Quantification of insulin by mass spectrometry

Abstract

Methods for determining the amount of insulin in a sample are described. Mass spectrometry methods for detecting and quantifying insulin in a biological sample using purification methods coupled with tandem mass spectrometry or high resolution / high accuracy mass spectrometry techniques are described. The methods comprise subjecting the sample to solid phase extraction (SPE) and high performance liquid chromatography (HPLC) to obtain a fraction enriched for sample-derived insulin; (b) subjecting the enriched insulin to an ionization source under conditions suitable to generate one or more insulin ions that are detectable by mass spectrometry; (c) determining the amount of one or more insulin ions by tandem mass spectrometry, wherein the sample is not subjected to immunopurification prior to ionization. The amount of the one or more ions determined in step (c) is used to determine the amount of insulin in the sample.

Description

[0001] This application is a divisional application of the patent application filed on December 27, 2011, with application number 201611246793.0 and invention title: "Quantification of Insulin by Mass Spectrometry".

[0002] Related patent applications

[0003] This application claims the benefit of U.S. Provisional Application No. 61 / 427,749, filed December 28, 2010, which is incorporated herein by reference in its entirety for all purposes. Invention Field

[0004] This invention relates to the quantitative measurement of insulin. In a specific aspect, this invention relates to a method for the quantitative measurement of insulin by mass spectrometry. Background of the Invention

[0005] The following description of the background of the invention is provided merely to help understand the invention and does not constitute prior art describing or forming the invention.

[0006] Insulin is a small peptide composed of 51 amino acids in two chains, represented as chain A and chain B, linked by disulfide bonds between cysteine ​​residues. The molar mass of human insulin is approximately 5607.4 amu. Chain A has 21 amino acids, while chain B has 30 amino acids.

[0007] Insulin is a crucial hormone for regulating the metabolism of fats and steroids in the body. When blood sugar levels rise after a meal, insulin is released into the bloodstream, allowing glucose to be transported from the circulation into cells.

[0008] Defects in insulin production or utilization lead to diabetes. Insulin is often administered to treat diabetes. Diabetes and its complications represent a major public health problem. Therefore, quantification of insulin in samples from patients with diabetes and prediabetes is important as a diagnostic tool and for monitoring patient treatment.

[0009] Immunological techniques have been widely used for insulin quantification (see, for example, Manley et al., Clin Chem., 2007, 53:922-32), and several mass spectrometry methods for detecting and / or quantifying insulin have been reported. See, for example, R. et al., Diabetes 1997, 46:44-50 (reported quantification of insulin in serum samples by immunoaffinity chromatography-solid phase extraction-HPLC-single mass spectrometry); Darby, SM et al., J. Anal Toxicol 2001, 25:8-14 (reported SPE-HPLC-MS quantification of insulin in plasma at high physiological levels); Fierens, C. et al., Rapid Commun. Mass Spectrom. 2001, 15:1433-41 (reported detection of insulin in aqueous solution by HPLC-(ESI)MS / MS); Magnes, C. et al., 52nd ASMS meeting, May 2004 (reported quantification of insulin in serum by high resolution / high precision mass spectrometry); Thevis, M. et al., Anal. Chem., 2005, 77:3579-85 (reported a method for quantifying insulin from plasma and detecting the insulin B chain). Immunoaffinity chromatography-solid phase extraction-HPLC-tandem mass spectrometry (IPA-SPE-HPLC-MS / MS); Thevis, M. et al., Anal. Chem., 2006, 77:3579-85 and Thomas, A. et al., Anal. Chem., 2007, 79:2518-24 (reporting a SPE-IPA-SPE-HPLC-MS / MS method for quantifying insulin from plasma and detecting the insulin B chain); Uytfanghe, K. et al., Rapid Comm Mass Spectrom., 2007, 21:819-821 (reported an immunoaffinity chromatography-solid phase extraction-HPLC-tandem mass spectrometry method for quantifying insulin from serum); Rodríguez-Cabaleiro, D. et al., Clin Chem., 2007, 53:1462-69 (reported an immunoaffinity chromatography-solid phase extraction-HPLC-tandem mass spectrometry method for quantifying insulin from plasma and detecting the insulin B chain); Thevis, M. et al., Mass Spectrom. Reviews, 2008, 27:35-50 (reported an immunoaffinity chromatography-solid phase extraction-HPLC-tandem mass spectrometry method for quantifying insulin from plasma and detecting the insulin B chain); and Guedes, S., J. Am Soc Mass Spectrom, 2009, 20:1319-26. Invention Overview

[0010] This invention provides a method for determining the amount of insulin in a sample by mass spectrometry.

[0011] In one aspect, the method utilizes tandem mass spectrometry. In some tandem mass spectrometry embodiments, the method is used to determine the amount of insulin in a biological sample taken from a human. In some embodiments, the method includes: (a) subjecting the sample to solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) to obtain an insulin-rich component from the sample; (b) subjecting the enriched insulin to an ionization source under conditions suitable for generating one or more insulin ions detectable by mass spectrometry; and (c) determining the amount of one or more insulin ions by tandem mass spectrometry, wherein the sample has not undergone immunopurification prior to ionization. In these embodiments, the amount of insulin in the sample is determined using the amount of said one or more ions determined in step (c).

[0012] In some embodiments, the sample is subjected to acidic conditions before ionization in positive ion mode. In some related embodiments, subjecting the sample to acidic conditions includes subjecting the enriched insulin to formic acid. In some related embodiments, one or more ions determined in step (c) include insulin precursor ions selected from ions with a mass-to-charge ratio (m / z) of 1162.5 ± 0.5 and 968.9 ± 0.5. In other related embodiments, one or more ions determined in step (c) include one or more fragment ions selected from ions with m / z of 226.2 ± 0.5 and 135.9 ± 0.5. In still other related embodiments, one or more fragment ions include one or more fragment ions from insulin precursor ions with m / z of 1162.5 ± 0.5 and one or more fragment ions from insulin precursor ions with m / z of 968.9 ± 0.5. In the relevant embodiments, one or more fragment ions from each precursor ion include one or more fragment ions selected from ions with m / z of 226.2±0.5 and 135.9±0.5.

[0013] In some embodiments, the sample is subjected to alkaline conditions before ionization in positive ion mode. In some related embodiments, subjecting the sample to alkaline conditions includes subjecting the sample to ammonia. In some related embodiments, one or more ions determined in step (c) include insulin precursor ions selected from ions with m / z of 1453.8 ± 0.5 and 1163.0 ± 0.5. In other related embodiments, one or more ions determined in step (c) include one or more fragment ions selected from ions with m / z of 226.2 ± 0.5 and 135.9 ± 0.5. In still other related embodiments, one or more fragment ions include one or more fragment ions from insulin precursor ions with m / z of 1453.8 and one or more fragment ions from insulin precursor ions with a mass-to-charge ratio of 1163.0 ± 0.5.

[0014] In some embodiments of determining the amount of insulin in a sample using tandem mass spectrometry, the method includes: (a) enriching insulin in the sample by an extraction technique; (b) subjecting the purified insulin from step (a) to high-performance liquid chromatography (HPLC) to obtain an insulin-rich component from the sample; (c) subjecting the enriched insulin to an ionization source under conditions suitable for generating insulin precursor ions detectable by mass spectrometry, wherein the m / z of the insulin precursor ions is 1162.5 ± 0.5; (d) subjecting the insulin precursor ions to collision-induced dissociation at a collision energy in the range of about 40-70 eV to generate one or more fragment ions detectable by mass spectrometry; and (e) determining the amount of one or more of said fragment ions by mass spectrometry. In these embodiments, the amount of ions determined in step (e) is related to the amount of insulin in the sample. In some embodiments, the extraction technique is solid-phase extraction (SPE).

[0015] In some embodiments, the sample is subjected to acidic conditions before ionization in positive ion mode. In some related embodiments, subjecting the sample to acidic conditions includes subjecting the sample to formic acid. In alternative embodiments, the sample is subjected to alkaline conditions before ionization. In some related embodiments, subjecting the sample to alkaline conditions includes ammonia.

[0016] In some embodiments, the collision energy is in the range of about 40-60 eV, for example, in the range of about 40-50 eV. In some embodiments, the one or more fragment ions generated in step (d) include one or more ions selected from ions with m / z of 226.2 ± 0.5 and 135.9 ± 0.5.

[0017] In some embodiments of determining the amount of insulin in biological samples taken from humans using tandem mass spectrometry, the method includes: (a) subjecting the sample to conditions suitable for the formation of insulin A chains from insulin; (b) subjecting the sample from step (a) to solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) to obtain a component rich in insulin A chains; (c) subjecting the enriched insulin A chains to an ionization source under conditions suitable for generating one or more insulin A chain ions detectable by mass spectrometry; and (d) determining the amount of one or more insulin A chain ions by tandem mass spectrometry. In these embodiments, the amount of ions determined in step (d) is related to the amount of insulin in the sample.

[0018] In some related 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 includes subjecting the sample to formic acid. In some embodiments, the insulin A chain generated in step (a) is not chemically modified prior to ionization. In some related embodiments, the one or more ions determined in step (d) include insulin A chain precursor ions selected from ions with m / z of 1192.9 ± 0.5 and 795.4 ± 0.5. In some related embodiments, the one or more ions determined in step (d) include one or more fragment ions selected from ions with m / z of 513.0 ± 0.5, 399.0 ± 0.5, 236.0 ± 0.5, and 133.0 ± 0.5. In some related embodiments, the one or more ions determined in step (d) include one or more fragment ions from an insulin A-chain precursor ion with an m / z of 1192.9 ± 0.5 and one or more fragment ions from an insulin A-chain precursor ion with an m / z of 795.4 ± 0.5. In some related embodiments, the one or more fragment ions from each precursor ion include one or more fragment ions selected from ions with m / z of 513.0 ± 0.5, 399.0 ± 0.5, 236.0 ± 0.5, and 133.0 ± 0.5.

[0019] In alternative embodiments, the method further includes the insulin A chain generated in step (a) prior to ionization. In some embodiments, the chemical modification includes alkylation of the insulin A chain. In other related embodiments, one or more ions determined in step (d) include alkylated insulin A chain precursor ions selected from ions with m / z of 1306.0±0.5 and 871.0±0.5. In other related embodiments, one or more ions determined in step (d) include one or more fragment ions selected from ions with m / z of 570.0±0.5, 456.0±0.5, 293.0±0.5, and 133.0±0.5. In other related embodiments, one or more ions determined in step (d) include one or more fragment ions from an alkylated insulin A chain precursor ion with m / z of 1306.0±0.5 and one or more fragment ions from an alkylated insulin A chain precursor ion with m / z of 871.0±0.5. In some related embodiments, one or more fragment ions from each alkylation precursor ion include one or more fragment ions selected from ions with m / z of 570.0±0.5, 456.0±0.5, 293.0±0.5, and 133.0±0.5.

[0020] In some related embodiments, tandem mass spectrometry is used to determine the amount of insulin in a biological sample. In these embodiments, the method includes: (a) subjecting the sample to conditions suitable for the formation of insulin B chains from insulin; (b) processing the sample from step (a) to obtain a component rich in insulin B chains; (c) subjecting the enriched insulin B chains to an ionization source under conditions suitable for generating one or more insulin B chain ions detectable by mass spectrometry; and (d) determining the amount of one or more insulin B chain ions by tandem mass spectrometry. In these embodiments, the amount of ions determined in step (d) is related to the amount of insulin in the sample.

[0021] In some implementations, step (b) involves enriching the insulin B chain by solid-phase extraction (SPE), high-performance liquid chromatography (HPLC), or both. In some relevant implementations using SPE and HPLC, both enrichment techniques can be performed online.

[0022] In some implementations, the biological sample includes a human plasma or serum sample. In some related implementations, when taken from a human, the amount of insulin measured is the amount of insulin present in the sample.

[0023] In some implementations, the ionization source is an electrospray ionization (ESI) source, such as a heated ESI source.

[0024] In some embodiments, the sample is subjected to acidic conditions before ionization in positive ion mode. In some related embodiments, subjecting the sample to acidic conditions includes subjecting the sample to formic acid.

[0025] In some embodiments, the insulin B chain is unmodified prior to ionization. In some related embodiments, one or more ions determined in step (d) include insulin B chain precursor ions selected from ions with m / z of 1144.2±0.5, 858.3±0.5, and 686.8±0.5. In some related embodiments, one or more ions determined in step (d) include ions selected from ions with m / z of 906.0±0.5, 825.0±0.5, 768.5±0.5, 753.0±0.5, 703.0±0.5, 345.0±0.5, and 226.2±0.5, such as ions with m / z of 768.5±0.5, 753.0±0.5, 345.0±0.5, and 226.2±0.5, such as ions with m / z of 768.5±0.5 and 753.0±0.5. In some embodiments, the one or more ions determined in step (d) include two or more fragment ions selected from: fragment ions from insulin B-chain precursor ions with m / z of 1144.2 ± 0.5, fragment ions from insulin B-chain precursor ions with m / z of 858.3 ± 0.5, and fragment ions from insulin B-chain precursor ions with m / z of 686.8 ± 0.5.

[0026] In some embodiments, tandem mass spectrometry includes generating a human insulin B-chain precursor ion with a mass-to-charge ratio (m / z) of 686.8 ± 0.5 and breaking the precursor ion into one or more fragment ions selected from ions with m / z of 906.0 ± 0.5, 825.0 ± 0.5, 768.5 ± 0.5, 753.0 ± 0.5, 703.0 ± 0.5, 345.0 ± 0.5, and 226.2 ± 0.5, for example, one or more fragment ions selected from 768.5 ± 0.5 and 753.0 ± 0.5.

[0027] In some implementations, tandem mass spectrometry includes breaking down precursor ions with a collision energy in the range of 10-25V, including both 10V and 25V.

[0028] In alternative embodiments, the insulin B chain is chemically modified prior to ionization. In some embodiments, the chemical modification includes alkylation of the insulin B chain. In some embodiments, one or more ions determined in step (d) comprise alkylated insulin B chain precursor ions selected from ions with m / z of 1181.9 ± 0.5, 886.9 ± 0.5, and 709.8 ± 0.5. In some embodiments, one or more ions determined in step (d) comprise one or more fragment ions selected from ions with m / z of 345.0 ± 0.5 and 226.2 ± 0.5. In some embodiments, one or more ions determined in step (d) comprise fragment ions selected from two or more of the following: fragment ions from alkylated insulin B chain precursor ions with a mass-to-charge ratio (m / z) of 1181.9 ± 0.5, fragment ions from alkylated insulin B chain precursor ions with m / z of 886.9 ± 0.5, and fragment ions from alkylated insulin B chain precursor ions with m / z of 709.8 ± 0.5. In some related embodiments, fragment ions from each precursor ion include ions selected from ions with m / z values ​​of 345.0 ± 0.5 and 226.2 ± 0.5.

[0029] In a second aspect, certain methods proposed herein utilize high-resolution / high-precision mass spectrometry to determine the amount of insulin in a sample. In some embodiments utilizing high-resolution / high-precision mass spectrometry, the method includes: (a) subjecting insulin from a sample to an ionization source under conditions suitable for generating polycharged insulin ions, wherein insulin ions can be detected by mass spectrometry; and (b) determining the amount of one or more polycharged insulin ions by high-resolution / high-precision mass spectrometry. In these embodiments, the amount of one or more ions determined in step (b) is related to the amount of insulin in the sample. In some embodiments, high-resolution / high-precision mass spectrometry is performed at a FWHM of 10,000 or higher and a mass precision of 50 ppm. In some embodiments, the high-resolution / high-precision mass spectrometry is performed using a high-resolution / high-precision time-of-flight (TOF) mass spectrometer. In some embodiments, the ionization conditions include ionizing insulin under acidic conditions. In some related embodiments, the acidic conditions include treating the sample with formic acid prior to ionization. In some embodiments, the polycharged insulin ions are selected from 4+, 5+, and 6+ charged insulin ions.

[0030] In some embodiments, one or more insulin ions in a 6+ charge state include one or more ions with an m / z in the range of about 968.0 ± 1.5. One or more insulin ions in a 6+ charge state include one or more ions selected from those with m / z of 968.28 ± 0.1, 968.45 ± 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, and 969.61 ± 0.1; for example, ions with an m / z of 968.95 ± 0.1.

[0031] In some embodiments, one or more insulin ions in a 5+ charged state include one or more ions with an m / z in the range of about 1162.5 ± 1.0. In some embodiments, one or more insulin ions in a 5+ charged state include one or more ions selected from ions with 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 1163.32 ± 0.1; for example, ions with an m / z of 1162.54 ± 0.1.

[0032] In some embodiments, one or more insulin ions in a 4+ charge state include one or more ions with an m / z range of about 1452.9 ± 0.8.

[0033] In any of the methods described herein, the sample may include a biological sample. In some embodiments, the biological sample may include a biological fluid, such as urine, plasma, or serum. In some embodiments, the biological sample may include a sample from a human being; for example, from an adult male or female, or an adolescent male or female, wherein the adolescent is under 18 years of age, under 15 years of age, under 12 years of age, or under 10 years of age. Human samples may be analyzed to diagnose or monitor a disease state or symptom, or to monitor the effectiveness of treatment for a disease state or symptom. In some related embodiments, the methods described herein can be used to determine the amount of insulin in a biological sample taken from a human being.

[0034] In embodiments utilizing tandem mass spectrometry, tandem mass spectrometry can be performed by any method known in the art, including, for example, multiple reaction monitoring, precursor ion scanning, or product ion scanning.

[0035] In some embodiments, tandem mass spectrometry includes breaking down precursor ions into one or more fragment ions. In embodiments that determine the amount of two or more fragment ions, these amounts can be subjected to any mathematical operation known in the art to correlate the measured ion amount with the amount of insulin in the sample. For example, the amounts of two or more fragment ions can be summed as part of the determination of the amount of insulin in the sample.

[0036] In any of the methods described herein, the target analyte (e.g., insulin, chemically modified or unmodified insulin A chain, or chemically modified or unmodified insulin B chain) can be purified from the sample by high-performance liquid chromatography (HPLC) prior to ionization. In any of the methods described herein, the target analyte can be purified from the sample by extraction techniques, such as passing the sample through a solid-phase extraction (SPE) column. In some embodiments, the extraction technique is not an immunopurification technique. Specifically, in some embodiments, the SPE column is not an immunoaffinity column. In some embodiments, immunopurification is not applied at any point in the methods described. In some embodiments, extraction techniques and HPLC can be performed online to allow for automated sample processing and analysis.

[0037] In some embodiments, high-resolution / high-precision mass spectrometry is performed at a resolution (FWHM) greater than or equal to about 10,000, for example, greater than or equal to about 15,000, for example, greater than or equal to about 20,000, for example, greater than or equal to about 25,000. In some embodiments, high-resolution / high-precision mass spectrometry is performed at a precision less than or equal to about 50 ppm, for example, less than or equal to about 20 ppm, for example, less than or equal to about 10 ppm, for example, less than or equal to about 5 ppm, for example, less than or equal to about 3 ppm. In some embodiments, high-resolution / high-precision mass spectrometry is performed at a resolution (FWHM) greater than or equal to about 10,000 and a precision less than or equal to about 50 ppm. In some embodiments, the resolution is greater than about 15,000 while the precision is less than or equal to about 20 ppm. In some embodiments, the resolution is greater than or equal to about 20,000 and the accuracy is less than or equal to about 10 ppm; preferably, the resolution is greater than or equal to about 20,000 and the accuracy is less than or equal to about 5 ppm, for example, less than or equal to about 3 ppm.

[0038] In some implementations, high-resolution / high-precision mass spectrometry is performed using an orbital trap mass spectrometer, a time-of-flight (TOF) mass spectrometer, or a Fourier transform ion cyclotron resonance mass spectrometer (sometimes called a Fourier transform mass spectrometer).

[0039] In some embodiments, the one or more insulin ions detectable by high-resolution / high-precision mass spectrometry are selected from one or more ions with m / z in the ranges of about 1452.9 ± 0.8, 1162.5 ± 1, and 968.8 ± 1.5. Ions in these ranges correspond to insulin ions with 4+, 5+, and 6+ charges, respectively. Single isotopic ions with these charges mainly fall within the mentioned m / z ranges. However, low-abundance naturally occurring isotopic variants may appear outside these ranges. Insulin ions in the range of 1162.5±1 preferably include insulin ions with m / z of about 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 1163.32±0.1; for example, ions with m / z of 1162.54±0.1. Insulin ions in the range of 968.8 ± 1.5 preferably include insulin ions with m / z of about 968.28 ± 0.1, 968.45 ± 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, and 969.61 ± 0.1; for example, ions with m / z of 968.95 ± 0.1. In some embodiments, linking the amount of one or more insulin ions detected by mass spectrometry to the amount of insulin protein in the sample includes comparison with an internal standard; for example, human or non-human insulin protein. The internal standard may optionally be isotopically labeled.

[0040] In any of the methods proposed herein, the sample may include a biological sample; preferably a bodily fluid sample, including, for example, plasma or serum.

[0041] Mass spectrometry can be performed in positron emission tomography (PET) mode (tandem or high resolution / high precision). Alternatively, it can be performed in electron-negative mode. Various ionization sources can be used, including, for example, atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) sources. In some embodiments, insulin and / or chemically modified or unmodified insulin A or B chains are ionized by ESI in PET mode.

[0042] In any of the methods presented herein, a detectable internal standard can be provided in the sample, and the amount of the internal standard in the sample is also determined. In embodiments utilizing a detectable internal standard, all or part of the target analyte and the internal standard present in the sample are ionized to generate multiple ions detectable in a mass spectrometer, and the generated one or more ions are detected by mass spectrometry. In these embodiments, the presence or amount of ions generated by the target analyte can be correlated with the presence or amount of the target analyte in the sample by comparing it with the amount of detected internal standard ions.

[0043] Alternatively, the amount of insulin in a sample can be determined by comparison with one or more external reference standards. Exemplary external reference standards include blank plasma or serum traced with human or non-human insulin, synthetic insulin analogs, or isotopically labeled variants thereof.

[0044] In some embodiments, the method is capable of determining the amount of insulin in a sample at levels ranging from about 10 μIU / mL to 500 μIU / mL (equivalent to about 60 pmol / L to 3000 pmol / L, or about 0.35 ng / mL to 17.4 ng / mL), including the range of 10 μIU / mL and 500 μIU / mL.

[0045] As used herein, unless otherwise stated, the singular forms “a,” “an,” and “the” include a plural indicator. Thus, for example, reference to “a protein” includes multiple protein molecules.

[0046] As used herein, the terms “purification” and “enrichment” do not refer to the removal of all substances from a sample except for the target analyte. Rather, these terms refer to the process of enriching one or more target analytes relative to other components in the sample that may interfere with the detection of the target analyte. Purifying a sample in various ways can relatively reduce one or more interfering substances, such as substances that may or may not interfere with the detection of selected precursor or daughter ions by mass spectrometry. When using the term, relative reduction does not require the complete removal of any substances present in the material to be purified along with the target analyte through purification.

[0047] As used herein, the term "immunopurification" refers to a purification process that uses antibodies, including polyclonal or monoclonal antibodies, to enrich one or more target analytes. Immunopurification can be performed using immunopurification methods well known in the art. Typically, immunopurification processes utilize antibodies that are bound, coupled, or otherwise attached to a carrier, such as a column, well, tube, gel, capsule, particle, etc. As used herein, immunopurification includes, but is not limited to, processes commonly referred to in the art as immunoprecipitation, and processes commonly referred to in the art as affinity chromatography or immunoaffinity chromatography.

[0048] As used herein, the term "immunoparticle" refers to capsules, beads, gel particles, etc., on or / or attached to their surface by antibodies, conjugated to them, or otherwise attached to them. In some preferred embodiments, the immune particles are agarose gels or agarose beads. In alternative preferred embodiments, the immune particles include glass beads, plastic beads, or silicone beads or silica gel.

[0049] As used herein, the term "anti-insulin antibody" refers to any polyclonal or monoclonal antibody that has an affinity for insulin. In various embodiments, the specificity of the insulin antibody for chemicals other than insulin may differ; for example, in some preferred embodiments, the anti-insulin antibody is specific for insulin and therefore has little or no affinity for other chemicals, while in other preferred embodiments, the anti-insulin antibody is non-specific and therefore binds to certain chemicals other than insulin.

[0050] As used herein, the term "sample" refers to any sample that may contain the target analyte. As used herein, the term "body fluid" refers to any fluid that can be separated from an individual body. For example, "body fluid" may include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc. In a preferred embodiment, the sample includes a body fluid sample from a human being; preferably plasma or serum.

[0051] As used herein, the term "solid-phase extraction" or "SPE" refers to the process by which a chemical mixture is separated into components due to the affinity of components dissolved or suspended in a solution (i.e., the mobile phase) for a solid (i.e., the solid phase) through which the solution passes or surrounds. In some cases, the solid phase may retain components unwanted by the mobile phase as the mobile phase passes through or surrounds it, resulting in the purification of the analyte in the mobile phase. In other cases, the solid phase may retain the analyte while allowing components unwanted by the mobile phase to pass through or surround it. In these cases, a second mobile phase is then used to elute the retained analyte from the solid phase for further processing or analysis. SPE, including TFLC, can be operated via single-mode or mixed-mode mechanisms. Mixed-mode mechanisms utilize ion exchange and hydrophobic retention within the same column; for example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention, or it may exhibit strong cation exchange and hydrophobic retention.

[0052] Typically, the affinity of SPE column packing material for the analyte can be due to any of a variety of mechanisms, such as one or more chemical interactions or immunoaffinity interactions. In some embodiments, SPE of insulin is not performed using immunoaffinity column packing materials. That is, in some embodiments, insulin is purified from the sample using an SPE column that is not an immunoaffinity column.

[0053] As used herein, the term “chromatography” refers to the process of separating a mixture of chemicals carried by a liquid or gas into components due to the differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0054] As used herein, the term "liquid chromatography" or "LC" refers to the process by which a fluid selectively impedes one or more components of a fluid solution as it passes uniformly through a column of fine material or through a capillary channel. Impedement is caused by the distribution of the mixture components between the stationary phase(s) and the bulk fluid (i.e., the mobile phase) as the fluid moves relative to the stationary phase. Examples of "liquid chromatography" include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and turbulent-flow liquid chromatography (TFLC) (sometimes referred to as high-turbulence liquid chromatography (HTLC) or high-throughput liquid chromatography).

[0055] As used herein, the term “high performance liquid chromatography” or “HPLC” (sometimes referred to as “high pressure liquid chromatography”) refers to liquid chromatography that improves separation by forcing the mobile phase through a stationary phase, typically a densely packed column, under pressure.

[0056] As used herein, the term "turbulent liquid chromatography" or "TFLC" (sometimes referred to as high-turbulence liquid chromatography or high-throughput liquid chromatography) refers to a form of chromatography that utilizes turbulence through the measuring material in the column packing as the basis for separation. TFLC has been applied to prepare samples containing two unknown drugs prior to analysis by mass spectrometry. See, for example, Zimmer et al., J Chromatogr A 854:23-35 (1999); see also U.S. Patent Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further elaborate on TFLC. Those skilled in the art understand "turbulence." When a fluid flows slowly and steadily, the flow is referred to as "laminar flow," for example, fluid flowing through an HPLC column at a low velocity in a laminar manner. In laminar flow, the motion of fluid particles is orderly coordinated and generally occurs as particles moving in a generally linear fashion. At higher rates, the inertia of water overcomes fluid friction and results in turbulence. Fluid that does not contact irregular boundaries "exceeds" fluid that is slowed by friction or deflected by rough surfaces. When fluid flows turbulently, it exhibits rotating vortex flow (vortexes), and the "dragging force" is greater than when the flow is laminar. When the fluid flow is laminar or turbulent, many references can be used to aid in determination (e.g., Turbulent Flow Analysis: Measurement and Prediction ,PSBernard&J.M.Wallace,John Wiley&Sons,Inc.,(2000); An Introduction to Turbulent Flow ,Jean Mathieu&Julian Scott,Cambridge University Press(2001)).

[0057] As used herein, the term “gas chromatography” or “GC” refers to the chromatographic method of vaporizing a sample mixture and injecting it through a carrier gas stream (such as nitrogen or helium) through a column containing a stationary phase consisting of liquid or solid particles, and separating the constituent compounds according to their affinity for the stationary phase.

[0058] As used herein, the terms "large particle column" or "extraction column" refer to chromatographic processes containing particles with an average particle size greater than about 50 μm. As used in this context, the term "about" means ±10%.

[0059] As used herein, the term "analytical column" refers to a chromatographic column with sufficient plates to separate substances eluted from the column from a sample, sufficient to allow determination of the presence or amount of an analyte. Such columns are often distinguished from "extraction columns," which have the general purpose of separating from non-retaining substances or extracting retained substances to obtain a purified sample for further analysis. As used in this context, the term "about" means ±10%. In a preferred embodiment, the analytical column contains particles with a diameter of about 5 μm.

[0060] As used herein, the terms "on-line" and "inline," as in "online automated form" or "online extraction," refer to processes that do not require operator intervention. Conversely, the term "off-line," as used herein, refers to processes that require manual operator intervention. Therefore, if the sample is precipitated and then the supernatant is manually loaded into the autosampler, the precipitation and loading steps are offline from subsequent steps. In various embodiments of the method, one or more steps can be performed in an online automated form.

[0061] As used herein, the term “mass spectrometry” or “MS” refers to an analytical technique for identifying compounds by their mass. MS refers to a method of filtering, detecting, and measuring based on their mass-to-charge ratio, or “m / z”. MS techniques typically involve (1) ionizing the compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating its mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A “mass spectrometer” typically includes an ion generator, a mass analyzer, and an ion detector. Generally, one or more target molecules are ionized, and the ions are subsequently introduced into the mass spectrometer, where the ions spatially follow a path that depends on their mass (“m”) and charge (“z”) due to a combination of magnetic and electric fields. See, for example, U.S. Patent No. 6,204,500 entitled "Mass Spectrometry From Surfaces"; U.S. Patent No. 6,107,623 entitled "Methods and Apparatus for Tandem Mass Spectrometry"; U.S. Patent No. 6,268,144 entitled "DNA Diagnostics Based On Mass Spectrometry"; U.S. Patent No. 6,124,137 entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes"; Wright et al., Prostate Cancer and Prostatic Diseases 1999, 2:264-76; and Merchant and Weinberger, Electrophoresis 2000, 21:1164-67.

[0062] As used herein, "high-resolution / high-precision mass spectrometry" refers to mass spectrometry performed using a mass analyzer capable of measuring the mass-to-charge ratio of charged classes with sufficient accuracy and precision to identify unique chemical ions. A unique chemical ion can be identified when the individual isotopic peaks from that ion are readily distinguishable. The specific resolution and mass precision required to identify a unique chemical ion vary with the ion's mass and charge state.

[0063] As used herein, the term "resolution" or "resolution (FWHM)" (also referred to in the art as "m / Δm") is used. 50％ The half-maximum width (FWHM) refers to the observed mass-to-charge ratio divided by the mass peak width at 50% of the maximum height. Figure 1A The effect of resolution differences is explained in section -C, which shows the theoretical mass spectrum of an ion with an m / z of approximately 1093. Figure 1AThe theoretical mass spectrum from a mass analyzer with a resolution of approximately 3000 (typical operating conditions for a conventional quadrupole mass analyzer) is shown. Figure 1A As observed, the peaks of each isotope are indistinguishable. Through comparison, Figure 1B The theoretical mass spectrum from a mass analyzer with a resolution of approximately 10,000 is shown, with each isotope peak clearly distinguishable. Figure 1C The theoretical mass spectra from a mass analyzer with a resolution of approximately 12,000 are shown. At this highest resolution, each isotopic peak contains less than 1% contribution from the baseline.

[0064] As used herein, the term "unique chemical ion" in mass spectrometry refers to a single ion consisting of a single atom. A single ion may carry one or more charges.

[0065] As used herein, the term "precision" (or "mass precision") in mass spectrometry refers to the possible deviation between the instrument response and the true m / z of the investigated ion. Precision is typically expressed in parts per million (ppm). Figure 2A -D illustrates the impact of differences in quality accuracy, showing the possible boundary between the measured m / z and the actual m / z for a theoretical peak value of 1093.52094. Figure 2A The possible range of m / z measured with an accuracy of 120 ppm is shown. Conversely, Figure 2B The possible range of m / z measured with an accuracy of 50 ppm is shown. Figure 2C and 2D The possible range of m / z, or even narrower, measured at 20ppm and 10ppm accuracy is shown.

[0066] The high-resolution / high-precision mass spectrometry method of the present invention can be performed on instruments capable of mass analysis at FWHM greater than 10,000, 15,000, 20,000, 25,000, 50,000, 100,000 or even higher. Similarly, the method of the present invention can be performed on instruments capable of mass analysis at precisions less than 50 ppm, 20 ppm, 15 ppm, 10 ppm, 5 ppm, 3 ppm or even lower. Instruments with these performance characteristics can be incorporated into certain orbital trap mass analyzers, time-of-flight (“TOF”) mass analyzers, or Fourier transform ion cyclotron resonance mass analyzers. In a preferred embodiment, the method is performed using an instrument including an orbital trap mass analyzer or a TOF mass analyzer.

[0067] The term "orbit trap" describes an ion trap consisting of a barrel-shaped outer electrode and a coaxial inner electrode. Ions are injected tangentially into an electric field between the electrodes, and are captured because the electrostatic interaction between the ions and the electrodes is balanced by centrifugal force as the ions travel along the orbit of the coaxial inner electrode. As the ions travel along the orbit of the coaxial inner electrode, the trajectory of the captured ions oscillates along the axis of the central electrode at a resonant frequency relative to the ion's mass-to-charge ratio. Detection of the orbital oscillation frequency allows the orbit trap to be used as a mass analyzer with high accuracy (down to 1-2 ppm) and high resolution (FWHM) (up to approximately 200,000). An orbit trap-based mass analyzer is described in detail in U.S. Patent No. 6,995,364, which is incorporated herein by reference in its entirety. Qualitative and quantitative analysis of various analytes has been reported using orbit trap analyzers. See, for example, U.S. Patent Application Publication No. 2008 / 0118932 (filed November 9, 2007); et al., Rapid Commun. Mass Spectrom., 2008, 22:477-485; Le Breton et al., Rapid Commun. Mass Spectrom., 2008, 22:3130-36; Thevis et al., Mass Spectrom. Reviews, 2008, 27:35-50; Thomas et al., J. Mass Spectrom., 2008, 43:908-15; Schenk et al., BMC Medical Genomics, 2008, 1:41; and Olsen et al., Nature Methods, 2007, 4:709-12.

[0068] As used herein, the term "operating in negative ion mode" refers to those mass spectrometry methods that generate and detect negative ions. As used herein, the term "operating in positive ion mode" refers to those mass spectrometry methods that generate and detect positive ions. In a preferred embodiment, the mass spectrometry is performed in positive ion mode.

[0069] As used herein, the term "ionization" refers to the process of generating analyte ions with a net charge equal to or greater than one or more electron units. A negative ion is an ion with a net negative charge of one or more electron units, while a positive ion is an ion with a net positive charge of one or more electron units.

[0070] As used herein, the term "electron ionization" or "EI" refers to a method in which a target analyte interacts with a stream of electrons in the gas or vapor phase. Collisions between electrons and the analyte generate analyte ions that can then be used for mass spectrometry.

[0071] As used herein, the term “chemical ionization” or “CI” refers to a method in which a reagent gas (e.g., ammonia) is subjected to electron collisions and analyte ions are formed through the interaction between the reagent gas ions and the analyte molecules.

[0072] As used herein, the term "fast atom bombardment" or "FAB" refers to a method in which a beam of high-energy atoms (often Xe or Ar) collides with a non-volatile sample, desorbing and ionizing the molecules contained within the sample. The sample is dissolved in a viscous liquid matrix, such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6-crown ether, 2-nitrophenyl octyl ether, sulfolane, diethanolamine, and triethanolamine. Selecting an appropriate matrix for the compound or sample is an empirical process.

[0073] As used herein, the term "matrix-assisted laser desorption / ionization" or "MALDI" refers to a method that exposes a non-volatile sample to laser irradiation, thereby desorbing and ionizing analytes in the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. In MALDI, the sample is mixed with an energy-absorbing matrix to promote the desorption of analyte molecules.

[0074] As used herein, the terms "surface-enhanced laser desorption / ionization" or "SELDI" refer to another method of desorbing and ionizing analytes in a non-volatile sample by exposing it to laser irradiation and through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more target analytes. Similar to MALDI, this process can also employ energy-absorbing materials to promote ionization.

[0075] As used herein, the term "electrospray ionization" or "ESI" refers to a method of passing a solution through a short capillary tube with a high positive or negative potential applied to its end. This causes the solution reaching the end of the tube to evaporate (atomize) into a jet or spray of very small solution droplets in solvent vapor. These droplets flow through an evaporation chamber. As the droplets become smaller, the electrical surface charge density increases until the natural repulsion between like charges causes the release of ions and neutral molecules.

[0076] As used herein, the terms "atmospheric pressure chemical ionization" or "APCI" refer to a mass spectrometry method similar to ESI; however, APCI generates ions through ion-molecule reactions occurring within plasma at atmospheric pressure. The plasma is held in place by a discharge between a spray capillary and a counter electrode. The ions are then typically extracted into the mass analyzer using a set of differential pump skimmer stages. Countercurrent drying and preheating of N2 gas can be used to enhance solvent removal. Gas-phase ionization in APCI can be more efficient than ESI for analyzing low-polarity compounds.

[0077] As used herein, the term "atmospheric pressure photoionization" or "APPI" refers to the mass spectrometric form in which the ionization mechanism of molecule M is photon absorption and electron emission to form the molecular ion M+. Because the photon energy is usually only slightly above the ionization potential, the molecular ion is not easily affected by dissociation. In many cases, the analysis of the sample may not require chromatography, thus saving considerable time and money. In the presence of water vapor or proton solvents, the molecular ion can absorb H to form MH+. This often occurs when M has a high proton affinity. Because the sum of M+ and MH+ is constant, this does not affect the accuracy of quantification. Drug compounds in proton solvents are usually observed to be MH+, while nonpolar compounds, such as naphthalene or testosterone, usually form M+. See, for example, Robb et al., Anal. Chem. 2000, 72(15):3653-3659.

[0078] As used herein, the term “inductively coupled plasma” or “ICP” refers to a method in which a sample interacts with a partially ionized gas at a sufficiently high temperature, causing most of the elements to break down into atoms and become ionized.

[0079] As used herein, the term “field desorption” refers to a method of placing a non-volatile sample on an ionization surface and using a strong electric field to generate analyte ions.

[0080] As used herein, the term "desorption" refers to the transfer of an analyte from its surface and / or inlet into the gas phase. Laser desorption / thermal desorption is a technique in which a sample containing the analyte is thermally desorbed into the gas phase using a laser pulse. The laser strikes the back of a specially designed 96-well plate with a metal base. The laser pulse heats the base, and the heat causes the sample to transfer into the gas phase. The gas phase sample is then drawn into a mass spectrometer.

[0081] As used in this article, the term "selective ion monitoring" is a detection mode of a mass spectrometer in which only ions with a relatively narrow mass range, typically about one mass unit, are detected.

[0082] As used in this article, the term "multi-reaction mode," sometimes referred to as "selective reaction monitoring," is a detection mode of a mass spectrometer in which precursor ions and one or more fragment ions are selectively detected.

[0083] As used herein, the terms “lower limit of quantitation,” “lower limit of quantitation,” or “LLOQ” refer to the point at which a measurement becomes quantitatively meaningful. At this LOQ, the analyte response is identifiable, discontinuous, and reproducible, with a relative standard deviation (RSD%) of less than 20% and an accuracy of 85%–115%.

[0084] As used herein, the term “limit of detection” or “LOD” refers to the point at which a measured value exceeds the uncertainty associated with it. LOD is the point at which a value exceeds the inaccuracy associated with the measurement and is defined as three times the mean RSD at zero concentration.

[0085] As used herein, the “amount” of an analyte in a body fluid sample typically refers to the absolute amount of detectable analyte in the sample volume. However, amount also encompasses a relative amount compared to the amount of another analyte. For example, the amount of analyte in a sample may be greater than the control or normal level of the analyte typically present in the sample.

[0086] As used herein, the term "about" for quantitative measurements (excluding measurements of ion mass) refers to an indicated value plus or minus 10%. The mass spectrometer may vary slightly in determining the mass of a specified analyte. In the context of ion mass or ion mass / charge ratio, the term "about" refers to + / - 0.50 atomic mass units.

[0087] The above summary of the invention is non-limiting, and other features and advantages of the invention will become apparent from the following detailed description and claims.

[0088] Detailed description of the attached diagram

[0089] Figure 1A -C shows the resolution of approximately 3000 ( Figure 1A ), approximately 10,000 ( Figure 1B ) and approximately 12,000 ( Figure 1C The theoretical mass spectra of ions with an m / z of approximately 1093 were analyzed by the mass analyzer.

[0090] Figure 2A -D indicates the mass accuracy at 120 ppm with a m / z of 1093.52094. Figure 2A ), 50ppm quality accuracy ( Figure 2B ), 20ppm quality accuracy ( Figure 2C ) and 10ppm quality accuracy ( Figure 2D Under these conditions, the possible deviation between the instrument reaction and the actual m / z of the ions at the theoretical peak value is considered.

[0091] Figure 3A Examples of collection spectra of human insulin by ionization in positive ion mode via ESI ions are shown in Figures B and B, respectively, under acidic and alkaline conditions. Details are described in Example 3.

[0092] Figure 4 A shows an example spectrum of human insulin in the range of approximately 900–1200 m / z generated using a QTOF mass spectrometer. Figure 4B shows the contaminant peaks generated from the back-extracted serum sample matrix using QTOF mass spectrometry. Details are described in Example 4.

[0093] Figure 5 A shows an exemplary high-resolution / high-precision spectrum of human insulin in the approximately 1154–1177 m / z range generated using a QTOF mass spectrometer. Figure 5 B shows the unfolded graph for the range of approximately 1159-1166 m / z.

[0094] Details are described in Example 4.

[0095] Figure 6 A shows an exemplary high-resolution / high-precision spectrum of human insulin in the approximately 964-973 m / z range generated using a QTOF mass spectrometer. Figure 6 B shows a plot for the range of approximately 967-971 m / z. Details are described in Example 4.

[0096] Figure 7 A linear graph of human insulin quantification in tracer-simulated serum standards, measured using high-resolution / high-precision MS of insulin, is shown. Details are described in Example 4.

[0097] Figure 8 A linear graph of human insulin quantification in tracer-retrieved serum standards, measured using high-resolution / high-precision MS, is shown. Details are described in Example 4.

[0098] Figure 9 The Q1 tandem mass spectrometry scans showing the generation of human insulin in 5+ and 6+ charge states are shown.

[0099] Details are described in Example 5.

[0100] Figure 10 The product ion scan is shown, displaying the fragmented human insulin precursor ion in a 6+ charge state. Details are discussed in Example 5.

[0101] Figure 11 The product ion scan is shown, displaying the fragmented human insulin precursor ions in a 5+ charged state. Details are discussed in Example 5.

[0102] Figure 12 The relative intensities of selective fragment ions generated by breaking up 6+ and 5+ human insulin precursor ions at different collision energies are shown. Details are discussed in Example 5.

[0103] Figure 13 Composite spectra of two possible human insulin A-chain precursor ions (in 2+ and 3+ charge states) and two possible human insulin B-chain precursor ions (in 3+ and 4+ charge states) are shown. Details are discussed in Example 6.

[0104] Figure 14 The tandem mass spectrometry Q1 scans showing the generation of possible human insulin B-chain precursor ions in 3+, 4+, and 5+ charge states are illustrated. Details are described in Example 6.

[0105] Figure 15 The relative intensities of selective fragment ions generated by breaking 3+ human insulin A chain precursor ions at different collision energies are shown. Details are discussed in Example 6.

[0106] Figure 16 The product ion scan from the fragmented human insulin B-chain precursor ion in a 4+ charged state is shown. Details are discussed in Example 6.

[0107] Figure 17 The product ion scan from the fragmented human insulin B-chain precursor ion in a 3+ charged state is shown. Details are discussed in Example 10.

[0108] Figure 18 The relative intensities of selective fragment ions generated by breaking 3+ human insulin B-chain precursor ions at different collision energies are shown. Details are discussed in Example 10.

[0109] Figure 19 A graph showing the LLOQ of human insulin in patient serum samples for assessment by tandem mass spectrometry of the human insulin B chain is presented. Details are discussed in Example 12.

[0110] Figure 20 A linear graph of the quantification of human insulin in tracer-extracted serum samples measured by tandem mass spectrometry of the human insulin B chain is shown. Details are described in Example 13. Invention Details

[0111] A method for determining the amount of insulin in a sample is described. More specifically, a mass spectrometry method for detecting and quantifying insulin in a sample is described. The method utilizes solid-phase extraction (SPE) and / or liquid chromatography (LC) to purify the selected analyte, combined with mass spectrometry (MS), thereby enabling the detection and quantification of insulin in a sample by an assay system. Preferred embodiments are particularly suitable for automated insulin quantification assays in large clinical laboratories.

[0112] Suitable samples for use in the methods of the present invention include any sample that may contain the target analyte. In some preferred embodiments, the sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, cell culture, or organ culture. In some preferred embodiments, the sample is obtained from a mammal, such as a dog, cat, horse, etc. Particularly preferred mammals are primates, and most preferably males or females. Preferred samples include bodily fluids, such as blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples; plasma and serum are preferred. For example, such samples can be obtained from a patient; that is, a living man or woman in a clinical setting undergoing diagnosis, prognosis, or treatment of a disease or condition. In embodiments where the sample includes a biological sample, when the sample is obtained from a biological source, the method can be used to determine the amount of insulin in the sample.

[0113] This invention also covers kits for insulin quantification. Kits for insulin quantification may include kits containing the compositions provided herein. For example, a kit may include packaging material and an isotope-labeled internal standard sufficient for at least one assay. Typically, the kit will also include instructions for using the packaged reagents for insulin quantification, recorded in tangible form (e.g., on paper or electronic media).

[0114] The calibration and QC mixed serum used in embodiments of the present invention is preferably prepared using a matrix similar to the intended sample matrix, provided that insulin is substantially absent.

[0115] Sample preparation for mass spectrometry analysis

[0116] In the preparation for mass spectrometry analysis, insulin can be enriched relative to one or more other components in the sample by various methods known in the art, including (e.g.) liquid chromatography, filtration, centrifugation, thin-layer chromatography (TLC), electrophoresis (including capillary electrophoresis), affinity separation (including immunoaffinity separation), extraction (including ethyl acetate or methanol extraction), and the use of a dissociative agent or a combination thereof.

[0117] One sample purification method that can be used prior to mass spectrometry analysis involves applying the sample to a solid-phase extraction (SPE) column under conditions where the column packing material reversibly retains the target analyte without retaining one or more other substances. In this technique, a first mobile phase condition can be used to retain the target analyte, followed by a second mobile phase condition to remove the retained substances from the column once the unretained substances have been washed away.

[0118] In some embodiments, insulin in the sample can be reversibly retained on an SPE column having a packing material containing an alkyl-binding surface. For example, in some embodiments, insulin can be enriched using a C-8 online SPE column (e.g., Oasis HLB online SPE column / box (2.1 mm × 20 mm) or equivalent from Phenomenex, Inc.) prior to mass spectrometry analysis. In some embodiments, the SPE column is used with HPLC-grade 0.2% aqueous formic acid as the washing buffer and 0.2% formic acid in acetonitrile as the eluent.

[0119] In some implementations, insulin is not purified by immunoaffinity techniques. Some of these implementations utilize SPE columns. In these implementations, the SPE column is not an immunoaffinity column.

[0120] In other embodiments, the method includes immunopurifying insulin prior to mass spectrometry analysis. Any immunopurification method well-known in the art can be used for the immunopurification step. Immunopurification processes often utilize antibodies that are bound, conjugated, or attached to a carrier, such as a column, well, tube, capsule, particle, etc. Typically, immunopurification methods include (1) culturing a sample containing the target analyte with an antibody so that the analyte binds to the antibody, (2) performing one or more washing steps, and (3) eluting the analyte from the antibody.

[0121] In some embodiments, an immunopurification culture step is performed using free antibodies in solution, followed by the antibody binding to or attaching to a solid surface prior to a washing step. In some embodiments, this can be accomplished using a primary antibody as an anti-insulin antibody and a secondary antibody attached to a solid surface with affinity for the primary anti-insulin antibody. In alternative embodiments, the primary antibody binds to the solid surface prior to the culture step.

[0122] Suitable carriers include, but are not limited to, tubes, glass slides, columns, beads, capsules, granules, gels, etc. In some preferred embodiments, the carrier is a multi-well plate, such as a 96-well plate, a 384-well plate, etc. In some embodiments, the carrier is an agarose gel or agarose beads or gel. There are many methods well known in the art by which antibodies (e.g., insulin antibodies or secondary antibodies) can bind, attach, immobilize, or couple to a carrier, for example, through covalent or non-covalent adsorption, affinity binding, ionic bonding, etc. In some embodiments, CNBr-coupled antibodies are used, for example, antibodies can be coupled to CNBr-activated agarose gel. In other embodiments, antibodies are attached to the carrier by binding to proteins, such as protein A, protein G, protein A / G, or protein L.

[0123] The washing step in immunopurification typically includes washing the carrier so that insulin remains bound to the anti-insulin antibodies on the carrier. The elution step in immunopurification typically includes adding a solution that disrupts the binding of insulin to the anti-insulin antibodies. Exemplary eluents include organic solutions, salt solutions, and high or low pH solutions.

[0124] Another sample purification method that can be used prior to mass spectrometry analysis is liquid chromatography (LC). In liquid chromatography, analytes are purified by applying a sample to a chromatographic column under flow-through conditions, wherein the target analyte elutes at a differential rate compared to one or more other substances. Such a process can enrich the amount of one or more target analytes relative to one or more other components of the sample.

[0125] Some liquid chromatography methods, including HPLC, rely on relatively slow laminar flow techniques. Traditional HPLC analysis depends on column packing, where laminar flow of the sample through the column is the basis for separating the target analyte from the sample. Those skilled in the art will understand that separation in such a column is a fractionating process and that suitable LC (including HPLC) instruments and columns can be selected for use with C-peptides. Chromatographic columns typically include a medium (i.e., packing material) that facilitates the separation (i.e., fractionation) of chemical components. The medium may include fine particles. The particles typically include bound surfaces that interact with various chemical components to facilitate their separation. A suitable bound surface is a hydrophobic bound surface, such as an alkyl bound surface or a cyano bound surface. Alkyl bound surfaces may include C-4, C-8, C-12, or C-18 bound alkyl groups. In some embodiments, the chromatographic column is a monolithic C-18 column. The chromatographic column includes an inlet for receiving the sample and an outlet for discharging the effluent containing the fractionated sample. The sample may be supplied directly to the inlet or from an SPE column, such as an inline SPE column or a TFLC column. In some implementations, an in-line filter may be used in front of the SPE column and / or HPLC column to remove particulates and phospholipids from the sample before it reaches the SPE and / or TFLC and / or HPLC column.

[0126] In one implementation, the sample is applied to the inlet of the LC column, eluted with a solvent or solvent mixture, and discharged at the outlet. Different solvent modes can be selected to elute the target analyte. For example, gradient mode, constant composition mode, or multi-mode (i.e., mixed) mode can be used for liquid chromatography. During chromatography, the separation of substances is affected by variables such as the selection of the eluent (also known as the "mobile phase"), elution mode, gradient conditions, temperature, etc.

[0127] In some embodiments, insulin in the sample is enriched using HPLC. This HPLC can be performed using a monolithic C-18 column chromatography system, such as the Onyx monolithic C-18 column (50 × 2.0 mm) from Phenomenex Inc., or an equivalent. In some embodiments, HPLC is performed using HPLC-grade 0.2% aqueous formic acid as solvent A and 0.2% formic acid in acetonitrile as solvent B.

[0128] By carefully selecting valves and connectors, two or more chromatographic columns can be connected when needed to transfer material from one column to the next without any manual steps. In a preferred embodiment, the selection of valves and pipes can be computer-controlled and pre-programmed to perform the necessary steps. Most preferably, the chromatographic system is also connected to a detector system, such as an MS system, in this online manner. Thus, the operator can place a tray of samples in an autosampler, and the remaining operations are performed under computer control, resulting in the purification and analysis of all selected samples.

[0129] In some implementations, TFLC can be used to purify insulin prior to mass spectrometry analysis. In such implementations, a TFLC column capturing the analyte can be used to extract the sample. The analyte is then eluted and transferred online to an analytical HPLC column. For example, sample extraction can be achieved using a TFLC extraction cartridge with a large particle size (50 μm) packing. The sample eluted from this column can then be transferred online to an HPLC analytical column for further purification prior to mass spectrometry analysis. Because these steps involved in the chromatographic process can be automated, the need for operator involvement during analyte purification is minimized. This feature results in time and cost savings and eliminates the chance of operator error.

[0130] In some implementations, one or more of the above purification techniques can be used in parallel to purify insulin to allow for the simultaneous processing of multiple samples. In some implementations, the purification techniques employed do not include immunopurification techniques, such as immunoaffinity chromatography.

[0131] Insulin was detected and quantified by mass spectrometry.

[0132] Mass spectrometry analysis is performed using a mass spectrometer comprising an ion source for ionizing and fractionating the sample and generating charged molecules for further analysis. In various embodiments, insulin can be ionized by any method known to those skilled in the art. For example, insulin can be ionized by electron ionization, chemical ionization, electrospray ionization (ESI), proton ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), laser diode thermal desorption (LDTD), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix-assisted laser desorption / ionization (MALDI), field ionization, field desorption, thermal spray / plasma spray ionization, surface-enhanced laser desorption / ionization (SELDI), inductively coupled plasma (ICP), and particle beam ionization. Those skilled in the art will understand that the choice of ionization method can be determined based on the analyte, sample type, detector type, and the selection of positive or negative modes, and insulin can be ionized in either positive or negative modes. In a preferred embodiment, insulin can be ionized by ESI in positive ion mode.

[0133] In mass spectrometry, after ionization of a sample, the resulting positively or negatively charged ions are analyzed to determine the mass-to-charge ratio (m / z). Various analyzers used for determining m / z include quadrupole analyzers, ion trap analyzers, time-of-flight analyzers, Fourier transform ion cyclotron resonance mass analyzers, and orbital trap analyzers. Some exemplary ion trap methods are described in Bartolucci et al., Rapid Commun. Mass Spectrom. 2000, 14:967-73.

[0134] Several detection modes can be used to detect ions. For example, selective ion monitoring (SIM) can be used to detect selected ions, or alternatively, mass phase transitions caused by collision-induced dissociation or loss of neutrality can be monitored, such as multiple reaction monitoring (MRM) or selective reaction monitoring (SRM). In some embodiments, a quadrupole analyzer is used to determine the mass-to-charge ratio. In a quadrupole or quadrupole ion trap instrument, ions in an oscillating radio frequency field experience forces proportional to the DC potential applied between the electrodes, the RF signal amplitude, and the mass-to-charge ratio. The voltage and amplitude can be selected such that only ions with a specific mass-to-charge ratio move along the length of the quadrupole, while all other ions are excluded. Therefore, a quadrupole instrument can be used as a "mass filter" and "mass detector" for ions injected into an instrument.

[0135] When ions collide with the detector, they generate electronic pulses that are converted into digital signals. The desired data is forwarded to a computer, which plots the collected ion counts against time. The resulting mass chromatogram is similar to that produced by conventional HPLC-MS methods. The area under the curve (AUC) or the amplitude of such peaks corresponding to a specific ion can be measured and correlated with the amount of the target analyte. In some embodiments, the AUC or peak amplitude of fragment ions and / or precursor ions is measured to determine the amount of insulin. A calibration standard curve can be used, based on the peak values ​​of one or more ions from internal or external molecular standards, to convert the relative abundance of a specified ion into the amount of the original analyte.

[0136] The resolution of MS techniques employing certain mass spectrometers can be improved through tandem mass spectrometry or MS / MS. In this technique, precursor ions (also called parent ions) generated by a target molecule are filtered in the MS instrument, followed by fragmentation to produce one or more fragment ions (also called daughter ions or product ions), which are then analyzed in a second MS process. By carefully selecting the precursor ions, only ions generated by certain analytes are allowed to pass through the fragmentation chamber, where they collide with inert gas atoms to produce fragment ions. Because precursor and fragment ions are generated in a reproducible manner under a series of specified ionization / fragmentation conditions, MS / MS techniques provide an extremely powerful analytical tool. For example, the combination of filtration / fragmentation can be used to eliminate interfering substances and may be particularly useful in complex samples, such as biological samples. In some embodiments, tandem mass spectrometry analysis is performed using a mass spectrometer with multiple quadrupole analyzers (e.g., a three-stage quadrupole instrument).

[0137] In some implementations using MS / MS technology, precursor ions are separated for further fragmentation, and fragment ions generated from the precursor ions are further detected using collisional activated dissociation (CAD). In CAD, the precursor ions gain energy through collisions with an inert gas and are subsequently fragmented through a process called "unimolecular decomposition." Sufficient energy must accumulate in the precursor ions so that some bonds in the ions can be broken due to the increased vibrational energy.

[0138] In some implementations, insulin in a sample is detected and / or quantified using MS / MS as follows. Insulin is enriched in the sample by first subjecting it to SPE followed by liquid chromatography, preferably HPLC; a liquid solvent stream from the chromatographic column enters the heated nebulizer port of the MS / MS analyzer; and the solvent / analyte mixture is vaporized within a heated, electrically charged conduit at the port. During these processes, the analyte (i.e., insulin) is ionized. Ions, such as precursor ions, pass through the instrument port and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters that allow selection of ions based on their mass-to-charge ratio (m / z) (i.e., selection of “precursor” and “fragment” ions in Q1 and Q3, respectively). Quadrupole 2 (Q2) is a collision chamber where ions are broken up. The first quadrupole (Q1) of the mass spectrometer selects molecules with the m / z of insulin ions. Precursor ions with the appropriate m / z are introduced into the collision chamber (Q2), while undesirable ions with any other m / z collide with the sides of the quadrupole and are eliminated. The precursor ions entering Q2 collide with and break up neutral gas molecules (e.g., argon molecules). The resulting fragment ions enter quadrupole 3 (Q3), where fragment ions are selected for detection.

[0139] Ionization of insulin can generate multi-charged precursor ions (e.g., 4+, 5+, 6+ precursor ions, etc.). Ionization conditions, particularly the pH of the buffer solution used in electrospray ionization, significantly affect the identity and quantity of the generated insulin precursor ions. For example, under acidic conditions, positive electrospray ionization may primarily generate 5+ and 6+ charged insulin precursor ions with m / z values ​​of 1162.5 ± 0.5 and 968.5 ± 0.5, respectively. However, under alkaline conditions, positive electrospray ionization may primarily generate 4+ and 5+ charged insulin precursor ions with m / z values ​​of 1453.75 ± 0.5 and 1162.94 ± 0.5, respectively. The method can utilize acidic or alkaline conditions; acidic conditions are preferred.

[0140] The method may include MS / MS performed in positive or negative ion mode; positive ion mode is preferred. In some embodiments, the electrospray buffer is acidic and Q1 is selected for insulin precursor ions with an m / z of about 1162.5 ± 0.5 or 968.5 ± 0.5. Fragmentation of any of these insulin precursor ions generates fragment ions with an m / z of about 226.21 ± 0.5 and / or 135.6 ± 0.5. Therefore, in an embodiment where Q1 is selected for one or more insulin precursor ions selected from ions with an m / z of about 1162.5 ± 0.5 and 968.5 ± 0.5, Q3 may select one or more fragment ions selected from ions with an m / z of about 226.21 ± 0.5 and 135.6 ± 0.5. In some embodiments, the relative abundance of a single fragment ion from a single precursor ion may be measured. Alternatively, the relative abundance of two or more fragment ions from a single precursor ion may be measured. In these embodiments, the relative abundance of each fragment ion can be subjected to any known mathematical processing to quantitatively assess insulin in the initial sample. In other embodiments, one or more fragment ions from two or more precursor ions can be measured and used as described above to quantitatively assess insulin in the initial sample.

[0141] Alternative operating modes for the tandem mass spectrometer in some implementations include product ion scanning and precursor ion scanning. For a description of these operating modes, see, for example, E. Michael Thurman et al., Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (edited by Amadeo R. Fernandez-Alba, Elsevier 2005) (387).

[0142] In other embodiments, a high-resolution / high-precision mass spectrometer can be used for the quantitative analysis of insulin according to the method of the present invention. To achieve acceptable accuracy in the quantitative results, the mass spectrometer must be able to exhibit a resolution of 10,000 or greater (FWHM) for the target ion with an accuracy of about 50 ppm or less; preferably, the mass spectrometer exhibits a resolution of 18,000 or better (FWHM) with an accuracy of about 5 ppm or less; for example, a resolution of 20,000 or better (FWHM) and an accuracy of about 3 ppm or less; for example, a resolution of 25,000 or better (FWHM) and an accuracy of about 3 ppm or less. Three example analyzers capable of exhibiting the required performance levels for insulin ions are orbital trap mass analyzers, certain TOF mass analyzers, and Fourier transform ion cyclotron resonance mass analyzers.

[0143] Elements found in bioactive molecules, such as carbon, oxygen, and nitrogen, exist naturally in many different isotopic forms. For example, most carbon exists as... 12 C exists, but only about 1% of all naturally occurring carbon is... 13 C exists. Therefore, a certain fraction of naturally occurring molecules containing at least one carbon atom will contain at least one carbon atom. 13 C atoms. Molecules contain naturally occurring elemental isotopes, resulting in multiple molecular isotopic forms. The mass difference between molecular isotopic forms is at least one atomic mass unit (amu). This is because elemental isotopes differ by at least one neutron (the mass of one neutron ≈ 1 amu). When molecular isotopic forms are ionized into multi-charged states, the mass difference between isotopic forms can become difficult to distinguish because mass spectrometry detects the mass-to-charge ratio (m / z). For example, two isotopic forms with a mass difference of 1 amu, both ionized to the 5+ state, will show a difference of only 0.2 in their m / z. High-resolution / high-precision mass spectrometers can distinguish isotopic forms of highly multi-charged ions (e.g., ions with ±2, ±3, ±4, ±5, or higher charges).

[0144] Due to naturally occurring elemental isotopes, each molecular ion typically exists in multiple isotopic forms (each isotopic form produces a mass spectrometric peak that can be detected individually if analyzed with a sufficiently sensitive mass spectrometer). The m / z ratios and relative abundances of multiple isotopic forms collectively constitute the isotopic characterization of the molecular ion. In some embodiments, the identity of the molecular ion under study can be confirmed using the m / z ratios and relative abundances of two or more molecular isotopic forms. In some embodiments, the molecular ion is quantified using mass spectrometric peaks from one or more isotopic forms. In some related embodiments, the molecular ion is quantified using a single mass spectrometric peak from one isotopic form. In other related embodiments, the molecular ion is quantified using multiple isotopic peaks. In these latter embodiments, the multiple isotopic peaks can be subjected to any appropriate mathematical manipulation. Several mathematical manipulations are known in the art, including, but not limited to, summing the areas under multiple peaks, or averaging the reactions from multiple peaks. Figure 4-6 Example spectra showcasing multiple isotopic forms of insulin ions, including 5+ and 6+, were observed. Figure 5 As observed in AB, peak values ​​for various isotopic forms of the 5+ insulin ion were observed at approximately 1161.72, 1161.92, 1162.12, 1162.32, 1162.52, 1162.72, 1162.92, 1163.12, and 1163.32. Figure 6As observed in AB, peaks were observed for various isotopic forms of the 6+ insulin ion at approximately 968.28, 968.45, 968.62, 968.79, 968.95, 969.12, 969.28, 968.45, and 969.61. However, it should be noted that the precise mass observed for any isotopic variant of the ion may vary slightly due to instrument variations.

[0145] In some embodiments, the relative abundance of one or more ions is measured using a high-resolution / high-precision mass spectrometer to quantitatively assess the amount of insulin in a sample. In some embodiments, the one or more ions measured by high-resolution / high-precision mass spectrometry are multiply charged insulin ions. These multiply charged ions may include one or more ions with m / z in the range of about 1453 ± 0.8 (i.e., one or more monoisotope peaks from 4+ ions) and / or 1162 ± 1 (i.e., one or more monoisotope peaks from 5+ ions) and / or about 968.8 ± 1.5 (i.e., one or more monoisotope peaks from 6+ ions).

[0146] Qualitative and quantitative analysis of various analytes has been reported using high-resolution orbital trap analyzers. See, for example, U.S. Patent Application Publication No. 2008 / 0118932 (filed November 9, 2007); et al., Rapid Commun. Mass Spectrom., 2008, 22:477-485; Le Breton et al., Rapid Commun. Mass Spectrom., 2008, 22:3130-36; Thevis et al., Mass Spectrom. Reviews, 2008, 27:35-50; Thomas et al., J. Mass Spectrom., 2008, 43:908-15; Schenk et al., BMC Medical Genomics, 2008, 1:41; and Olsen et al., Nature Methods, 2007, 4:709-12.

[0147] The results of analyte determinations can be correlated with the amount of analyte in the original sample using many methods known in the art. For example, with careful control of sampling and analytical parameters, the relative abundance of a specified ion can be compared with a table that converts the relative abundance into the absolute amount of the original molecule. Alternatively, an external standard can be used with the sample, and a standard curve can be generated based on the ions generated by those standards. Using such a standard curve, the relative abundance of a specified ion can be converted into the absolute amount of the original molecule. In some preferred embodiments, an internal standard is used to generate a standard curve for calculating insulin levels. Methods for generating and using such standard curves are well known in the art, and those skilled in the art can select an appropriate internal standard. For example, in a preferred embodiment, one or more forms of isotopically labeled insulin can be used as an internal standard. Many other methods for correlating the amount of ions with the amount of the original molecule will be well known to those skilled in the art.

[0148] As used in this article, when analyzed by mass spectrometry, "isotopic labeling" produces a mass shift in labeled molecules relative to unlabeled molecules. Examples of suitable labeling materials include deuterium (…). 2 H) 13 C and 15 N. One or more isotopic labels may be incorporated at one or more positions in a molecule, and one or more isotopic labels may be used on the same isotopically labeled molecule.

[0149] Insulin is quantified by mass spectrometry of unmodified insulin A and / or B chains.

[0150] In other embodiments, insulin may be chemically treated prior to mass spectrometry analysis to generate the constituent chains of insulin. The A and B chains of insulin can be separated by any chemical treatment known in the art to induce disulfide reduction. For example, insulin can be treated with TCEP (tris(2-carboxyethyl)phosphine) to reduce the disulfide bridges of insulin and separate the A and B chains.

[0151] Chains A and B can then undergo one or more of the purification steps described above for insulin purification. In a preferred embodiment, chain A and / or chain B are purified by HPLC prior to mass spectrometry analysis.

[0152] Once purified, the A and / or B chains are subjected to an ionization source. As with insulin, those skilled in the art will understand that the choice of ionization method can be determined based on the analyte, sample type, detector type, and the selection of positive or negative modes. Insulin A and B chains can be ionized in either positive or negative modes. In a preferred embodiment, insulin A and / or B chains are ionized via ESI in positive mode.

[0153] Ionizing the insulin A chain can generate multi-charged A chain precursor ions (e.g., 2+, 3+ precursor ions, etc.). For example, positive electrospray ionization of insulin A chain molecules can generate 2+ and 3+ charged A chain precursor ions with m / z values ​​of 1192.0 ± 0.5 and 795.0 ± 0.5, respectively. Similar to insulin, the identity and amount of multi-charged substances generated by ionizing the insulin A chain are affected by the ionization conditions used. In a preferred embodiment, the insulin A chain is ionized under acidic conditions.

[0154] In an embodiment for performing tandem mass spectrometry analysis of the insulin A chain, Q1 can be selected for one or more insulin A chain precursor ions with m / z of about 1192.0 ± 0.5 and 795.0 ± 0.5. Breaking any of these insulin A chain precursor ions can generate fragment ions with m / z of about 513.0 ± 0.5, 399.0 ± 0.5, 236.0 ± 0.5, and 133.0 ± 0.5. Therefore, in an embodiment where Q1 selects one or more insulin A chain precursor ions selected from ions with m / z of about 1192.0 ± 0.5 and 795.0 ± 0.5, Q3 can select one or more fragment ions selected from ions with m / z of about 513.0 ± 0.5, 399.0 ± 0.5, 236.0 ± 0.5, and 133.0 ± 0.5. In some embodiments, the relative abundance of individual fragment ions from a single precursor ion can be measured. Alternatively, the relative abundance of two or more fragment ions from a single precursor ion can be measured. In these embodiments, the relative abundance of each fragment ion can be subjected to any known mathematical processing to quantitatively assess insulin in the initial sample. In other embodiments, one or more fragment ions from two or more precursor ions can be measured and used as described above to quantitatively assess insulin in the initial sample.

[0155] Similarly, ionization of the insulin B chain can generate multi-charged B chain precursor ions (e.g., 3+, 4+, 5+ precursor ions, etc.). For example, positive electrospray ionization of insulin B chain molecules can generate 3+, 4+, and 5+ charged B chain precursor ions with m / z values ​​of 1144.2±0.5, 858.3±0.5, and 686.8±0.5, respectively. Similar to insulin, the identity and amount of multi-charged compounds generated by ionizing the insulin B chain are affected by the ionization conditions employed. In a preferred embodiment, the insulin B chain is ionized under acidic conditions.

[0156] In an embodiment for performing tandem mass spectrometry analysis of the insulin B chain, Q1 can be selected for one or more insulin B chain precursor ions with m / z of approximately 1144.2 ± 0.5, 858.3 ± 0.5, and 686.8 ± 0.5. Fragmentation of these three insulin B chain precursor ions generates fragment ions with m / z of approximately 825.4 ± 0.5, 768.5 ± 0.5, 753.2 ± 0.5, 345.0 ± 0.5, and 226.2 ± 0.5. Therefore, in embodiments where Q1 selects one or more insulin B-chain precursor ions selected from ions with m / z of about 1144.2 ± 0.5, 858.3 ± 0.5, and 686.8 ± 0.5, Q3 may select one or more fragment ions selected from ions with m / z of about 825.4 ± 0.5, 768.5 ± 0.5, 753.2 ± 0.5, 345.0 ± 0.5, and 226.2 ± 0.5; preferably ions with m / z of about 345.0 ± 0.5 and 226.2 ± 0.5. In some embodiments, the relative abundance of a single fragment ion from a single precursor ion may be measured. Alternatively, the relative abundance of two or more fragment ions from a single precursor ion may be measured. In these embodiments, the relative abundance of each fragment ion may be subjected to any known mathematical processing to quantitatively assess insulin in the initial sample. In other embodiments, one or more fragment ions from two or more precursor ions may be measured and used as described above to quantitatively assess insulin in the initial sample.

[0157] Insulin is quantified by mass spectrometry to measure the chemically modified insulin A and / or B chains.

[0158] In alternative embodiments, the individual insulin A and B chains may undergo one or more chemical modification steps prior to ionization and / or purification. For example, upon separation, the insulin A and B chain molecules may undergo urea methylation to achieve complete alkylation of the constitutive cysteine ​​residues. For instance, urea methylation can be achieved by reacting the insulin A and / or B chains with iodoacetamide after reduction with DTT (1,4-dithiothreitol). Urea methylation of the insulin A chain results in the methylation of four cysteine ​​residues, leading to a mass increase of approximately 228.08 amu (approximately 57.02 per cysteine). Urea methylation of the insulin B chain results in the methylation of two cysteine ​​residues, leading to a mass increase of approximately 114.04 amu (approximately 57.02 per cysteine).

[0159] Once purified, the chemically modified (e.g., alkylated) A and / or B chains are subjected to an ionization source. As with insulin, those skilled in the art will understand that the choice of ionization method can be determined based on the analyte, sample type, detector type, and the selection of positive or negative modes. Alkylated insulin A and B chains can be ionized in either positive or negative modes. In a preferred embodiment, alkylated insulin A and / or B chains are ionized via ESI in positive mode.

[0160] Ionization of alkylated insulin A chains can generate multi-charged alkylated A chain precursor ions (e.g., 2+, 3+, etc.). For example, positive electrospray ionization of alkylated insulin A chain molecules can generate 2+ and 3+ charged alkylated A chain precursor ions with m / z of 1306.0 ± 0.5 and 871.0 ± 0.5, respectively. Similar to insulin, the identity and amount of multi-charged compounds generated by ionization of alkylated insulin A chains are affected by the ionization conditions employed. In a preferred embodiment, alkylated insulin A chains are ionized under acidic conditions.

[0161] In an embodiment of performing tandem mass spectrometry analysis on alkylated insulin A chains, Q1 can be selected for one or more alkylated insulin A chain precursor ions with m / z of about 1306.0 ± 0.5 and 871.0 ± 0.5. Breaking any of these alkylated insulin A chain precursor ions can generate fragment ions with m / z of about 570.0 ± 0.5, 456.0 ± 0.5, 293.0 ± 0.5, and 133.0 ± 0.5. Therefore, in an embodiment where Q1 is selected for one or more alkylated insulin A chain precursor ions selected from ions with m / z of about 1192.0 ± 0.5 and 795.0 ± 0.5, Q3 can be selected for one or more fragment ions selected from ions with m / z of about 570.0 ± 0.5, 456.0 ± 0.5, 293.0 ± 0.5, and 133.0 ± 0.5. In some embodiments, the relative abundance of individual fragment ions from a single precursor ion can be measured. Alternatively, the relative abundance of two or more fragment ions from a single precursor ion can be measured. In these embodiments, the relative abundance of each fragment ion can be subjected to any known mathematical processing to quantitatively assess insulin in the initial sample. In other embodiments, one or more fragment ions from two or more precursor ions can be measured and used as described above to quantitatively assess insulin in the initial sample.

[0162] Similarly, ionization of alkylated insulin B chains can generate multi-charged alkylated B chain precursor ions (e.g., 3+, 4+, 5+ precursor ions, etc.). For example, positive electrospray ionization of alkylated insulin B chain molecules can generate 3+, 4+, and 5+ charged alkylated B chain precursor ions with m / z values ​​of 1181.9±0.5, 886.9±0.5, and 709.8±0.5, respectively. Similar to insulin, the identity and amount of multi-charged compounds generated by ionizing alkylated insulin B chains are affected by the ionization conditions employed. In a preferred embodiment, alkylated insulin B chains are ionized under acidic conditions.

[0163] In an embodiment of performing tandem mass spectrometry analysis on alkylated insulin B-chain, Q1 can be selected from one or more insulin B-chain precursor ions with m / z of about 1181.9 ± 0.5, 886.9 ± 0.5, and 709.8 ± 0.5. Fragmentation of these three alkylated insulin B-chain precursor ions generates fragment ions with m / z of about 345.0 ± 0.5 and 226.2 ± 0.5. Therefore, in an embodiment where Q1 selects one or more alkylated insulin B-chain precursor ions selected from ions with m / z of about 1144.2 ± 0.5, 858.3 ± 0.5, and 686.8 ± 0.5, Q3 can select one or more fragment ions selected from ions with m / z of about 345.0 ± 0.5 and 226.2 ± 0.5. In some embodiments, the relative abundance of a single fragment ion from a single precursor ion can be measured. Alternatively, the relative abundance of two or more fragment ions from a single precursor ion can be measured. In these embodiments, the relative abundance of each fragment ion can be subjected to any known mathematical processing to quantitatively assess insulin in the initial sample. In other embodiments, one or more fragment ions from two or more precursor ions can be measured and used as described above to quantitatively assess insulin in the initial sample.

[0164] One or more steps of any of the above methods can be performed using automated machines. In some embodiments, one or more purification steps are performed online, and more preferably, all purification and mass spectrometry analysis steps are performed online.

[0165] The following examples are provided to illustrate the present invention. These examples are by no means intended to limit the scope of the method. Example

[0166] Example 1 : Sample preparation

[0167] Simulated serum samples containing different amounts of insulin were prepared by tracing human insulin in simulated serum at various concentrations (40 mg / mL bovine serum albumin (BSA) in AEBSF phosphate buffer (PBS) with 0.002% protease inhibitor) to evaluate the linear response (discussed below in Example 4).

[0168] Human insulin was also traced at various concentrations in serum back-extracted from dual activated charcoal obtained from Golden West Biologicals, Inc. to assess response linearity (discussed below in Example 4).

[0169] Example 2 : Insulin enrichment prior to mass spectrometry analysis

[0170] The human insulin tracer simulation and back-extracted serum sample injections prepared above were performed using a Cohesive Technologies Aria TX-420 system with Aria OS V 1.6 or later software.

[0171] A 75 μL sample was introduced into a Waters Oasis HLB (25 μm, 2.1 × 20 mm) online solid-phase extraction (SPE) column. The SPE column retained human insulin while allowing other serum proteins and macromolecules to flow through.

[0172] Insulin was eluted from the extraction column to the analytical column (a monolithic C18 analytical column from Phenomenex Inc., 5 μm particle size, 50 × 2.1 mm) using 0.2% formic acid in 40% acetonitrile. An HPLC gradient was applied to the analytical column to separate insulin from other analytes contained in the sample. Mobile phase A was 0.2% formic acid in water, and mobile phase B was 0.2% formic acid in acetonitrile. The HPLC gradient started at 28.5% organic gradient and increased to 37% within approximately 90 s.

[0173] The insulin-enriched sample was then subjected to high-resolution / high-precision MS or MS / MS to quantify insulin.

[0174] Example 3 : The effect of pH on insulin ionization

[0175] Insulin was ionized using an ESI source in positive ion mode. When generating positive insulin ions using this ionization source, the pH of the electrospray carrier solution was observed to affect the amount and characteristics of the generated insulin ions.

[0176] Under acidic conditions, multicharged insulin ions were observed at m / z of 968.5 ± 0.50 (for 6+ ions) and 1162.3 ± 0.50 (for 5+ ions). Figure 3A An exemplary spectrum of insulin collected under acidic conditions is shown.

[0177] Under alkaline conditions, multicharged insulin ions were observed at m / z of 1163.0 ± 0.50 (for 5+ ions) and 1453.8 ± 0.50 (for 4+ ions). Figure 3B An exemplary spectrum of insulin collected under alkaline conditions is shown.

[0178] Sufficient signals are generated under both acidic and alkaline conditions, allowing for quantitative analysis under either condition.

[0179] Example 4 : Insulin was detected and quantified using high-resolution / high-precision MS.

[0180] High-resolution / high-precision MS was performed using the Agilent TOF MS system (Agilent Technologies, Inc.). This system employs an MS analyzer capable of performing high-resolution / high-precision MS. While measuring insulin, the instrument exhibits a resolution of approximately 25,000 FWHM and a mass accuracy of approximately 1 ppm.

[0181] Ionization was performed using an ESI source in positive ion mode. As discussed in Example 3, the pH of the electrospray carrier solution affected the amount and identity of the generated insulin ions. The sample prepared in Example 1 was acidified before ionization when eluting it from the SEP column with formic acid solution. As described in Example 3, multicharged insulin ions were observed to exist in 6+ and 5+ charge states.

[0182] Observe the pollutant peaks eluted from the back-extracted serum sample. Figure 4 A shows an example spectrum of insulin in a simulated serum sample in the range of approximately 900–1200 m / z, generated using a QTOF mass spectrometer. Figure 4 B shows the contaminant peaks generated by QTOF mass spectrometry from the back-extracted serum sample matrix. The contaminant peaks were observed to originate from elution at times different from the insulin peak (data not shown).

[0183] exist Figure 5 A spectral analysis shows exemplary high-resolution / high-precision spectra of individual isotopic peaks of the 5+ ion in the range of approximately 1155–1176 m / z. Figure 5 A close-up of the portion of the spectrum between approximately 1159 and 1166 is observed in B. As seen in the spectrum, individual exemplary isotope peaks are observed at m / z of approximately 1161.72, 1161.92, 1162.12, 1162.32, 1162.52, 1162.72, 1162.92, 1163.12, and 1163.34.

[0184] exist Figure 6 A spectral analysis shows the individual isotopic peaks of the 6+ ion in an exemplary high-resolution / high-precision spectrum in the range of approximately 964–973 m / z. Figure 6 A close-up of the portion of the spectrum between approximately 967 and 971.4 is observed in B. As seen in the spectrum, individual exemplary isotope peaks are observed at approximately 968.28, 968.45, 968.62, 968.79, 968.95, 968.12, 968.28, 968.45, and 968.61 m / z.

[0185] Ion data at m / z 1162.54 ± 0.10 were collected to quantify insulin in tracer-simulated and back-extracted serum samples to assess quantification linearity. Both sample types showed linearity in the concentration range of approximately 1.22 ng / mL to 1250 ng / mL. Figure 7 and 8 The linearity of insulin detection data in tracer-simulated serum samples and tracer-retrieved serum samples is shown separately. The goodness of fit (R0) for insulin quantification by high-resolution / high-precision mass spectrometry was determined. 2 The value was 0.9981 in tracer simulated serum and 0.9979 in tracer back-extracted serum.

[0186] Example 5 : Insulin was detected and quantified by tandem MS.

[0187] MS / MS was performed using a Thermo TSQ Vantage MS / MS system (Thermo Electron Corporation). The following software programs from Thermo Electron were used in the embodiments described herein: TSQ Vantage V2.0.0 or later, Xcalibur V 2.0 or later, and LCQuan V 2.5 or later. Liquid solvent / analyte streams exiting the analytical column flowed into the ESI source interface of the MS / MS analyzer. The solvent / analyte mixture was vaporized within the heated tubing of the interface. The analyte was ionized by ESI under acidic conditions in positive ion mode.

[0188] The ions enter the first quadrupole (Q1). Several possible insulin precursor ions were observed at Q1. Figure 9 Exemplary Q1 spectra were observed. Fragmentation studies were conducted on multi-charged insulin precursor ions with m / z values ​​of approximately 1163.32 ± 0.50 (5+ ions) and approximately 969.56 ± 0.50 (6+ ions). Figure 10 and 11 Exemplary product ion scans from each of the broken precursor ions are shown in the figure.

[0189] The effect of collision energy on the fragmentation modes of 5+ and 6+ precursor ions was investigated. Each precursor ion was fragmented at collision energies ranging from about 7 eV to about 80 eV, and the relative intensities of three selected fragment ions (m / z of about 135.9 ± 0.50, 226.2 ± 0.50, and 345.3 ± 0.50) were monitored. Figure 12 The results of these studies are presented in the document. For example... Figure 12 As observed, the relative intensities of fragment ions vary significantly with collision energy. Table 1 shows the optimal collision energy values ​​for each monitored phase transition.

[0190] Table 1. Optimal collision energies for exemplary mass phase transitions observed in insulin (positive polarity-acidic conditions)

[0191] Precursor ions (m / z) Product ions (m / z) Optimal collision energy 969.56±0.50(6+) 135.9±0.50 30eV 226.2±0.50 32eV 345.4±0.50 28eV 1163.32±0.50(5+) 135.9±0.50 45eV 226.2±0.50 42eV 345.4±0.50 44eV

[0192] For the quantitative analysis of insulin by breaking down 6+ ions, the precursor ions entering the quadrupole 2 (Q2) collide with argon gas at a collision energy of 30 eV to generate ion fragments, which then enter the quadrupole 3 (Q3) for further selection. The following mass phase transition was observed when 969.56 ± 0.50 precursor ions were broken down. Figure 10 An exemplary fragmentation spectrum collected from a Q3 scan (product ion scan) is shown in the figure.

[0193] Two observed phase transitions were monitored in MRM mode and summed for quantitative analysis: precursor ions of 969.56±0.50-135.9±0.50 and 226.2±0.50 (see Table 2). Although quantification was achieved by monitoring two mass phase transitions, quantification could also be achieved by monitoring only a single mass phase transition. Conversely, other mass phase transitions (including, for example, ...) could be selected. Figure 10 Any other fragment ions observed can be substituted for or amplified in any combination of the phase transitions monitored above. Similarly, while quantification is performed at a collision energy of 30 eV, any collision energy that produces a sufficient ion signal can be used and may depend on the identity of the fragment ions being monitored. For example, for the two fragment ions indicated above, the collision energy can be in the range of about 20 to about 50 eV, for example in the range of about 25 to about 40 eV, for example about 28-32 eV.

[0194] To quantify insulin by breaking down 5+ ions, precursor ions entering quadrupole 2 (Q2) collide with argon gas at a collision energy of 49 eV to generate ion fragments, which then enter quadrupole 3 (Q3) for further selection. The following mass phase transition was observed when 1163.32 ± 0.50 precursor ions were broken down. Figure 11 An exemplary fragmentation spectrum collected from a Q3 scan (product ion scan) is shown in the figure.

[0195] Two observed phase transitions were monitored in MRM mode and summed for quantitative analysis: precursor ions of 1163.32±0.50-135.9±0.50 and 226.2±0.50 (see Table 2). Although quantification was achieved by monitoring two mass phase transitions, quantification could also be achieved by monitoring only a single mass phase transition. Conversely, other mass phase transitions (including, for example, ...) could be selected. Figure 10Any other fragment ions observed can be substituted for or amplified in any combination of the phase transitions monitored above. Similarly, while quantification is performed at a collision energy of 49 eV, any collision energy that produces a sufficient ion signal can be used and may depend on the identity of the fragment ions being monitored. For example, for the two fragment ions indicated above, the collision energy may be in the range of about 25 to about 70 eV, for example in the range of about 30 to about 60 eV, for example about 35-50 eV.

[0196] Table 2. Exemplary mass phase transitions (positive polarity-acidic conditions) for quantitative insulin monitoring

[0197] Precursor ions (m / z) Product ions (m / z) Collision energy 969.56±0.50(6+) 135.9±0.50,226.2±0.50 30eV 1163.32±0.50(5+) 135.9±0.50,226.2±0.50 49eV

[0198] Example 6 : Insulin A and B chains were detected and quantified by tandem MS.

[0199] Insulin tracer mimic serum and back-extracted serum samples prepared as described in Example 1 were treated with TCEP (tris(2-carboxyethyl)phosphine) to reduce the disulfide bridges of insulin and separate the A and B chains. After disulfide reduction, the samples containing the separated A and B chains were subjected to the same purification process described in Example 2. The resulting insulin A and B chains were analyzed by MS / MS as described in Example 5. In positive mode, the two analytes were ionized by ESI under acidic conditions.

[0200] Several possible insulin A-chain and B-chain precursor ions were observed at Q1. Figure 13 The composite spectra of two possible A-chain precursor ions (in 2+ and 3+ charge states) and a possible B-chain precursor ion (in 3+ and 4+ charge states) are shown. The m / z values ​​of the two A-chain precursor ions are observed to be approximately 1192.86 ± 0.50 (2+ ion) and 795.43 ± 0.50 (3+ ion). The m / z values ​​of the two B-chain precursor ions are observed to be approximately 1144.09 ± 0.50 (3+ ion) and 858.40 ± 0.50 (4+ ion). A third possible B-chain precursor ion (…) is also observed. Figure 14 The m / z (shown in the figure) is approximately 686.83 ± 0.50 (for 5+ ions). Fragmentation studies were conducted on all the A-chain and B-chain precursor ions mentioned above.

[0201] The effect of collision energy on A-chain 3+ precursor ions (m / z = approximately 795.43 ± 0.50) was investigated. Precursor ions were fragmented at collision energies ranging from approximately 7 eV to approximately 80 eV, and the relative intensities of four selected fragment ions (m / z = approximately 513.0 ± 0.50, 399.0 ± 0.50, 236.0 ± 0.50, and 133.0 ± 0.50) were determined. Figure 15 The results of these studies are presented in the document. For example... Figure 15As observed, the relative intensities of fragment ions vary significantly with collision energy. Table 3 shows the optimal collision energy values ​​for each monitored phase transition.

[0202] Table 3. Optimal collision energies for exemplary mass phase transitions observed in insulin A-chain 3+ precursor ions (positive polarity-acidic conditions)

[0203] Precursor ions (m / z) Product ions (m / z) Optimal collision energy 795.4±0.50(3+) 133.0±0.50 28eV 236.0±0.50 23eV 399.0±0.50 16eV 513.0±0.50 12eV

[0204] Insulin quantification was performed using A-chain precursor ions with m / z / of approximately 1192.86 ± 0.50 (2+ ions) and 795.43 ± 0.50 (3+ ions). Quantification experiments were performed with each precursor ion. In these experiments, either a 2+ ion (m / z / of approximately 1192.86 ± 0.50) or a 3+ ion (m / z / of approximately 795.43 ± 0.50) was selected as the precursor ion and fragmented at the collision energies shown in Table 3. Regardless of the selected precursor ion, the following fragment ions were monitored: 513.0 ± 0.50, 399.0 ± 0.50, 236.0 ± 0.50, and 133.0 ± 0.50. Although quantification was performed by monitoring four mass phase transitions, it could also be performed by monitoring only a single mass phase transition. Conversely, additional mass phase transitions (including, for example, any other fragment ions observed) could be selected in any combination to replace or increase any of the phase transitions monitored above.

[0205] Table 4. Exemplary mass phase transitions (positive polarity-acidic conditions) for quantitative insulin A-chain monitoring.

[0206] Precursor ions (m / z) Product ions (m / z) 1192.86±0.50 (2+ ions) 513.0±0.50, 399.0±0.50, 236.0±0.50 and 133.0±0.50 795.43±0.50 (3+ ions) 513.0±0.50, 399.0±0.50, 236.0±0.50 and 133.0±0.50

[0207] Insulin quantification was also performed using B-chain precursor ions with m / z / values ​​of approximately 1144.09 ± 0.50 (3+ ions), 858.40 ± 0.50 (4+ ions), and 686.83 ± 0.50 (5+ ions). Quantification experiments were performed using each precursor ion. In these experiments, 3+ ions (m / z / approximately 1144.09 ± 0.50), 4+ ions (m / z / approximately 795.43 ± 0.50), or 5+ ions (m / z / approximately 686.83 ± 0.50) were selected as precursor ions and fragmented at a collision energy of 30 eV. Regardless of the selected precursor ion, the following fragment ions were monitored: 226.2 ± 0.50 and 345.0 ± 0.50. Figure 16 An exemplary spectrum of the broken B-chain 4+ ions at a collision energy of 30 eV (i.e., product ion scan) is shown. While quantification was achieved by monitoring two mass phase transitions, quantification can be achieved by monitoring only a single mass phase transition. Conversely, additional mass phase transitions (including, for example, ...) can be selected. Figure 16 (Any other fragment ions observed in the study) can be replaced or amplified in any combination with any of the phase transitions monitored above.

[0208] Table 5. Exemplary mass phase transitions (positive polarity-acidic conditions) for quantitative insulin B-chain monitoring.

[0209] Precursor ions (m / z) Product ions (m / z) 1144.09±0.50 (3+ ions) 226.2±0.50、345.0±0.50 858.40±0.50 (4+ ions) 226.2±0.50、345.0±0.50 686.83±0.50 (5+ ions) 226.2±0.50、345.0±0.50

[0210] Example 7: Detection and quantification of insulin A-chain (alkylated) and B-chain (alkylated) by tandem MS

[0211] Insulin tracer mimic serum and back-extracted serum samples were treated with DTT (1,4-dithiothreitol) to generate mimic and back-extracted serum samples containing separated A and B chains. Prior to purification, insulin A and B chain molecules were urea-methylated to fully alkylate each constitutive cysteine ​​present in the molecule. In the A chain, this process alkylated four cysteines, resulting in a mass increase of approximately 228.08 amu. In the B chain, this process alkylated two cysteines, resulting in a mass increase of approximately 114.04 amu.

[0212] Following cysteine ​​alkylation, the sample containing alkylated A and alkylated B chains was subjected to the same purification process described in Example 2. The resulting alkylated A and alkylated B chains were then analyzed by MS / MS as described in Example 5. In positive mode, both analytes were ionized by ESI under acidic conditions.

[0213] Several possible alkylated A-chain and alkylated B-chain precursor ions were observed at Q1. Two possible A-chain precursor ions with m / z of approximately 1306.0 ± 0.50 (2+ ion) and 871.0 ± 0.50 (3+ ion) were selected for fragmentation and quantification. Three possible alkylated B-chain precursor ions with m / z of approximately 1181.9 ± 0.50 (3+ ion), 886.40 ± 0.50 (4+ ion), and 709.80 ± 0.50 (5+ ion) were selected for fragmentation and quantification.

[0214] Insulin quantification was performed using alkylated A-chain precursor ions with m / z values ​​of approximately 1306.0 ± 0.50 (2+ ions) and 871.0 ± 0.50 (3+ ions). Quantification experiments were performed with each precursor ion. In these experiments, either the 2+ ion (m / z value of approximately 1306.0 ± 0.50) or the 3+ ion (m / z value of approximately 871.0 ± 0.50) was selected as the precursor ion and fragmented at a collision energy of 30 eV. Regardless of the selected precursor ion, the following fragment ions were monitored: 133.0 ± 0.50, 293.0 ± 0.50, 456.0 ± 0.50, and 570.0 ± 0.50. Although quantification was performed by monitoring four mass phase transitions, it could also be performed by monitoring only a single mass phase transition. Conversely, additional mass phase transitions could be selected in any combination to replace or increase any of the phase transitions monitored above.

[0215] Table 6. Exemplary mass phase transitions (positive polarity-acidic conditions) for quantitative monitoring of alkylated insulin A-chain

[0216] Precursor ions (m / z) Product ions (m / z) 1306.0±0.50 (2+ ions) 133.0±0.50, 293.0±0.50, 456.0±0.50 and 570.0±0.50 871.0±0.50 (3+ ions) 133.0±0.50, 293.0±0.50, 456.0±0.50 and 570.0±0.50

[0217] Insulin quantification was also performed using alkylated B-chain precursor ions with m / z values ​​of approximately 1181.9 ± 0.50 (3+ ions), 886.9 ± 0.50 (4+ ions), and 709.8 ± 0.50 (5+ ions). Quantification experiments were performed using each precursor ion. In these experiments, 3+ ions (m / z approximately 1181.9 ± 0.50), 4+ ions (m / z approximately 886.9 ± 0.50), or 5+ ions (m / z approximately 709.8 ± 0.50) were selected as precursor ions and fragmented at a collision energy of 30 eV. Regardless of the selected precursor ion, the following fragment ions were monitored: 226.2 ± 0.50 and 345.0 ± 0.50. Although quantification was achieved by monitoring two mass phase transitions, it could also be achieved by monitoring only a single mass phase transition. Conversely, additional mass phase transitions could be selected in any combination to replace or increase any of the phase transitions monitored above.

[0218] Table 7. Exemplary mass phase transitions (positive polarity-acidic conditions) for quantitative monitoring of the B chain of alkylated insulin.

[0219] Precursor ions (m / z) Product ions (m / z) 1144.09±0.50 (3+ ions) 226.2±0.50、345.0±0.50 858.40±0.50 (4+ ions) 226.2±0.50、345.0±0.50 686.83±0.50 (5+ ions) 226.2±0.50、345.0±0.50

[0220] Example 8 : Preparation of human samples for insulin quantification by quantifying the insulin B chain

[0221] Two internal standard solutions were used in the quantification of insulin in human samples. A first internal standard solution with a concentration of 10 pmol / μL was prepared using bovine insulin dissolved in 0.2% formic acid in water. This solution was then diluted by 30 μL with 500 mL of an alkali / extraction buffer containing 1.5 M Tris base and ethanol in a 15:85 ratio. The isotopically labeled human insulin B chain (with 5... 13 C and 1 15 The second internal standard solution was prepared by dissolving 1 mg of the peptide in 1 mL of 0.2% formic acid in water. This concentrated solution was then diluted with 1000 μL of water to a concentration of 5 μL to prepare the second internal standard solution.

[0222] Thaw previously frozen human serum samples to room temperature and vortex thoroughly. Once thawed, add 150 μL of each sample to 350 μL of alkaline / extraction buffer traced by bovine insulin. Vortex the resulting mixture at 1000 rpm for 2 min and incubate at -20°C for 60 ± 5 min to allow precipitation. After incubation, centrifuge the samples at 5500 rpm for 10 min. Then transfer 250 μL of supernatant from each sample to a 96-microtiter plate. Mix 2 mL of TCEP reduction solution (Thermo Scientific catalog number #77720) with 100 μL of a second internal standard solution, and add 20 μL of this mixture to each sample in the 96-microtiter plate. Vortex the samples again at 1000 rpm for 2 min and incubate at 37°C for 60 ± 5 min to reduce insulin in the samples, separating any intact insulin present in the samples into A and B chains. The samples were then incubated at -20°C for 10 min to allow precipitation. The precipitated samples were then centrifuged again at 5500 rpm for 10 min. Prior to MS / MS analysis, 225 μg of the supernatant from each sample was enriched by SPE and HPLC.

[0223] Example 9: Enriching the insulin B chain from human samples prior to mass spectrometry.

[0224] Sample injection of the treated serum sample prepared in Example 8 was performed using a Cohesive Technologies Aria TX-420 system with Aria OS V 1.6 or later software.

[0225] A 225 μL sample was introduced into a Waters Oasis HLB (25 μm, 2.1 × 20 mm) online solid-phase extraction (SPE) column. The SPE column retains the insulin B chain while allowing other serum proteins and macromolecules to flow through. The retained insulin B chain was washed with 0.2% formic acid.

[0226] Insulin B chain was eluted from the extraction column to an analytical column equipped with a protective box (Michrom Bioresources 300 Armstrong Magic C4 (2.1 × 50 mm, 5 μm particle size) analytical column and Phenomenex safety protection box (Phenomenex P / N AHO-4286)) using acetonitrile in 35% formic acid in 0.2% formic acid. An HPLC gradient was applied to the protective / analytical column to separate insulin from other analytes contained in the sample. Mobile phase A was 0.2% formic acid in water, and mobile phase B was 0.2% formic acid in acetonitrile (containing 2.5% isopropanol). The HPLC gradient started at 12.0% organic gradient and increased to 42% within approximately 90 seconds.

[0227] The insulin-enriched sample was then subjected to MS / MS to quantify the insulin.

[0228] Example 10: Detection and quantification of insulin B chain from human serum by tandem MS

[0229] MS / MS was performed using a Thermo TSQ Vantage MS / MS system (Thermo Electron Corporation). The following software programs from Thermo Electron were used in the embodiments described herein: TSQ Vantage V2.0.0 or later, Xcalibur V 2.0 or later, and LCQuan V 2.5 or later. Liquid solvent / analyte streams exiting the analytical column flowed into the ESI source interface of the MS / MS analyzer. The solvent / analyte mixture was vaporized within the heated tubing of the interface. The analyte was ionized by ESI under acidic conditions in positive ion mode.

[0230] As described in Example 6 above, several possible insulin B-chain precursor ions were observed at Q1. An insulin B-chain precursor ion with an m / z of approximately 686.83 ± 0.50 (5+ ion) was selected for fragmentation. Fragmentation studies revealed numerous insulin B-chain fragment ions. Figure 17 An exemplary fragmentation spectrum is shown in the figure.

[0231] The effect of collision energy on the fragmentation mode of human insulin B-chain 5+ precursor ions (m / z approximately 686.9 ± 0.50) was investigated. Precursor ions were fragmented at collision energies ranging from approximately 7 V to approximately 80 V, and the relative intensities of five selected fragment ions (m / z approximately 906.0 ± 0.50, 825.0 ± 0.50, 768.5 ± 0.50, 753.0 ± 0.50, and 703.0 ± 0.50) were monitored. Figure 18 The results of these studies are presented in the document. For example... Figure 18 As observed, the relative intensities of fragment ions vary significantly with collision energy. Table 8 shows the approximate optimal collision energy values ​​for each monitored phase transition.

[0232] Table 8. Optimal collision energies for exemplary mass phase transitions observed in human insulin B-chain 5+ precursor ions (positive polarity-acidic conditions)

[0233] Precursor ions (m / z) Product ions (m / z) Optimal collision energy (approximate) 686.9±0.50(5+) 703.0±0.50 17V 753.0±0.50 16V 768.5±0.50 20V 825.0±0.50 16V 906.0±0.50 14V

[0234] Human insulin B-chain fragment ions with m / z values ​​of approximately 768.5 ± 0.50 and 753.2 ± 0.50 were selected for quantification. Similar studies were performed using bovine insulin (internal standard 1) and isotopically labeled insulin B-chain (internal standard 2), both described in Example 8. Table 9 shows the mass phase transitions monitored for each type of insulin B-chain selected for further quantification.

[0235] Table 9. Exemplary mass phase transitions (positive polarity-acidic conditions) for quantitative insulin B-chain monitoring

[0236]

[0237] While quantification can be accomplished by monitoring the two mass phase transitions of each insulin B chain as shown in Table 8, quantification of any of the analytes shown can be accomplished by monitoring only a single mass phase transition. Alternatively, additional mass phase transitions (including, for example, ...) can be selected. Figure 17 (Any other human insulin B-chain fragment ions observed in the study) can be replaced or amplified in any combination with any of the phase transitions monitored above.

[0238] Example 11 : Intra- and inter-measurement accuracy, reproducibility, and precision studies

[0239] Intra-assay and inter-assay accuracy, reproducibility, and precision studies of the assays described in Examples 8-10 were performed using five QC pools prepared from back-extracted serum traced with 8, 12, 20, 40, and 80 μIU / mL human insulin (Biocell Laboratories Inc., 1131-00, batch HHP03) to cover the assumed reportable assay range.

[0240] Eight replicates from each of the five QC pooled sera were analyzed in a single assay to determine the coefficient of variation (CV) of the samples in the assay. Data from these studies are shown in Table 10. Statistical analysis of the results demonstrated that the reproducibility (CV) of the five QC pooled sera ranged from 3.0% to 7.9%, all within acceptable levels (i.e., ≤15% CV, except for the acceptable LOQ level of ≤20% CV). Further analysis of the data presented in Table 10 revealed intra-assay precision for each pooled sera within an acceptable range of 80–120%.

[0241] Table 10. Measurement of internal variation and precision

[0242]

[0243] To investigate inter-assay variability, eight replicates from each of the five QC pooled sera were analyzed at different 5-day intervals. Data from these studies are shown in Table 11. The inter-assay variability (%CV) of the pooled sera ranged from 7.1% to 14.0%. The total variability at the targeted insulin levels of 8, 12, 20, 40, and 80 μIU / mL was 14.0%, 10.2%, 10.0%, 7.5%, and 7.1%, respectively. Analysis of all pooled sera met the requirement of acceptable reproducibility ≤15%CV, except for LOQ levels with acceptable ≤20%CV. Further analysis of the data presented in Table 11 revealed inter-assay precision for each pooled sera within an acceptable range of 80–120%.

[0244] Table 11. Inter-measurement variability and precision

[0245]

[0246]

[0247] Example 12 : Analytical sensitivity: Limit of blank (LOB), limit of detection (LOD), and limit of quantitation (LOQ)

[0248] Selectivity is the ability of an analytical method to distinguish and quantify an analyte in the presence of other components in a sample. Both LOB and LOD are indicators of a measured value exceeding the uncertainty associated with that measurement. LOB is defined as two standard deviations from zero concentration. LOD is defined as four standard deviations from zero concentration. For selectivity tests, blank samples (re-extracted serum) with appropriate biological matrix were obtained, interferences were tested, and analyzed using the methods described in Examples 8-10. The blank back-extracted serum samples were measured 14 times. The results of these studies were statistically analyzed, given an LOB of 1.4 μIU / mL and an LOD of 1.8 μIU / mL.

[0249] LLOQ is the point at which the measurement becomes quantitatively significant. At this LLOQ, the analyte response is identifiable, discontinuous, and reproducible with an accuracy of 20% and a precision of 80%–120%. LLOQ was determined by measuring six back-extracted serum samples traced with human insulin at concentrations close to the expected LLOQ (1.25, 2.5, 5, 10, 15, and 25 μIU / mL), and then the intra-assay reproducibility was estimated for seven measurements. Data from these studies were analyzed and plotted (shown in…). Figure 19 (in the middle) and the LLOQ was determined to be 3 μIU / mL by the curve, which is the lowest concentration to obtain acceptable performance, at which point the 95% confidence interval of CV is still less than 20%.

[0250] Example 13 : Determine the reportable range and linearity

[0251] To determine the linear range of the assays described in Examples 8-10, eight tracer-back-extracted serum samples (human insulin concentrations of 5, 10, 15, 25, 50, 100, 200, and 300 μIU / mL) were prepared and analyzed separately over 5 days. Five consecutive weighted (1 / X) linear regressions yielded correlation coefficients of 0.995 or higher with an accuracy of ±20%, revealing a quantifiable range of 5–300 μIU / mL. Figure 20 An exemplary calibration curve is shown in the figure.

[0252] Example 14 : Sample type study

[0253] Available in 6 different types of BD Vacutainer TM Ten human patient mixed serum samples (normal serum, SST, EDTA plasma, heparin sodium plasma, heparin lithium plasma, and sodium citrate plasma) were collected in tubes to estimate matrix specificity. Insulin was then extracted and analyzed from samples of each mixed serum sample according to the methods described in Examples 8-10. These studies indicate that sodium citrate plasma samples are unacceptable for analysis, but all other sample types are acceptable.

[0254] Example 15 : Interference research

[0255] The effect of hemolytic interference on insulin assays was estimated by tracing different levels of insulin in samples from patients with low, moderate, and high hemolysis. Insulin was then extracted and analyzed according to the methods described in Examples 8-10. The results of these studies indicate that acceptable results (i.e., within 80-120% accuracy) were obtained for samples with high and moderate hemolysis. Samples with high hemolysis were unacceptable.

[0256] The effect of lipemia interference on insulin measurement was estimated by tracing different levels of insulin in samples from patients with low, moderate, and high lipemia. Insulin was then extracted and analyzed according to the methods described in Examples 8-10. The results of these studies indicate that acceptable results (i.e., within 80-120% accuracy) were obtained for all levels of lipemia.

[0257] The effect of bilirubin interference on insulin measurement was estimated by tracing different levels of insulin in samples from patients with low, moderate, and high jaundice. Insulin was then extracted and analyzed according to the methods described in Examples 8-10. The results of these studies indicate that acceptable results (i.e., within 80-120% accuracy) were obtained for all levels of bilirubin.

[0258] All papers, patents, and patent applications mentioned or cited herein, as well as all other documents and information provided electronically, are incorporated herein by reference in their entirety, as if specifically and separately each individual publication were incorporated by reference. The applicant reserves the right to incorporate in its entirety any material from any such papers, patents, patent applications, or other physical and electronic documents into this application.

[0259] The methods described herein are adaptable to be practiced in the absence of any elements or limitations not specifically disclosed herein. Therefore, for example, the terms "comprising," "including," "containing," etc., should be interpreted broadly and without limitation. Furthermore, the terminology and expressions used herein have been used as descriptive rather than restrictive terms, and it is not intended that any equivalent form or portion thereof excluding the features shown and described be used. It should be recognized that various modifications may be possible within the scope of the claimed invention. Therefore, it should be understood that while the invention has been particularly disclosed by way of preferred embodiments and optional features, those skilled in the art may take modifications and variations of the invention embodied herein, and such modifications and variations are considered to be within the scope of the invention.

[0260] This invention has been described broadly and generally. Every narrow class and subgroup belonging to the general disclosure also constitutes part of the method. This includes a general description of the method with limiting clauses, or a general description of the method with negative limitations that remove any subject matter from the class, whether or not such removed subject matter is specifically referenced herein.

[0261] Other embodiments are within the scope of the following claims. Furthermore, when the features or aspects of the method are described according to the Markush group, those skilled in the art will recognize that the invention is also described according to any individual member or subgroup member of the Markush group.

[0262] This invention includes the following:

[0263] 1. A method for determining the amount of insulin in a biological sample by tandem mass spectrometry, the method comprising:

[0264] (a) subjecting the sample to conditions suitable for the generation of the insulin B chain from insulin;

[0265] (b) Process the sample from step (a) to obtain a component rich in insulin B chain;

[0266] (c) subjecting the enriched insulin B chains to an ionization source under conditions suitable for generating one or more insulin B chain ions detectable by mass spectrometry; and

[0267] (d) The amount of one or more insulin B-chain ions was determined by tandem mass spectrometry.

[0268] The amount of ions measured in step (d) is related to the amount of insulin in the sample.

[0269] 2. The method according to embodiment 1, wherein the treatment in step (b) includes enriching the insulin B chain by solid phase extraction (SPE).

[0270] 3. The method according to embodiment 1, wherein the treatment in step (b) includes enriching the insulin B chain by high performance liquid chromatography (HPLC).

[0271] 4. The method according to embodiment 1, wherein the biological sample includes human plasma or serum samples.

[0272] 5. The method according to embodiment 4, wherein when taken from humans, the amount of insulin measured is the amount of insulin present in the sample.

[0273] 6. The method according to embodiment 1, wherein the ionization source is an electrospray (ESI) ionization source.

[0274] 7. The method according to embodiment 1, wherein the sample is subjected to acidic conditions before ionization in positive ion mode.

[0275] 8. The method according to embodiment 7, wherein subjecting the sample to acidic conditions includes subjecting the sample to formic acid.

[0276] 9. The method according to embodiment 1, wherein the insulin B chain is not chemically modified prior to ionization.

[0277] 10. The method according to embodiment 9, wherein the one or more ions determined in step (d) include insulin B-chain precursor ions selected from ions with mass-to-charge ratios (m / z) of 1144.2 ± 0.5, 858.3 ± 0.5, and 686.8 ± 0.5.

[0278] 11. The method according to embodiment 9, wherein the one or more ions determined in step (d) include one or more fragment ions selected from ions with mass-to-charge ratios (m / z) of 906.0±0.5, 825.0±0.5, 768.5±0.5, 753.0±0.5, 703.0±0.5, 345.0±0.5, and 226.2±0.5.

[0279] 12. The method according to embodiment 9, wherein the one or more ions determined in step (d) include one or more fragment ions selected from: fragment ions from insulin B-chain precursor ions with a mass-to-charge ratio (m / z) of 1144.2 ± 0.5, fragment ions from insulin B-chain precursor ions with a m / z of 858.3 ± 0.5, and fragment ions from insulin B-chain precursor ions with a m / z of 686.8 ± 0.5.

[0280] 13. The method according to embodiment 1, wherein the tandem mass spectrometry comprises generating a human insulin B-chain precursor ion with a mass-to-charge ratio (m / z) of 686.8 ± 0.5 and breaking the precursor ion into one or more fragment ions selected from ions with m / z of 906.0 ± 0.5, 825.0 ± 0.5, 768.5 ± 0.5, 753.0 ± 0.5, 703.0 ± 0.5, 345.0 ± 0.5, and 226.2 ± 0.5.

[0281] 14. The method according to embodiment 13, wherein the ions determined in step (d) include one or more ions from ions with m / z of 768.5 ± 0.5 and 753.0 ± 0.5.

[0282] 15. The method according to embodiment 13, wherein the breakage is carried out with an impact energy in the range of 10-25V, including 10V and 25V.

[0283] 16. The method according to embodiment 1, wherein the insulin B chain is chemically modified prior to ionization.

[0284] 17. The method according to embodiment 16, wherein the chemical modification comprises alkylating the insulin B chain.

[0285] 18. The method according to embodiment 17, wherein the one or more ions determined in step (d) include alkylated insulin B-chain precursor ions selected from ions with mass-to-charge ratios (m / z) of 1181.9 ± 0.5, 886.9 ± 0.5, and 709.8 ± 0.5.

[0286] 19. The method according to embodiment 17, wherein the one or more ions determined in step (d) comprise one or more fragment ions selected from ions with mass-to-charge ratios (m / z) of 345.0 ± 0.5 and 226.2 ± 0.5.

[0287] 20. The method according to embodiment 17, wherein the one or more ions determined in step (d) comprise two or more fragment ions selected from: fragment ions from alkylated insulin B-chain precursor ions with a mass-to-charge ratio (m / z) of 1181.9 ± 0.5, fragment ions from alkylated insulin B-chain precursor ions with a m / z of 886.9 ± 0.5, and fragment ions from alkylated insulin B-chain precursor ions with a m / z of 709.8 ± 0.5.

[0288] 21. The method according to embodiment 20, wherein the fragment ions from each precursor ion include ions selected from ions with m / z of 345.0 ± 0.5 and 226.2 ± 0.5.

[0289] 22. A method for determining the amount of insulin in a biological sample taken from a human body by tandem mass spectrometry, the method comprising:

[0290] (a) subjecting the sample to solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) to obtain an insulin-rich component from the sample;

[0291] (b) subjecting the enriched insulin to an ionization source under conditions suitable for generating one or more insulin ions detectable by mass spectrometry; and

[0292] (c) Determine the amount of one or more insulin ions by tandem mass spectrometry.

[0293] The sample was not immunopurified prior to ionization; and the amount of the ion determined in step (c) was related to the amount of insulin in the sample.

[0294] 23. The method according to embodiment 22, wherein the biological sample includes a plasma or serum sample.

[0295] 24. The method according to embodiment 22, wherein the ionization source is an electrospray (ESI) ionization source.

[0296] 25. The method according to embodiment 22, wherein the sample is subjected to acidic conditions before ionization in positive ion mode.

[0297] 26. The method according to embodiment 25, wherein subjecting the sample to acidic conditions includes subjecting the sample to formic acid.

[0298] 27. The method according to embodiment 25, wherein the one or more ions determined in step (c) include insulin precursor ions selected from ions with mass-to-charge ratios (m / z) of 1162.5 ± 0.5 and 968.9 ± 0.5.

[0299] 28. The method according to embodiment 27, wherein the one or more ions determined in step (c) include one or more fragment ions selected from ions with m / z of 226.2 ± 0.5 and 135.9 ± 0.5.

[0300] 29. The method according to embodiment 27, wherein the one or more fragment ions comprise one or more fragment ions from an insulin precursor ion with m / z of 1162.5 ± 0.5 and one or more fragment ions from an insulin precursor ion with m / z of 968.9 ± 0.5.

[0301] 30. The method according to embodiment 29, wherein the one or more fragment ions from each precursor ion include one or more fragment ions selected from ions with m / z of 226.2 ± 0.5 and 135.9 ± 0.5.

[0302] 31. The method according to embodiment 21, wherein the sample is subjected to alkaline conditions before ionization in positive ion mode.

[0303] 32. The method according to embodiment 31, wherein subjecting the sample to alkaline conditions includes subjecting the sample to ammonia.

[0304] 33. The method according to embodiment 31, wherein the one or more ions determined in step (c) include insulin precursor ions selected from ions with mass-to-charge ratios (m / z) of 1453.8 ± 0.5 and 1163.0 ± 0.5.

[0305] 34. The method according to embodiment 33, wherein the one or more ions determined in step (c) include one or more fragment ions selected from ions with m / z of 226.2 ± 0.5 and 135.9 ± 0.5.

[0306] 35. The method according to embodiment 33, wherein the one or more fragment ions comprise one or more fragment ions from insulin precursor ions with m / z of 1163.0 ± 0.5 and one or more fragment ions from insulin precursor ions with m / z of 968.9 ± 0.5.

[0307] 36. A method for determining the amount of insulin in a sample by tandem mass spectrometry, the method comprising:

[0308] (a) subjecting the sample to solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) to obtain an insulin-rich component from the sample;

[0309] (b) The enriched insulin is subjected to an ionization source under conditions suitable for generating insulin precursor ions detectable by mass spectrometry, wherein the mass-to-charge ratio (m / z) of the insulin precursor ions is 1162.5 ± 0.5.

[0310] (c) The insulin precursor ions are subjected to collision-induced dissociation at a collision energy in the range of approximately 40-70 eV to generate one or more fragment ions detectable by mass spectrometry; and

[0311] (d) Determine the amount of one or more of the fragment ions by mass spectrometry.

[0312] The amount of the ion measured in step (d) is related to the amount of insulin in the sample.

[0313] 37. The method according to embodiment 36, wherein the sample comprises a biological sample.

[0314] 38. The method according to embodiment 36, wherein the sample comprises a plasma or serum sample.

[0315] 39. The method according to embodiment 36, wherein the sample comprises a biological sample taken from a human, and the method is used to determine the amount of insulin in the biological sample taken from a human.

[0316] 40. The method according to embodiment 36, wherein the ionization conditions include subjecting the sample to acidic conditions prior to ionization.

[0317] 41. The method according to embodiment 40, wherein subjecting the sample to acidic conditions includes subjecting the sample to formic acid.

[0318] 42. The method according to embodiment 36, wherein the ionization conditions include subjecting the sample to alkaline conditions prior to ionization.

[0319] 43. The method according to embodiment 42, wherein subjecting the sample to alkaline conditions includes subjecting the sample to ammonia.

[0320] 44. The method according to embodiment 36, wherein the ionization is performed using an electrospray ionization (ESI) source in positive ion mode.

[0321] 45. The method according to embodiment 36, wherein the impact energy is in the range of about 40-60 eV.

[0322] 46. ​​The method according to embodiment 36, wherein the impact energy is in the range of about 40-50 eV.

[0323] 47. The method according to embodiment 36, wherein the one or more fragment ions comprise one or more ions selected from ions with m / z of 226.2 ± 0.5 and 135.9 ± 0.5.

[0324] 48. A method for determining the amount of insulin in a biological sample taken from a human body by tandem mass spectrometry, the method comprising:

[0325] (a) subjecting the sample to conditions suitable for the formation of insulin A chain from insulin;

[0326] (b) subject the sample from step (a) to solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) to obtain a component rich in insulin A chain;

[0327] (c) subjecting the enriched insulin A chains to an ionization source under conditions suitable for generating one or more insulin A chain ions detectable by mass spectrometry; and

[0328] (d) The amount of one or more insulin A chain ions was determined by tandem mass spectrometry.

[0329] The amount of ions measured in step (d) is related to the amount of insulin in the sample.

[0330] 49. The method according to embodiment 48, wherein the biological sample includes a plasma or serum sample.

[0331] 50. The method according to embodiment 48, wherein the ionization source is an electrospray (ESI) ionization source.

[0332] 51. The method according to embodiment 48, wherein the sample is subjected to acidic conditions before ionization in positive ion mode.

[0333] 52. The method according to embodiment 41, wherein subjecting the sample to acidic conditions includes subjecting the sample to formic acid.

[0334] 53. The method according to embodiment 48, wherein the insulin A chain generated in step (a) is not chemically modified prior to ionization.

[0335] 54. The method according to embodiment 53, wherein the one or more ions determined in step (d) include insulin A chain precursor ions selected from ions with mass-to-charge ratios (m / z) of 1192.9 ± 0.5 and 795.4 ± 0.5.

[0336] 55. The method according to embodiment 53, wherein the one or more ions determined in step (d) comprise one or more fragment ions selected from ions with mass-to-charge ratios (m / z) of 570.0±0.5, 456.0±0.5, 293.0±0.5, and 133.0±0.5.

[0337] 56. The method according to embodiment 53, wherein the one or more ions determined in step (d) include one or more fragment ions from an insulin A chain precursor ion with a mass-to-charge ratio (m / z) of 1192.9 ± 0.5 and one or more fragment ions from an insulin A chain precursor ion with a m / z of 795.4 ± 0.5.

[0338] 57. The method according to embodiment 56, wherein the one or more fragment ions from each precursor ion include one or more fragment ions selected from ions with m / z of 570.0±0.5, 456.0±0.5, 293.0±0.5 and 133.0±0.5.

[0339] 58. The method according to embodiment 48 further includes the insulin A chain generated in the chemical modification step (a) prior to ionization.

[0340] 59. The method according to embodiment 58, wherein the chemical modification comprises alkylating the insulin A chain.

[0341] 60. The method according to embodiment 59, wherein the one or more ions determined in step (d) include alkylated insulin A chain precursor ions selected from ions with mass-to-charge ratios (m / z) of 1306.0 ± 0.5 and 871.0 ± 0.5.

[0342] 61. The method according to embodiment 59, wherein the one or more ions determined in step (d) include one or more fragment ions selected from ions with mass-to-charge ratios (m / z) of 570.0±0.5, 456.0±0.5, 293.0±0.5 and 133.0±0.5.

[0343] 62. The method according to embodiment 59, wherein the one or more ions determined in step (d) include one or more fragment ions from an alkylated insulin A-chain precursor ion with a mass-to-charge ratio (m / z) of 1306.0 ± 0.5 and one or more fragment ions from an alkylated insulin A-chain precursor ion with a m / z of 871.0 ± 0.5.

[0344] 63. The method according to embodiment 62, wherein the one or more fragment ions from each alkylation precursor ion comprise one or more fragment ions selected from ions with m / z of 570.0±0.5, 456.0±0.5, 293.0±0.5 and 133.0±0.5.

[0345] 64. A method for determining the amount of insulin in a sample by high-resolution / high-precision mass spectrometry, the method comprising:

[0346] (a) Under conditions suitable for generating multiply charged insulin ions, insulin from the sample is subjected to an ionization source, wherein the multiply charged insulin ions can be detected by mass spectrometry;

[0347] (b) Determine the amount of one or more multiply charged insulin ions by high-resolution / high-precision mass spectrometry;

[0348] The amount of the ion measured in step (b) is related to the amount of insulin in the sample.

[0349] 65. The method according to embodiment 64, wherein the high-resolution / high-precision mass spectrometry is performed at an FWHM of 10,000 or higher and a mass precision of 50 ppm or lower.

[0350] 66. The method according to embodiment 64, wherein the high-resolution / high-precision mass spectrometry is performed at an FWHM of 15,000 or higher and a mass accuracy of 20 ppm or lower.

[0351] 67. The method according to embodiment 64, wherein the high-resolution / high-precision mass spectrometry is performed at an FWHM of 20,000 or higher and a mass precision of 5 ppm or lower.

[0352] 68. The method according to embodiment 64, wherein the high-resolution / high-precision mass spectrometry is performed using a high-resolution / high-precision time-of-flight (TOF) mass spectrometer.

[0353] 69. The method according to embodiment 64, wherein the ionization source is an electrospray (ESI) ionization source.

[0354] 70. The method according to embodiment 64, wherein the ionization conditions include ionizing insulin under acidic conditions.

[0355] 71. The method according to embodiment 69, wherein the acidic conditions include treating the sample with formic acid prior to ionization.

[0356] 72. The method according to embodiment 64, wherein the one or more multicharged insulin ions comprise one or more ions selected from 4+, 5+ and 6+ charged insulin ions.

[0357] 73. The method according to embodiment 72, wherein the one or more multicharged insulin ions include 6+ charged insulin ions.

[0358] 74. The method according to embodiment 73, wherein the one or more 6+ charged insulin ions comprise one or more ions with a mass-to-charge ratio (m / z) in the range of about 968.0 ± 1.5.

[0359] 75. The method according to embodiment 73, wherein the one or more insulin ions in a 6+ charge state comprise one or more ions selected from those with a mass-to-charge ratio (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.

[0360] 76. The method according to embodiment 72, wherein the one or more multicharged insulin ions include 5+ charged insulin ions.

[0361] 77. The method according to embodiment 76, wherein the one or more insulin ions in a 5+ charged state comprise one or more ions with a mass-to-charge ratio (m / z) in the range of about 1162.5 ± 1.0.

[0362] 78. The method according to embodiment 76, wherein the one or more insulin ions in a 5+ charge state comprise one or more ions selected from those with a mass-to-charge ratio (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.

[0363] 79. The method according to embodiment 76, wherein the one or more insulin ions in a 5+ charge state include ions with a mass-to-charge ratio (m / z) of 1162.54 ± 0.1.

[0364] 80. The method according to embodiment 72, wherein the one or more multicharged insulin ions include 4+ charged insulin ions.

[0365] 81. The method according to embodiment 80, wherein the one or more insulin ions in a 4+ charge state comprise one or more ions with a mass-to-charge ratio (m / z) in the range of about 1452.9 ± 0.8.

[0366] 82. The method according to embodiment 64, wherein insulin is purified from the sample by high performance liquid chromatography (HPLC) prior to ionization.

[0367] 83. The method according to embodiment 81, wherein the sample is subjected to solid-phase extraction (SPE) prior to HPLC.

[0368] 84. The method according to embodiment 64, wherein the sample comprises a biological sample.

[0369] 85. The method according to embodiment 64, wherein the sample is derived from humans.

[0370] 86. The method according to embodiment 64, wherein the sample comprises a plasma or serum sample.

[0371] 87. The method according to embodiment 64, wherein the sample is a biological sample from a human, and the method is used to determine the amount of insulin in the sample when taken from a human.

Claims

1. A method for determining the amount of insulin in human plasma or serum samples by high-resolution / high-precision mass spectrometry, the method comprising: (a) subjecting the sample to solid-phase extraction to form an extracted insulin sample; (b) The extracted insulin sample was purified by high performance liquid chromatography to form a purified insulin sample; (c) The purified insulin sample is subjected to an ionization source under acidic conditions suitable for generating multiply charged insulin ions, wherein the multiply charged insulin ions can be detected by mass spectrometry. (d) Determination of the amount of one or more polycharged insulin ions by high-resolution / high-precision mass spectrometry, wherein the high-resolution / high-precision mass spectrometry is performed at an FWHM of 10,000 or higher and a mass precision of 50 ppm or lower, wherein the polycharged insulin ions include 6+ charged insulin ions having a mass-to-charge ratio (m / z) of 968.0 ± 1.5; and (e) The amount of insulin in the sample is determined using the amount of the 6+ charged insulin ions.

2. The method of claim 1, wherein the high-resolution / high-precision mass spectrometry is performed at an FWHM of 15,000 or higher and a mass accuracy of 20 ppm or lower.

3. The method of claim 1, wherein the high-resolution / high-precision mass spectrometry is performed at an FWHM of 20,000 or higher and a mass accuracy of 5 ppm or lower.

4. The method of 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 of claim 1, wherein the acidic conditions comprise treating the sample with formic acid prior to ionization.

7. The method of claim 1, wherein the one or more insulin ions in a 6+ charge state comprise one or more ions selected from those with a mass-to-charge ratio (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.

8. The method of claim 1, wherein the one or more multicharged insulin ions further comprises 5+ charged insulin ions.

9. The method of claim 8, wherein the one or more insulin ions in a 5+ charge state comprise one or more ions with a mass-to-charge ratio (m / z) in the range of 1162.5 ± 1.

0.

10. The method of claim 8, wherein the one or more insulin ions in a 5+ charge state comprise one or more ions selected from those with a mass-to-charge ratio (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.

11. The method of claim 8, wherein the one or more insulin ions in a 5+ charge state comprise ions with a mass-to-charge ratio (m / z) of 1162.54 ± 0.1.

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