Method for the quantification of insulin and c-peptide

Abstract

There is a need for a reliable and accurate method of identifying insulin resistance.SOLUTION: A method of measuring insulin resistance in a diabetic or pre-diabetic patient by mass spectrometry, comprising: (a) subjecting insulin and C-peptide from a sample to an ionization source under conditions suitable for generating one or more insulin and C-peptide ions detectable by mass spectrometry; and (b) determining the amount of one or more insulin and C-peptide ions by mass spectrometry, wherein the amount of the one or more ions determined is used to determine the amount of insulin and C-peptide in the sample; Using the amounts of insulin and C-peptide determined by mass spectrometry to determine an insulin resistance score or a probability of insulin resistance occurring.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Cross-reference to related patent applications This application was filed on March 1, 2017, the contents of which are incorporated by reference in their entirety into this disclosure. U.S. Provisional Application No. 62 / 480,029 filed on March 31, 2018 and U.S. Provisional Application No. 62 / 480,029 filed on March 16, 2018 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 644,378 It claims benefits in the [Background technology]

[0002] Insulin resistance varies several-fold in apparently healthy populations, and the risk of death among these individuals Approximately one-quarter to one-third of the most insulin-resistant individuals have a group of metabolic disorders and There is an increased risk of developing associated clinical syndromes. It is not practical to directly estimate the plasma insulin concentration at the clinical level. is highly correlated with a direct measure of plasma glucose disposal, which indicates that plasma Several surrogate estimates of insulin resistance based on measurements of insulin and glucose concentrations have been proposed. Unfortunately, there are no standardized insulin assays. As a result, those who are sufficiently insulin resistant are at high risk for multiple adverse clinical outcomes. A universally applicable numerical cut-point for identifying healthy individuals has been proposed. It was not possible to establish a

[0003] In response to this dilemma, multiple surrogates based on commonly measured metabolic abnormalities associated with IR have been proposed. The markers may help identify individuals with insulin resistance before the onset of overt disease. For example, the diagnosis of metabolic syndrome (MetS) is an example of this approach. The diagnosis of tS is associated with direct measurements of insulin-mediated glucose disposal, but Only about 50% of the most insulin-resistant population, one-third of healthy people, or did not meet the MetS diagnosis. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need for reliable and accurate methods to identify insulin resistance. [Means for solving the problem]

[0005] In one aspect, provided herein are methods for treating diabetes and pre-diabetes. A method for diagnosing or prognosing insulin resistance, comprising: detecting insulin and C-peptide in a sample; The present invention measures insulin and c-peptide levels in patients by determining the amount of The method includes the steps of:

[0006] In certain embodiments, the methods provided herein involve determining the amino acid sequence in a sample by mass spectrometry. Some embodiments include a multiplex assay that simultaneously measures the amount of thrombin and C-peptide. In the method, the method comprises: (a) detecting one or more insulins and C- The insulin and C-peptide from the sample are extracted under conditions suitable for generating peptide ions. (b) subjecting the solution to an ionization source; and (b) identifying one or more insulins and and determining the amount of C-peptide ions.

[0007] In some embodiments, the in vivo assay is performed using a mass spectrometric method described herein. A insulin level of 7 μIU / mL or higher is diagnostic of insulin resistance. In embodiments, the insulin level determined by the mass spectrometry methods described herein is 8 μg / mL. IU / mL or greater is diagnostic of insulin resistance. , insulin levels of 9 μIU / mL or greater as determined by mass spectrometry as described herein In some embodiments, insulin resistance is diagnosed if If the insulin level determined by the mass spectrometry method used is 10 μIU / mL or higher, In some embodiments, insulin resistance is diagnosed. Analytical insulin levels of 11 μIU / mL or higher indicate insulin resistance. In some embodiments, the sex is diagnosed by the mass spectrometry methods described herein. If the determined insulin level is 12 μIU / mL or higher, insulin resistance is diagnosed. In some embodiments, the inclusion mass determined by the mass spectrometry methods described herein is Insulin levels above 13 μIU / mL are diagnostic of insulin resistance. In some embodiments, the insulin levels determined by the mass spectrometry methods described herein are A level of 14 μIU / mL or greater is diagnostic of insulin resistance. In the present study, insulin levels were 15 μIU / mL or more is diagnostic of insulin resistance.

[0008] In a preferred embodiment, the insulin concentration determined by the mass spectrometry methods described herein is A phosphorus level of 15 μIU / mL or higher is diagnostic of insulin resistance.

[0009] In some embodiments, C- as determined by mass spectrometry methods described herein Peptide levels above 1.4 ng / mL are diagnostic of insulin resistance. In an embodiment of the present invention, the C-peptide value determined by the mass spectrometry method described herein If the IL-10 level is 1.5 ng / mL or higher, insulin resistance is diagnosed. The C-peptide value determined by the mass spectrometry method described herein is 1.6n g / mL or higher diagnoses insulin resistance. C-peptide levels of 1.7 ng / mL or greater as determined by the mass spectrometry method described herein If the blood glucose level is above 100 mg / kg, insulin resistance is diagnosed. If the C-peptide level determined by the mass spectrometry method described is 1.8 ng / mL or higher In some embodiments, insulin resistance is diagnosed. C-peptide levels ≥ 1.9 ng / mL as determined by mass spectrometry are considered to be insulin-resistant. In some embodiments, the mass spectrometry methods described herein are used to diagnose cancer resistance. C-peptide levels of 2 ng / mL or higher determined by MRI are diagnostic of insulin resistance. In some embodiments, the nucleotide sequence determined by the mass spectrometry methods described herein is A C-peptide level of 2.1 ng / mL or higher is diagnostic of insulin resistance. In some embodiments, the C-peptide as determined by the mass spectrometry methods described herein A blood glucose level of 2.2 ng / mL or higher is diagnostic of insulin resistance. In some embodiments, the C-peptide value determined by the mass spectrometry methods described herein is 2 A level of 0.3 ng / mL or higher is diagnostic of insulin resistance. C-peptide values ​​determined by the mass spectrometry method described herein were 2.4 ng / mL. mL or more is diagnostic of insulin resistance.

[0010] In a preferred embodiment, the C-peptide is determined by the mass spectrometry methods described herein. A peptide level of 2.4 ng / mL or higher is diagnostic of insulin resistance.

[0011] In some embodiments, the insulin resistance score (RS) and / or insulin The probability of resistance development, P(IR), is the index of resistance measured by the methods provided herein. Based on phosphorus and C-peptide levels, as provided herein.

[0012] In some embodiments, the insulin resistance score (RS) and / or insulin The probability of resistance development, P(IR), is determined by: RS = (insulin x 0.0295) + (C-peptide x 0.00372)

[0013]

number

[0014] In some embodiments, the insulin resistance score (RS) and / or insulin The probability of resistance development, P(IR), is the probability of resistance development measured by the methods provided herein. Based on phosphorus and C-peptide, and creatine levels measured by standard methods are provided herein.

[0015] In some embodiments, the insulin resistance score (RS) and / or insulin The probability of resistance development, P(IR), is determined by: RS = (insulin x 0.0265) + (C-peptide x 0.00511) + (Clear Chinine x -3.2641)

[0016]

number

[0017] In some embodiments, the insulin resistance score (RS) and / or insulin The probability of resistance development, P(IR), is the probability of resistance development measured by the methods provided herein. Phosphorus and C-peptide, as well as creatine, triglycerides measured by standard methods The present invention provides a method for determining the level of cholesterol in the blood based on the level of cholesterol (TG) / HDL-C, and BMI.

[0018] In some embodiments, the insulin resistance score (RS) and / or insulin The probability of resistance development, P(IR), is determined by: RS = (insulin x 0.0227) + (C-peptide x 0.0046) + (creatinine) Nin × -3.5553) + (TG / HDL-C × 0.101) + (BMI × 0.0711 )

[0019]

number

[0020] In some embodiments, the steady state plasma glucose (SSPG) concentration is determined by a predetermined concentration. If the patient falls in the top tertile of the cluster, insulin resistance is diagnosed. If the SSPG concentration is ≥ 190 mg / dL, insulin resistance is diagnosed. In some embodiments, an SSPG concentration of ≧195 mg / dL indicates insulin resistance. In some embodiments, if the SSPG concentration is ≥ 198 mg / dL, In some embodiments, insulin resistance is diagnosed if the SSPG concentration is ≥ 2 If the blood glucose level is above 100 mg / dL, insulin resistance is diagnosed. If the SSPG concentration is ≥ 205 mg / dL, insulin resistance is diagnosed.

[0021] In some embodiments, insulin resistance is measured using mass spectrometry methods described herein. Diagnosis is by a combination of insulin and C-peptide levels determined by In embodiments, insulin resistance is determined by mass spectrometry methods described herein. In some embodiments, the diagnosis is made by a combination of insulin and C-peptide levels. Insulin resistance is determined by the mass spectrometry method described herein. The diagnosis is made by a combination of serum ATP and C-peptide levels and SSPG concentrations.

[0022] In some embodiments, the determined amount of one or more ions is used to determine the amount of ions in the sample. In some embodiments, the amount of insulin and C-peptide in the sample is determined. The amount of insulin and C-peptide is related to the amount of insulin in the patient.

[0023] In some embodiments, the method further comprises: (a) subjecting the sample to a concentration step to concentrate insulin and (b) obtaining a fraction enriched in C-peptide and C-peptide, and (c) obtaining a fraction enriched in C-peptide and C-peptide that can be detected by mass spectrometry. and concentrating the insulin and C-peptide ions under conditions suitable for generating one or more insulin and C-peptide ions. (c) subjecting the isolated insulin and C-peptide to an ionization source; and (d) subjecting the isolated insulin and C-peptide to mass spectrometry. and determining the amount of one or more insulin and C-peptide ions by In some embodiments, the determined amount of one or more ions is used to determine the amount of ions in the sample. In some embodiments, the amount of insulin and C-peptide in the sample is determined. The amount of insulin and C-peptide is related to the amount of insulin in the patient. In an embodiment, the amount of insulin and C-peptide in the sample is used to determine the level of insulin in the patient. The insulin to C-peptide ratio is determined.

[0024] In some embodiments, the enrichment steps provided herein involve antibody-based enrichment. In some embodiments, the method comprises immunocapture of phospholipid and C-peptide. (b) immunocapturing insulin and C-peptide; and (b) detecting them by mass spectrometry. under conditions suitable to generate one or more insulin and C-peptide ions, (c) subjecting the immunocaptured insulin and C-peptide to an ionization source; determining the amount of one or more insulin and C-peptide ions by quantitative analysis. Includes:

[0025] In some embodiments, the immunocapturing step provided herein comprises administering an anti-inflammatory drug to a subject. In some embodiments, the method includes using a thrombin antibody and an anti-C-peptide antibody. Thus, the antibodies provided herein are monoclonal antibodies. In some embodiments, the antibodies provided herein are murine monoclonal antibodies. In some embodiments, the antibodies provided herein are monoclonal IgG antibodies. In some embodiments, the antibodies provided herein are polyclonal antibodies.

[0026] In some embodiments, the anti-insulin antibody and the anti-C-peptide antibody are attached to magnetic beads. In some embodiments, the immunocaptured antibody on the magnetic beads is immobilized on the magnetic beads. Insulin and C-peptide are washed and eluted.

[0027] In some embodiments, the serum is delipidated prior to quantification by mass spectrometry. In embodiments, one or more delipidation reagents are used to remove lipids from the sample. In some embodiments, the degreasing reagent is CLEANASCITE®.

[0028] In some embodiments, the methods provided herein involve purifying the sample prior to mass spectrometry analysis. In some embodiments, the method comprises preparing a liquid chromatography In some embodiments, the method further comprises purifying the sample using a liquid chromatograph. The flow is performed by high performance liquid chromatography (HPLC) or high turbulence liquid chromatograph ( In some embodiments, the method comprises subjecting the sample to solid phase extraction (SPE). This includes the step of applying.

[0029] In some embodiments, the mass spectrometry comprises tandem mass spectrometry. In some embodiments, the mass spectrometry is high-resolution mass spectrometry. The quantitative analysis is high resolution / high accuracy mass spectrometry.

[0030] In some embodiments, ionization is by electrospray ionization (ESI). In some embodiments, ionization is by atmospheric pressure chemical ionization (APCI). In some embodiments, the ionization is in positive ion mode.

[0031] In some embodiments, the methods provided herein include adding an internal standard to the sample. In some embodiments, the internal standard for insulin is bovine insulin. In some embodiments, the internal standard for C-peptide is C-peptide. In some embodiments, the internal standard is labeled. In some embodiments, the internal standard is deuterated or isotopically labeled. do.

[0032] In some embodiments, the patient sample is a serum sample. In some embodiments, the patient sample is a plasma sample. In some embodiments, the patient sample is blood, Saliva or urine samples.

[0033] In some embodiments, the sample is subjected to acidic conditions prior to ionization. In an embodiment, subjecting the sample to acidic conditions results in concentrated insulin and C-peptide. to formic acid.

[0034] In some embodiments, the sample is subjected to basic conditions prior to mass spectrometry. In an embodiment, the step of subjecting the sample to basic conditions comprises exposing the sample to Trizma. In some embodiments, the step of subjecting the sample to basic conditions comprises subjecting the sample to The method includes exposing the cells to Trizma and ethanol.

[0035] In some embodiments, one or more ions have a mass charge of 968.7±0.5. In some embodiments, the insulin precursor ions have a charge ratio (m / z) of 1. The ions or ions were 136.0±0.5, 226.1±0.5 and 345.2±0. one or more fragment ions selected from the group consisting of ions having m / z of 5 In some embodiments, insulin has an m / z of 226.1±0.5. The fragment ions are quantifier ions. The one or more ions are bovine lecithin ions having a mass-to-charge ratio (m / z) of 956.8±0.5. In some embodiments, the one or more ions include insulin precursor ions. Ions with m / z of 36.0±0.5, 226.1±0.5, and 315.2±0.5 In some embodiments, the fragment ions include one or more fragment ions selected from the group consisting of: In this state, the bovine insulin fragment ion with m / z of 136.0±0.5 , the quantifier ion.

[0036] In some embodiments, the one or more ions have a mass of 1007.7±0.5. In some embodiments, the C-peptide precursor ion has a charge ratio (m / z) , one or more ions are 533.3±0.5, 646.4±0.5 and 927.5± One or more fragment ions selected from the group consisting of ions having an m / z of 0.5 In some embodiments, 533.3±0.5, 646.4±0.5, and and either the C-peptide fragment ion with m / z of 927.5±0.5, or The sum of these intensities can be used for quantification. The multiple ions were C-peptide with a mass-to-charge ratio (m / z) of 1009.5 ± 0.5. In some embodiments, one or more ions include a heavy internal standard precursor ion. have m / z of 540.3±0.5, 653.4±0.5 and 934.5±0.5 ions. In an embodiment, 540.3±0.5, 653.4±0.5 and 934.5±0.5 Any of the C-peptide heavy internal standard fragment ions with m / z The total intensity can be used for quantification.

[0037] In some embodiments, provided herein are methods for detecting insulin and C in a sample. - utilizing mass spectrometry to determine the amount of peptides, the method comprising: (a) an extraction technique; (b) concentrating insulin and C-peptide in the sample by a method; a) Purified insulin and C-peptide from the sample were subjected to liquid chromatography. (c) obtaining a fraction enriched in insulin and C-peptide from the fraction; and (d) analyzing the fraction by mass spectrometry. The concentrated insulin is then injected under conditions suitable to generate insulin precursor ions detectable by the (d) subjecting the sample to a mass spectrometry source to generate one or more fragment ions. and determining the amount of one or more of the determined The amount of several ions is used to determine the amount of insulin and C-peptide in the sample. In some embodiments, the amount of insulin and C-peptide in the sample is compared to the insulin concentration in the patient. In some embodiments, the amount of insulin and C-peptide in the sample is correlated with the amount of phosphorus. The amount of insulin is used to determine the insulin to C-peptide ratio in the patient.

[0038] In some embodiments, the extraction techniques provided herein involve antibody-based injections. In some embodiments, the immunocapture of phospholipids and C-peptides provided herein. Extraction techniques used include solid phase extraction (SPE).

[0039] In some embodiments, the collision energy is in the range of about 40 to 60 V. In some embodiments, the collision energy is in the range of about 40-50V.

[0040] In another aspect, provided herein are methods for detecting insulin or erythrocytes in a sample by mass spectrometry. A method for determining the amount of C-peptide, comprising: (a) administering insulin or C-peptide to a subject by immunization; and (b) capturing one or more insulin or C molecules detectable by mass spectrometry. - Immunocaptured insulin or C-peptide under conditions suitable for generating peptide ions (c) subjecting the insulin to an ionization source; and (d) isolating one or more insulins or and determining the amount of C-peptide ions. In the present invention, a method for determining the amount of insulin in a sample by mass spectrometry is provided. (a) immunocapturing insulin; and (b) detecting insulin by mass spectrometry. The immunocapture insulin is then reacted with the immunocapture insulin under conditions suitable to generate one or more insulin ions that can be released. (c) subjecting the insulin to an ionization source; and (d) identifying the one or more insulins by mass spectrometry. and determining the amount of phosphorus ions. Provided herein are methods for determining the amount of C-peptide in a sample by mass spectrometry. (a) immunocapturing C-peptide; and (b) detecting it by mass spectrometry. The immunocaptured C-peptide is subjected to conditions suitable for generating one or more C-peptide ions. (c) subjecting the peptide to an ionization source; and (d) identifying one or more C-peptides by mass spectrometry. and determining the amount of tide ion. The immunocapturing step may include a step of using an anti-insulin antibody or an anti-C-peptide antibody. In some embodiments, the anti-insulin antibody or anti-C-peptide antibody is a magnetic In some embodiments, the immunocapture on the magnetic beads is The resulting insulin or C-peptide is washed and eluted.

[0041] In another aspect, provided herein are methods for treating diabetes and its complications in patients with diabetes and prediabetes. In some embodiments, the methods provided herein are methods for diagnosing other glycemic disorders. The method for quantifying endogenous insulin and C-peptide is used to diagnose diabetes. In some embodiments, the endogenous insulin and C-peptide Quantitative methods to distinguish between insulin-secreting tumors and exogenous insulin administration as causes of hypoglycemia In some embodiments, the endogenous insulin provided herein is used to Methods for quantifying C-peptide and C-peptide are used to distinguish between type 1 and type 2 diabetes. In some embodiments, the endogenous insulin and C-peptide provided herein The method for the quantification of is used to assess the risk of diabetes in pre-diabetic patients.

[0042] In some embodiments, the mass spectrometry comprises tandem mass spectrometry. In some embodiments, the mass spectrometry is high-resolution mass spectrometry. The quantitative analysis is high resolution / high accuracy mass spectrometry.

[0043] In another aspect, certain methods provided herein involve determining the amount of insulin in a sample. High resolution / high accuracy mass spectrometry is used to In some embodiments, the method comprises (a) providing a solution suitable for generating multivalent insulin ions; subjecting insulin from the sample to an ionization source under conditions (insulin ions are mass (b) detectable by high-resolution / high-accuracy mass spectrometry with one or more multivalent and determining the amount of insulin ions. and relating the amount of one or more ions determined in step (b) to the amount of insulin in the sample. In some embodiments, high resolution / high accuracy mass spectrometry is performed with a FWH of 10,000. In some embodiments, high resolution / high accuracy quality is used. Quantitative analysis is performed using a high-resolution / high-precision time-of-flight (TOF) mass spectrometer. In embodiments, the ionization conditions include ionization of 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 multivalent insulin ions include 4+, 5+, and 6+ valent insulin ions. The ion is selected from the group consisting of threonine ions.

[0044] In some embodiments, the one or more insulin ions in the 6+ charge state are about Contains one or more ions with m / z in the range of 968.8±1.5. In an embodiment, one or more insulin ions in the 6+ charge state are 968.28± 0.1, 968.45±0.1, 968.62±0.1, 968.79±0.1, 968 0.95±0.1, 969.12±0.1, 969.28±0.1, 969.45±0.1 , ions having m / z of 969.61±0.1 ions, such as ions with m / z of 968.95±0.1.

[0045] In some embodiments, one or more insulin ions in the 5+ charge state are about Contains one or more ions with m / z in the range of 1162.5±1.0. In this embodiment, one or more insulin ions in the 5+ charge state are 1161.7 2±0.1, 1161.92±0.1, 1162.12±0.1, 1162.32±0. 1, 1162.52±0.1, 1162.72±0.1, 1162.92±0.1, 11 ions having m / z values ​​of 63.12±0.1, 1163.32±0.1, and 1163.32±0.1. One or more ions selected, for example, an ion having an m / z of 1162.54±0.1. This includes:

[0046] In some embodiments, the one or more insulin ions in the 4+ charge state are about It contains one or more ions with m / z in the range of 1452.9±0.8.

[0047] In any of the methods described herein, the sample may comprise 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 is from a human, e.g., an adult male or female, or The sample may include samples from young men or women, where young means under 18 years of age, under 15 years of age, Human samples are used to diagnose or monitor a disease state or condition. or to monitor the efficacy of treatment of a disease state or condition. In some related embodiments, the methods described herein involve extracting from a human. The method can be used to determine the amount of insulin in a biological sample when

[0048] In embodiments utilizing tandem mass spectrometry, the tandem mass spectrometry may be, for example, a multiple reaction. reaction monitoring, precursor ion scanning, or product ion scanning. This can be done by methods known in the art.

[0049] In some embodiments, tandem mass spectrometry involves separating precursor ions into one or more fractions. The method includes the step of fragmenting the two or more fragment ions. In the embodiment where the amount is determined, the measured amount of ions is related to the amount of insulin in the sample. The amounts can be subjected to mathematical manipulations known in the art to determine the amount of sample As part of the step of determining the amount of insulin in The amounts can be summed.

[0050] In any of the methods described herein, the analyte of interest (e.g., insulin, or (chemically modified or unmodified insulin) was analyzed by high performance liquid chromatography prior to ionization. It can be purified from the sample by high performance liquid chromatography (HPLC). In the method, the analyte of interest is extracted by, for example, applying the sample to a solid phase extraction (SPE) column. In some embodiments, the protein can be purified from the sample by an extraction technique such as The technique is not an immunopurification technique. Specifically, in some embodiments, the SPE column In some embodiments, immunopurification is performed using a method other than immunoaffinity column. In some embodiments, extraction techniques and HPLC are not used. can be performed online to allow automated sample processing and analysis. can.

[0051] In some embodiments, high resolution / high accuracy mass spectrometry is performed at about 10,000 or more, e.g. For example, about 15,000 or more, for example, about 20,000 or more, for example, about 25,000 In some embodiments, high resolution (FWHM) is performed. / High-precision mass spectrometry is about 50 ppm or less, for example, about 20 ppm or less, about 10 ppm The accuracy is about 5 ppm or less, about 3 ppm or less, etc. In this form, high-resolution / high-precision mass spectrometry has a resolution (FWHM) of approximately 10,000 or more. and performed to an accuracy of about 50 ppm or less. In some embodiments, the resolution is about 1 5,000 or greater, with an accuracy of about 20 ppm or less. , the resolution is about 20,000 or more, and the accuracy is about 10 ppm or less, and preferably , the resolution is about 20,000 or more, and the accuracy is about 5 ppm or less, for example, about 3 ppm The following are some examples:

[0052] In some embodiments, the high resolution / high accuracy mass spectrometry is performed using an orbitrap mass spectrometry. mass spectrometer, time-of-flight (TOF) mass spectrometer or Fourier transform ion cyclotron resonance mass spectrometer This can be done using a mass spectrometer (sometimes known as a Fourier transform mass spectrometer). can.

[0053] In some embodiments, one or more molecules detectable by high resolution / high accuracy mass spectrometry are The number of insulin ions was approximately 1452.9±0.8, 1162.5±1, and 968.8± one or more ions selected from the group consisting of ions having m / z within the range of 1.5 Ions within these ranges are insulins with 4+, 5+, and 6+ charges, respectively. These monoisotopic ions with charge correspond to the phosphorus ion. However, naturally occurring isotopic variants of lower abundance are also present. The insulin ion in the range of 1162.5±1 is preferably 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± Insulin with m / z of 0.1, 1163.12±0.1, and 1163.32±0.1 ions, such as those with m / z of 1162.54±0.1. Insulin ions within the range of 968.28±0.1, 968.28±0.1, 968.28±0.1 are preferred. 0.45±0.1, 968.62±0.1, 968.79±0.1, 968.95±0.1 , 969.12±0.1, 969.28±0.1, 969.45±0.1, 969.61 Insulin ions with m / z of 968.95 ± 0.1, e.g., an m / z of 968.95 ± 0.1 In some embodiments, one ion detected by mass spectrometry is or a step of relating the amount of insulin ions to the amount of insulin protein in the sample. The cross-over involves comparison to an internal standard, such as human or non-human insulin protein. can optionally be isotopically labeled.

[0054] In any of the methods described herein, the sample is a biological sample, preferably a For example, the sample may include a body fluid sample including plasma or serum.

[0055] Mass spectrometry (tandem or high resolution / high accuracy) should be performed in positive ion mode. Alternatively, mass spectrometry can be performed in negative ion mode. For example, atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) A variety of ionization sources can be used to ionize insulin, including: In the form, insulin and / or chemically modified or unmodified insulin B chains are The sample is ionized in positive ion mode.

[0056] In any of the methods described herein, a separately detectable internal standard is added to the sample. The amount of the internal standard can be determined in the sample. In this embodiment, all or part of both the analyte of interest and the internal standard present in the sample are The ions are ionized to generate multiple ions that can be detected by a mass spectrometer, and each ion generates 1 In these embodiments, the ion or ions of interest are detected by mass spectrometry. The presence or amount of ions produced from the analyte is determined by comparison with the amount of the detected internal standard ions. This can be related to the amount of analyte of interest present in the sample.

[0057] Alternatively, the amount of insulin in the sample is determined by comparison to one or more external reference standards. Exemplary external reference standards include human or non-human insulin, synthetic blank plasma or serum spiked with synthetic insulin analogs or their isotope-labeled variants. do.

[0058] In some embodiments, the method comprises administering a dose in the range of about 10 μIU / mL to 500 μIU / mL. The amount of insulin in the sample within the range of levels can be determined.

[0059] The above summary of the invention is not intended to be limiting, and other features and advantages of the invention may be found in the accompanying drawings, in which: These and other aspects of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0060] [Figure 1A] 1 is a graph showing odds ratios for those in the top quartile for insulin or C-peptide levels versus those not in the top quartile. The odds ratios are from models adjusted for age, sex, fasting plasma glucose, insulin, C-peptide, HDL-C, LDL-C, triglycerides, creatinine, alanine aminotransferase, body mass index, systolic and diastolic blood pressure. [Figure 1B] 1 is a graph showing odds ratios for those in the top quartile for insulin or C-peptide levels versus those not in the top quartile. The odds ratios are from models adjusted for age, sex, fasting plasma glucose, insulin, C-peptide, HDL-C, LDL-C, triglycerides, creatinine, alanine aminotransferase, body mass index, systolic and diastolic blood pressure. [Figure 1C] 1 is a graph showing odds ratios for those in the top quartile for insulin or C-peptide levels versus those not in the top quartile. The odds ratios are from models adjusted for age, sex, fasting plasma glucose, insulin, C-peptide, HDL-C, LDL-C, triglycerides, creatinine, alanine aminotransferase, body mass index, systolic and diastolic blood pressure. [Figure 2]Boxplots showing fasting insulin levels in patients with fasting glucose <90 mg / dL (left), 90 to <100 mg / dL (center), and 100 to 125 mg / dL (right). Differences in insulin levels between categories were assessed by parametric (ANOVA) and nonparametric (Kruksal-Wallis) methods. [Figure 3] Box plots showing fasting insulin levels in normoglycemic participants (fasting glucose <100 mg / dL). Left: BMI <26; Right: BMI ≥ 26. Differences in insulin levels between categories were assessed by t-test. [Figure 4] 1 is a graph showing the relationship between fasting blood glucose measurements and fasting insulin levels. [Figure 5] 1 is a graph showing the relationship between fasting blood glucose measurements and fasting C-peptide levels. [Figure 6] Graph showing insulin levels by BMI category, sex, and fasting glucose. [Figure 7] FIG. 1 shows an overview of the methods presented herein. [Figure 8] FIG. 1 shows the fragmentation of intact insulin and the mass-to-charge ratios of the measured ions. [Figure 9] FIG. 1 shows the fragmentation of C-peptide and the mass-to-charge ratios of the measured ions. [Figure 10-1] 1 is a graph showing the chromatography of insulin and C-peptide. [Figure 10-2] Continued from Figure 10-1. [Figure 11-1] 1 is a graph showing standard curves for insulin and C-peptide. [Figure 11-2] Continued from Figure 11-1. [Figure 12] FIG. 1 shows the accuracy of controls versus calibrators adjusted for peptide content. [Figure 13] FIG. 1 is a graph showing insulin correlation (n=117) of the method presented herein versus the Beckman assay. [Figure 14] FIG. 1 is a graph showing C-peptide correlation (n=121) of the method presented herein versus the Centaur ICMA assay. [Figure 15] 10 is a graph showing C-peptide calibrators in the Centaur ICMA. [Figure 16] FIG. 1 shows an overview of the verification results for insulin. [Figure 17] FIG. 1 shows an overview of the verification results for C-peptide. [Figure 18] 1 is a graph showing the relationship between creatinine and C-peptide. [Figure 19] FIG. 1 shows the insulin resistance scores of samples based on insulin and C-peptide levels. DETAILED DESCRIPTION OF THE INVENTION

[0061] As used herein, unless otherwise indicated, the singular forms "a," "an," and "the" Thus, for example, a reference to "a protein" includes multiple referents. It contains several protein molecules.

[0062] As used herein, the terms "purify," "purify," and "enrich" refer to It does not mean that all substances other than the analyte(s) are removed from the sample. Instead, these terms refer to other substances in a sample that may interfere with the detection of the analyte of interest. means a procedure that increases the amount of one or more analytes of interest relative to the components of the Purification of a sample by means of one or more interfering substances, e.g., by mass spectrometry, can be performed. one or more that may or may not interfere with the detection of parent or daughter ions The relative reduction in the amount of material to be purified is referred to as the relative reduction in the amount of material to be purified. It is necessary that the purification completely removes substances that are present in the raw material along with the analyte of interest. Not necessary.

[0063] As used herein, the term "immunopurification" or "immunopurifying" refers to the process of immunopurifying one of the following: or antibodies, including polyclonal or monoclonal antibodies, for enrichment of multiple analytes. Immunopurification refers to a purification procedure utilizing any of the immunopurification methods known in the art. Immunopurification procedures can often be carried out using a solid support, e.g., a column, Conjugated particles attached to wells, tubes, gels, capsules, particles, or the like. Immunopurification, as used herein, is the use of antibodies attached or otherwise bound to the target protein. In this case, a procedure often referred to in the art as immunoprecipitation, as well as a procedure often referred to in the art as affinity chromatography, A procedure called affinity chromatography or immunoaffinity chromatography Includes without limitation:

[0064] As used herein, the term "immunoparticle" refers to a particle that has a surface (on and / or within the particle) Capsules, beads having antibodies bound to, conjugated to, or otherwise attached to , gel particles or the like. In certain preferred embodiments, the immunoparticles are Sepharose or agarose beads. In an alternative preferred embodiment, the immunoparticles are , glass, plastic or silica beads or silica gel.

[0065] As used herein, the term "anti-insulin antibody" refers to an antibody that has an affinity for insulin. In various embodiments, the present invention refers to a polyclonal or monoclonal antibody having the following properties: The specificity of insulin antibodies for species other than insulin may vary, e.g. For example, in certain preferred embodiments, the anti-insulin antibody is specific for insulin. and therefore has little or no affinity for chemical species other than insulin. However, in other preferred embodiments, the anti-insulin antibodies are non-specific and do not target insulin. It binds to certain chemical species other than phosphorus.

[0066] As used herein, the term "sample" refers to a sample that may contain an analyte of interest. As used herein, the term "body fluid" refers to any fluid that can be separated from the body of an individual. For example, "body fluid" means blood, plasma, serum, bile, saliva, urine, tears, It may include sweat and the like. In a preferred embodiment, the sample is a bodily fluid sample from a human. , preferably plasma or serum.

[0067] As used herein, the term "solid phase extraction" or "SPE" refers to the process by which a solution passes through or surrounds a sample. Dissolved or suspended in a solution (i.e., mobile phase) for a freely flowing solid (i.e., solid phase) It refers to a method of separating a mixture of chemicals into components as a result of the affinity of the components. In some cases, undesirable components of the mobile phase may be released as the mobile phase flows through or around the solid phase. may be retained by the solid phase, resulting in the purification of the analyte in the mobile phase. In this case, the analyte is retained by the solid phase and the undesired components of the mobile phase pass through or In these cases, the liquid may be allowed to flow around it for further processing or analysis. A second mobile phase is then used to elute the retained analyte from the solid phase. SPE can function in a single or mixed mode mechanism. A mixed mode mechanism involves multiple steps on the same column. It utilizes ion exchange and hydrophobic retention in a mixed-mode SPE column. The solid phase may exhibit strong anion exchange and hydrophobic retention, or strong cation exchange and hydrophobic retention. May show sexual preservation.

[0068] Generally, the affinity of the SPE column packing material for the analyte is determined by one or more chemical This can be due to any of a variety of mechanisms, such as immunoaffinity or immunoaffinity interactions. In embodiments, SPE of insulin is performed without the use of immunoaffinity column packing material. That is, in some embodiments, insulin is purified by S It is purified from the sample using a PE column.

[0069] As used herein, the term "chromatography" refers to the process of chromatography carried out by liquid or gas. The difference in the chemicals when a mixture of chemicals flows around or over a stationary liquid or solid phase. It refers to the process by which a substance is separated into its components as a result of differential partitioning.

[0070] As used herein, the term "liquid chromatography" or "LC" refers to a method for When the particles uniformly penetrate a column or capillary passage of fine material, one or more particles of the fluid solution This refers to a method by which components are selectively retarded. Retardation is achieved by separating one or more stationary phases from the bulk fluid ( the stationary phase(s) as this fluid moves relative to the stationary phase(s). It results from the partitioning of the components of a mixture. Examples of "liquid chromatography" include reversed-phase liquid chromatography. RPLC, High Performance Liquid Chromatography (HPLC) and Turbulent Flow Liquid Chromatography (TFLC) (sometimes called high turbulence liquid chromatography (HTLC) or known as high-throughput liquid chromatography).

[0071] As used herein, "high performance liquid chromatography" or "HPLC" (sometimes referred to as "high performance liquid chromatography" or "HPLC") The term "high pressure liquid chromatography" refers to the process of transferring a mobile phase under pressure to a stationary phase. A method of increasing the degree of separation by forcing a liquid through a column, typically a tightly packed column. It means chromatographic analysis.

[0072] As used herein, "turbulent flow liquid chromatography" or "TFLC" (sometimes referred to as "high turbulence liquid chromatography" or "TFLC") This is known as high-speed liquid chromatography or high-performance liquid chromatography. The term utilizes the turbulent flow of the substance being assayed through a column packing as the basis for separation. TFLC refers to a form of chromatography. TFLC is a method for separating two atomic fractions of a sample prior to analysis by mass spectrometry. This has been applied to the preparation of samples containing drugs. ogr, Vol. A854, pp. 23-35 (1999). For further explanation of TFLC, see U.S. Patent Nos. 5,968,367, 5,919,368, 5,795, See also U.S. Pat. Nos. 469 and 5,772,874. Those skilled in the art understand "turbulent flow." When a fluid flows slowly and smoothly, the flow is called "laminar flow." For example, Fluid moving at a low flow rate through an HPLC column is laminar. The motion of the particles is regular, with the particles generally moving in a substantially straight line. In this case, the inertia of the water overcomes the frictional force of the fluid, resulting in turbulence. The fluid "overtakes" it, slowed by friction or deflected by uneven surfaces. When a fluid is flowing turbulently, it experiences greater resistance than when the flow is laminar. The fluid flows in a spiral (or vortex) due to the resistance. The fluid flow can be laminar or turbulent. Many references are available to help determine the case (e.g., Tu rbulent Flow Analysis:Measurement and Pr ediction, PS Bernard & JMWallace, John Wiley & Sons, Inc. (2000), An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001) ).

[0073] As used herein, the term "gas chromatography" or "GC" refers to the process of analyzing a sample. The mixture is evaporated and the carrier moves through a column containing a stationary phase consisting of a liquid or particulate solid. Inject into a stream of gas (as nitrogen or helium) to measure the affinity of the compound to the stationary phase. This refers to chromatography, which separates a compound into its component compounds according to the following formula.

[0074] As used herein, the term "large particle column" or "extraction column" refers to a column having a particle size of approximately 50 μm. In this context, a chromatographic column containing particles with a mean particle diameter greater than m is referred to. When used herein, the term "about" means ±10%.

[0075] As used herein, the term "analytical column" refers to a column that is used to determine the presence or amount of an analyte. Enough chloroform to allow sufficient separation of the substances in the sample to be eluted from the column. Such a column may further comprise a chromatographic column. Separating or separating retained material from unretained material to obtain a purified sample for further analysis It is often distinguished from "extraction columns" which have the general purpose of extracting When used herein, the term "about" means ±10%. Thus, the analytical column contains particles with a diameter of approximately 5 μm.

[0076] As used herein, the terms "online" and "inline" refer to, e.g., "online automated fashion" or "online automated fashion" As used in "online extraction," it refers to a procedure that is performed without the need for operator intervention. In contrast, the term "offline" as used herein refers to a process that is not manually performed by an operator. It refers to a procedure that requires intervention by industry. Thus, the sample is subjected to precipitation and then If the supernatant is manually loaded into the autosampler, the precipitation and loading steps are performed in the subsequent steps. In various embodiments of the method, one or more steps It can be carried out online automatically.

[0077] As used herein, the term "mass spectrometry" or "MS" refers to the analysis of compounds by their mass. MS refers to an analytical technique for identifying molecules by their mass-to-charge ratio or "m / z" of ions. MS technology is a method of filtering, detecting, and measuring ions based on the Generally, the method includes the steps of (1) ionizing a compound to produce a charged compound; and detecting the molecular weight of the compound and calculating the mass-to-charge ratio. The mass spectrometer generally refers to an ionization device that can ionize and detect the ions. , mass spectrometers, and ion detectors. Generally, one or more molecules of interest are ionized. The ions are then introduced into a mass analyzer where they are separated by a combination of magnetic and electric fields. The ions follow paths in space that depend on their mass ("m") and charge ("z"). For example, "Mass Spectrometry From Surface" U.S. Patent No. 6,204,500, entitled "Method and Apparatus for Tandem Mass Spectrometry," Apparatus(Methods and Apparatus for Tandem Mass No. 6,107,623, entitled "Mass Spectrometry-Based DNA Diagnostics Based On Mass Spec No. 6,268,144, entitled "Methods for Desorption and Detection of Analytes," Surface-Enhanced Photosensitive Binding and Release for abile Attachment And Release For Desopti No. 6,124 entitled "On and Detection of Analytes" ,137, Wright et al., Prostate Cancer and Prosta tic Diseases, 1999, Vol. 2, pp. 264-76 and Merchant and Weinberger, Electrophoresis, 2000, Vol. 21, No. 1 See pages 164-67.

[0078] As used herein, "high resolution / accurate mass spectrometry" refers to the analysis of unique chemical ions. The quality of the charged species can be determined with sufficient precision and accuracy to confirm the chemical ion Mass spectrometry refers to mass spectrometry performed using a mass spectrometer capable of measuring the mass-to-charge ratio. Identification of a given chemical ion is possible when the individual isotopic peaks of that ion are easily distinguishable. The specific resolution and quality required to identify specific chemical ions is possible. The quantitative accuracy depends on the mass and charge state of the ions.

[0079] As used herein, "resolution" or "resolution (FWHM)" (known in the art as "m / s") refers to a Δm 50% ") is the width of the mass peak at 50% of its maximum height (half maximum It refers to the observed mass-to-charge ratio divided by the full width at half maximum (FWHM).

[0080] As used herein, a "unique chemical ion" with respect to mass spectrometry is an ion that is composed of a single atom. A single ion may be singly or multiply charged.

[0081] As used herein, "accuracy" (or "mass accuracy") in relation to mass spectrometry refers to the accuracy of the analysis. Accuracy refers to the possible deviation of the instrument response from the true m / z of the ion being measured. It is generally expressed in parts per million (ppm).

[0082] The high-resolution / high-accuracy mass spectrometry of the present invention can be used to detect 10,000, 15,000, 20,000, FW greater than 25,000, 50,000, 100,000 or even greater The method of the present invention can be carried out on an instrument capable of performing mass spectrometry in HM. is 50 ppm, 20 ppm, 15 ppm, 10 ppm, 5 ppm, less than 3 ppm or These can be performed on instruments capable of performing mass spectrometry with an accuracy of even smaller magnitudes. Instruments capable of these performance characteristics include certain Orbitrap mass spectrometers, time-of-flight (" Incorporating a TOF mass spectrometer or a Fourier transform ion cyclotron resonance mass spectrometer In a preferred embodiment, the method is carried out using an Orbitrap mass spectrometer or a Time of Flight mass spectrometer. This is carried out by instruments including a quantitative analyzer.

[0083] The term "orbitrap" refers to an ion trap consisting of a barrel-like outer electrode and a coaxial inner electrode. Describe the trap: Ions are injected tangentially into the electric field between the electrodes, and the The electrostatic interaction between the ions balances with the centrifugal force when the ions orbit the coaxial inner electrode. When the ions orbit the coaxial inner electrode, the trajectory of the trapped ions The electron vibrates along the axis of the central electrode at a harmonic frequency depending on the mass-to-charge ratio of the electron. This allows the Orbitrap to be used with high accuracy (as low as 1-2 ppm) and high resolution (FWHM) (up to It will be possible to use it as a mass spectrometer with a mass of approximately 200,000. A mass spectrometer based on the ion beam is disclosed in U.S. Pat. No. 6,423,699, which is incorporated herein by reference in its entirety. This is described in detail in US Pat. No. 6,995,364.

[0084] As used herein, the term "operating in negative ion mode" refers to the "Operating in positive ion mode" refers to a mass spectrometry method that generates and detects ions. The term "mass spectrometry" as used herein refers to a mass spectrometry method that generates and detects positive ions. In a preferred embodiment, mass spectrometry is performed in positive ion mode.

[0085] As used herein, the term "ionization" or "ionize" refers to the act of ionizing one or more electrons. Negative ions refer to a method for generating analyte ions with a net charge equal to the molecular unit. An ion is one that has a net negative charge of one or more electron units, while a positive ion is one that has a net negative charge of one or more electron units. has a net positive charge of electron units.

[0086] As used herein, the term "electron ionization" or "EI" refers to gas-phase or vapor-phase The term refers to the way in which the analyte of interest in a sample interacts with the flow of electrons. The collisions produce analyte ions that can then be subjected to mass spectrometry techniques.

[0087] As used herein, the term "chemical ionization" or "CI" refers to the process of ionizing a reagent gas (e.g., Ammonia (e.g., ammonia) is subjected to electron bombardment, resulting in the interaction of reagent gas ions with analyte molecules. This refers to the process by which analyte ions are generated.

[0088] As used herein, the term "fast atom bombardment" or "FAB" refers to high energy A beam of atoms (often Xe or Ar) bombards a non-volatile sample, dissolving the atoms contained in the sample. This refers to a method in which the test sample is dissolved in glycerol, thioglycerol, or Benzyl alcohol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitro Phenyloctyl ether, sulfolane, diethanolamine and triethanolamine The compound or sample is dissolved in a viscous liquid matrix such as Selection is an empirical process.

[0089] As used herein, "matrix-assisted laser desorption ionization" or "MALDI" The term "" refers to the process of photoionizing, protonating, deprotonating and clustering a non-volatile sample. A laser that desorbs and ionizes analytes in a sample through various ionization pathways, including decay For MALDI, the sample is exposed to radiation that promotes desorption of the analyte molecules. It is mixed with an energy absorbing matrix that accelerates the process.

[0090] As used herein, "surface-enhanced laser desorption ionization" or "SELDI" The term "" refers to the process of photoionizing, protonating, deprotonating and clustering a non-volatile sample. A laser that desorbs and ionizes analytes in a sample through various ionization pathways, including decay For SELDI, the sample is generally exposed to one of the following radiation sources: Alternatively, multiple analytes can be bound to a surface that preferentially retains them. Similar to MALDI, this method The method may also employ an energy absorbing material to facilitate ionization.

[0091] As used herein, the term "electrospray ionization" or "ESI" This refers to a method in which a solution is passed through a short capillary tube to the ends of which a high positive or negative potential is applied. The solution that reaches the target vaporizes (atoms) into a jet or stream of very small droplets of the solution in solvent vapor. This spray of droplets flows through the evaporation chamber. As the droplets get smaller, When the electrons are released, the natural repulsion between the like-charged molecules causes the release of ions and neutral molecules. The surface charge density increases until

[0092] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to ES APCI is a mass spectrometry technique similar to I, but APCI is an ion- Ions are generated by molecular reactions. The plasma is maintained by an electrical discharge between the atomizing capillary and the counter electrode. The ions are then pumped, typically using a pair of differentially pumped skimmer stages. The solvent is removed using countercurrent dry, preheated N2 gas. Gas phase ionization in APCI allows for the analysis of less polar species. may be more effective than ESI in this regard.

[0093] The term "atmospheric pressure photoionization" or "APPI" as used herein refers to the photoionization of molecules M Mass spectrometry in which the ionization mechanism is the absorption of a photon and the emission of an electron to produce the molecular ion M+ Since the photon energy is generally just above the ionization potential, In many cases, samples can be analyzed without the need for chromatography. This can save considerable time and money. In the presence of an ionic solvent, the molecular ion can abstract H to form MH+. This tends to happen if the molecule has rotonophilicity. The sum of M+ and MH+ is constant. Therefore, this does not affect the accuracy of the quantification. Drug compounds in protic solvents are usually Although observed as MH+, non-polar compounds such as naphthalene or testosterone are usually M+. See, e.g., Robb et al., Anal. Chem., 2000, 72(1) No. 5, pp. 3653-3659.

[0094] As used herein, the term "inductively coupled plasma" or "ICP" refers to most The sample is partially ionized at a temperature high enough that the elements are atomized and ionized. This refers to the way in which the gas interacts with the object.

[0095] As used herein, the term "field desorption" refers to the process of applying a non-volatile test sample to an ionizing surface. This refers to a method in which a sample is placed on a substrate and a strong electric field is used to generate analyte ions.

[0096] As used herein, the term "desorption" refers to the removal and / or cleavage of an analyte from a surface. Laser desorption / thermal desorption refers to the entry of the analyte into the gas phase. The laser is a technique for thermally desorbing atoms into the gas phase using a laser pulse. The backside of a specially designed 96-well plate is irradiated. The laser pulse heats the bottom, and the heat is transferred to the sample. The gas phase sample is then drawn into a mass spectrometer.

[0097] As used herein, the term "selected ion monitoring" refers to the monitoring of a relatively narrow mass range. detection mode of a mass spectrometer where only ions within a range of, typically about 1 mass unit, are detected It is Do.

[0098] As used herein, "multiple reaction monitoring," sometimes known as "selective reaction monitoring," refers to In the "reaction mode," precursor ions and one or more fragment ions are selectively detected. This is the detection mode of the mass spectrometer.

[0099] As used herein, the terms "lower limit of quantitation," "lower limit of quantitation," or "LLOQ" , the point at which the measurement becomes quantitatively meaningful. The responses of the substances are identifiable and individual, with relative standard deviations (RSD%) of less than 20%. It is reproducible with an accuracy of 85% to 115%.

[0100] As used herein, the term "limit of detection" or "LOD" refers to the amount of a measurement that is related to The LOD is the point at which a value is greater than the uncertainty associated with that measurement. The point above which the RSD is exceeded is defined as three times the RSD of the mean value at zero concentration.

[0101] As used herein, the "amount" of an analyte in a body fluid sample generally refers to the amount of analyte in a volume of sample. However, the amount is not necessarily the same as other analytes. Relative amounts are also contemplated, for example, the amount of an analyte in a sample compared to the amount normally present in the sample. The amount may be greater than a control or normal level of the analyte.

[0102] The term "about" is used herein to refer to quantitative measurements that do not include measurement of the mass of an ion. Where applicable, this means the stated value plus or minus 10%. Determining the mass of an ion can vary slightly depending on the mass or mass / charge ratio of the ion. The term "about" in this context means + / - 0.50 atomic mass units.

[0103] Quantitative determination of serum insulin is useful in the evaluation of insulin resistance syndrome and diabetes. It is primarily used to diagnose glycemic disorders in pre-morbid patients. C-peptide is a serotonin receptor that regulates insulin secretion. It is a peptide that connects the two peptide chains and is released from proinsulin during processing. It is secreted from pancreatic beta cells and then co-secreted. Therefore, peripheral blood levels of C-peptide and insulin are no longer equimolar, but still In this embodiment, the methods provided herein include: (1) hypotension; (2) insulin-secreting tumors as a cause of diabetes and exogenous insulin administration, and (3) type 1 diabetes and type 2 diabetes. It measures endogenous insulin and C-peptide to distinguish between type 2 diabetes and type 3 diabetes.

[0104] In one aspect, provided herein are methods for detecting insulin and erythrocytes in a sample using mass spectrometry. METHOD FOR MEASURING INSULIN LEVELS IN A PATIENT BY DETERMINING THE AMOUNT OF INSULIN AND C-PEPTIDE - Patent application In some embodiments, the methods provided herein involve analyzing the analytes by mass spectrometry. It includes a multiplex assay that simultaneously measures the amount of insulin and C-peptide in a sample. In some embodiments, the method comprises (a) detecting one or more indicators detectable by mass spectrometry. Insulin and C-peptide are isolated from the sample under conditions suitable for generating phosphorus and C-peptide ions. (b) subjecting the C-peptide and the C-peptide to an ionization source; and (b) identifying one or more and determining the amount of insulin and C-peptide ions. In one embodiment, the amount of one or more ions determined is used to determine the amount of insulin and pentaerythritol in the sample. In some embodiments, the amount of insulin and C-peptide in the sample is determined. In some embodiments, the amount of insulin administered is correlated to the amount of insulin in the patient. The amount of insulin and C-peptide in the sample was used to assess the insulin vs. C-peptide ratio in patients. Determine the ratio of

[0105] In some embodiments, the method further comprises: (a) subjecting the sample to a concentration step to concentrate insulin and (b) obtaining a fraction enriched in C-peptide and C-peptide, and (c) obtaining a fraction enriched in C-peptide and C-peptide that can be detected by mass spectrometry. and concentrating the insulin and C-peptide ions under conditions suitable for generating one or more insulin and C-peptide ions. (c) subjecting the isolated insulin and C-peptide to an ionization source; and (d) subjecting the isolated insulin and C-peptide to mass spectrometry. and determining the amount of one or more insulin and C-peptide ions by In some embodiments, the determined amount of one or more ions is used to determine the amount of ions in the sample. In some embodiments, the amount of insulin and C-peptide in the sample is determined. The amount of insulin and C-peptide is related to the amount of insulin in the patient. In an embodiment, the amount of insulin and C-peptide in the sample is used to determine the level of insulin in the patient. The ratio of insulin to C-peptide is determined. In some embodiments, the insulin to C-peptide ratios provided herein are The enrichment process used involves immunocapture of insulin and C-peptide using antibodies. In some embodiments, the method includes (a) immunocapturing insulin and C-peptide. and (b) one or more of insulin and C-peptide detectable by mass spectrometry. Immunocaptured insulin and C-peptide were incubated under conditions suitable for generating ions. (c) subjecting the one or more insulins and C to an ionization source; and - determining the amount of peptide ions. The immunocapturing step provided herein comprises detecting anti-insulin antibodies and anti-C-peptide antibodies. In some embodiments, the antibodies provided herein are In some embodiments, the antibodies provided herein are monoclonal antibodies. In some embodiments, the antibody provided herein is a mouse monoclonal antibody. The antibody is a monoclonal IgG antibody. The antibody provided is a polyclonal antibody. In some embodiments, the anti-C-peptide antibody and the anti-C-peptide antibody are immobilized on magnetic beads. Immunocaptured insulin and C-peptide on magnetic beads are washed and eluted. do.

[0106] In some embodiments, the serum is delipidated prior to quantification by mass spectrometry. In embodiments, one or more delipidation reagents are used to remove lipids from the sample. In some embodiments, the degreasing reagent is CLEANASCITE®.

[0107] In some embodiments, the methods provided herein involve purifying the sample prior to mass spectrometry analysis. In some embodiments, the method comprises preparing a liquid chromatography In some embodiments, the method further comprises purifying the sample using a liquid chromatograph. The flow is performed by high performance liquid chromatography (HPLC) or high turbulence liquid chromatograph ( In some embodiments, the method comprises subjecting the sample to solid phase extraction (SPE). This includes the step of applying.

[0108] In some embodiments, the mass spectrometry comprises tandem mass spectrometry. In some embodiments, the mass spectrometry is high-resolution mass spectrometry. The mass analysis is high resolution / high accuracy mass spectrometry. In some embodiments, the ionization is , by electrospray ionization (ESI). In some embodiments, ions In some embodiments, the ionization is by atmospheric pressure chemical ionization (APCI). Ionization was in positive ion mode.

[0109] In some embodiments, the methods provided herein include adding an internal standard to the sample. In some embodiments, the internal standard for insulin is bovine insulin. In some embodiments, the internal standard for C-peptide is C-peptide. In some embodiments, the internal standard is labeled. In some embodiments, the internal standard is deuterated or isotopically labeled. do.

[0110] In some embodiments, the patient sample is a serum sample. In some embodiments, the patient sample is a plasma sample. In some embodiments, the patient sample is blood, Saliva or urine samples.

[0111] In some embodiments, the sample is subjected to acidic conditions prior to ionization. In an embodiment, subjecting the sample to acidic conditions results in concentrated insulin and C-peptide. In some embodiments, the sample is exposed to basic conditions prior to the addition of formic acid. In some embodiments, the step of subjecting the sample to basic conditions comprises subjecting the sample to In some embodiments, the sample is exposed to Trizma under basic conditions. The step of placing includes exposing the sample to Trizma and ethanol.

[0112] In some embodiments, one or more ions have a mass charge of 968.7±0.5. In some embodiments, the insulin precursor ions have a charge ratio (m / z) of 1. The ions or ions were 136.0±0.5, 226.1±0.5 and 345.2±0. one or more fragment ions selected from the group consisting of ions having m / z of 5 In some embodiments, insulin has an m / z of 226.1±0.5. The fragment ions are quantifier ions. The one or more ions are bovine lecithin ions having a mass-to-charge ratio (m / z) of 956.8±0.5. In some embodiments, the one or more ions include insulin precursor ions. Ions with m / z of 36.0±0.5, 226.1±0.5, and 315.2±0.5 In some embodiments, the fragment ions include one or more fragment ions selected from the group consisting of: In this state, the bovine insulin fragment ion with m / z of 136.0±0.5 , a quantifier ion. In some embodiments, one or more ions The C-peptide precursor ion had a mass-to-charge ratio (m / z) of 1007.7 ± 0.5. In some embodiments, the one or more ions are 533.3±0.5, 6 ions selected from the group consisting of ions having m / z of 46.4±0.5 and 927.5±0.5 In some embodiments, the fragment ions include one or more fragment ions that are 533. C-peptides with m / z of 3±0.5, 646.4±0.5 and 927.5±0.5 Any of the fragment ions can be used as a quantifier ion. In some embodiments, one or more ions have a mass charge of 1009.5±0.5. The C-peptide heavy internal standard precursor ion with a charge ratio (m / z) of 1000 m / s is included. In the form, one or more ions are 540.3±0.5, 653.4±0.5, and One or more ions selected from the group consisting of ions having an m / z of 934.5±0.5 In some embodiments, 540.3±0.5, 653. C-peptide heavy internal standard flag with m / z of 4±0.5 and 934.5±0.5 Any of the ment ions can be used as quantifier ions.

[0113] In some embodiments, provided herein are methods for detecting insulin and C in a sample. - utilizing mass spectrometry to determine the amount of peptides, the method comprising: (a) an extraction technique; (b) concentrating insulin and C-peptide in the sample by a method; a) Purified insulin and C-peptide from the sample were subjected to liquid chromatography. (c) obtaining a fraction enriched in insulin and C-peptide from the fraction; and (d) analyzing the fraction by mass spectrometry. The concentrated insulin is then injected under conditions suitable to generate insulin precursor ions detectable by the (d) subjecting the sample to a mass spectrometry source to generate one or more fragment ions. and determining the amount of one or more of the determined The amount of several ions is used to determine the amount of insulin and C-peptide in the sample. In some embodiments, the amount of insulin and C-peptide in the sample is compared to the insulin concentration in the patient. In some embodiments, the amount of insulin and C-peptide in the sample is correlated with the amount of phosphorus. The amount of insulin used to determine the insulin to C-peptide ratio in a patient. In embodiments, the extraction techniques provided herein involve the extraction of insulin and C- In some embodiments, the extraction techniques provided herein include immunocapture of peptides. The technique involves solid phase extraction (SPE).

[0114] In some embodiments, the collision energy is in the range of about 40-60 eV. In some embodiments, the collision energy is in the range of about 40-50 eV.

[0115] In another aspect, provided herein are methods for detecting insulin or erythrocytes in a sample by mass spectrometry. A method for determining the amount of C-peptide, comprising: (a) administering insulin or C-peptide to a subject by immunization; and (b) capturing one or more insulin or C molecules detectable by mass spectrometry. - Immunocaptured insulin or C-peptide under conditions suitable for generating peptide ions (c) subjecting the insulin to an ionization source; and (d) isolating one or more insulins or and determining the amount of C-peptide ions. In the present invention, a method for determining the amount of insulin in a sample by mass spectrometry is provided. (a) immunocapturing insulin; and (b) detecting insulin by mass spectrometry. The immunocapture insulin is then reacted with the immunocapture insulin under conditions suitable to generate one or more insulin ions that can be released. (c) subjecting the insulin to an ionization source; and (d) identifying the one or more insulins by mass spectrometry. and determining the amount of phosphorus ions. Provided herein are methods for determining the amount of C-peptide in a sample by mass spectrometry. (a) immunocapturing C-peptide; and (b) detecting it by mass spectrometry. The immunocaptured C-peptide is subjected to conditions suitable for generating one or more C-peptide ions. (c) subjecting the peptide to an ionization source; and (d) identifying one or more C-peptides by mass spectrometry. and determining the amount of tide ion. The immunocapturing step may include a step of using an anti-insulin antibody or an anti-C-peptide antibody. In some embodiments, the anti-insulin antibody or anti-C-peptide antibody is a magnetic In some embodiments, the immunocapture on the magnetic beads is The resulting insulin or C-peptide is washed and eluted.

[0116] In another aspect, provided herein are methods for treating blood clotting in diabetic and prediabetic patients. A method for diagnosing a glucose disorder or insulin resistance syndrome. In some embodiments, The methods for quantifying endogenous insulin and C-peptide provided herein can be used to diagnose diabetes. In some embodiments, the endogenous insulin provided herein is used to The method for quantifying C-peptide was used to investigate the relationship between insulin-secreting tumors and exogenous insulin as causes of hypoglycemia. In some embodiments, the methods provided herein are used to differentiate between insulin administration. A method for quantifying endogenous insulin and C-peptide to distinguish between type 1 and type 2 diabetes In some embodiments, the endogenous insulin provided herein is used to Methods for quantifying C-peptide and C-peptide to assess the risk of diabetes in prediabetic patients Used for this purpose.

[0117] Suitable test samples for use in the methods of the present invention are test samples that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample, i.e., an animal, a cell culture Samples obtained from biological sources such as cultures, organ cultures, etc. In certain preferred embodiments, In this case, the sample is obtained from a mammal such as a dog, cat, horse, etc. Particularly preferred mammals are The subject is a primate, most preferably a male or female human. Preferred samples are blood, plasma, plasma, or plasma. Such samples include body fluids such as serum, saliva, cerebrospinal fluid, or tissue samples, preferably plasma and serum. Such samples may be, for example, samples from patients, i.e., clinically relevant individuals for the diagnosis, prognosis, or treatment of a disease or condition. The sample may be obtained from a living male or female individual presenting to the facility. In an embodiment, the method comprises determining the concentration of insulin in a sample when the sample is obtained from a biological source. can be used to determine the amount.

[0118] The present invention also contemplates a kit for the quantitative assay of insulin. Assay kits can include kits containing the compositions provided herein. The package contains packaging materials and a quantity of isotopically labeled internal standard sufficient for at least one assay. Generally, the use of prepackaged reagents for quantitative assays of insulin is This also includes instructions recorded in any form (e.g., contained on paper or electronic media).

[0119] The calibration and QC pools used in embodiments of the present invention preferably contain essentially The preparation was carried out using a matrix similar to the target sample matrix, provided that the matrix was not present. To manufacture.

[0120] Preparation of samples for mass spectrometry In preparation for mass spectrometry, e.g., immunocapture, liquid chromatography, filtration, Centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, immunoassay Affinity separation, including ethyl acetate or methanol extraction Extraction methods and the use of chaotropic agents or any combination of the above or the like Insulin can be isolated from one or more samples by various methods known in the art, including by combining It can be concentrated compared to other components of the

[0121] One method of sample purification that can be used prior to mass spectrometry is to ensure that the analytes of interest are not present in the column. The sample is reversibly retained on the polymer packing material, but one or more other substances are not retained. The technique involves adding a sample to a solid phase extraction (SPE) column. If the column retains the first mobile phase condition, the second mobile phase condition can be used, and if the non-retained Once the material has been washed away, a second transfer is then performed to remove the retained material from the column. Mobile phase conditions can be used.

[0122] In some embodiments, insulin in a sample is bound to a loading material comprising an alkyl-bonded surface. For example, in some embodiments, the hydroxybenzoate can be reversibly retained on an SPE column containing In this study, a C-8 online SPE column (Oasis manufactured by Phenomenex, Inc.) was used. s HLB online SPE column / cartridge (2.1mm x 20mm) or equivalent ) can be used to enrich insulin prior to mass spectrometry. In an embodiment, the use of an SPE column involves the use of 0.2% aqueous HPLC grade gel as a wash solution. The acid is carried out using 0.2% formic acid in acetonitrile as the elution solution.

[0123] In other embodiments, the method includes immunopurifying insulin prior to mass spectrometry. The immunopurification step is carried out using any of the immunopurification methods known in the art. Often, immunopurification procedures involve the use of solid supports, e.g., columns, wells, tubes, etc. , capsules, particles or the like, bound, conjugated, immobilized or otherwise Generally, immunopurification involves the following steps: (1) extracting the analyte from the sample; A sample containing the analyte of interest is incubated with the antibody so that the antibody binds to the sample. (2) performing one or more washing steps; and (3) removing the antibody. and eluting the analyte.

[0124] In certain embodiments, the incubation step of the immunopurification involves the removal of free ATP in solution. This is performed using an antibody, which is then bound or attached to a solid surface before a washing step. In certain embodiments, this is a primary antibody that is an anti-insulin antibody and a primary anti-insulin antibody. This is achieved by using a secondary antibody bound to a solid surface that has affinity for the thrombin antibody. In an alternative embodiment, the primary antibody is applied to a solid surface prior to the incubation step. Attach to the surface.

[0125] Suitable solid supports include, without limitation, tubes, slides, columns, beads, capsules, particles, In some preferred embodiments, the solid support is, for example, For example, multi-well plates such as 96-well plates, 384-well plates, and the like. In some embodiments, the solid support is sepharose or agarose. The antibody (e.g., insulin antibody or secondary antibody) is bound to a solid support, such as beads or a gel. The molecules can be attached, immobilized or linked by many methods well known in the art, e.g., covalent Covalent or non-covalent adsorption, affinity binding, ionic binding, etc. In this case, the antibody is conjugated using CNBr. For example, the antibody is conjugated using CNBr-activated cephalosporin. In another embodiment, the antibody can be linked to protein A, protein B, or a combination thereof. via antibody-binding proteins such as Protein G, Protein A / G, or Protein L on a solid support Attach to.

[0126] The wash steps of immunopurification generally involve the removal of insulin from the solid support by anti-insulin antibodies. It is necessary to wash the solid support so that the antibody remains bound to the antibody. This step generally requires the addition of a solution that disrupts the binding of insulin to anti-insulin antibodies. Exemplary elution solutions include organic solutions, salt solutions, and high or low pH solutions. nothing.

[0127] Another method of sample purification that can be used prior to mass spectrometry is liquid chromatography. (LC). In liquid chromatography techniques, one or more analytes of interest are The sample is chromatographically analyzed under mobile phase conditions that cause it to elute at a different rate than other substances in the Such a procedure can purify the analyte by adding it to a sample. The amount of one or more analytes of interest may be enriched relative to one or more other components.

[0128] Certain methods of liquid chromatography, including HPLC, rely on relatively slow laminar flow techniques. In traditional HPLC analysis, laminar flow of the sample through the column separates the target analytes from the sample. The separation of precipitates relies on the column packing, which is the basis for the separation of precipitates. Understanding that the separation is a partitioning process, HP suitable for use with C-peptide You can choose the LC, instrument, and column, including LC. Chromatographic analytical columns are , generally a medium (i.e., The medium may contain fine particles. The particles are generally made up of various compounds. It contains a binding surface that interacts with the component to facilitate separation of the compound's components. The surface is a hydrophobic bonded surface, such as an alkyl-bonded or cyano-bonded surface. The faces may contain C-4, C-8, C-12, or C-18 bonded alkyl groups. In an embodiment, the chromatographic analytical column is a monolithic C-18 column. The chromatography column has an inlet for receiving the sample and an outlet for discharging the effluent containing the fractionated sample. The sample can be introduced directly into the inlet or into an SPE column, such as an online SPE column. In some embodiments, the eluate may be delivered from a pulp or TFLC column. Particles and particles in the sample are analyzed before the sample reaches the SPE and / or TFLC and / or HPLC column. An online filter was used on the SPE column and / or HPLC column to remove phospholipids and phospholipids. It can be used before the ram.

[0129] In one embodiment, the sample is applied to the LC column at the inlet port and a solvent or solvent mixture is added. The analyte(s) of interest can be eluted by the compound and discharged at the outlet. Various solvent modes for eluting the chromatographic components can be selected. The analysis can be performed using gradient, isocratic, or polymorphic (i.e., mixed) modes. During chromatography, separation of substances occurs via an eluent (known as the "mobile phase"). (also known), are affected by variables such as elution mode, gradient conditions, temperature selection, etc.

[0130] In some embodiments, the insulin in the sample is concentrated by HPLC. PLC is a monolithic C-18 column chromatographic system, e.g., Phenomen Onyx monolith C-18 column (50x2.0mm) manufactured by ex Inc. or equivalent In certain embodiments, HPLC can be performed using H as solvent A. PLC was performed using 0.2% aqueous formic acid and 0.2% formic acid in acetonitrile as solvent B. To carry out.

[0131] Careful selection of valves and fittings allows for one chromatic Two or more chromatography columns are used to pass material from one column to the next. In a preferred embodiment, the valves and piping are The selection is made by a computer preprogrammed to perform the necessary steps. Most preferably, the chromatography system is controlled in such an online manner. It is also connected to a detection system, e.g., an MS system. The tray can be attached to the autosampler and the rest of the operation is computer controlled. This is carried out under the conditions described above, resulting in the purification and analysis of all selected samples.

[0132] In some embodiments, TFLC is used for purification of insulin prior to mass spectrometry. In such an embodiment, the TFLC column that captures the analyte can be The sample can then be extracted using the HPLC method. The analytes are then eluted and analyzed online. Transfer the sample to an LC column. For example, the sample is extracted using a TFLC column containing a large particle size (50 μm) packing material. This can be achieved using an extraction cartridge. The sample eluted from this column is It can be transferred online to an analytical HPLC column for further purification prior to analysis. The steps involved in these chromatographic procedures can be linked together in an automated fashion. This feature minimizes the need for operator intervention during the purification of the analyte. This can result in time and cost savings and eliminate opportunities for operator error.

[0133] In some embodiments, one or more of the above-described purification techniques may be used for the simultaneous processing of multiple samples. It can be used in parallel for the purification of insulin to allow for the processing of In some embodiments, the purification technique used is immunoaffinity chromatography. Excluding immunopurification techniques such as

[0134] Detection and quantification of insulin by mass spectrometry Mass spectrometry is a method for ionizing fractionated samples to produce charged molecules for further analysis. In various embodiments, the insulin is The insulin can be ionized by methods known to those skilled in the art. For example, insulin can be ionized by methods known to those skilled in the art. electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization Atmospheric Pressure Chemical Ionization (APCI), Photoionization, Atmospheric Pressure Photoionization (APPI), Laser diode thermal desorption (LDTD), fast atom bombardment (FAB), liquid secondary ionization (L SI), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption Desorption, thermospray / plasma spray ionization, surface-enhanced laser desorption ionization ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization Those skilled in the art will appreciate that the choice of ionization method depends on the analyte being measured, the type of sample, and the detector. It is understood that the decision can be based on the type, choice of positive vs. negative mode, etc. Therefore, insulin is ionized in the positive mode but not in the negative mode. In a preferred embodiment, insulin may be ionized by ESI. The electrons are ionized in the ionization mode.

[0135] In mass spectrometry, the sample is generally ionized and then the resulting positive or negative ions are analyzed. The negatively charged ions can be analyzed to determine their mass-to-charge ratio (m / z). Various analyzers for determining ion beam radiation include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. spectrometer, Fourier transform ion cyclotron resonance mass spectrometer and Orbitrap spectrometer Some exemplary ion trapping methods include those described by Bartolucci et al. Rapid Commun.Mass Spectrom., 2000, Vol. 14, 96 It is described on pages 7-73.

[0136] Ions can be detected using several detection modes. For example, The ions can be detected using selected ion monitoring mode (SIM), i.e. or alternatively due to collision-induced dissociation or neutral loss Mass transitions can be monitored, for example, by multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). In some embodiments, the mass-to-charge ratio can be monitored by quadruple NMR. Determined using a quadrupole analyzer. In a "quadrupole" or "quadrupole ion trap" instrument Therefore, ions in an oscillating radio frequency field are generated by the DC potential applied between the electrodes, the amplitude of the RF signal, The voltage and amplitude are proportional to the width and mass-to-charge ratio of the particle. Only ions of interest can be selected to traverse the quadrupole, while all other ions are deflected. Thus, quadrupole instruments act as a "mass filter" and "filter" for the ions injected into the instrument. The detector can function as both a mass detector and a detector of interest.

[0137] When ions hit the detector, they emit a pulse of electrons that is converted into a digital signal. The acquired data is transferred to a computer, which then analyzes the collected information. The on counts are plotted against time. The resulting mass chromatogram is similar to the traditional HP Similar to a chromatogram obtained by LC-MS. Peaks corresponding to specific ions The area under or amplitude of such peaks can be measured and related to the amount of analyte of interest. In certain embodiments, the fragment ion(s) and / or precursor ion(s) can be The area under the curve or amplitude of the ON peak is measured to determine the amount of insulin. The relative abundance of ions is determined by a calibration based on one or more ion peaks of internal or external molecular standards. A standard curve can be used to convert to absolute amounts of the original analyte.

[0138] The resolution of MS techniques using specific mass spectrometers is referred to as "tandem mass spectrometry" or "MS / MS In this technique, precursor ions derived from the molecule of interest can be used to enhance the The precursor ions (also called parent ions) can be filtered by the MS instrument. The ions are then fragmented to produce one or more fragments that are analyzed in a second MS step. Precursor ions (also called daughter ions or product ions) are generated. Deep selection allows only ions produced by specific analytes to reach the fragmentation chamber where fragment ions are produced by collision with atoms of an inert gas. Both precursor and fragment ions are reproducible under a set of defined ionization / fragmentation conditions. Because of this high reproducibility, MS / MS techniques can be extremely powerful analytical tools. , filtration / fragmentation combinations are used to remove interfering substances. It can be used for a wide range of applications and can be particularly useful for complex samples such as biological samples. In an embodiment, a mass spectrometer including a multiple quadrupole analyzer (e.g., a triple quadrupole analyzer) Tandem mass spectrometry is performed using a tandem mass spectrometer (e.g.,

[0139] In certain embodiments using MS / MS techniques, precursor ions can be further fragmented. Isolate precursor ions for subsequent detection using collision-activated dissociation (CAD) for synthesis. In CAD, precursor ions are generated by collision with an inert gas. The molecule gains energy through collisions and then fragments into molecules through a process called "unimolecular decomposition." The increased vibrational energy creates enough Energy must be deposited in the precursor ions.

[0140] In some embodiments, insulin in a sample can be determined using MS / MS as follows: The sample is first subjected to SPE and then to liquid chromatography. By subjecting the sample to a chromatography, preferably HPLC, insulin is concentrated and chromatographically The liquid solvent flow from the chromatographic analytical column is directed to the heated nebulizer interface of the MS / MS analyzer. The solvent / analyte mixture is vaporized in the heated and charged tube of the interface. During these processes, the analyte (i.e., insulin) is ionized. Ions, e.g., precursor ions, pass through an aperture in the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) separate ions based on their mass-to-charge ratio (m / z). Selection (i.e., of "precursor" and "fragment" ions in Q1 and Q3, respectively) Quadrupole 2 (Q2) is a mass filter that allows ions to be fragmented. The first quadrupole (Q1) of the mass spectrometer is the collision cell where insulin ions are condensed. Select a molecule with the correct m / z. The precursor ion with the correct m / z is selected by the collision The unwanted ions with other m / z are passed into the chamber (Q2), but are trapped on the side of the quadrupole. The precursor ions that enter Q2 collide with neutral gas molecules (such as argon molecules) and are removed. The fragment ions are then passed through quadrupole 3 (Q3). where fragment ions are selected for detection.

[0141] Insulin ionization occurs through the formation of multiply charged precursor ions (such as 4+, 5+, 6+, etc.). The ionization conditions, especially the p of the buffers used in the electrospray technique, can H has a significant effect on the identity and quantity of insulin precursor ions produced. Under neutral conditions, positive electrospray ionization mainly resulted in 1162 5+ and 6+ insulin precursors with m / z of 968.5±0.5 and 968.5±0.5 However, under basic conditions, positive electrospray ions can be generated. The compounds were mainly identified as m / z 1453.75±0.5 and 1162.94±0.5, respectively. The method can generate 4+ and 5+ charged insulin precursor ions having an acidic or basic Neutral conditions, preferably acidic conditions, may be used.

[0142] The method can be carried out in either positive or negative ion mode, preferably in positive ion mode. In certain embodiments, this may include MS / MS performed in an electrospray buffer. The buffer solution is acidic, and Q1 has an m / z of about 1162.5±0.5 or 968.5±0.5. Select an insulin precursor ion having any of these insulin precursor ions. Fragmentation results in m / z of about 226.21±0.5 and / or 135.6±0.5 Therefore, a fragment ion having Q1 of about 1162.5±0.5 is generated. and one or more selected from the group consisting of an ion having an m / z of 968.5±0.5 In an embodiment where an insulin precursor ion of about 226.21±0.5 is selected, Q3 is about 226.21±0.5 and one or more fragments selected from the group of ions having m / z of 135.6±0.5. In certain embodiments, a single fragment ion from a single precursor ion may be selected. Alternatively, the relative abundance of two fragment ions from a single precursor ion can be measured. The relative abundance of one or more fragment ions can be measured. In this case, the relative abundance of each fragment ion is subjected to known mathematical processing to determine the initial abundance of each fragment ion in the sample. In another embodiment, two or more precursors of insulin can be quantitatively assessed. One or more fragment ions from the ions are measured and utilized as described above to identify the fragment ions in the sample. The initial insulin can be qualitatively assessed.

[0143] Alternative modes of operating a tandem mass spectrometry instrument that can be used in certain embodiments include: These operation modes include product ion scan and precursor ion scan. For a description of the method, see, for example, E. Michael Thurman et al., Chromatography graphic-Mass Spectrometric Food Analysis s for Trace Determination of Pesticide R esidues, Chapter 8 (Amadeo R.Fernandez-Alb a, Elsevier 2005)(387).

[0144] In another embodiment, the high resolution / high accuracy mass spectrometer is To achieve acceptable accuracy of the quantitative results, The mass spectrometer measures 10,000 ions of interest with an accuracy of about 50 ppm or better. It must be possible to show a resolution (FWHM) of 1000 or more. The analyzer has a resolution (FW) of 18,000 or better with an accuracy of about 5 ppm or better. FWHM), for example, a resolution of 20,000 or more and about 3 ppm or less Accuracy such as 25,000 or more resolution (FWHM) and approximately 3pp The accuracy of the ion concentration is 1000 m or less. Three specific spectrometers that can be used are the Orbitrap mass spectrometer, a specific TO F mass spectrometer and Fourier transform ion cyclotron resonance mass spectrometer.

[0145] Elements found in biologically active molecules, such as carbon, oxygen, and nitrogen, are naturally occurring in many different isotopes. For example, most carbon atoms exist in the 12 C, but not all naturally occurring About 1% of the carbon present is 13 C. Therefore, at least one carbon atom A portion of a naturally occurring molecule containing at least one 13 Contains C atoms Naturally occurring elemental isotopes are included in molecules, resulting in multiple molecular isotopes. The difference in mass between molecular isotopes is at least 1 atomic mass unit (amu). This is because elemental isotopes differ by at least one neutron (the mass of one neutron ≈ 1 amu) When molecular isotopes are ionized into multiply charged states, their detection in mass spectrometry is based on the mass-to-charge ratio (m / z), mass differences between isotopes can be difficult to distinguish. For example, two isotopes differing in mass by 1 amu that both ionize to the 5+ state are roughly The high resolution / high accuracy mass spectrometry shows a difference in m / z between them of only 0.2. Identify isotopes of ions (e.g., ions with ±2, ±3, ±4, ±5 or higher charges) It is possible.

[0146] Naturally occurring elemental isotopes allow for the determination of all molecular ions (with sufficiently sensitive mass spectrometers) (each of which may produce a separately detectable spectral peak when analyzed by an instrument) Multiple isotopes are commonly present in a molecule. The m / z ratios and relative abundances of multiple isotopes are In some embodiments, two or more molecules collectively comprise the isotopic signature of the ions. The m / z ratios and relative abundances of the molecular isotopes are used to confirm the identity of the molecular ion under consideration. In some embodiments, one or more isotopic mass spectrometric peaks can be used. In some related embodiments, the molecular ion is quantified using a single isotope. A single mass spectrometric peak is used to quantify the molecular ion. The isotopic peak is used to quantify the molecular ion. The isotope peaks can be subjected to appropriate mathematical treatments. known in the art and include the sum of the areas under multiple peaks or the average of the responses from multiple peaks. However, the present invention is not limited to these.

[0147] In some embodiments, one is used to qualitatively assess the amount of insulin in a sample. Alternatively, the relative abundance of multiple ions is measured using a high-resolution / high-accuracy mass spectrometer. In the embodiment, one or more ions measured by high resolution / high accuracy mass spectrometry are multivalent insulin ions. These multivalent ions are approximately 1453±0.8 (i.e. , one or more monoisotopic peaks of the 4+ ion) and / or 1162±1 (i.e., 5+ ion) and / or 968.8±1.5 (i.e., one or more monoisotopic peaks of one or more monoisotopic peaks of the 6+ ion) It may contain multiple ions.

[0148] The results of the analyte assay can be analyzed by a number of methods known in the art to determine the amount of analyte in the original sample. For example, if sampling and analytical parameters are carefully considered, the amount of precipitate can be related to the amount of If the relative abundance of a given ion is deeply controlled, the relative abundance can be calculated by first Alternatively, an external standard can be run with the sample. A standard curve can be generated based on the ions obtained from those standards. Using a standard curve, the relative abundance of a given ion can be converted to the absolute amount of the original molecule. In certain preferred embodiments, an internal standard can be used to calculate the amount of insulin. Methods for generating and using such standard curves are well known in the art. It is well known that the internal standard can be selected by those skilled in the art. For example, in a preferred embodiment In the method, one or more forms of isotopically labeled insulin can be used as an internal standard. Many other methods for relating the amount of an ion to the amount of the original molecule are known to those skilled in the art.

[0149] As used herein, "isotopically labeled" refers to a compound that, when analyzed by mass spectrometry techniques, appears to be unlabeled. This results in a mass shift of the labeled molecule relative to the molecule. Examples of suitable labels include deuterium ( 2 H ), 13 C and 15 N. One or more isotope labels can be added to one or more molecules. One or more types of isotopic labels can be incorporated into the same isotopically labeled molecule. It can be used for.

[0150] In another embodiment, insulin is used to obtain its component chains prior to mass spectrometry analysis. can be subjected to chemical treatment. The B chain of insulin undergoes disulfide reduction. These can be separated by chemical treatments known in the art. The insulin was treated with TCEP (tris(2-carboxyethyl)phosphine) to The disulfide bridges can be reduced to separate the A and B chains.

[0151] The following examples serve to illustrate the invention. These examples are provided to illustrate the scope of the present method. It is not a restriction. [Example]

[0152] Example 1: Human insulin resistance studies Human subjects were apparently healthy, self-described non-Hispanic white individuals with no history of cardiovascular disease. All individuals provided written informed consent to participate in the study. submitted the proposal.

[0153] Subjects with fasting glucose ≥ 126 mg / dL or those taking hypoglycemic medications Subjects with a diagnosis of diabetes were excluded from the analysis.

[0154] Race and ethnicity were determined during medical history. Weight and height were measured while the individual was lightly dressed and without shoes. Body length was measured by dividing weight in kilograms by height in meters squared. The blood pressure was measured using an automatic blood pressure recorder. Before the test, subjects sat quietly in a chair for 5 minutes with both feet on the floor and arms supported at heart level. Patients were seated. Three blood pressure readings were taken at 1-minute intervals using an appropriately sized cuff and the average was calculated. Metabolic syndrome was present if three of the following characteristics were present: BMI > 30 kg / m 2; FG > 100 mg / dL; hypertension (SBP ≥ 130 mmHg or DBP ≥ 85 mmH g), low HDL-C (<50 mg / dL, female; <40 mg / dL, male), TG ≥15 0 mg / dL.

[0155] Insulin-mediated glucose disposal was measured using the insulin suppression test (IST). The insulin suppression test was used to evaluate insulin-mediated glucose disposal. One catheter was used to draw a blood sample and the other catheter Octreotide (0.27 μg / m 2 / min), insulin (32mU / m 2 / min) and glucose (267 mg / m 2 A 180-minute infusion of 100 mg / min was administered. Blood was sampled at 10-minute intervals from 150 to 180 minutes to measure steady-state plasma glucose (S Steady-state plasma insulin (SPG) and steady-state plasma insulin (SSPI) concentrations were determined. Since SSPI concentrations are similar in individuals, SSPG concentrations may affect insulin delivery and This provides a direct measure of the ability of the cell to mediate the disposal of an input glucose load. Therefore, the higher the SSPG concentration, the more insulin resistant the individual is. Insulin-mediated glucose disposal, as determined by the euglycemic-hyperinsulinemic clamp technique, was This is highly correlated with the resulting insulin-mediated glucose disposal. R is defined as falling in the top tertile of measured insulin resistance (SSPG ≥ 198 mg / dL). The serum samples used for measuring insulin and C-peptide were prepared using the IST protocol. It was derived from a fasting baseline sample obtained before the start of the call.

[0156] The analysis included 335 subjects (39% with a complete set of biochemical and anthropometric measurements) included men).

[0157] In this population, 118 of 335 subjects had metabolic syndrome.

[0158] The clinical characteristics of the classified study patient population are shown in Table 1, where subjects were insulin resistant. They are classified according to their condition. People with insulin resistance are more likely to be men, Fasting plasma glucose (FPG), insulin, C-peptide, triglycerides, araniol aminotransferase, body mass index (BMI), and systolic blood pressure HDL-C and LDL-C were higher in those with insulin resistance. It was small.

[0159] [Table 1]

[0160] Example 2: Insulin and C-peptide measurements We used SSPG to identify non-diabetic (FG < 125 mg / dL and no diagnosis of diabetes) IR was evaluated in 632 non-Hispanic white participants. IR was associated with a significant improvement in this population. The upper tertile of SSPG (≥ 201 mg / dL) was defined as

[0161] Insulin and C-peptide were assessed by multiplex tandem mass spectrometry assay.

[0162] The serum was delipidated and then insulin and C- The peptide was immunocaptured. The beads were washed and the peptide was immunocaptured using acidified acetonitrile in water. The peptide was eluted from the beads. Trizma base was added to increase the stability of the peptide. Preparation of plater, addition of internal standard, delipidation, bead deposition, immunocapture The washing and elution steps of peptides from the beads were carried out using a Hamilton STAR® The procedure was automated using a robotic liquid handler.

[0163] The elution plate was placed on a ThermoFisher TurboFlow Aria TX4 The sample was transferred to an HTLC system and injected onto a hydrophilic / lipophilic balance (HLB) trapping column. This allowed insulin and C-peptide to be further enriched from background contaminants. A solvent is used to release the peptides from the extraction cartridge and transfer them to a reversed-phase analytical column. The remaining insulin and C-peptide were purified by acetonitrile gradient chromatography. were separated from background contaminants and from each other.

[0164] The solvent flow from the HPLC column was fed into the heated electrochemical chamber of an Agilent 6490 mass spectrometer. In the mass spectrometer, only ions with the desired mass-to-charge ratio are selected. It was passed through the quadrupole 1 (Q1) region and into the collision chamber (Q2). The accelerated ions collide with neutral argon gas molecules and break down into small fragments. In this case, only the selected ions were selected to reach the detector. The peak intensities were proportional to the number of molecules that entered the mass spectrometer. A calibration curve is established by calculating for known calibrators. Then, using the calibration formula: , the concentrations of insulin and C-peptide in patient samples can be determined.

[0165] 968.7 (precursor) and 136.0, 226.1, and 345.2 (fragments) The insulin m / z values ​​were 1007.7 (precursor) and 533.3, 646.4. , and C-peptide m / z of 927.5 (fragment) were used.

[0166] Example 3: Intra- and inter-assay precision Intra-assay precision is defined as the reproducibility of measurements within an assay and is used for QCL, QCM, and QCH. The coefficient of variation of the five replicates of the sample was obtained by assaying five replicates from the same sample. The CV (conversion coefficient) was used to determine whether the reproducibility was acceptable (≤15%). Statistical analysis of the QCs revealed that the reproducibility (CV) of insulin was 6. ranged from 0.2 to 11.5% for β-peptide and 5.1 to 6.3% for C-peptide. The intra-assay precision was also calculated across all assays (Table 2). (930TP5319: Assay Validation Calculation For insulin, the intra-run CV ranged from 4.7 to 9.6%, and the C- For peptides, the intra-run CVs ranged from 4.7 to 7.0%.

[0167] Inter-assay variability is defined as the reproducibility of measurements between assays. CH was evaluated over 5 days. The inter-assay variability (%CV) of the pool was The range for C-peptide was 6.2-9.0%. All QC pools for insulin and C-peptide showed acceptable reproducibility of ≤15% CV. The requirements were met (Table 3).

[0168] Example 4: Analytical sensitivity (detection limit) Blank Limit (LOB): The LOB is the limit at which a measurement is greater than its associated uncertainty. The selectivity is the concentration of the sample in a sample, arbitrarily defined as two standard deviations (SD) from zero concentration. Selectivity is the ability of an analytical method to distinguish and quantify an analyte in the presence of other components. For this purpose, the analysis of blank samples of the appropriate biological matrix (strip serum) is obtained. The selectivity guaranteed in terms of interference and the lower limit of quantification was tested. The blank was measured 20 times. , and the resulting area ratio was back-calculated.

[0169] LOB was 0.9 μIU / mL for insulin and 0.06 n for C-peptide. was determined to be g / mL.

[0170] Limit of Detection (LOD): The LOD is the point at which the measurement is greater than its associated uncertainty. is arbitrarily defined as four standard deviations (SD) from zero concentration. Selectivity is the Selectivity is the ability of an analytical method to identify and quantify an analyte in the presence of other components. , the analysis of a blank sample of the appropriate biological matrix (stripped serum) is obtained, and interferences The selectivity guaranteed at the lower limit of quantitation was tested. The area ratio was calculated backwards.

[0171] The LODs were 1.5 μIU / mL for insulin and 0.10 nM for C-peptide. was determined to be g / mL.

[0172] Limit of Quantitation (LOQ): The LOQ is the point at which a measurement becomes quantitatively meaningful. Insulin and C-peptide responded at this LOQ, were discriminative, and separated. It is reproducible with a precision of 0% and accuracy of 80% to 120%. The LOQ is the predicted LOQ. Five samples with concentrations close to 1.25, 2.5, 5, 10, and 20 μI for insulin U / mL, C-peptide 0.11, 0.22, 0.44, 0.85, 0.17n (µg / mL) was assayed, followed by intra-assay reproducibility in 7 runs and a further 8 runs. The inter-assay reproducibility was determined by evaluating the insulin and C-peptide 2.5 μIU / mL and 0.11 ng / mL, respectively, with a 95% confidence interval of CV. This is the minimum concentration that results in acceptable performance, with the difference remaining below 20%.

[0173] The LOQ was 2.5 or 3 μIU / mL for insulin and 0.5 or 3 μIU / mL for C-peptide. It was set at 11 ng / mL.

[0174] Example 5: Analyte Measurement Range (AMR) Calibration verification: 10 spiked strip serum sample pools (calibrator concentrations The insulin levels are 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 2 40 and 320 μIU / mL, and 0.11, 0.21, 0.43, 0.85, 1.7 0, 3.40, 6.80, 13.60, 20.40 and 27.20 ng / mL) They were prepared and analyzed 18 times on 13 separate days.

[0175] Accuracy of ±20% by weighted (1 / X) quadratic regression (ignoring the origin) from 18 curves 0.989 or greater for insulin and 0.99 for C-peptide A correlation coefficient of 2 or more was obtained, and the values ​​for insulin were 5 to 320 μIU / mL and C- The peptide showed a linear range of 0.11 to 27.20 ng / mL (Table 6). .11~27.20ng / mL.

[0176] Example 6: Diagnosis of insulin resistance Anthropomorphic measurements (age, sex, SBP, DBP and BMI) and biomarkers (FG, insulin, C-peptide, H DL-C, LDL-C, TG, creatinine, and alanine aminotransferase ( ALT)) All were available. Among these 335 participants, we found FG, IN urin, C-peptide, HDL-C, TG, and BMI (all P<0.0001), A LT (P = 0.002) and SBP (P = 0.008) were significantly higher after adjusting for age and sex. We found that the IR was related to the model selected. Using this method, we found that an IR model including only insulin, C-peptide, and BMI yielded an AUC When model selection was restricted to biomarkers, the index was found to have a correlation coefficient of 0.89. The model including only phospholipid and c-peptide had an AUC of 0.88. In this study of non-Hispanic whites with diabetes, fasting serum insulin and C-peptide Both peptide concentrations were associated with measures of IR and, when combined, provided a meaningful indication of the prevalence of IR. This provided highly accurate information.

[0177] Differences in traditional risk factors between those with and without IR are shown for discrete variables. The Wilcoxon rank sum test was used for evaluation, and the chi-square test was used for continuous variables. The association between phosphorus and C-peptide and IR was evaluated using age, sex, SBP, DBP, BMI, F Logistic regression analysis adjusted for G, HDL-C, LDL-C, TG, creatinine, and ALT. Risk score 1 was calculated using a logistic regression model. Risk score 2 included insulin and C-peptide. All probability values ​​are two-sided, with 95% confidence intervals (CIs) shown. All analyses were performed using SAS The experiment was carried out using version 9.2.

[0178] The associations between insulin resistance and biochemical and anthropometric measures are shown in Table 2 After adjusting for age, sex, and ethnicity, all but creatinine were associated with insulin resistance. However, the model included all biochemical and anthropometric measures. When included in the analysis, only insulin, C-peptide, creatinine, and BMI were associated with insulin resistance. was associated with resistance.

[0179] [Table 2]

[0180] [Table 3]

[0181] Insulin, C-peptide, creatinine, and BMI were the most important variables in our modeling. These were also important variables, so we combined them into a single risk score. (Model 1). This analysis revealed that using this method, age, sex, ethnicity, and fasting plasma Glucose, LDL-C, HDL-C, triglycerides, alanine aminotransferase In models adjusted for blood pressure, systolic, and diastolic blood pressure, this risk score Individuals in the top quartile of the serotonin test were more likely to be insulin resistant than those outside the top quartile. demonstrated that patients were >15 times more likely to have HIV (OR=15.1, 95% CI 8.7 ~26.3) (Table 3).

[0182] Recognizing that incorporating clinical variables into laboratory diagnosis can present practical challenges, we: Insulin, C-peptide, and creatinine only (Model 2), or insulin and C We also tested the performance of a risk score that included only insulin and C-peptides (Model 3). In the risk score incorporating peptide and creatinine (Model 2), the top For those in the quartile, the odds of being in the IR are in the top quartile for this risk score. The ratio for participants versus non-participants decreased slightly to 13.6 (95% CI 7.9 to 23.6). Finally, using only insulin and C-peptide results (Model 3), this risk Those in the top quartile of scores had higher odds of being insulin resistant than those outside the top quartile was nine times more likely (OR=9.9, 95% CI 5.8-17.0).

[0183] Metabolic syndrome has long been strongly associated with insulin resistance and the risk of future type 2 diabetes. In this study population, we compared age, sex, ethnicity, LDL cholesterol, and -C, creatinine, alanine aminotransferase, systolic and diastolic blood pressure found that metabolic syndrome was associated with insulin resistance even in models adjusted for In contrast, insulin and C-peptide After further adjustment for insulin resistance, metabolic syndrome was not associated with insulin resistance. (OR = 1.1, 95% CI 0.6-1.9) (Table 1). All three risk scores are associated with insulin resistance, regardless of whether the syndrome is present or not. (Table 4).

[0184] [Table 4]

[0185] Information from these models can be used to define the probability that an individual is insulin resistant. Table 5 shows the results of 1) C-peptide and insulin, 2) C-peptide and insulin , and creatinine, BMI, and 3) C-peptide, insulin, creatinine, and Individuals at various percentiles of three different risk scores, including either ≥100 or ≥100% BMI, were assessed. Although there are small differences between the models, most of the information is It is clear that the effects of C-peptide and insulin are included in the model.

[0186] [Table 5]

[0187] Figure 1 shows the relationship between insulin and C-peptide levels in individuals with and without metabolic syndrome. These findings suggest that thiazides are associated with insulin resistance (IR).

[0188] We investigated the effects of GCRCs on the survival of a multiethnic cohort of patients studied in GCRC settings over a 12-year period. Insulin resistance levels derived from formal measurements of insulin resistance using SSPG The ability of clinical parameters and laboratory results to predict risk was examined. Consistent with this, even after adjusting for age, sex, and ethnicity, FPG, insulin, and C-pe Protein, HDL-C, LDL-C, triglycerides, alanine aminotransferase A wide range of clinical parameters, including blood pressure, body mass index (BMI), and It was observed that there were associations with formal measures of insulin resistance. Most of the data remained non-significant after adjusting for insulin and C-peptide results. These are themselves a reflection of the underlying state of insulin resistance, which The inclusion of phosphorus and C-peptide levels accounts for almost the entire adjustment of the model. It was suggested that when the model included insulin and C-peptide measurements, BMI and Only creatinine and creatinine remained marginally significant.

[0189] A surprising finding from this study was that both insulin and C-peptide measurements revealed SSPG. contributes significantly to the ability to accurately predict insulin resistance levels measured using It was an observation.

[0190] In this study, we used a multiplex assay to quantify high-throughput liquid chromatography proteins. Intact insulin and C-peptide were measured using NMR mass spectrometry. This allows for the definition of specific thresholds that persist over time.

[0191] There are many methods available that can convey information about the estimated level of insulin resistance. However, we believe one of the most useful ways to represent this data is to determine whether an individual has insulin resistance. The goal is to provide a probability of having a particular threshold of resistance. We define this as insulin, defined here as SSPG ≥ 198 mg% (upper tertile). The prediction tool was based on creatinine and BMI, and was expressed as a probability of resistance. Although insulin and C-peptide account for the majority of the information.

[0192] In summary, we demonstrate that a model incorporating fasting and C-peptide measurements can predict SSPG. The results showed that the formal measurement of insulin resistance levels used can be predicted with good accuracy. The model is simple and does not depend on clinical parameters or other laboratory values. A strength of this study is that it is valid regardless of whether or not a medical device is available. The authors argue that such risk scores may be used to assess an individual's insulin level, regardless of whether clinical signs are present. These findings suggest that the use of methicillin-resistant strains in the treatment of HIV-1 infection may be useful in assessing the level of resistance to methicillin. The measurements may be used to assess whether lifestyle or pharmacological interventions to reduce insulin resistance are effective. This could be beneficial for longitudinal assessment of subjects receiving treatment, an assessment that is currently difficult outside of a research setting. This also suggests that...

[0193] Insulin and C-peptide are key regulators of traditional risk factors, including each other and fasting glucose. It is associated with insulin resistance independently of other factors.

[0194] Insulin and C-peptide in people with and without metabolic syndrome , the probability of insulin resistance formally assessed using the SSPG method can be assessed. .

[0195] C-peptide and insulin (standardized, traceable C-peptide and insulin The risk score, which combines the measured values, tells patients whether they have insulin resistance. can be derived and used to provide the probability that

[0196] A high-throughput mass spectrometry assay for simultaneous quantification of intact insulin and C-peptide concentrations This technique will serve as a reference point and final standard for the standardization of insulin and C-peptide measurements. This will serve as a universally accepted quantitative method for the identification of insulin resistance. In the current study, intact insulin and C-peptide were measured using this method. Insulin-mediated glucose disposal in apparently healthy, non-diabetic individuals The usefulness of these standardized measures for assessing insulin resistance measured by In the current analysis, we evaluated the initial cohort (self-identified) for which all measurements were available. This methodology is applied to individuals with a high prevalence of HIV / AIDS (non-Hispanic white individuals).

[0197] Example 6: Intact insulin and C-peptide levels measured by multiplex mass spectrometry Bell Elevated insulin levels have been shown to be associated with an increased risk of developing diabetes. Although clinical trials for insulin and C-peptide have been available for decades, This is due, at least in part, to the wide range of immunoassays available and the Do not compare the results obtained from the Platform with those obtained from other platforms. Because of the difficulty in correlating insulin measurements, insulin measurements are not widely used in clinical practice. This drawback has been noted in the literature for both insulin and C-peptide. Therefore, we measure both intact insulin and C-peptide. We developed a multiplexed mass spectrometry-based assay. Insulin, C-peptide, and glucose levels in fasting serum samples from the study subjects The relationship between them was examined.

[0198] Apparently healthy subjects provided informed consent (WIRB#201219 40) Fasting venous blood samples were obtained. Glucose levels were measured using an Olympus AU270 0™ chemistry-immunoanalyzer (Melville, NY) and insulin and and C-peptide levels were determined by a multiplex mass spectrometry assay. phic) measurements were obtained at the time of blood collection.

[0199] The study involved 103 apparently healthy volunteers (46.7% male, median age = 35, median BMI = 26.1). The value was 8.07 μIU / ml (IQR 5.38-12.55), which was observed in 19.4% of subjects. Insulin was elevated (≥15 μIU / ml). Among those with a serum creatinine level between 90 and <100 mg / dL, the incidence rates were 50% and 40%, respectively. Those with elevated thoracic acid levels and, in contrast, fasting glucose <90 mg / dL Only 9.6% of patients had elevated insulin levels. The median insulin level was In 10 subjects with impaired fasting blood glucose, the mean blood glucose level was 14.78 μIU / ml (IQR 6.44-42%). .29), and 9.79 in 20 subjects with fasting glucose 90 to <100 mg / dL. μIU / ml (8.43-17.70), versus those with fasting glucose <90 mg / dL In 73 subjects, the median was 7.26 μIU / ml (4.49-9.47). P = 0.0004). Insulin levels in those with a BMI > 26 (median = 9.17 μIU / ml, IQR 6.96-17.33) than those with a BMI of 26 or less. was significantly higher (median = 6.92 μIU / ml, IQR 4.08-9.09; P = 0.00 03). Insulin and C-peptide levels were highly correlated (r=0.88).

[0200] Clinical sample collection and preparation Blood was obtained from apparently healthy adult volunteers (WIRB protocol #1085 473). Anthropomorphic measurements were taken at the time of sampling. No barriers were included. Blood was collected using serum preparation tubes (red top) and allowed to clot. The obtained serum was immediately The samples were processed into a 500-ml tube and then stored at -80°C until analysis. The clonal antibody was used to measure the concentration of insulin and C-peptide from patient serum (150 μL). The samples were transferred to a robotic liquid handler (Microlab STAR, Hamidi). The samples were processed at a facility in Reno, NV.

[0201] Assay TurboFlow Aria TLX-4 (Thermo-Fisher, San Jose, CA) using a fully automated online two-dimensional liquid chromatography system. , to separate intact insulin and C-peptide from remaining matrix components prior to MS. Analytical separation of 6490 Triple Quadr with iFunnel was achieved. Upole Mass Spectrometer (Agilent, Santa Cl A chromatograph (Ira, CA) served as the MS / MS detector. The description was given previously 9 Glucose levels were measured using an Olympus AU2700™ Determined using a chemi-immunoanalyzer (Melville, NY).

[0202] statistical analysis Low (<90 mg / dL), medium (90 to <100 mg / dL) and high (100 to 125 mg / dL) The difference in insulin levels between participants with fasting glucose levels (g / dL) was calculated using the parameter Parametric (ANOVA) and nonparametric (Kruksal-Wallis) tests Insulin levels in patients with low (<26) and high (≥26) BMI were assessed. Differences in insulin levels were assessed using unpaired t-tests. The results were evaluated in multivariate regression models adjusted for age, sex, and fasting glucose level. It was worth it.

[0203] Median insulin levels were 0.01 in 73 subjects with fasting glucose <90 mg / dL. was 7.26 μIU / ml (4.49-9.47), and fasting glucose was 90-<100 mg. / dL, 9.79 μIU / ml (8.43-17.70), fasting In 10 subjects with dysglycemia, the mean blood glucose level was 14.78 μIU / ml (IQR 6.44-42.29). ) (Figure 2).

[0204] Insulin levels in those with BMI > 26 (median = 9.17 μIU / ml, The mean age at onset was 6 years (IQR 6.96-17.33) compared with those with a BMI ≤ 26 (median = 6 .92μIU / ml, IQR4.08~9.09; P=0.0003) (Figure 3).

[0205] Insulin and C-peptide levels were found to increase in response to fasting glucose. (Figures 4 and 5).

[0206] In multivariate regression models, after adjusting for age, sex, and fasting glucose, BM I was associated with fasting insulin (P = 0.00002). fasting insulin was associated with an increase of 0.59 μIU (95% CI 0.33 ~0.85) (Figure 6).

[0207] Consideration This study used standardized S Traceable assay for measuring intact insulin and C-peptide We demonstrate the application of a multiplexed mass spectrometry-based assay for C-peptide. The amount of ATP was carefully quantified using calibrators determined by quantitative amino acid analysis. These findings suggest that (1) the incidence of insulin resistance in a significant number of individuals with fasting blood glucose levels within the normal range is high; (2) elevated glucose levels within the normal range It shows that the proportion showing an increase in insulin gradually increases.

[0208] conclusion We used a multiplex intact insulin and C-peptide assay to measure both analytes. A standard range was defined for

[0209] Individuals with normal fasting glucose, normal hemoglobin A1C, and BMI < 26 were used. Therefore, if we look at the definition of normal ranges, we see that for insulin, <16 μIU / m The normal range for L and C-peptides is 0.68 to 2.16 ng / ml.

[0210] Fasting levels of insulin and C-peptide increase as fasting glucose increases. It gradually increased.

[0211] Fasting insulin levels were strongly influenced by BMI.

[0212] Using well-characterized assessments of insulin resistance, fasting insulin in individuals urin and C-peptide, as well as fasting glucose and anthropomorphic measurements This tool allows for easy assessment of insulin sensitivity levels by defining the relationship between insulin sensitivity and insulin resistance. It is possible to define rules.

[0213] Example 7: Measuring insulin and C-peptide levels by mass spectrometry Identifying insulin resistance in apparently healthy individuals We measured insulin, C-peptide, TG / HDL ratio, creatinine, and BMI. The risk scores for insulin resistance in both individuals with and without metabolic syndrome were The authors determined that this could help identify individuals with certain sexes.

[0214] All study participants were apparently healthy and had no history of cardiovascular disease. Individuals with a blood glucose ≥ 126 mg / dL at baseline or taking hypoglycemic medications Individuals with this condition were excluded from the analysis.

[0215] Race and ethnicity were determined during medical history. Weight and height were measured while the individual was lightly dressed and without shoes. Body length was measured by dividing weight in kilograms by height in meters squared. The blood pressure was measured using an automatic blood pressure recorder. Before the test, subjects sat quietly in a chair for 5 minutes with both feet on the floor and arms supported at heart level. Patients were seated. Three blood pressure readings were taken at 1-minute intervals using an appropriately sized cuff and the average was calculated. Metabolic syndrome was present if three of the following characteristics were present: BMI > 30 kg / m 2; FG > 100 mg / dL; hypertension (SBP ≥ 130 mmHg or DBP ≥ 85 mmH g), low HDL-C (<50 mg / dL, female; <40 mg / dL, male), TG ≥15 0 mg / dL.

[0216] After an overnight fast, intravenous catheters were placed in each arm. One catheter was used to collect blood samples. The other catheter was used to administer octreotide (0.27 μg / m 2 / min), Insulin (32 mU / m 2 / min) and glucose (267 mg / m 2 / min) for 180 minutes Blood samples were taken at 10-minute intervals from 150 to 180 minutes after the infusion to determine the steady-state blood flow. Determine steady-state plasma glucose (SSPG) and steady-state plasma insulin (SSPI) concentrations Since SSPI concentrations were similar in all individuals during IST, the SSPG concentrations Thus, a direct measure of insulin's ability to mediate the disposal of an infused glucose load is Therefore, the higher the SSPG concentration, the more insulin resistant the individual is. Insulin-mediated glucose disposal determined by IST is highly resistant to euglycemic hyperinsulinemia. This is highly correlated with glucose disposal obtained using the insulin clamp technique. IR was defined as the upper tertile of measured insulin resistance (SSPG ≥ 198 mg / dL). The serum samples used for insulin and C-peptide measurements were These were derived from fasting baseline samples obtained before the start of the protocol. - Measurement of peptides was carried out as described herein.

[0217] [Table 6]

[0218] Differences in biochemical and anthropometric measurements between individuals with and without IR were assessed as continuous variables. Wilcoxon rank sum test or t-test for statistical significance, and chi-square test for discrete variables The associations between study variables and IR were assessed using logistic regression models adjusted for the covariates shown in the table. The SSPG distribution histogram was evaluated in non-Hispanic whites. (n=335), Hispanic (n=42), and Other (n=158) Therefore, ethnicity was coded as a categorical variable for these three groups. Core components were selected in stepwise regression. All available variables were included in the model. After optimal fitting, the significance selection cutoff was specified. Add available variables that satisfy the condition to the model, and then add all candidate variables in the model. If the significance of the results increases beyond a specified exclusion level, the results are removed. until no variables are added or removed for the inclusion and exclusion cutoffs given in the text. Repeat with the following: Risk score coefficients are calculated based on age, sex, ethnicity, insulin, C-peptide, and creatine. Alanine, BMI, TG / HDLC, FG, SBP, DBP, LDL-C, and alanine Aminotransferases, except for variables that are part of the risk score, were not investigated. All p-values ​​are two-sided and 95% confidence intervals are shown. All analyses were performed using SAS version 9.2.

[0219] Biochemical and anthropometric measurements of study participants according to insulin resistance status are shown in Table 1 Participants with insulin resistance had higher levels of FG, insulin, and C-peptide. The subjects had blood cholesterol, HDL-C, TG, TG / HDL, AAT, BMI, and SBP. Participants with insulin resistance had lower levels of HDL-C and LDL-C.

[0220] We compared age, sex, ethnicity, FG, insulin, C-peptide, LDL-C, TG / H The study was conducted while adjusting for DL, creatinine, AAT, BMI, and SBP and DBP. The associations between the study variables and insulin resistance were examined (Table 2). The analysis showed that insulin, C-peptide, creatinine, BMI, and TG / HDL were significantly correlated with the HR. was associated with insulin resistance, regardless of levels of C-peptide and other study variables. For every 10 pmol / L increase in insulin, there is an increased risk of insulin resistance (SSPG ≥ 198 The odds of having a blood glucose level of 1.2 mg / dL were 1.2 times higher (95% CI 1.1-1.4). For every 100 pmol / L increase in C-peptide, the odds of being insulin resistant was 1.6 times higher (95% CI 1.3-2.0).

[0221] Since insulin and C-peptide are highly correlated (r=0.85), we The prevalence of IR by tertile of insulin and C-peptide was examined (Figure 18). In the thrombolytic tertiles, the fraction of IR individuals increased according to C-peptide tertile level. Conversely, for each C-peptide tertile, the fraction of IR individuals increased with insulin tertile level. Great.

[0222] [Table 7]

[0223] [Table 8]

[0224] We used a stepwise model selection procedure to select the variables in Table 2 as well as age and gender. We then identified variables for the IR risk model. Using P<0.001 as the criterion for not being removed from the blood, insulin, C-peptide, and creatinine (in that order). The top four risk scores including these variables For individuals in the top quartile (vs. individuals outside the top quartile), the unadjusted average for IR was The risk ratio was 18.7 (95% CI 11.4-30.7, Table 3). After adjusting for variables not included, the odds ratio was 10.8 (95% CI 6 .2-19.0). Insulin and C-peptide can be measured in a single multiplex test. , we also tested a model including only insulin and C-peptide, unadjusted for IR. We found that the odds ratio for the variable was 12.8 (95% CI 8.0-20.4). Using a more lenient selection criterion (P<0.05) for inclusion and non-exclusion, 5 Four variables were included in the final model: insulin, C-peptide, creatinine, BMI, and TG / HDL (in that order). Those in the top quartile of risk scores that include these variables For individuals, the unadjusted odds ratio for IR was 20.0 (95% CI 12.1 ~33.0). After adjusting for variables not included in the risk score, the odds ratio was 16. The mean age at onset was 0.1 (95% CI 9.5-27.3).

[0225] We also investigated HOMA-IR, a commonly used method to estimate insulin resistance. Tested and in those in the top quartile of HOMA-IR (vs. those not in the top quartile) , with an unadjusted odds ratio for IR of 10.3 (95% CI 6.6-16.1). After adjusting for variables not included in the risk score, the odds ratio was The mean age at onset was 1.5 (95% CI 0.8-2.7).

[0226] Even when the study population was limited to individuals with metabolic syndrome, these risk scores were not associated with IR. remained unchanged after adjusting each score for variables not included in that score. For insulin and C-peptide scores, the OR was 9.1 (95% CI 4.2-19.5), and For phosphorus, C-peptide, and creatinine scores, OR = 13.3 (95% CI 5.9 ~30.1), insulin, C-peptide, creatinine, BMI, and TG / HDL levels For core, OR = 13.7 (95% CI 6.3–29.9).

[0227] The clinical characteristics of the classified study patient population are shown in Table 1, where subjects were insulin resistant. They are classified according to their condition. People with insulin resistance are more likely to be men, Fasting plasma glucose (FPG), insulin, C-peptide, triglycerides, araniol aminotransferase, body mass index (BMI), and systolic blood pressure HDL-C and LDL-C were higher in those with insulin resistance. It was small.

[0228] We also used these risk scores to estimate the probability that an individual had IR (Table 4). Therefore, a single cut point (e.g., whether a patient falls in the top quartile of the risk score) Rather than assigning a likelihood of IR based on whether For the three risk scores in Tables 3 and 4, the probability of having an IR can be calculated. For each, we calculate the probability of IR from the measurements of the risk score components. We provide a formula that can be used for

[0229] The associations between insulin resistance and biochemical and anthropometric measures are shown in Table 2 After adjusting for age, sex, and ethnicity, all but creatinine were associated with insulin resistance. However, the model included all biochemical and anthropometric measures. When included in the analysis, only insulin, C-peptide, creatinine, and BMI were associated with insulin resistance. was associated with resistance.

[0230] Insulin, C-peptide, creatinine, and BMI were the most important variables in our modeling. These were also important variables, so we combined them into a single risk score. (Model 1). This analysis revealed that using this method, age, sex, ethnicity, and fasting plasma Glucose, LDL-C, HDL-C, triglycerides, alanine aminotransferase In models adjusted for blood pressure, systolic, and diastolic blood pressure, this risk score Individuals in the top quartile of the serotonin test were more likely to be insulin resistant than those outside the top quartile. demonstrated that patients were >15 times more likely to have HIV (OR=15.1, 95% CI 8.7 ~26.3) (Table 3).

[0231] Recognizing that incorporating clinical variables into laboratory diagnosis can present practical challenges, we: Insulin, C-peptide, and creatinine only (Model 2), or insulin and C We also tested the performance of a risk score that included only insulin and C-peptides (Model 3). In the risk score incorporating peptide and creatinine (Model 2), the top For those in the quartile, the odds of being in the IR are in the top quartile for this risk score. The ratio for participants versus non-participants decreased slightly to 13.6 (95% CI 7.9 to 23.6). Finally, using only insulin and C-peptide results (Model 3), this risk Those in the top quartile of scores had higher odds of being insulin resistant than those outside the top quartile was nine times more likely (OR=9.9, 95% CI 5.8-17.0).

[0232] In this study population, we compared age, sex, ethnicity, LDL-C, creatinine, and alanine Models adjusted for steroid aminotransferase, systolic and diastolic blood pressure also showed We found that diabetes syndrome was associated with insulin resistance (OR = 3.7, 95% confidence interval [CI]). In contrast, further adjustments for insulin and C-peptide Later, metabolic syndrome was not associated with insulin resistance (OR = 1.1, 95% confidence interval [CI]). I 0.6–1.9) (Table 1). Notably, regardless of whether metabolic syndrome was present or not, All three risk scores were associated with insulin resistance (Table 4). The probability that an individual is insulin resistant at various percentiles is shown.

[0233] [Table 9]

[0234] Risk score calculation and probability of IR Risk score 1: insulin (pmol / L), C-peptide (pmol / L), creatine Atinine (mg / dL) (1.1)RS = (insulin × 0.0265) + (C-peptide × 0.00511) +(creatinine x -3.2641)

[0235]

number

[0236]

number

[0237]

number

[0238] Surprisingly, the findings of this study show that the measurement of both insulin and C-peptide contributes significantly to the ability to accurately predict insulin resistance levels measured using SSPG This was the observation.

[0239] We believe that a model incorporating fasting and C-peptide measurements is useful for instability using SSPG. demonstrated that formal measurements of phosphorus resistance levels can be predicted with good accuracy. .

[0240] Top quartile risk score including insulin, C-peptide, creatinine, and BMI Those who placed in the top quartile were more likely to be IR compared to those who did not place in the top quartile ( OR=15.1, 95% CI 8.7-26.3). This association was also observed in patients with metabolic syndrome. those with metabolic syndrome (OR=17.7, 95% CI 7.8-40.5) and those without metabolic syndrome (OR=16.9 95%CI 7.3-39.2) was observed in both groups.

[0241] All articles, patents and patent applications, and all other literature and electronic publications mentioned or cited herein. The content of publicly available information is subject to the exclusion of all representations, warranties, and conditions, unless each individual publication is specifically and individually incorporated by reference. No. 6,399,423, filed on Oct. 1, 2003, and incorporated herein by reference in its entirety to the same extent as if set forth herein. Applicants reserve the right to withdraw any and all copies of any such articles, patents, patent applications, or other physical and electronic documents. The right to physically incorporate into this application any and all materials and information from any and all sources is reserved.

[0242] The methods illustratively described herein may not include any element or components not specifically disclosed herein. may be suitably practiced in the absence of any element, limitation, or limitation. Thus, for example, the terms "comprising," "including," ) and "containing" are read broadly and without limitation. Further, the terms and phrases used herein are to be regarded as terms of description and not of limitation. and in the use of such terms and expressions This does not exclude any equivalents or portions of the features described herein that fall within the scope of the invention as claimed. It is recognized that various modifications are possible in the present invention. Although specifically disclosed by embodiments and optional features, the present disclosure and Variations and modifications of the invention embodied in the present invention may be used by those skilled in the art, and such variations may be made. It is to be understood that all such variations and modifications are considered to be within the scope of the present invention.

[0243] The invention has been described broadly and generically herein. Each of the narrower species and subspecies also forms part of the method. This includes the selection of any pair from a class. A general description of the method with conditions or negative limitations for removing material is provided to clarify whether the removed material is in accordance with the present invention. This includes whether or not it is specifically listed in the schedule.

[0244] Other embodiments are within the scope of the following claims. is described by a Markush group, one skilled in the art will understand that the present invention also It is understood that the term "group" may be described by any individual member or subgroup of members of the group. It will be.

Claims

1. 1. A method for measuring insulin resistance in a diabetic or prediabetic patient, comprising: (a) providing one or more insulin and C-peptide ions detectable by mass spectrometry; Insulin and C-peptide from the sample are introduced into the ionization source under conditions suitable for generating The steps to take, (b) determining the amount of one or more insulin and C-peptide ions by mass spectrometry; Steps and A method comprising:

2. 10. The method of claim 1, further comprising measuring creatinine levels.

3. 10. The method of claim 1, further comprising measuring a body mass index (BMI). How to do it.

4. 10. The method of claim 1, further comprising measuring triglyceride (TG) levels. Law.

5. The method further comprises measuring high density lipoprotein C (HDL-C) levels. Item 1. The method according to item 1.

6. The method of claim 1 further comprising measuring BMI, TG, and HDL-C levels. How to post.

7. The method of claim 1 , which results in an insulin resistance score.

8. The method of claim 1, wherein the method results in the probability of developing insulin resistance.

9. 10. The method of claim 1, wherein the biological sample comprises a plasma or serum sample.

10. 10. The method of claim 1, wherein the ionization source is an electrospray (ESI) ionization source. method.

11. The method of claim 1 , wherein the sample is subjected to acidic conditions prior to mass spectrometry.

12. 10. The method of claim 9, wherein the step of subjecting the sample to acidic conditions comprises exposing the sample to formic acid. Item 12. The method according to item 11.

13. The method of claim 1 , wherein the sample is subjected to basic conditions prior to mass spectrometry.

14. The step of placing the sample under basic conditions comprises treating the sample with Trizma and / or ethanol. The method of claim 13 , comprising exposing to

15. The one or more ions have a mass-to-charge ratio (m / z) of 968.9±0.

5.

2. The method of claim 1, comprising administering to said patient a steroid hormone precursor ion.

16. The one or more ions are 136.0±0.5, 226.1±0.5 and 345. one or more insulins selected from the group consisting of ions with m / z of 2±0.5 The method of claim 1 , comprising fragment ions.

17. the one or more ions have a mass-to-charge ratio (m / z) of 1007.7±0.5 The method of claim 1, comprising a C-peptide precursor ion.

18. The one or more ions are 533.3±0.5, 646.4±0.5 and 927. one or more C-peptides selected from the group consisting of ions having an m / z of 5±0.5; The method of claim 1 , wherein the fragment ions are fragment ions.

19. 10. The method of claim 1, wherein the sample is delipidated prior to quantification by mass spectrometry.

20. The method of claim 1 , further comprising purifying the sample prior to mass spectrometry.

21. The step of purifying comprises subjecting the sample to liquid chromatography.

21. The method of claim 20.

22. The liquid chromatography is performed using high performance liquid chromatography (HPLC) or high turbulence liquid chromatography.

22. The method of claim 21, comprising chromatographic (HTLC).

23. 2. The method of claim 1 , wherein the purifying step comprises subjecting the sample to solid phase extraction (SPE).

20. The method according to claim 20.

24. The mass spectrometry is tandem mass spectrometry, high-resolution mass spectrometry, or high-resolution / high-precision mass spectrometry. The method of claim 1 .

25. The method of claim 1 , wherein the ionization is electrospray ionization (ESI).

26. The method of claim 1 , wherein the ionization is in positive ion mode.

27. 10. The method of claim 1, wherein internal standards for insulin and C-peptide are added to the sample. 。

28. 28. The method of claim 27, wherein the internal standard for insulin is bovine insulin.

29. The bovine insulin is prepared by reacting with a precursor ion having a mass-to-charge ratio (m / z) of 956.8±0.

5. and m / z values ​​of 136.0±0.5, 226.1±0.5, and 315.2±0.

5.

29. The method of claim 28, wherein the fragment ions are selected from the group consisting of ions Law.

30. 28. The method of claim 27, wherein the internal standard for C-peptide is a C-peptide heavy internal standard. method.

31. The C-peptide heavy internal standard has a mass-to-charge ratio (m / z) of 1009.5±0.

5. Precursor ions with 540.3±0.5, 653.4±0.5 and 934.5±0.

5. The method of claim 1, further comprising:

30. The method according to claim 30.

32. The determined amount of one or more ions is used to determine the amount of insulin and C- The method of claim 1, wherein the amount of a peptide is determined.

33. The amounts of insulin and C-peptide in the sample are used to determine the ratio of insulin to C-peptide.

33. The method of claim 32, wherein a ratio is determined.

34. The sample is subjected to a concentration step to obtain a fraction enriched in insulin and C-peptide. The method of claim 1 further comprising:

35. 35. The method of claim 34, wherein the enrichment step comprises immunocapture of insulin and C-peptide. method.

36. the immunocapture comprises using an anti-insulin antibody and an anti-C-peptide antibody; 36. The method of claim 35.

37. 37. The method of claim 36, wherein the antibody is a monoclonal antibody.

38. 38. The method of claim 37, wherein the antibody is an IgG.

39. The anti-insulin antibody and the anti-C-peptide antibody are immobilized on magnetic beads.

37. The method of claim 36.

40. Washing and eluting immunocaptured insulin and C-peptide on magnetic beads. Item 36. The method according to item 35.

41. The mass spectrometry is tandem mass spectrometry, high-resolution mass spectrometry, or high-resolution / high-precision mass spectrometry. The method of claim 35.

42. C-peptide levels of 2.4 ng / mL or higher diagnose insulin resistance. Item 1. The method according to item 1.

43. Insulin resistance is diagnosed when insulin levels are 15 μIU / mL or greater.

1. The method according to claim 1.

44. C-peptide level is 2.4 ng / mL or higher and insulin level is 15 μIU / mL or higher.

2. The method of claim 1, wherein insulin resistance is diagnosed if

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