The laser pulse heats the bottom, and the heat is transferred to the sample. The sample is then transferred to the gas phase. The gaseous sample is then drawn into the mass spectrometer.

[0079] As used herein, the term "selective ion monitoring" refers to a relatively narrow mass range. Within the range, the detection mode of a mass spectrometer generally detects only ions within a range of approximately 1 mass unit. It is Do.

[0080] In this specification, "selective reaction monitoring" is sometimes referred to as "multiple reaction monitoring," which is a known term. The "responsive mode" selectively detects precursor ions and one or more fragment ions. This is the detection mode of a mass spectrometer.

[0081] As used herein, the terms “lower limit of quantification,” “lower limit of quantification,” or “LLOQ” are used in this specification. This refers to the point at which the measurement becomes quantitatively meaningful. The responses of objects are identifiable, individual, and have a relative standard deviation (RSD%) of less than 20%. It is reproducible with an accuracy of 85% to 115%.

[0082] As used herein, the terms “Limit of Detection” or “LOD” refer to the measurement value relative to the limit of detection. It is a point greater than the associated uncertainty. LOD is the uncertainty associated with the measurement of a value. This is a point that exceeds a certain threshold and is defined as three times the average RSD at zero concentration.

[0083] As used herein, the "amount" of the analyte in a bodily fluid sample generally refers to the volume of the sample. This refers to the absolute value that reflects the mass of the analyte that can be detected. However, the quantity is the same as other analytes. The relative amount is also intended to be compared to the amount of the sample. For example, the amount of the analyte in the sample is the amount of the substance that is normally present in the sample. The amount of the analyte being analyzed may be greater than the control or normal levels.

[0084] The term "approximately" is used herein in reference to quantitative measurements that do not involve the measurement of ion mass. If present, it means plus or minus 10% of the displayed value. Mass spectrometers analyze the specified substance. There may be slight differences in how the mass of an object is determined. The mass of an ion or the mass of an ion / The term "approximately" in relation to charge ratios means + / - 0.50 atomic mass units.

[0085] The above summary of the present invention is not limiting, and other features and advantages of the present invention are as described above. This is evident from the following detailed description and claims. [Brief explanation of the drawing]

[0086] [Figure 1A] This figure shows the theoretical mass spectrum of an ion with a z-frequency of approximately 1093 m / z, as analyzed by a mass spectrometer with a resolution of approximately 3000. [Figure 1B] This figure shows the theoretical mass spectrum of an ion with a frequency of approximately 10⁹³ m / z, as analyzed by a mass spectrometer with a resolution of approximately 10,000. [Figure 1C] This figure shows the theoretical mass spectrum of an ion with a z frequency of approximately 10⁹³ when analyzed by a mass spectrometer with a resolution of approximately 12,000. [Figure 2A] This figure shows the possible bias in the instrument's response from the true m / z of the ions, considering the theoretical peak at 1093.52094 m / z with a mass accuracy of 120 ppm. [Figure 2B] This figure shows the possible bias in the instrument's response from the true m / z of the ions, considering the theoretical peak at 1093.52094 m / z with a mass accuracy of 50 ppm. [Figure 2C] This figure shows the possible bias in the instrument's response from the true m / z of the ions, considering the theoretical peak at 1093.52094 m / z with a mass accuracy of 20 ppm. [Figure 2D] This figure shows the possible bias in the instrument's response from the true m / z of the ions, considering the theoretical peak at 1093.52094 m / z with a mass accuracy of 10 ppm. [Figure 3A]Figures 3A and 3B show specific spectra collected by ESI ionization of human insulin in positive ion mode under acidic conditions. Further details are described in Example 3. [Figure 3B] Figures 3A and 3B show specific spectra collected by ESI ionization of human insulin in positive ion mode under basic conditions. Further details are described in Example 3. [Figure 4] Figure 4A shows a specific example spectrum of human insulin obtained by QTOF mass spectrometry over a range of approximately 900-1200 m / z. Figure 4B shows the contaminant peaks from the strip serum sample matrix obtained by QTOF mass spectrometry. Further details are described in Example 4. [Figure 5] Figure 5A shows a high-resolution / high-precision spectrum of human insulin obtained by QTOF mass spectrometry, specifically in the m / z range of approximately 1154–1177. Figure 5B is a magnified view of the m / z range of approximately 1159–1166. Further details are described in Example 4. [Figure 6] Figure 6A shows a high-resolution / high-precision spectrum of human insulin obtained by QTOF mass spectrometry, specifically in the m / z range of approximately 964–973. Figure 6B is a magnified view of the m / z range of approximately 967–971. Further details are described in Example 4. [Figure 7] Figure 7 shows a linear plot of the quantification of human insulin in spiked simulated serum standards, measured by high-resolution / high-precision MS of insulin. Further details are described in Example 4. [Figure 8] Figure 8 shows a linear plot of the quantification of human insulin in spiked strip serum standards measured by high-resolution / high-precision MS of insulin. Further details are described in Example 4. [Figure 9] Figure 9 shows a tandem mass spectrometry Q1 scan illustrating the generation of 5+ and 6+ charged human insulin precursor ions. Further details are provided in Example 5. [Figure 10]Figure 10 shows the product ion scan resulting from the fragmentation of 6+ charged human insulin precursor ions. Further details are described in Example 5. [Figure 11] Figure 11 shows the product ion scan resulting from the fragmentation of 5+ charged human insulin precursor ions. Further details are described in Example 5. [Figure 12] Figure 12 shows the relative intensities of selected fragment ions generated by fragmenting 6+ and 5+ human insulin precursor ions with various collision energies. Further details are described in Example 5. [Figure 13] Figure 13 shows a composite spectrum illustrating two possible human insulin A chain precursor ions (in 2+ and 3+ charged states) and two possible human insulin B chain precursor ions (in 3+ and 4+ charged states). Further details are provided in Example 6. [Figure 14] Figure 14 shows a tandem mass spectrometry Q1 scan illustrating the generation of human insulin B chain precursor ions capable of 13+, 4+, and 5+ charged states. Further details are provided in Example 6. [Figure 15] Figure 15 shows the relative intensities of selected fragment ions generated by fragmenting 3+ human insulin A chain precursor ions with various collision energies. Further details are described in Example 6. [Figure 16] Figure 16 shows the product ion scan resulting from the fragmentation of 4+ charged human insulin B chain precursor ions. Further details are described in Example 6. [Figure 17] Figure 17 shows the product ion scan resulting from the fragmentation of 3+ charged human insulin B-chain precursor ions. Further details are described in Example 10. [Figure 18] Figure 18 shows the relative intensities of selected fragment ions generated by fragmenting 3+ human insulin B chain precursor ions with various collision energies. Further details are described in Example 10. [Figure 19]Figure 19 shows a plot used to evaluate the LLOQ of human insulin in patient serum samples by tandem mass spectrometry of human insulin B chains. Further details are described in Example 12. [Figure 20] Figure 20 shows a plot of linearity in the quantification of human insulin in spiked strip serum samples, measured by tandem mass spectrometry of human insulin B chains. Further details are described in Example 13. [Modes for carrying out the invention]

[0087] Detailed description of the invention This document describes a method for determining the amount of insulin in a sample. More specifically, it describes how to determine the amount of insulin in a sample. This paper describes a mass spectrometry method for detecting and quantifying [a substance]. The method involves purifying the selected analyte. Solid-phase extraction (SPE) and / or liquid chromatography (LC) to be performed by mass fraction This method is used in combination with MS (mass spectroscopy) to detect and determine insulin in the sample. An assay system for quantification may be provided. A preferred embodiment is an automated insulin quantification system. It is particularly well-suited for application in large-scale clinical facilities for assays.

[0088] A suitable test sample for use in the method of the present invention is a test sample that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample, i.e., an animal, a cell culture. The sample is obtained from biological sources such as nutrient and organ cultures. In a specific preferred embodiment... The samples are obtained from mammals such as dogs, cats, and horses. Particularly preferred mammals The sample is a primate, most preferably a male or female human. Preferred samples are blood, plasma, and blood It contains body fluids or tissue samples such as clear water, saliva, cerebrospinal fluid, preferably plasma and serum. Such samples are, for example, used in patients, i.e., for the diagnosis, prognosis, or treatment of a disease or condition. It can be obtained from living men or women who report to a bed facility. The sample contains biological samples. In one embodiment, the method involves the insulin in the sample when the sample is obtained from a biological source. It can be used to determine the quantity.

[0089] This invention also intends to provide a kit for insulin quantitative assay. A kit for use may include a kit containing the compositions provided herein. For example, The packaging material and a fixed amount of isotope-labeled internal standard sufficient for at least one assay. This may include. Generally, there are concerns about the use of packaged reagents for insulin quantitative assays. This also includes instruction manuals recorded in a tangible form (for example, on paper or electronic media).

[0090] The calibration and QC pool used in embodiments of the present invention is preferably such that insulin is essentially Under the condition that it does not exist, we use a matrix similar to the target sample matrix to perform the analysis. To manufacture.

[0091] Preparation of samples for mass spectrometry In preparation for mass spectrometry, for example, liquid chromatography, filtration, centrifugation, Thin-layer chromatography (TLC), electrophoresis including capillary electrophoresis, immunoaffinity - Affinity separation including separation, extraction methods including ethyl acetate or methanol extraction and This includes the use of chaotropic agents or any combination of the above or similar agents. In addition, insulin is extracted from one or more other components in a sample by various methods known in the art. It can be concentrated compared to [another method].

[0092] One method of purifying a sample that can be used before mass spectrometry is to ensure that the analyte is colorless. a sample is added to a solid phase extraction (SPE) column under conditions where it is reversibly retained by the packing material and one or more other substances are not retained. In this technique, when the analyte of interest is retained by the column, a first mobile phase condition can be used, and if the non-retained substances are washed away, a second mobile phase condition can then be used to remove the retained substance from the column. and when the analyte of interest is retained by the column, a first mobile phase condition can be used, and if the non-retained substances are washed away, a second mobile phase condition can then be used to remove the retained substance from the column. and when the analyte of interest is retained by the column, a first mobile phase condition can be used, and if the non-retained substances are washed away, a second mobile phase condition can then be used to remove the retained substance from the column. substances are washed away, a second mobile phase condition can then be used to remove the retained substance from the column. and if the non-retained substances are washed away, a second mobile phase condition can then be used to remove the retained substance from the column.

[0093] In some embodiments, insulin in a sample can be reversibly retained on an SPE column containing a packing material with an alkyl-bonded surface. For example, in some embodiments, a C-8 online SPE column (such as the Oasi s HLB online SPE column / cartridge (2.1 mm x 20 mm) manufactured by Phenomenex, Inc. or equivalents) can be used to concentrate insulin prior to mass spectrometry. In some embodiments, the use of the SPE column is carried out using 0.2% aqueous formic acid for HPLC as the wash solution and 0.2% formic acid in acetonitrile as the elution solution. In some embodiments, insulin in a sample can be reversibly retained on an SPE column containing a packing material with an alkyl-bonded surface. For example, in some embodiments, a C-8 online SPE column (such as the Oasi s HLB online SPE column / cartridge (2.1 mm x 20 mm) manufactured by Phenomenex, Inc. or equivalents) can be used to concentrate insulin prior to mass spectrometry. In some embodiments, the use of the SPE column is carried out using 0.2% aqueous formic acid for HPLC as the wash solution and 0.2% formic acid in acetonitrile as the elution solution. In some embodiments, insulin in a sample can be reversibly retained on an SPE column containing a packing material with an alkyl-bonded surface. For example, in some embodiments, a C-8 online SPE column (such as the Oasi s HLB online SPE column / cartridge (2.1 mm x 20 mm) manufactured by Phenomenex, Inc. or equivalents) can be used to concentrate insulin prior to mass spectrometry. In some embodiments, the use of the SPE column is carried out using 0.2% aqueous formic acid for HPLC as the wash solution and 0.2% formic acid in acetonitrile as the elution solution. In some embodiments, insulin in a sample can be reversibly retained on an SPE column containing a packing material with an alkyl-bonded surface. For example, in some embodiments, a C-8 online SPE column (such as the Oasi s HLB online SPE column / cartridge (2.1 mm x 20 mm) manufactured by Phenomenex, Inc. or equivalents) can be used to concentrate insulin prior to mass spectrometry. In some embodiments, the use of the SPE column is carried out using 0.2% aqueous formic acid for HPLC as the wash solution and 0.2% formic acid in acetonitrile as the elution solution. In some embodiments, insulin in a sample can be reversibly retained on an SPE column containing a packing material with an alkyl-bonded surface. For example, in some embodiments, a C-8 online SPE column (such as the Oasi s HLB online SPE column / cartridge (2.1 mm x 20 mm) manufactured by Phenomenex, Inc. or equivalents) can be used to concentrate insulin prior to mass spectrometry. In some embodiments, the use of the SPE column is carried out using 0.2% aqueous formic acid for HPLC as the wash solution and 0.2% formic acid in acetonitrile as the elution solution. In some embodiments, insulin in a sample can be reversibly retained on an SPE column containing a packing material with an alkyl-bonded surface. For example, in some embodiments, a C-8 online SPE column (such as the Oasi s HLB online SPE column / cartridge (2.1 mm x 20 mm) manufactured by Phenomenex, Inc. or equivalents) can be used to concentrate insulin prior to mass spectrometry. In some embodiments, the use of the SPE column is carried out using 0.2% aqueous formic acid for HPLC as the wash solution and 0.2% formic acid in acetonitrile as the elution solution. In some embodiments, insulin in a sample can be reversibly retained on an SPE column containing a packing material with an alkyl-bonded surface. For example, in some embodiments, a C-8 online SPE column (such as the Oasi s HLB online SPE column / cartridge (2.1 mm x 20 mm) manufactured by Phenomenex, Inc. or equivalents) can be used to concentrate insulin prior to mass spectrometry. In some embodiments, the use of the SPE column is carried out using 0.2% aqueous formic acid for HPLC as the wash solution and 0.2% formic acid in acetonitrile as the elution solution.

[0094] In some embodiments, insulin is not purified by immunoaffinity techniques. Some of these embodiments utilize an SPE column. In these embodiments, the SPE column is not an immunoaffinity column. In some embodiments, insulin is not purified by immunoaffinity techniques. Some of these embodiments utilize an SPE column. In these embodiments, the SPE column is not an immunoaffinity column. In some embodiments, insulin is not purified by immunoaffinity techniques. Some of these embodiments utilize an SPE column. In these embodiments, the SPE column is not an immunoaffinity column.

[0095] In other embodiments, the method includes the step of immuno-purifying insulin prior to mass spectrometry. The immuno-purification step can be carried out using any of the immuno-purification methods well known in the art. Often, immuno-purification procedures are carried out on a solid support, such as a column, well, tube. In other embodiments, the method includes the step of immuno-purifying insulin prior to mass spectrometry. The immuno-purification step can be carried out using any of the immuno-purification methods well known in the art. Often, immuno-purification procedures are carried out on a solid support, such as a column, well, tube. In other embodiments, the method includes the step of immuno-purifying insulin prior to mass spectrometry. The immuno-purification step can be carried out using any of the immuno-purification methods well known in the art. Often, immuno-purification procedures are carried out on a solid support, such as a column, well, tube. , conjugated, immobilized or otherwise bound to capsules, particles or similar objects This method utilizes antibodies bound in this state. Generally, immunopurification is performed by (1) the analyte The sample containing the target analyte is incubated with the antibody so that it binds to the antibody. (1) a step of performing one or more washing steps, and (2) a step of removing the antibody The process includes the step of eluting the analyte.

[0096] In certain embodiments, the incubation step of immunopurification involves free in solution The procedure is carried out using antibodies, which are then bound or attached to the solid surface before the washing step. In certain embodiments, this refers to a primary antibody which is an anti-insulin antibody and a primary anti-insulin antibody. This is achieved by using a secondary antibody conjugated to a solid surface having affinity for the srin antibody. This can be done. In an alternative embodiment, the primary antibody is solidified before the incubation step. To bond to a surface.

[0097] Suitable solid carriers can be used without limitation in tubes, slides, columns, beads, capsules, and granules. This includes children, gels and similar materials. In some preferred embodiments, the solid carrier is, for example, For example, multi-well plates such as 96-well plates, 384-well plates, and similar types. In some embodiments, the solid carrier is Sepharose or agarose. It is a bead or gel. Antibodies (e.g., insulin antibodies or secondary antibodies) are bound to a solid carrier. Many methods known in the art can be used to attach, immobilize, or connect, for example, sharing These include bonding or non-covalent adsorption, affinity bonding, ionic bonding, etc. In some embodiments... Antibodies are linked using CNBr. For example, antibodies are linked using CNBr-activated cephalometric radiopharmaceuticals. It can be linked to a case. In another embodiment, the antibody is protein A, prote Solid carriers via antibody-binding proteins such as In G, Protein A / G, or Protein L Attach it to the surface.

[0098] The washing step in immunopurification generally involves removing insulin from a solid carrier containing anti-insulin antibodies. It is necessary to wash the solid carrier so that it remains bound. Elution of immunopurification TEP generally requires the addition of a solution that disrupts the binding of insulin to anti-insulin antibodies. This is essential. Specific examples of elution solutions include organic solutions, salt solutions, and high or low pH solutions.

[0099] Other methods of purifying a sample that can be used before mass spectrometry include liquid chromatography. (LC) is a liquid chromatography technique in which the target analytes are one or more. Under mobile phase conditions that elute at a different rate compared to other substances, the sample was subjected to chromatographic analysis. By adding it to the sample, the analyte can be purified. Such a procedure is used for the sample. It may increase the amount of one or more analytes compared to one or more other components.

[0100] Certain liquid chromatography methods, including HPLC, rely on relatively slow laminar flow techniques. Traditional HPLC analysis involves the laminar flow of the sample through the column, which separates the target from the sample. It relies on column packing, which is the basis for the separation of precipitates. Those skilled in the art will know that in such a column Understanding that separation is a distribution process, HPL is suitable for use with C peptides. LC, instruments, and columns containing C can be selected. Chromatographic analysis columns are: Generally, a medium (i.e., a medium) that facilitates the separation (i.e., fractionation) of compound components. It contains a filler. The medium may contain fine particles. The particles generally contain a binding surface that interacts with various compound components to promote the separation of the compound components. One suitable binding surface is a hydrophobic binding surface such as an alkyl binding or cyano binding surface. The alkyl binding surface may contain a C-4, C-8, C-12 or C-18 linked alkyl group. In some embodiments the chromatographic analysis column is a monolithic C-18 column. The chromatographic analysis column includes an inlet for receiving the sample and an outlet for discharging the eluate containing the fractionated sample. The sample can be supplied directly to the inlet or from an SPE column such as an on-line SPE column or a TFLC column. In some embodiments, an on-line filter can be used before the SPE column and / or HPLC column to remove particles and phospholipids in the sample before the sample reaches the SPE and / or TFLC and / or HPLC column. phospholipids in the sample before the sample reaches the SPE and / or TFLC and / or HPLC column. In one embodiment, the sample can be added to the LC column at the inlet, eluted with a solvent or solvent mixture, and discharged at the outlet. Various solvent modes for eluting the analyte(s) of interest can be selected. For example, liquid chromatography can be performed using a gradient mode, an isocratic mode or a polymorphic (i.e., mixed) mode. During chromatography, the separation of substances is affected by variables such as the eluent (also known as the "mobile phase"), the elution mode, the gradient conditions, the choice of temperature, etc. In some embodiments, insulin in the sample is concentrated by HPLC. This H

[0101] In one embodiment, the sample can be added to the LC column at the inlet, eluted with a solvent or solvent mixture, and discharged at the outlet. The analyte(s) of interest can be eluted. Various solvent modes for eluting the analyte(s) of interest can be selected. For example, liquid chromatography can be performed using a gradient mode, an isocratic mode or a polymorphic (i.e., mixed) mode. During chromatography, the separation of substances is affected by variables such as the eluent (also known as the "mobile phase"), the elution mode, the gradient conditions, the choice of temperature, etc. can be performed using a gradient mode, an isocratic mode or a polymorphic (i.e., mixed) mode. During chromatography, the separation of substances is affected by variables such as the eluent (also known as the "mobile phase"), the elution mode, the gradient conditions, the choice of temperature, etc. During chromatography, the separation of substances is affected by variables such as the eluent (also known as the "mobile phase"), the elution mode, the gradient conditions, the choice of temperature, etc. also known), the elution mode, the gradient conditions, the choice of temperature, etc.

[0102] In some embodiments, insulin in the sample is concentrated by HPLC. This H ​PLC is a monolithic C-18 column chromatography system, for example, Phenomen ex Inc.'s Onyx Monolith C-18 column (50x2.0mm) or equivalent It can be carried out using this method. In a particular embodiment, HPLC is performed using H as solvent A. Using 0.2% aqueous formic acid for PLC and 0.2% formic acid in acetonitrile as solvent B, To administer.

[0103] Careful selection of valves and fittings for piping eliminates the need for manual steps, resulting in a single chrominance. Two or more chromatography columns are used to allow a substance to pass from one column to the next. Rams can be connected as needed. In a preferred embodiment, valves and piping selection The selection is performed by a computer pre-programmed to carry out the necessary steps. Controlled. Most preferably, the chromatography system is such an online formula It is also connected to a detection system, such as an MS system. Therefore, the operator can sample The tray can be attached to the autosampler, and the rest of the operation is computer controlled. The process is carried out under these conditions, resulting in the purification and analysis of all selected samples.

[0104] In some embodiments, TFLC is used for the purification of insulin before mass spectrometry. It is possible to have a TFLC column that captures the analyte. The sample can be extracted using this method. Then, the analyte is eluted and analyzed online via HP. Transfer to an LC column. For example, sample extraction is performed using a TFLC column containing large particle size (50 μm) packing material. This can be achieved using an extraction cartridge. The sample eluted from this column is by mass. The sample can be transferred online to an analytical HPLC column for further purification before analysis. The steps involved in these chromatography procedures can be linked together automatically. Therefore, the need for operator involvement during the purification of the analyte can be minimized. This can lead to significant time and cost savings, and potentially eliminate opportunities for operator error.

[0105] In some embodiments, one or more of the above-described purification techniques are used for the simultaneous processing of multiple samples. To make this possible, it can be used in parallel for insulin purification. In that embodiment, the purification technique used is immunoaffinity chromatography. Excluding immunopurification techniques such as those mentioned above.

[0106] Detection and quantification of insulin by mass spectrometry Mass spectrometry is used to ionize fractionated samples and generate charged molecules for further analysis. This is performed using a mass spectrometer that includes an ion source. In various embodiments, insulin It can be ionized by methods known to those skilled in the art. For example, the ionization of insulin These include electron ionization, chemical ionization, electrospray ionization (ESI), and photon ionization. Chemical ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), Laser diode thermal desorption (LDTD), fast atomic bombardment (FAB), liquid secondary ionization (L SI), Matrix-assisted laser desorption / ionization (MALDI), Field ionization, Field desorption Desorption, thermospray / plasma spray ionization, surface-enhancing laser desorption ionization This is done by ionization (SELDI), inductively coupled plasma (ICP), and particle beam ionization. This is possible. Those skilled in the art will know that the selection of the ionization method depends on the analyte being measured, the type of sample, and the detector. It can be determined based on the type, the selection of positive versus negative modes, etc. Therefore, even if insulin is ionized in positive mode, it is also ionized in negative mode. It may be done. In a preferred embodiment, insulin is positively ionized by ESI. It is ionized in 2D mode.

[0107] In mass spectrometry, generally, after ionizing the sample, the resulting positive or By analyzing negatively charged ions, the mass-to-charge ratio (m / z) can be determined. Various analyzers for determining time of flight include quadrupole analyzers, ion trap analyzers, and time of flight analyzers. Type analyzers, Fourier transform ion cyclotron resonance mass spectrometers, and orbitrap type analyzers This includes the calculation. Some specific examples of ion trap methods include Bartolucci et al., Rapid Comm This is described in un. Mass Spectrom., 2000, Vol. 14, pp. 967-963.

[0108] Ions can be detected using several detection modes. For example, selected ions This means that it can be detected using Selective Ion Monitoring Mode (SIM). or alternatively, collision-induced dissociation or neutral loss resulting from Mass transitions can be monitored, for example, using multiple reaction monitoring (MRM) or selective reaction monitoring (S). It can be monitored by RM). In some embodiments, the mass-to-charge ratio is 4 Determined using a multipolar analyzer. A "quadrupole" or "quadrupole ion trap" instrument is used to determine odor. In a vibrating high-frequency electric field, ions are affected by the DC potential applied between the electrodes and the vibration of the RF signal. It is subjected to a force proportional to its width and mass / charge ratio. The voltage and amplitude have a specific mass / charge ratio. Only certain ions traverse the quadrupole, while all other ions are selectively deflected. Therefore, the quadrupole instrument has a "mass filter" and " It can function as both a "mass detector" and a "mass detector."

[0109] When ions collide with the detector, they produce pulses of electrons that are converted into digital signals. It occurs. The acquired data is transferred to a computer, and the computer processes the collected data. The ON count is plotted against time. The resulting mass chromatogram is obtained using traditional HP The chromatogram is similar to that obtained by LC-MS. Peaks corresponding to specific ions. The area below or the amplitude of such a peak can be measured and correlated with the amount of the analyte being examined. In certain embodiments, fragment ions (one or more) and / or precursor ions The amount of insulin is determined by measuring the area under the curve or amplitude of the ON peak. The relative abundance of ion is the relative abundance of one or more internal or external molecular standards. Using a calibration standard curve based on the peak of the initial analyte, it is possible to convert it to the absolute amount of the initial analyte. ru.

[0110] The resolution of MS techniques using a specific mass spectrometer is referred to as "tandem mass spectrometry" or "MS / MS". This can be improved by ". In this technology, the precursor iodine obtained from the target molecule The parent ion (also called the parent ion) can be filtered by an MS instrument, and the precursor ion These are then fragmented and analyzed in a second MS procedure, one or more fragments. It produces a precursor ion (also called a daughter ion or product ion). Note on the precursor ion. Through careful selection, only ions generated by the specific analyte are collected in the fragmentation chamber. It passes through a barrier, where it collides with atoms of an inert gas, generating fragment ions. Both the precursor and fragment ions are re-evolved under a series of predetermined ionization / fragmentation conditions. Because it generates data with high fidelity, MS / MS technology can be an extremely powerful analytical tool. For example, The combination of filtration / fragmentation removes interfering substances. It can be used for this purpose and may be particularly useful for complex samples such as biological samples. In terms of application methods, mass spectrometers including multiple quadrupole analyzers (triple quadrupole analyzers) Tandem mass spectrometry is performed using (etc.).

[0111] In certain embodiments using MS / MS technology, precursor ions are further fragmented. For subsequent detection, the precursor ion was isolated and subjected to collision-activated dissociation (CAD). Fragment ions are generated from this. In CAD, the precursor ions are in contact with the inert gas. Energy is gained through the thrust, and then fragmentation occurs through a process called "single-molecule decomposition". This is because the increase in vibrational energy allows for the breaking of specific bonds within the ion, so Energy must be stored in the precursor ions.

[0112] In some embodiments, insulin in the sample is analyzed using MS / MS as follows: It is detected and / or quantified. First, the sample is placed in an SPE, then in a liquid chromatograph. Preferably, by subjecting the sample to HPLC, the insulin in the sample is concentrated, and the chromatograph The flow of liquid solvent from the tograph analysis column to the heated nebulizer interface of the MS / MS analyzer - The solvent / analyte mixture enters the interface and vaporizes in the heated charged tube. It is converted into gas. In these processes, the analyte (i.e., insulin) is ionized. The ions, for example, precursor ions, pass through the opening of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are based on the mass-to-charge ratio (m / z) of the ions. Selection (i.e., the "precursor" and "fragment" ions in Q1 and Q3, respectively) It is a mass filter that enables selection. Quadrupole 2 (Q2) allows ions to fragment It is a collision cell that is transformed. The first quadrupole (Q1) of the mass spectrometer is insulin io Select a molecule with the correct m / z. The precursor ion with the correct m / z is the collision ion. Although passed through the chamber (Q2), unwanted ions with other m / z values ​​are located on the sides of the quadrupole. They collide and are removed. The precursor ions that enter Q2 collide with neutral gas molecules (such as argon molecules). They collide and become fragments. The generated fragment ions are passed through quadrupole 3 (Q3). Here, fragment ions are selected for detection.

[0113] Insulin ionization involves polyvalent precursor ions (such as 4+, 5+, 6+ precursor ions). This can result in ionization conditions, particularly the p of buffers used in electrospray technology. H significantly affects the identity and quantity of the insulin precursor ions produced. For example, acid Under sexual conditions, positive electrospray ionization is primarily performed by 1162 each. 5+ and 6+ valent insulin precursors with m / z values ​​of 0.5±0.5 and 968.5±0.5 It can generate ON. However, under basic conditions, positive electrospray ions The chemistry is primarily based on m / z values ​​of 1453.75±0.5 and 1162.94±0.5, respectively. This method can generate 4+ and 5+ valent insulin precursor ions having the properties of acid or base. Acidic conditions, preferably, can be utilized.

[0114] This method uses either a positive or negative ion mode, preferably a positive ion mode. This may include MS / MS performed in a galvanic. In certain embodiments, electrospray grading may be used. The cinder is acidic, and Q1 is approximately 1162.5 ± 0.5 or 968.5 ± 0.5 m / z Select an insulin precursor ion that has any of these insulin precursor ions. Fragmentation results in a m / z of approximately 226.21±0.5 and / or 135.6±0.5. A fragment ion having the following is generated. Therefore, Q1 is approximately 1162.5 ± 0.5 and one or more selected from the group consisting of ions having an m / z of 968.5 ± 0.5 In an embodiment in which an insulin precursor ion is selected, Q3 is approximately 226.21 ± 0.5 and one or more furcations selected from the group of ions having a m / z of 135.6 ± 0.5 A single precursor ion can be selected. In certain embodiments, a single fraction from a single precursor ion is obtained. The relative abundance of the precursor ion can be measured. Alternatively, two precursor ions can be obtained from a single precursor ion. The relative abundance of one or more fragment ions can be measured. In these embodiments In this process, the relative abundance of each fragment ion is subjected to any known mathematical treatment, and the sample is... The initial insulin dose can be quantitatively evaluated. In other embodiments, two or more Measure one or more fragment ions from the precursor ion and use them as described above. The initial insulin content in the sample can be quantitatively evaluated.

[0115] Alternative mode for operating tandem mass spectrometers that can be used in specific embodiments The method includes product ion scanning and precursor ion scanning. These operating modes For an explanation of the term, see, for example, E. Michael Thurman et al., Chromatographic-Mass Spectrometer tric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (Ama See deo R. Fernandez-Alba (eds.), Elsevier 2005) (387).

[0116] In other embodiments, a high-resolution / high-precision mass spectrometer is used by an insulin spectrometry method according to the present invention. It can be used for quantitative analysis of [the subject]. In order to achieve an acceptable accuracy of quantitative results, The mass spectrometer measures the target ion with an accuracy of approximately 50 ppm or less, up to 10,000. It must be able to exhibit a resolution (FWHM) or higher. Preferably, mass The analyzer has an accuracy of approximately 5 ppm or less and a resolution of 18,000 or higher (FW). HM), for example, resolution of 20,000 or more (FWHM) and about 3 ppm or less. Accuracy such as the following, for example, a resolution of 25,000 or higher (FWHM) and approximately 3pp. This indicates accuracy of m or less. It shows the required level of performance for insulin ions. Three specific examples of analyzers that can perform this are an orbital mass spectrometer, a specific TO These are F-mass spectrometers and Fourier transform ion cyclotron resonance mass spectrometers.

[0117] Elements found in biologically active molecules such as carbon, oxygen, and nitrogen exist in many isotopes and are ethereal. It exists naturally. For example, most carbon is 12It exists as C, but is not present in all natural forms. Approximately 1% of the carbon present is 13 It exists as C. Therefore, at least one carbon atom A portion of naturally occurring molecules containing at least one 13 Contains a carbon atom. This occurs when naturally occurring elemental isotopes are included in a molecule, resulting in the formation of multiple molecular isotopes. The difference in mass between molecular isotopes is at least one atomic mass unit (amu). This is because, This is because elemental isotopes differ by at least one neutron (mass of one neutron ≈ 1 amu ). When molecular isotopes are ionized to a highly charged state, detection by mass spectrometry is possible using the mass-to-charge ratio (m). Because it is based on ( / z), the mass differences between isotopes can be difficult to distinguish. For example, two isotopes with different masses, both of which are ionized to the 5+ state, are These show a difference of about 0.2 m / z. High-resolution / high-precision mass spectrometry is highly effective for highly charged atoms. Identifying ON isotopes (such as ions with ±2, ±3, ±4, ±5, or higher charges). It is possible.

[0118] Because they are naturally occurring elemental isotopes, all molecular ions (a sufficiently sensitive mass spectrometer) When analyzed using different instruments, each may produce a spectral peak that can be detected individually. Multiple isotopes generally exist. The m / z ratio and relative abundance of multiple isotopes are determined by the molecule. It collectively contains the isotopic signatures of ions. In some embodiments, two or more components The m / z ratio and relative abundance of the isotopes are useful for confirming the identity of the molecular ion under consideration. It can be used. In some embodiments, the mass spectrometry of one or more isotopes A meter is used to quantify molecular ions. In some related embodiments, one isotope A single mass spectrometry peak is used to quantify molecular ions. In other related embodiments, multiple Molecular ions are quantified using isotopic peaks. In these latter embodiments, multiple Isotope peaks can be subjected to any appropriate mathematical processing. This is known in the art, and is the sum of the areas under multiple peaks or the average of the response due to multiple peaks. This includes, but is not limited to, the multiple isotopes of 5+ and 6+ insulin ions. Spectral examples are shown in Figures 4-6. As can be seen in Figures 5A-B, 5+ The peaks for various isotopes of the insulin ion are approximately 1161.72, 1161.92, and 11 62.12, 1162.32, 1162.52, 1162.72, 1162.92, 11 This is observed in 63.12 and 1163.32. As can be seen in Figures 6A and 6B, 6+ The peaks for various isotopes of the thrin ion are approximately 968.28, 968.45, and 968.62. , 968.79, 968.95, 969.12, 969.28, 968.45 and 969 It is found at 0.61. However, the exact mass observed for the isotopic variants of the ion is Note that these values ​​may vary slightly due to equipment fluctuations.

[0119] In some embodiments, one is used to qualitatively evaluate the amount of insulin in a sample. Alternatively, the relative abundance of multiple ions is measured using a high-resolution / high-precision mass spectrometer. In this embodiment, one or more ions are measured by high-resolution / high-precision mass spectrometry. These are polyvalent insulin ions. These polyvalent ions are approximately 1453 ± 0.8 (i.e.) , one or more single isotope peaks of the 4+ ion) and / or 1162±1 (i.e., (One or more single isotope peaks of the 5+ ion) and / or 968.8±1.5 (that is, (One or more single isotope peaks of the 6+ ion) with m / z within the range of one or It may contain multiple ions.

[0120] The use of high-resolution orbital analyzers is beneficial for the qualitative and quantitative analysis of various analytes. It was reported. For example, U.S. Patent Application Publication No. 2008 / 0118932 (November 2007) (Filing dated 9th of the month), Bredehoft et al., Rapid Commun. Mass Spectrom., 2008, Vol. 22, pp. 477-485. Page, Le Breton et al., Rapid Commun. Mass Spectrom., 2008, Vol. 22, pp. 3130-3136, Thevis Thomas et al., J. Mass Spectrom. Reviews, 2008, Vol. 27, pp. 35-50. , 2008, Vol. 43, pp. 908-9015, Schenk et al., BMC Medical Genomics, 2008, Vol. 1, p. 41 and See Olsen et al., Nature Methods, 2007, Vol. 4, pp. 709-7012.

[0121] The results of the analyte assay are obtained by many methods known in the art, which determine the initial analyte in the sample. This can be related to the amount of precipitate. For example, the sampling and analysis parameters can be carefully considered. If closely controlled, the relative abundance of a given ion is equal to the initial molecular weight of that relative abundance. It can be compared with a table that converts to absolute quantities. Alternatively, an external standard can be used along with the sample. It is possible to create standard curves based on ions obtained from those standards. Using a standard curve, the relative abundance of a given ion can be converted to the absolute amount of the initial molecule. In a particular preferred embodiment, the amount of insulin is calculated using an internal standard. To create a standard curve. Methods for creating and using such a standard curve are available in the art. This is knowledge, and those skilled in the art can select appropriate internal standards. For example, preferred embodiments In this, one or more forms of isotope-labeled insulin can be used as an internal standard. Many other methods for relating the amount of ions to the amount of the initial molecule are well known to those skilled in the art.

[0122] As used herein, "isotope labeling" refers to unlabeled labeling when analyzed by mass spectrometry. This results in a mass shift of the labeled molecule compared to the original molecule. An example of a suitable label is deuterium ( 2 H ), 13 C and 15 Examples include N. One or more isotope labels are used in the molecule. It can be incorporated at multiple positions, and one or more types of isotopic labels can be placed on the same isotopically labeled molecule. It can be used for this purpose.

[0123] Determination of insulin by quantification of unmodified insulin A and / or B chains by mass spectrometry. In another embodiment, to obtain the insulin component chain before mass spectrometry, It can be subjected to chemical treatment. The A and B chains of insulin undergo disulfide reduction. It can be separated by any chemical treatment known in the art that can induce it. For example, if insulin is treated with TCEP (tris(2-carboxyethyl)phosphine) This reduces the disulfide crosslinks of insulin, allowing for the separation of the A and B chains.

[0124] Chains A and B are then used to purify one or more of the above-mentioned purified insulin chains. It can be used for stepping. In a preferred embodiment, the A chain and / or B chain are divided by mass The sample is purified by HPLC before precipitation.

[0125] Once purified, the A chain and / or B chain are then subjected to an ionization source. (In the case of insulin) Similarly, those skilled in the art will know that the selection of the ionization method depends on the analyte being measured, the type of sample, and the detector. It can be determined based on the type, the selection of positive versus negative modes, etc. It can be understood that insulin A and B chains can be ionized in positive or negative mode. In a preferred embodiment, insulin A chain and / or B chain are positively charged by ESI. It is ionized in live mode.

[0126] The ionization of insulin A chain is due to polyvalent A chain precursor ions (such as 2+, 3+, etc.). This can lead to positive electrospray iodine of insulin A chain molecules. The ionization has m / z values ​​of 1192.0±0.5 and 795.0±0.5 respectively, and 2+ It can generate 3+ valent A-chain precursor ions. Similar to insulin, the ions of the insulin A chain. The identity and quantity of the multivalent species produced by the ionization process are influenced by the ionization conditions used. In a preferred embodiment, the insulin A chain is ionized under acidic conditions.

[0127] In an embodiment where insulin A chains are subjected to tandem mass spectrometry, Q1 is approximately 1192. One or more insulin A chain precursors having m / z values ​​of 0±0.5 and 795.0±0.5 Ions can be selected. Fragmentation of any of these insulin A chain precursor ions Therefore, approximately 513.0±0.5, 399.0±0.5, 236.0±0.5, and 133.0 Fragment ions with a m / z of ±0.5 can be generated. Therefore, Q1 is approximately 11 Selected from the group consisting of ions having m / z values ​​of 92.0±0.5 and 795.0±0.5 In an embodiment in which one or more insulin A chain precursor ions are selected, Q3 is approximately 513.0±0.5, 399.0±0.5, 236.0±0.5, and 133.0±0.5 Select one or more fragment ions from the group of ions having m / z. Obtain. In certain embodiments, the relative presence of a single fragment ion from a single precursor ion. The amount can be measured. Alternatively, two or more fragments from a single precursor ion can be measured. The relative abundance of ON can be measured. In these embodiments, each fragment The relative abundance of ions is subjected to any known mathematical treatment to quantify the initial insulin in the sample. It can be evaluated objectively. In other embodiments, one or more precursor ions from two or more precursor ions It measures multiple fragment ions and uses them as described above to determine the first insulin in the sample. This can be evaluated qualitatively.

[0128] Similarly, the ionization of insulin B chains involves the introduction of polyvalent B chain precursor ions (3+, 4+, 5+, etc.). This can result in (such as ion removal). For example, the positive electre of the insulin B chain molecule The Loss Spray ionization values ​​were 1144.2±0.5, 858.3±0.5, and 686, respectively. It is possible to generate 3+, 4+, and 5+ valent B-chain precursor ions having an m / z of 0.8 ± 0.5. Similar to insulin, the identity and quantity of the polyvalent species produced by the ionization of the insulin B chain are It is affected by the ionization conditions used. In a preferred embodiment, insulin B The chain is ionized under acidic conditions.

[0129] In an embodiment where insulin B chains are subjected to tandem mass spectrometry, Q1 is approximately 1144. One or more m / z values ​​having 2±0.5, 858.3±0.5, and 686.8±0.5 The insulin B chain precursor ions can be selected from these three insulin B chain precursor ions. Due to fragmentation, the values ​​are approximately 825.4±0.5, 768.5±0.5, and 753.2±0. 5. Fragment ions having m / z values ​​of 345.0±0.5 and 226.2±0.5 It can be generated. Therefore, Q1 is approximately 1144.2±0.5, 858.3±0.5 and 68 One or more instruments selected from the group consisting of ions having a m / z of 6.8 ± 0.5 In an embodiment where a phosphorus B chain precursor ion is selected, Q3 is approximately 825.4 ± 0.5, 76 m Selected from the group of ions having / z, preferably about 345.0 ± 0.5 and 226. One or more fragment ions selected from the group of ions having a m / z of 2 ± 0.5 A single fragment ion can be selected. In certain embodiments, a single fragment ion can be selected from a single precursor ion. The relative abundance of ions can be measured. Alternatively, two or more fractions from a single precursor ion can be measured. The relative abundance of ion can be measured. In these embodiments, each The relative abundance of lgment ions is subjected to any known mathematical treatment, and the first ingredient in the sample is Phosphorus can be quantitatively evaluated. In other embodiments, two or more precursor ions or Measure one or more of their fragment ions and use them as described above to determine the first fragment ions in the sample. Insulin can be evaluated qualitatively.

[0130] Quantitative determination of chemically modified insulin A and / or B chains by mass spectrometry Quantitative determination of In an alternative embodiment, separate insulin A and B chains are ionized and / or purified. It may be subjected to one or more chemical modification steps beforehand. For example, if separated Furthermore, insulin A and B chain molecules completely alkylate their constituent cysteine. It can undergo carbamide methylation. For example, carbamide methylation can occur in insulin A chain. and / or the B chain is reduced with DTT (1,4-dithiothreitol) followed by iodoacetate. This can be achieved by subjecting the insulin A chain to a reaction with toamide. Midmethylation results in a mass increase of approximately 228.08 amu (approximately 57.02 per cysteine). This results in the alkylation of four cysteine ​​molecules in the insulin B chain. Chilling causes a mass increase of approximately 114.04 amu (approximately 57.02 per cysteine). This results in the alkylation of two cysteine ​​molecules.

[0131] Once purified, the chemically modified (e.g., alkylated) A and / or B chains It is used as an ionization source. As with insulin, those skilled in the art will know that the selection of the ionization method is measured The analyte to be analyzed, the type of sample, the type of detector, the selection of positive versus negative mode, etc. Understand that it can be determined based on the alkylated insulin A and B chains. It can be ionized in positive or negative mode. In a preferred embodiment, Furthermore, alkylated insulin A and / or B chains are activated by ESI in a positive mode. It will be turned on.

[0132] Ionization of alkylated insulin A chains involves polyvalent alkylated A chain precursor ions (2+, 3+ This can result in precursor ions such as those found in alkylated insulin A chain molecules. Ditive electrospray ionization yielded values ​​of 1306.0 ± 0.5 and 871.0, respectively. It is possible to generate 2+ and 3+ valent alkylated A-chain precursor ions having a m / z of ±0.5. Similar to insulin, the identity of the polyvalent species produced by the ionization of alkylated insulin A chains. The amount and other factors are affected by the ionization conditions used. In a preferred embodiment, Ionize lycylated insulin A chain under acidic conditions.

[0133] In an embodiment in which alkylated insulin A chains are subjected to tandem mass spectrometry, Q1 is approximately One or more alkyl groups having m / z values ​​of 1306.0±0.5 and 871.0±0.5. Alkylated insulin A chain precursor ions can be selected. These alkylated insulin A chain precursor ions Due to fragmentation of either of the components, approximately 570.0±0.5, 456.0±0.5, 2 Fragment ions with m / z values ​​of 93.0±0.5 and 133.0±0.5 are generated. Therefore, Q1 has m / z values ​​of approximately 1192.0±0.5 and 795.0±0.5. One or more alkylated insulin A chain precursor ions selected from the group consisting of ions In the embodiment where n is selected, Q3 is approximately 570.0±0.5, 456.0±0.5, Selected from the group of ions having m / z values ​​of 293.0±0.5 and 133.0±0.5. One or more fragment ions may be selected. In certain embodiments, a single precursor ion The relative abundance of a single fragment ion from ON can be measured. Alternatively, a single The relative abundance of two or more fragment ions from a precursor ion can be measured. In these embodiments, the relative abundance of each fragment ion is determined by any known mathematical formula. This allows for the quantitative evaluation of the initial insulin in the sample. In other embodiments... In this process, one or more fragment ions from two or more precursor ions are measured, and the above procedure is performed. This method can be used to qualitatively evaluate the initial insulin in a sample.

[0134] Similarly, the ionization of alkylated insulin B chains involves polyvalent alkylated B chain precursor ions (3 This can result in precursor ions such as +, 4+, 5+, etc. For example, alkylated insulin The positive electrospray ionization of the B-chain molecule is 1181.9 ± 0.5, respectively. 3+, 4+, and 5+ valent apheresis having m / z values ​​of 886.9±0.5 and 709.8±0.5 It can generate alkylated B-chain precursor ions, similar to insulin. The identity and quantity of the multivalent species produced by the ionization of the chain are influenced by the ionization conditions used. It is affected. In a preferred embodiment, alkylated insulin B chains are subjected to acidic conditions. To transform into.

[0135] In an embodiment in which alkylated insulin B chains are subjected to tandem mass spectrometry, Q1 is approximately 1 One or more insulin B chain precursor ions can be selected. These three alkylated insulins Fragmentation of the phosphorus B chain precursor ion results in approximately 345.0±0.5 and 226.2±0 A fragment ion with an m / z of 0.5 can be generated. Therefore, Q1 is approximately 1144 From ions with m / z values ​​of 0.2±0.5, 858.3±0.5, and 686.8±0.5 Select one or more alkylated insulin B chain precursor ions from the group. In the application configuration, Q3 has m / z values ​​of approximately 345.0±0.5 and 226.2±0.5. One or more fragment ions can be selected from the group of ions. In the application method, the relative abundance of a single fragment ion from a single precursor ion is measured. This is possible. Alternatively, the relative existence of two or more fragment ions from a single precursor ion. The quantity can be measured. In these embodiments, the relative abundance of each fragment ion The amount is subjected to any known mathematical process to quantitatively evaluate the initial insulin in the sample. This is possible. In other embodiments, one or more flags from two or more precursor ions are used. Menthon ions are measured and used as described above to qualitatively evaluate the initial insulin in the sample. It is possible.

[0136] One or more steps of the above method may be performed using an automated device. Yes, it is possible. In certain embodiments, one or more purification steps are performed online. More preferably, all purification and mass spectrometry steps can be performed online. ru.

[0137] The following examples serve to illustrate the present invention. These examples extend the scope of the method. It is not a restriction. [Examples]

[0138] Example 1: Sample preparation Simulated serum samples containing varying amounts of insulin are used to evaluate a linear response. Pseudoserum (phosphate-buffered saline containing 0.002% protease inhibitor AEBSF (PB) S) Add human insulin to bovine serum albumin (BSA) 40 mg / mL dissolved in buffer solution. It was prepared by spiking at various concentrations (described in Example 4 below).

[0139] Human insulin is also used to evaluate the linearity of the response, as seen in the Golden West B Serum that had been treated twice with activated carbon, obtained from iologicals, Inc., was subjected to various concentrations of [unclear]. We piked it (as described in Example 4 below).

[0140] Example 2: Insulin concentration before mass spectrometry The sample injection of the human insulin spike simulated and serum strips prepared above was performed by Coh With the esive Technologies Aria TX-420 system, The test was conducted using software running OS version 1.6 or later.

[0141] 75 μL of sample was extracted using an online solid-phase extraction (SPE) column, Waters Oasis. It was introduced into an HLB (25 μm, 2.1 × 20 mm) column. The SPE column retains human insulin. While the substance was retained, other serum proteins and macromolecules were released.

[0142] Insulin was eluted from the extraction column with 0.2% formic acid dissolved in 40% acetonitrile. , analytical column (integrated C18 analytical column manufactured by Phenomenex Inc. (particle size 5μ) It was added to a sample measuring 50 × 2.1 mm. Other analytes contained in the sample were used to extract the insulin. To separate phosphorus, an HPLC gradient was applied to the analytical column. Mobile phase A was 0.2% glycerin. In the acidic aqueous solution, mobile phase B was 0.2% formic acid dissolved in acetonitrile. HPLC gradient It started with an organic gradient of 28.5% and increased to 37% in about 90 seconds.

[0143] Next, the insulin-concentrated sample is subjected to high-resolution / high-precision MS or MS for insulin quantification. Submitted to / MS.

[0144] Example 3: Effect of pH on insulin ionization Insulin ionization was performed using an ESI source in positive ion mode. While using this ionization source to generate insulin ions, electrosp The pH of the insulin carrier solution affects the amount and identity of the insulin ions generated. The following was observed.

[0145] Under acidic conditions, polyvalent insulin ions are 968.5 ± 0.50 (6+ ions) and 1 Observed at m / z of 162.3 ± 0.50 (5+ ions). Insulin under acidic conditions. An example of a spectrum collected from the ionization is shown in Figure 3A.

[0146] Under basic conditions, polyvalent insulin ions are 1163.0 ± 0.50 (5+ ions) It was observed at m / z of 1453.8 ± 0.50 (4+ ions). Under basic conditions, An example of a spectrum collected from the ionization of thrin is shown in Figure 3B.

[0147] Sufficient signal is generated under both acidic and basic conditions, enabling quantitative analysis under both conditions. I was able to do it.

[0148] Example 4: Detection and quantification of insulin by high-resolution / high-precision MS High-resolution / high-precision MS is the Agilent TOF MS system (Agilent This was performed using (Technologies, Inc.). This system offers high resolution. We use an MS analyzer that enables high-precision MS. This instrument measures approximately 2 times during insulin measurement. It exhibits a resolution of 5,000 FWHM and a mass accuracy of approximately 1 ppm.

[0149] Ionization was performed in positive ion mode using an ESI source, as described in Example 3. Furthermore, the pH of the electrospray carrier solution depends on the amount of insulin ions generated and the same It affected uniformity. The sample prepared in Example 1 was analyzed by formic acid solution from an SPE column. Since elution occurred, the sample was acidified before ionization. As described in Example 3, polyvalent Insulin ions were observed to be in both 6+ and 5+ charged states.

[0150] The elution of contamination peaks from the strip serum sample was observed. Figure 4A shows QTOF. The approximately 900 to 1200 m / z range of insulin in simulated serum samples generated by a mass spectrometer. A spectral example is shown. Figure 4B shows strip serum generated by a QTOF mass spectrometer. This shows the contamination peaks of the sample matrix. The source of the contamination peaks is different from the insulin peak. Dissolution was observed during the process (data not shown).

[0151] The individual isotopic peaks of the 5+ ion are shown in the m / z range of approximately 1155 to 1176. An example of a high-resolution / high-precision spectrum is shown in Figure 5A. The spectrum is between approximately 1159 and 1166. A magnified view of the portion is shown in Figure 5B. As can be seen in the spectrum, individual isotope peaks Examples include approximately 1161.72, 1161.92, 1162.12, 1162.32, and 1162. 0.52, 1162.72, 1162.92, 1163.12 and 1163.34 m / z It is recognized as such.

[0152] High-resolution isotope peak of 6+ ions showing individual isotope peaks over the m / z range from about 964 to 973 An example of resolution / high-precision spectrum is shown in Fig. 6A. An enlarged view of the spectral portion between about 967 and 971.4 is shown in Fig. 6B. As can be seen in the spectrum, examples of individual isotope peaks are observed at m / z of about 968.28, 968.45, 968.62, 968.79, 968.95, 968 .12, 968.28, 968.45 and 968.61.

[0153] To evaluate the linearity of quantification, data on ions with m / z of 1162.54 ± 0.10 for insulin quantification in spiked simulated and stripped serum samples were collected . Both types of samples showed linearity over a concentration range from about 1.22 ng / mL to 1250 ng / mL. Graphs showing the linearity of data on insulin detection in spiked simulated serum samples and spiked stripped serum samples are shown in Figs. 7 and 8, respectively. When determining the goodness-of-fit test (R ) of insulin by high-resolution / high-precision mass spectrometry quantification, it was 0.9981 for spiked simulated serum and 0.9979 for spiked stripped serum . Example 5: Detection and Quantification of Insulin by Tandem MS 2 MS / MS was performed using a Thermo TSQ Vantage MS / MS system (Thermo Electron Corporation). All are the following software programs made by Thermo Electron, TSQ Van tage V2.0.0 or higher, Xcalibur V2.0 or higher and LCQuan V2 .

[0154] Example 5: Detection and Quantification of Insulin by Tandem MS MS / MS was performed using a Thermo TSQ Vantage MS / MS system (Thermo Electron Corporation). All are the following software programs made by Thermo Electron, TSQ Van tage V2.0.0 or higher, Xcalibur V2.0 or higher and LCQuan V2 which are all made by Thermo Electron. TSQ Van tage V2.0.0 or higher, Xcalibur V2.0 or higher and LCQuan V2 A concentration of 0.5 or higher was used in the examples described herein. The solvent / analyte mixture flowed to the ESI source interface of the MS / MS analyzer. The substance was converted into vapor within the heated tubing at the interface. The analyte was acidified by ESI. It was ionized in positive ion mode under sexual conditions.

[0155] The ions passed through the first quadrupole (Q1). Several possible insulin precursor ions. However, this was observed in Q1. An example of the Q1 spectrum is shown in Figure 9. Fragmentation The test yielded approximately 1163.32 ± 0.50 (5+ ions) and approximately 969.56 ± 0.50 (6 The experiment was conducted with polyvalent insulin precursor ions having m / z (+ ions). The fraction of each precursor ion Examples of product ion scanning from spectroscopy are shown in Figures 10 and 11, respectively. vinegar.

[0156] Collision energy for fragmentation patterns from 5+ and 6+ precursor ions The effects were investigated. Each precursor ion was subjected to collision energies ranging from approximately 7 eV to approximately 80 eV. Fragmentation is performed, and three types of selected fragment ions (approximately 135.9 ± 0.50, 2 The relative intensity at 26.2±0.50 and 345.3±0.50 m / z was monitored. The results of these tests are shown in Figure 12. As shown in Figure 12, the relative strength of the fragment ions The degree changes significantly depending on the collision energy. Each monitored transition is optimized for the optimal collision. The energy values ​​are shown in Table 1.

[0157] [Table 1]

[0158] To quantify insulin by fragmentation of 6+ ions, a quadrupole 2( Q2) The precursor ions entering collide with the argon gas with a collision energy of 30 eV. On fragments are generated, and these ion fragments are further selected by a quadrupole 3( Q3) was passed. The following mass transition occurred: 969.56±0.50 precursor ion fragmentation. Approved by the survey. Fractions collected from Q3 scan (product ion scan) An example of a spectroscopy spectrum is shown in Figure 10.

[0159] Two of the observed transitions were monitored in MRM mode and totaled for quantitative analysis: 96 Precursor ions from 9.56±0.50 to 135.9±0.50 and 226.2±0.50 ( (See Table 2). Quantification was performed by monitoring two mass transitions, Quantitative analysis may be performed by monitoring only one mass transition. Conversely, the above-mentioned mo To replace or increase any of the monitored transitions in any combination, additional mass transitions Select a transfer (for example, including any other fragment ions recognized in Figure 10). It is possible. Similarly, quantification was performed with a collision energy of 30 eV, but sufficient ionization Any collision energy that produces a gunal may be used, and the fragment io may be monitored. It may depend on the identity of n (singular or plural). For example, the two fragments shown above For toions, the collision energy is in the range of approximately 20 to 50 eV, for example, approximately 25 to 4 The range may be 0 eV, for example, approximately 28 to 32 eV.

[0160] To quantify insulin by fragmentation of 5+ ions, a quadrupole 2( Q2) The precursor ions entering collide with the argon gas with a collision energy of 49 eV. On fragments are generated, and these ion fragments are further selected by a quadrupole 3( Q3) was passed. The following mass transition occurred in the 1163.32±0.50 precursor ion fragment. Observed at the station. Data collected from Q3 scan (product ion scan) An example of a lagmentation spectrum is shown in Figure 11.

[0161] Two of the observed transitions were monitored in MRM mode and totaled for quantitative analysis: 11 Precursor ions ranging from 63.32±0.50 to 135.9±0.50 and 226.2±0.50 (See Table 2). Quantitative analysis was performed by monitoring two mass transitions. Quantitative analysis may be performed by monitoring only one mass transition. Conversely, the above To replace or increase any of the monitored transitions in any combination, an additional mass Select a transition (for example, including any other fragment ions observed in Figure 10) Similarly, quantification was performed with a collision energy of 49 eV, but sufficient ion signals Any collision energy that produces the fragment ion may be used, and the fragment ion to be monitored ( It may depend on the identity of singular or plural. For example, the two fragments shown above On, the collision energy is in the range of approximately 25 to 70 eV, for example, approximately 30 to 60 eV. The range of V may be, for example, approximately 35 to 50 eV.

[0162] [Table 2]

[0163] Example 6: Detection and quantification of insulin A and B chains by tandem MS Simulated serum and strips spiked with insulin prepared as described in Example 1. Serum samples are treated with TCEP (tris(2-carboxyethyl)phosphine) and insulin The disulfide bridges were reduced, and the A and B chains were separated. The same purification procedure described in Example 2 was performed on the sample containing chains A and B. The insulin A and B chains were subjected to MS / MS analysis as described in Example 5. The analytes were ionized in positive mode under acidic conditions using ESI.

[0164] Several possible insulin A and B chain precursor ions were observed in Q1. Possible A-chain precursor ions (2+ and 3+ charged states) and two possible B-chain precursor ions (3+ and Figure 13 shows the composite spectrum of these two A-chain precursor ions. These are approximately 1192.86±0.50 (2+ ions) and 795.43±0.50 (3+ ions). These were observed at m / z. These two B-chain precursor ions were approximately 1144.09±0. Observed at m / z values ​​of 50 (3+ ions) and 858.40±0.50 (4+ ions). A third possible B-chain precursor ion (shown in Figure 14) is also approximately 686.83 ± 0.50 (5+ The fragmentation was observed in m / z of the ions. The fragmentation test was performed on the A and B chains as described above. This was performed on all ion-deactivating devices.

[0165] Fragmentation from A-chain 3+ precursor ions (approximately 795.43 ± 0.50 m / z) The effect of collision energy on the ion pattern was investigated. Precursor ions ranged from approximately 7 eV to approximately 80 eV. Fragmentation occurs at collision energies in the eV range, and four types of selected fragment io n (approximately 513.0±0.50, 399.0±0.50, 236.0±0.50 and 133 The relative intensity (m / z) of 0 ± 0.50 was monitored. The results of these tests are shown in Figure 15. As shown in Figure 15, the relative intensity of fragment ions depends on the collision energy. The values ​​change significantly. Table 3 shows the optimal collision energy values ​​for each monitored transition.

[0166] [Table 3]

[0167] Insulin quantification was approximately 1192.86±0.50 (2+ ions) and 795.43± The experiment was conducted using an A-chain precursor ion with an m / z of 0.50 (3+ ion). The quantitative experiment was performed using the precursor. The experiment was conducted for each ion. In these quantitative experiments, the 2+ ion (approximately 1192.86 ± Precursor ions are 0.50 m / z or 3+ ions (approximately 795.43 ± 0.50 m / z). Selected as such, it was fragmented with the collision energies shown in Table 3. The following fragments Gumment ions, 513.0±0.50, 399.0±0.50, 236.0±0.50 And 133.0±0.50 was monitored regardless of the selected precursor ion. Quantitative analysis was performed. The analysis was performed by monitoring four mass transitions, but quantification was performed using only one mass transition. This may be done by monitoring. Conversely, any of the monitored transitions may be entrusted to the other party. In order to replace or increase in a combination of intents, additional mass transitions (e.g., any observed) You may also select (including other fragment ions).

[0168] [Table 4]

[0169] Insulin quantification is also approximately 1144.09 ± 0.50 (3+ ions), 858.40 It has m / z values ​​of ±0.50 (4+ ions) and 686.83±0.50 (5+ ions). The experiment was conducted using B-chain precursor ions. Quantitative experiments were performed for each precursor ion. In quantitative experiments, 3+ ions (approximately 1144.09 ± 0.50 m / z) or 4+ ions (approximately 7 (95.43 ± 0.50 m / z) or 5+ ions (approximately 686.83 ± 0.50 m / z) We selected it as a precursor ion and fragmented it with a collision energy of 30 eV. Fragment ions, 226.2±0.50 and 345.0±0.50, are selected precursors. The ion was monitored regardless of its characteristics. Flux of B-chain 4+ ions at collision energy of 30 eV Figure 16 shows an example of a spectrum from a spectroscopy (i.e., a product ion scan). As shown, quantification was performed by monitoring two mass transitions, but only one was quantified. This may be done by monitoring the mass transitions. Conversely, the monitored transitions may be performed To replace or increase any of the transitions in any combination, additional mass transitions (e.g., You may also select any other fragment ions observed in Figure 16.

[0170] [Table 5]

[0171] Example 7 Insulin A chain (alkylation) and B chain (alkylation) by tandem MS Detection and Quantification Simulated serum spiked with insulin and serum strip samples were used with DTT(1,4-dithio Simulated and strip serum samples containing separated A and B chains treated with slateol. A substance was produced. Before purification, the insulin A and B chain molecules were carbamide-methylated, and then... Each of the constituent cysteines present in the molecule was completely alkylated. In the A chain, four Cysteine ​​is alkylated by this method, resulting in a mass increase of approximately 228.08 amu. In the B chain, two cysteine ​​molecules were alkylated by this method, resulting in approximately 114.0 A mass increase of 4amu occurred.

[0172] After alkylating cysteine, it contains alkylated A chain and alkylated B chain. The same purification procedure as described in Example 2 was performed on the sample. The obtained alkylated A The chain and the alkylated B chain were subjected to MS / MS analysis as described in Example 5. Both were analyzed. The substance was ionized in positive mode under acidic conditions using ESI.

[0173] Several possible alkylated A-chain and alkylated B-chain precursor ions were observed in Q1. Approximately 1306.0 ± 0.50 (2+ ions) and 871.0 ± 0.50 (3+ ions) Two possible A-chain precursor ions with m / z values ​​are selected for fragmentation and quantification. Selected: Approximately 1181.9±0.50 (3+ ions) and 886.40±0.50 (4+ ions). Three possible alkyl groups having an on- and a m / z of 709.80±0.50 (5+ ion) We selected the ligated B-chain precursor ions for fragmentation and quantification.

[0174] Insulin levels were approximately 1306.0 ± 0.50 (2+ ions) and 871.0 ± 0. The experiment was conducted using an alkylated A-chain precursor ion with a m / z of 50(3+ ion). The quantitative experiment was performed using The experiments were carried out with each of the precursor ions. In these quantitative experiments, the 2+ ion (approximately 1306) was found. Precursor ions are 0±0.50 m / z or 3+ ions (approximately 871.0±0.50 m / z). It was selected as a component and fragmented with a collision energy of 30 eV. The following fragments Toion, 133.0±0.50, 293.0±0.50, 456.0±0.50 and 5 70.0±0.50 was monitored regardless of the selected precursor ion. Quantification was performed using four methods. The analysis was performed by monitoring mass transitions, but quantification was performed by monitoring only one mass transition. This may be done by doing so. Conversely, any pair of the monitored transitions mentioned above Additional mass transitions may be selected to replace or increase in combination.

[0175] [Table 6]

[0176] Insulin quantification is also approximately 1181.9 ± 0.50 (3+ ions), 886.9 ± 0 Alkylated B chain precursors of 0.50 (4+ ion) and 709.8±0.50 (5+ ion) The experiments were conducted with the system ON. Quantitative experiments were performed for each of the precursor ions. In these quantitative experiments... , 3+ ions (approximately 1181.9 ± 0.50 m / z), 4+ ions (approximately 886.9 ± 0. Using a precursor ion of 50 m / z or 5+ ion (approximately 709.8 ± 0.50 m / z) Selected and fragmented with a collision energy of 30 eV. The following fragment ions , 226.2±0.50 and 345.0±0.50 are regardless of the selected precursor ion. It was monitored. Quantification was performed by monitoring two mass transitions, but quantification was This can be done by monitoring only one mass transition. Conversely, the monitoring To replace or increase any of the transitions in any combination, select additional mass transitions. You may choose this option.

[0177] [Table 7]

[0178] Example 8: Preparation of human samples for insulin quantification by quantification of insulin B chains Two types of internal standard solutions were used for the quantification of insulin in human samples. The first internal standard solution The semi-solution is prepared by dissolving bovine insulin in a 0.2% formic acid aqueous solution at a concentration of 10 pmol / μL. Prepared. Next, 30 μL of this solution was mixed with 1.5 M Tris base and ethanol in a 15:85 ratio. The base / extraction solution was diluted in 500 mL of the solution containing the base in the given ratio. The second internal standard solution was prepared by Pepti Human iodine isotope-labeled by dissolving 1 mg of iodine in 1 mL of 0.2% formic acid aqueous solution. Srin B chain (5 proline units) 13 C and 1 piece 15 Prepared with (labeled with N) 5 μl of this concentrated solution was diluted with 1000 μl of water to prepare a second internal standard solution.

[0179] The previously frozen human serum sample was thawed, returned to room temperature, and thoroughly mixed. 150 μL of each sample was added to 350 μL of base / extract solution spiked with bovine insulin. The resulting mixture was stirred at a speed of 1000 rpm for 2 minutes and then stored in a -20°C freezer for 60 ± The sample was incubated for 5 minutes to allow a precipitate to form. After incubation, the sample was transferred to 550°C. The samples were centrifuged at 0 rpm for 10 minutes. Next, 250 μL of the supernatant from each sample was divided into 96 microliters. It was then transferred to the plate. After that, TCEP reducing solution (Thermo Scientific Catalog) Mix 2 mL of log number 77720 with 100 μL of the second internal standard solution, and this mixture 20 μL of the solution was added to each sample in a 96-microliter plate. The samples were then subjected to another 1000r cycle. Stir at a speed of pm for 2 minutes, then incubate in a 37°C incubator for 60 ± 5 minutes. This causes the reduction of insulin in the sample, and any complete insulin present in the sample. The A and B chains were then separated. Next, the sample was incubated in a -20°C freezer for 10 minutes. Then, a precipitate was formed. The precipitated sample was centrifuged again at 5500 rpm for 10 minutes. Next The supernatant of each sample was concentrated by SPE and HPLC before MS / MS analysis.

[0180] Example 9: Concentration of insulin B chains in human samples before mass spectrometry Sample injection of the processed serum sample prepared in Example 8 was performed using Cohesive Technolo gies Aria TX-420 system with Aria OS V1.6 or later software The procedure was carried out using apparel.

[0181] 225 μL of each sample was placed in Waters Oasis HLB (25 μm, 2.1 × 20). The sample (mm) was introduced into an online solid-phase extraction (SPE) column. The SPE column was insulin B The chain was retained, but other proteins and macromolecules were released. The retained insulin B The chain was washed with 0.2% formic acid.

[0182] Next, the insulin B chain was dissolved in 0.2% formic acid containing 0.025% isopropanol. Elute from the extraction column with 35% acetonitrile, and analyze the column with a guard cartridge. (Michrom Bioresources 300 Armstrong Magi c C4 (2.1 × 50 mm, particle size 5 μm) analytical column and Phenomenex Se Curity Guard Column Cartridge (Phenomenex P / N AHO-42) It was added to 86)). In order to separate insulin from other analytes contained in the sample Therefore, an HPLC gradient was applied to the guard / analytical column. Mobile phase A was a 0.2% formic acid aqueous solution. Mobile phase B is 0.2% formic acid dissolved in acetonitrile containing 2.5% isopropanol. The HPLC gradient was started at a 12.0% organic gradient and increased to 42% in approximately 90 seconds. Ta.

[0183] Next, the insulin-concentrated sample was subjected to MS / MS for insulin quantification.

[0184] Example 10: Detection and quantification of insulin B chain in human serum by tandem MS MS / MS refers to the Thermo TSQ Vantage MS / MS system (The This was done using RMO (Electron Corporation). The following software programs are manufactured by Hermo Electron: TSQ Van tage V2.0.0 or higher, Xcalibur V2.0 or higher and LCQuan V2 A value of 0.5 or higher was used in the examples described herein. Liquid discharged from the analytical column. The solvent / analyte flowed onto the heated ESI source interface of the MS / MS analyzer. The precipitate mixture was converted to vapor in a heated tubing at the interface. The analyte was subjected to ESI. It was ionized in positive ion mode under acidic conditions using [a specific method / tool].

[0185] As described above in Example 6, several possible insulin B chain precursor ions were observed in Q1. It was detected. The insulin B chain has an m / z of approximately 686.83 ± 0.50 (5+ ions). Ionizers were selected for fragmentation. The fragmentation test was performed as follows: Several insulin B-chain fragment ions were shown. Fragmentation Spectrum An example of a clef is shown in Figure 17.

[0186] Fragments of human insulin B chain 5+ precursor ions (approximately 686.9 ± 0.50 m / z) The effect of collision energy on the station pattern was investigated. Precursor ions start from approximately 7V. Fragmentation occurs with collision energies in the range of approximately 80V, and five types of selected fragments are produced. Ions (approximately 906.0±0.50, 825.0±0.50, 768.5±0.50, 75 The relative intensity (m / z) of 3.0±0.50 and 703.0±0.50 was monitored. The results of the tests are shown in Figure 18. As shown in Figure 18, the relative intensity of the fragment ions This changes significantly depending on the collision energy. Each monitored transition is nearly optimal for the collision. The thrust energy values ​​are shown in Table 8.

[0187] [Table 8]

[0188] Human insulin B with m / z values ​​of approximately 768.5±0.50 and 753.2±0.50 This method was selected for quantitative analysis of chain fragment ions. Similar tests were performed in all examples. Bovine insulin (internal standard 1) and isotope-labeled insulin B chain (internal standard 2) as described in 8. The study was conducted using various insulin B chains selected for use in further quantitative tests. The monitored mass transitions are shown in Table 9.

[0189] [Table 9]

[0190] Quantitative analysis involves monitoring the two mass transitions of each insulin B chain, as shown in Table 8. Therefore, the procedure was carried out, but the quantitative determination of any of the specified analytes was monitored by monitoring only one mass transition. This may be done by doing so. Conversely, any pair of the monitored transitions mentioned above Additional mass transitions (e.g., any observed in Figure 17) are required for substitution or augmentation in combination. Other options (including human insulin B chain fragment ions) may be selected.

[0191] Example 11: Testing of accuracy, reproducibility, and precision within and between assays The intra-assay and inter-assay accuracy, reproducibility, and correctness of the assays described in Examples 8-10 The accuracy tests should be 8, 12, 20, and 40 to cover the reportable estimate range of the assay. and serum strips spiked with 80 μIU / mL human insulin (Biocell Prepared from Laboratories Inc., 1131-00, Lot HHP03). The process was carried out in five QC pools.

[0192] Eight replicas from each of the five QC pools were analyzed in a single assay, and the results within the assay were analyzed. The coefficient of variation (CV) of the substance was determined. The data obtained from these tests are shown in Table 10. The statistical analysis performed revealed that the reproducibility (CV) of the five QC pools ranged from 3.0 to 7.9%. Within this range, all are within acceptable levels (i.e., ≤15%CV, ≤20%CV). It was shown that (excluding acceptable LOQ levels)... Further subdivision of the data shown in Table 10 Analysis revealed that the intra-assay accuracy for each pool was within an acceptable range of 80-120%. It became softer.

[0193] [Table 10]

[0194] To investigate inter-assay variability, eight replicas from each of the five QC pools were separately processed. Analysis was performed five times a day. The data obtained from these tests are shown in Table 11. Pool assay The inter-period variation (%CV) ranged from 7.1% to 14.0%. 8, 12, 20, 40 and 8 The overall variability in target insulin levels at 0 μIU / mL was 14.0% and 10%, respectively. The percentages were 2%, 10.0%, 7.5%, and 7.1%. Analysis of the entire pool showed that ≤20%CV was Except for the acceptable LOQ level, the requirements for acceptable reproducibility of ≤15%CV are met. Further analysis of the data shown in Table 11 revealed that the inter-assay accuracy for each pool was 8. It was found that the acceptable range was 0-120%.

[0195] [Table 11]

[0196] Example 12: Analytical sensitivity: Blank limit (LOB), limit of detection (LOD), and limit of quantification ( LOQ) Selectivity refers to the ability of an analytical method to distinguish and quantify the analyte in the presence of other components in the sample. It is a force. Both LOB and LOD are greater than the uncertainty associated with a given measurement. It is also a large indicator. LOB is defined as twice the standard deviation from zero concentration. LOD is It is defined as four times the standard deviation from zero concentration. To test selectivity, appropriate biological Prepare blank samples of target matrix (serum strips), test for interference, and then proceed to Example 8~ The analysis was performed using the method described in section 10. Blank strip serum samples were measured 14 times. Statistical analysis of the results of these tests revealed that LOB 1.4 μIU / mL and LOD1 The level was 0.8 μIU / mL.

[0197] The key feature of LLOQ is that the measured values ​​become quantitatively meaningful. The analyte's response at LLOQ. It is identifiable, clearly distinguishable, and reproducible with an accuracy of 20% and precision of 80% to 120%. There are LLOQs, predicted LLOQs (1.25, 2.5, 5, 10, 15 and 25). Six serum strips spiked with human insulin at a concentration close to μIU / mL were used. This was determined by performing the assay and then evaluating the intraassay reproducibility of seven measurements. When the data from these tests are analyzed and plotted (as shown in Figure 19), LLOQ is 3 from the curve. It was determined that the minimum concentration that produces acceptable performance is μIU / mL, and the 95% confidence interval of the CV is the minimum concentration that produces acceptable performance. The percentage remained below 20% during that period.

[0198] Example 13: Reportable range and linearity of the assay To establish the linear range of the assays described in Examples 8-10, eight spiked samples were taken. Trip serum samples (human insulin concentrations of 5, 10, 15, 25, 50, 100, 200 and A 300 μIU / mL solution was prepared and analyzed five times on separate days. The weights of the five consecutive measurements were used. A correlation coefficient of 0.995 or higher can be obtained with an accuracy of ±20% using (1 / X) linear regression. It was found that the quantifiable range is 5 to 300 μIU / mL. An example of a calibration curve is shown below. This is shown in Figure 20.

[0199] Example 14: Testing of specimen types Matrix specificity was determined by a pool of 10 human patients and six different types of BD Vacu. tainer(trademark) tube (raw serum, SST, EDTA plasma, heparin sodium) Evaluation by collecting in plasma (lithium heparin plasma and sodium citrate plasma). Next, insulin was extracted from the samples in each pool and followed the method described in Examples 8-10. This was the analysis. These tests showed that the sodium citrate plasma sample was not acceptable for analysis. However, all other types of samples were shown to be acceptable.

[0200] Example 15: Interference Test The effect of hemolytic interference on insulin determination is observed in patient samples with mild, moderate, and severe hemolysis. We evaluated it by spiking insulin at various levels. Next, insulin The samples were extracted and analyzed according to the methods described in Examples 8-10. Based on the data from these tests... Therefore, acceptable results (i.e., accuracy within 80-120%) are achieved for mild and moderate dissolution. It was shown that the result could be obtained from blood samples. Highly hemolyzed samples were not acceptable.

[0201] The influence of lipidemia on insulin determination is significant in patients with mild, moderate, and severe lipidemia. The insulin levels in the sample were evaluated by spiking them at various levels. Next, The nsrin was extracted and analyzed according to the methods described in Examples 8-10. Depending on the data, acceptable results (i.e., accuracy within 80-120%) are achieved across all levels. It has been shown that this can be obtained in patients with lipidemia.

[0202] The effect of bilirubin interference on insulin determination is observed in patients with mild, moderate, and severe jaundice. The insulin was evaluated by spiking various levels in the food. Next, The thrin was extracted and analyzed according to the methods described in Examples 8-10. The data from these tests Depending on the results, acceptable results (i.e., accuracy within 80-120%) are achieved across all levels of performance. It was shown that it can be obtained in lilbin.

[0203] All papers, patents and patent applications, as well as all other documents and electronic documents mentioned or cited herein. The content of the information available is incorporated specifically and individually by reference in each individual publication. It is incorporated herein by reference in whole to the same extent as it is indicated. The applicants may use any such paper, patent, patent application or other physical and electronic document. We reserve the right to physically incorporate all of these materials and information into this application.

[0204] The methods described herein exemplify any elements not specifically disclosed herein or This can be properly implemented in the absence of elements(s), limitations, or limitations(s). For example, the terms "comprising" and "including" Words like ")" and "contain" can be read broadly and without limitation. It should be taken. Furthermore, the terms and expressions used in this specification are descriptive, not limiting. It is used for the purpose of, and in the use of such terms and expressions, indicated and explained This does not preclude any equivalent or part thereof of the described features. Within the scope of the claimed invention. It is recognized that various modifications are possible in this. Therefore, the present invention is preferable While the embodiments and optional features have been specifically disclosed, the details disclosed herein include Modifications and variations of the present invention implemented in this manner can be used by those skilled in the art, and such modifications It should be understood that any modifications are considered to fall within the scope of the present invention.

[0205] The present invention has been described extensively and generally in this specification. Each of the narrower species and subspecies also forms part of this method. This includes any pair from the class. A general description of a method having conditions or negative limitations for deleting an object, where the deleted material is the original. It is included regardless of whether it is specifically stated in the detailed document or not.

[0206] Other embodiments are included in the following claims. Furthermore, features or aspects of the method. If described by the Markush group, then, as those skilled in the art will know, the present invention is also, Understand that a group of Kush groups can be described by any individual component or subgroup of components. It will probably happen. The present invention may encompass the following embodiments. [1] A method for determining the amount of insulin in a biological sample by tandem mass spectrometry, The method is, (a) A step of providing the sample to conditions suitable for obtaining insulin B chains from insulin, (b) To obtain the insulin B chain concentrated fraction, process the sample from step (a) Step, (c) To generate one or more insulin B chain ions that can be detected by mass spectrometry. The steps include providing concentrated insulin B chains to an ionization source under conditions suitable for the following: (d) Tandem mass spectrometry is used to determine the amount of one or more insulin B chain ions. Step Includes, The amount of ions determined in step (d) is related to the amount of insulin in the sample. method. [2] The processing step of step (b) is performed by solid-phase extraction (SPE) of insulin B chains The method according to [1] above, comprising the step of concentrating the [3] The processing step in step (b) is high-performance liquid chromatography (HPLC) The method according to [1] above, comprising the step of enriching the insulin B chain by means of [1]. [4] The method according to [1] above, wherein the biological sample comprises human plasma or serum sample. [5] The amount of insulin to be determined is the amount of insulin present in the sample when taken from a human. The quantity, as described in [4] above. [6] The ionization source is an electrospray (ESI) ionization source as described in [1] above. The method. [7] The above sample is subjected to acidic conditions before ionization in positive ion mode. The method described in [1]. [8] The step of subjecting the sample to acidic conditions includes the step of subjecting the sample to formic acid, The method described in [7]. [9] The insulin B chain described above is not chemically modified before ionization, as described in [1] above. method.

[10] The one or more ions determined in step (d) are 1144.2 ± 0.5, 8 It consists of ions having mass-to-charge ratios (m / z) of 58.3±0.5 and 686.8±0.5. The method according to [9] above, comprising an insulin B chain precursor ion selected from the group.

[11] The one or more ions determined in step (d) are 906.0 ± 0.5, 82 5.0±0.5, 768.5±0.5, 753.0±0.5, 703.0±0.5, 34 It consists of ions with mass-to-charge ratios (m / z) of 5.0±0.5 and 226.2±0.5. The method according to [9] above, comprising one or more fragment ions selected from the group.

[12] The one or more ions determined in step (d) have a quality of 1144.2 ± 0.5 Fragment ions from insulin B chain precursor ions with charge-to-weight ratio (m / z), 85 Fragment ions from insulin B chain precursor ions having a m / z of 8.3 ± 0.5 Fragments from insulin B chain precursor ions with a m / z of 686.8±0.5 The above [9] includes one or more fragment ions selected from the group consisting of ONs. Method of description.

[13] The aforementioned tandem mass spectrometry was performed on a human with a mass-to-charge ratio (m / z) of 686.8 ± 0.5. A step of generating a nsrin B chain precursor ion, and the precursor ion to 906.0±0 0.5, 825.0±0.5, 768.5±0.5, 753.0±0.5, 703.0±0 A group consisting of ions having m / z values ​​of 0.5, 345.0±0.5, and 226.2±0.5. The process includes the step of fragmenting into one or more fragment ions selected from the above, The method described in [1] above.

[14] The ions determined in step (d) are 768.5±0.5 and 753.0±0.5 m The ions described in

[13] above, which include one or more ions from the group consisting of ions having / z. The method.

[15] The fragmentation step is performed using collision energy in the range of 10 to 25V (including the endpoints). The method described in

[13] above, using ghee.

[16] The method according to [1] above, wherein the insulin B chain is chemically modified before ionization.

[17] The above

[16] describes a chemical modification which includes the step of alkylating the insulin B chain. Methods used.

[18] The one or more ions determined in step (d) are 1181.9 ± 0.5, 8 It consists of ions having mass-to-charge ratios (m / z) of 86.9±0.5 and 709.8±0.5. The alkylated insulin B chain precursor ion selected from the group described in

[17] above, comprising method.

[19] The one or more ions determined in step (d) are 345.0 ± 0.5 and 2 One or more ions selected from the group of ions having a mass-to-charge ratio (m / z) of 26.2 ± 0.5. The method described in

[17] above, comprising a number of fragment ions.

[20] The one or more ions determined in step (d) have a quality of 1181.9 ± 0.5 Fragments from alkylated insulin B chain precursor ions with a charge-to-weight ratio (m / z) ON, from alkylated insulin B chain precursor ions with m / z of 886.9±0.5 Fragment ions and alkylated insulin B chains having a m / z of 709.8±0.5 Two or more fragments selected from the group consisting of fragment ions from precursor ions The method described in

[17] above, comprising ions. [twenty one] The fragment ions from each precursor ion were 345.0±0.5 and 226.2±0. The above

[20] includes ions selected from the group consisting of ions having a m / z of 0.5. Method of loading. [twenty two] Tandem mass spectrometry can determine the amount of insulin in a biological sample taken from a human. A method for making a determination, the method is (a) To obtain an insulin-concentrated fraction from the sample, the sample is subjected to solid-phase extraction (SPE). and the step of subjecting to high-performance liquid chromatography (HPLC), (b) Suitable for generating one or more insulin ions detectable by mass spectrometry The steps include providing concentrated insulin to an ionization source under the following conditions, and (c) Steps to determine the amount of one or more insulin ions by tandem mass spectrometry. P Includes, The aforementioned sample was not subjected to immunopurification before ionization, and the ions determined in step (c) A method for relating the quantity to the amount of insulin in the sample. [twenty three] The method according to

[22] above, wherein the biological sample comprises plasma or serum. [twenty four] The ionization source is an electrospray (ESI) ionization source as described in

[22] above. Method of loading. [twenty five] The method according to

[0022] above, wherein the sample is subjected to acidic conditions before ionization in positive ion mode.

[26] The step of subjecting the sample to acidic conditions includes the step of subjecting the sample to formic acid, The method described in

[25] .

[27] The one or more ions determined in step (c) are 1162.5 ± 0.5 and Selected from the group consisting of ions having a mass-to-charge ratio (m / z) of 968.9 ± 0.5. The method according to

[25] above, comprising a nsrin precursor ion.

[28] The one or more ions determined in step (c) are 226.2 ± 0.5 and 1 One or more fractions selected from the group consisting of ions having a m / z of 35.9 ± 0.5 The method according to

[27] above, comprising a rument ion.

[29] The one or more fragment ions have a m / z of 1162.5 ± 0.5. One or more fragment ions from the nsrin precursor ion and 968.9±0.5 Containing one or more fragment ions from an insulin precursor ion having an m / z, The method described in

[27] above.

[30] The one or more fragment ions from each precursor ion are 226.2 ± 0.5 and One or more ions selected from the group consisting of ions having an m / z of 135.9 ± 0.5 The method according to

[29] above, comprising a fragment ion.

[31] The method according to

[0021] above, wherein the sample is subjected to basic conditions before ionization in positive ion mode.

[32] The step of subjecting the sample to basic conditions includes the step of subjecting the sample to ammonia. The method described in

[31] above.

[33] The one or more ions determined in step (c) are 1453.8±0.5 and Selected from the group consisting of ions having a mass-to-charge ratio (m / z) of 1163.0 ± 0.5. The method according to

[31] above, comprising an insulin precursor ion.

[34] The one or more ions determined in step (c) are 226.2 ± 0.5 and 1 One or more fractions selected from the group consisting of ions having a m / z of 35.9 ± 0.5 The method according to

[33] above, comprising a rument ion.

[35] The one or more of the aforementioned fragment ions have a m / z of 1163.0 ± 0.5. One or more fragment ions from the nsrin precursor ion and 968.9±0.5 Containing one or more fragment ions from an insulin precursor ion having an m / z, The method described in

[33] above.

[36] A method for determining the amount of insulin in a sample by tandem mass spectrometry, wherein the method is: (a) To obtain an insulin-concentrated fraction from the sample, the sample is subjected to solid-phase extraction (SPE) and and the step of subjecting to high-performance liquid chromatography (HPLC), (b) Under conditions suitable for generating insulin precursor ions detectable by mass spectrometry The step is to provide concentrated insulin to an ionization source, wherein the insulin precursor The ion has a mass-to-charge ratio (m / z) of 1162.5 ± 0.5, step, (c) To generate one or more fragment ions that can be detected by mass spectrometry. The insulin precursor ion is then subjected to collision induction with a collision energy in the range of approximately 40 to 70 eV. Steps for dissociation, and (d) A step of determining the amount of one or more of the fragment ions by mass spectrometry. Includes, The amount of ions determined in step (d) is related to the amount of insulin in the sample. method.

[37] The method according to

[36] above, wherein the sample includes a biological sample.

[38] The method according to

[36] above, wherein the sample comprises plasma or serum.

[39] The sample includes a biological sample taken from a human, and the method is used when the sample is taken from a human. The method described above

[36] is used to determine the amount of insulin in a biological sample. The method.

[40] The ionization conditions include a step of subjecting the sample to acidic conditions before ionization. The method described in

[36] .

[41] The step of subjecting the sample to acidic conditions includes the step of subjecting the sample to formic acid, The method described in

[40] .

[42] The ionization conditions include a step of subjecting the sample to basic conditions before ionization. The method described in

[36] .

[43] The step of subjecting the sample to basic conditions includes the step of subjecting the sample to ammonia. The method described in

[42] above.

[44] The aforementioned ionization is performed using an electrospray ionization (ESI) source to create positive ions. The method described in

[36] above, performed in a pedestrian zone.

[45] The method according to

[36] above, wherein the collision energy is in the range of approximately 40 to 60 eV.

[46] The method according to

[36] above, wherein the collision energy is in the range of approximately 40 to 50 eV.

[47] The aforementioned one or more fragment ions are 226.2±0.5 and 135.9±0.5 The method according to

[0036] above, comprising one or more ions selected from the group consisting of ions having m / z.

[48] Tandem mass spectrometry determines the amount of insulin in biological samples taken from humans. A method for determining, the method is (a) A step of providing the sample to conditions suitable for obtaining insulin A chains from insulin, (b) To obtain the insulin A chain concentrated fraction, the sample from step (a) is subjected to solid-phase extraction. Steps to subject to SPE and high-performance liquid chromatography (HPLC), (c) To generate one or more insulin A chain ions that can be detected by mass spectrometry. The steps include providing concentrated insulin A chain to an ionization source under conditions suitable for the following: (d) Tandem mass spectrometry is used to determine the amount of one or more insulin A chain ions. Step Includes, The amount of ions determined in step (d) is related to the amount of insulin in the sample. method.

[49] The method according to

[48] above, wherein the biological sample comprises plasma or serum.

[50] The ionization source is an electrospray (ESI) ionization source as described in

[48] above. Method of loading.

[51] The method according to

[0048] above, wherein the sample is subjected to acidic conditions before ionization in positive ion mode.

[52] The step of subjecting the sample to acidic conditions includes the step of subjecting the sample to formic acid, The method described in

[41] .

[53] The insulin A chain obtained in step (a) is chemically modified before ionization. No, the method described in

[48] above.

[54] The one or more ions determined in step (d) are 1192.9 ± 0.5 and Selected from the group consisting of ions having a mass-to-charge ratio (m / z) of 795.4 ± 0.5. The method according to

[53] above, comprising a nsrin A chain precursor ion.

[55] The one or more ions determined in step (d) are 570.0 ± 0.5, 45 The mass-to-charge ratios (m / z) of 6.0±0.5, 293.0±0.5, and 133.0±0.5 are used. The method according to

[0053] above, comprising one or more fragment ions selected from the group of ions having.

[56] The one or more ions determined in step (d) have a quality of 1192.9 ± 0.5 One or more fragments from insulin A chain precursor ions having a charge-to-weight ratio (m / z) One of the toion and one of the insulin A chain precursor ions having a m / z of 795.4±0.5 or the method described in

[53] above, comprising a plurality of fragment ions.

[57] The one or more fragment ions from each precursor ion are 570.0 ± 0.5, 4 Ions with m / z values ​​of 56.0±0.5, 293.0±0.5, and 133.0±0.5 The fragment ions include one or more selected from the group consisting of the above

[56] , as described above. Method of loading.

[58] A step to chemically modify the insulin A chain obtained in step (a) before ionization. The method described in

[48] above, further including the method described in

[48] above.

[59] The above

[58] describes a chemical modification which includes the step of alkylating the insulin A chain. Methods used.

[60] The one or more ions determined in step (d) are 1306.0 ± 0.5 and A selection from the group consisting of ions having a mass-to-charge ratio (m / z) of 871.0 ± 0.5. The method according to

[59] above, comprising a lylated insulin A chain precursor ion.

[61] The one or more ions determined in step (d) are 570.0 ± 0.5, 45 The mass-to-charge ratios (m / z) of 6.0±0.5, 293.0±0.5, and 133.0±0.5 are used. The method according to

[0059] above, comprising one or more fragment ions selected from the group of ions having.

[62] The one or more ions determined in step (d) have a quality of 1306.0 ± 0.5 One or more alkylated insulin A chain precursor ions having a charge-to-weight ratio (m / z) Fragment ions and alkylated insulin A chains having a m / z of 871.0 ± 0.5 The method described in

[59] above, comprising one or more fragment ions from a precursor ion. .

[63] The one or more fragment ions from each alkylated precursor ion are 570.0 m / z values ​​of ±0.5, 456.0±0.5, 293.0±0.5, and 133.0±0.5 The above includes one or more fragment ions selected from the group consisting of ions having The method described in

[62] .

[64] A method for determining the amount of insulin in a sample by high-resolution / high-precision mass spectrometry, the The method is, (a) Under conditions suitable for generating polyvalent insulin ions from the sample A step of providing an ionization source, wherein the polyvalent insulin ion is, Steps that can be detected through analysis, (b) Determine the amount of one or more polyvalent insulin ions by high-resolution / high-precision mass spectrometry. Steps to determine Includes, The amount of ions determined in step (b) is related to the amount of insulin in the sample. method.

[65] The aforementioned high-resolution / high-precision mass spectrometry uses FWHM of 10,000 or more and quality of 50 ppm or less. The method described in

[64] above, performed with quantitative precision.

[66] The aforementioned high-resolution / high-precision mass spectrometry uses FWHM of 15,000 or more and quality of 20 ppm or less. The method described in

[64] above, performed with quantitative precision.

[67] The aforementioned high-resolution / high-precision mass spectrometry uses FWHM of 20,000 or more and mass of 5 ppm or less. The method described in

[64] above, performed with precision.

[68] The above-mentioned high-resolution / high-precision mass spectrometry is based on a high-resolution / high-precision time-of-flight (TOF) mass spectrometer. The method described in

[64] above, performed using

[64] .

[69] The ionization source is an electrospray (ESI) ionization source as described in

[64] above. Method of loading.

[70] The ionization conditions include the ionization of insulin under acidic conditions as described in

[64] above. The method.

[71] The acidic conditions include treatment of the sample with formic acid before ionization, as described in

[69] above. The method.

[72] The one or more polyvalent insulin ions mentioned above are 4+, 5+, and 6+ valent insulin ions. The method according to

[64] above, comprising one or more ions selected from the group.

[73] The method according to

[0072] above, wherein the one or more polyvalent insulin ions include a 6+ valent insulin ion.

[74] The one or more of the aforementioned 6+ valent insulin ions are in a mass within the range of approximately 968.0 ± 1.5 The method according to

[73] above, comprising one or more ions having a charge ratio (m / z).

[75] One or more insulin ions in a 6+ charged state, 968.28±0.1, 96 8.45±0.1, 968.62±0.1, 968.79±0.1, 968.95±0. 1, 968.12±0.1, 968.28±0.1, 968.45±0.1, and 968. One selected from the group consisting of ions having a mass-to-charge ratio (m / z) of 61±0.1 The method according to

[73] above, comprising multiple ions.

[76] The method according to

[0072] above, wherein the one or more polyvalent insulin ions include a 5+ valent insulin ion.

[77] The one or more insulin ions in the 5+ charged state are in the range of approximately 1162.5 ± 1.0. The above

[76] includes one or more ions having a mass-to-charge ratio (m / z) within method.

[78] The aforementioned one or more insulin ions in a 5+ charged state are 1161.72±0.1, 1 161.92±0.1, 1162.12±0.1, 1162.32±0.1, 1162. 52±0.1, 1162.72±0.1, 1162.92±0.1, 1163.12±0 From the group consisting of ions having mass-to-charge ratios (m / z) of 0.1 and 1163.34±0.1 The method according to

[76] above, comprising one or more selected ions.

[79] The one or more insulin ions in the 5+ charged state have a mass of 1162.54 ± 0.1 The method according to

[76] above, comprising an ion having a charge ratio (m / z).

[80] The method according to

[0072] above, wherein the one or more polyvalent insulin ions include 4+ valent insulin ions.

[81] The one or more insulin ions in the 4+ charged state are in the range of approximately 1452.9 ± 0.8. The above

[80] includes one or more ions having a mass-to-charge ratio (m / z) within method.

[82] Before ionization, the sample was analyzed by high-performance liquid chromatography (HPLC) to extract insulin. The method described in

[64] above for purifying n.

[83] The method according to

[81] above, wherein the sample is subjected to solid-phase extraction (SPE) before HPLC.

[84] The method according to

[64] above, wherein the sample includes a biological sample.

[85] The method according to

[64] above, wherein the sample is obtained from a human.

[86] The method according to

[64] above, wherein the sample comprises plasma or serum.

[87] The above sample is a biological sample from a human, and the above method is used when the sample is taken from a human. The method described in

[64] above, used to determine the amount of insulin in a sample.

Claims

1. A method for determining the amount of insulin in a human plasma or serum sample by high-resolution / high-precision mass spectrometry, wherein the method is: (a) The step of subjecting the human plasma or serum sample to solid-phase extraction (SPE) to form an extracted insulin sample; (b) A step of purifying the extracted insulin sample by high-performance liquid chromatography (HPLC) to form a purified insulin sample; (c) A step of providing the purified insulin sample to an ionization source under acidic conditions suitable for generating polyvalent insulin ions, wherein the polyvalent insulin ions can be detected by mass spectrometry; and (d) A step of determining the amount of polyvalent insulin ions by high-resolution / high-precision mass spectrometry performed with a measurement value of 10,000 or more FWHM and a mass accuracy of 50 ppm or less, wherein the polyvalent insulin ions include 6+ valent insulin ions having a mass-to-charge ratio (m / z) in the range of approximately 968.8 ± 1.

5. Includes, A method for relating the amount of 6+ valent insulin ions determined in step (d) to the amount of insulin in the human plasma or serum sample.

2. The method according to claim 1, wherein the high-resolution / high-precision mass spectrometry is performed with 15,000 or more measurement values / FWHM and a mass accuracy of 20 ppm or less.

3. The method according to claim 1, wherein the high-resolution / high-precision mass spectrometry is performed with 20,000 or more measurement values / FWHM and a mass accuracy of 5 ppm or less.

4. The method according to claim 1, wherein the high-resolution / high-precision mass spectrometry is performed using a high-resolution / high-precision time-of-flight (TOF) mass spectrometer.

5. The method according to claim 1, wherein the ionization source is an electrospray (ESI) ionization source.

6. The method according to claim 1, wherein the acidic conditions include treatment of the sample with formic acid before ionization.

7. The method according to claim 1, wherein the polyvalent insulin ion further comprises one or more ions selected from the group consisting of 4+ and 5+ valent insulin ions.

8. The method according to claim 1, wherein the 6+ valent insulin ion comprises one or more ions selected from ions having mass-to-charge ratios (m / z) of 968.28±0.1, 968.45±0.1, 968.62±0.1, 968.79±0.1, 968.95±0.1, 968.12±0.1, 968.28±0.1, 968.45±0.1 and 968.61±0.

1.

9. The method according to claim 1, wherein the polyvalent insulin ion comprises a 5+ valent insulin ion.

10. The method according to claim 9, wherein the 5+ valent insulin ion comprises one or more ions having a mass-to-charge ratio (m / z) in the range of about 1162.5 ± 1.

0.

11. The method according to claim 9, wherein the 5+ valent insulin ion comprises one or more ions selected from ions having mass-to-charge ratios (m / z) of 1161.72±0.1, 1161.92±0.1, 1162.12±0.1, 1162.32±0.1, 1162.52±0.1, 1162.72±0.1, 1162.92±0.1, 1163.12±0.1 and 1163.34±0.

1.

12. The method according to claim 9, wherein the 5+ valent insulin ion comprises an ion having a mass-to-charge ratio (m / z) of 1162.54 ± 0.

1.

13. The method according to claim 1, wherein the polyvalent insulin ion includes a 4+ valent insulin ion.

14. The method according to claim 13, wherein the 4+ valent insulin ion comprises one or more ions having a mass-to-charge ratio (m / z) in the range of about 1452.9 ± 0.8.

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