Mass spectrometric determination of insulins and their degradation products in sports drug testing

Thevis, Mario; Thomas, Andreas; Schänzer, Wilhelm

doi:10.1002/mas.20154

Cited by 96 publications

(60 citation statements)

References 93 publications

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“…Multiplexed immunoaffinity separations prior to LC-MS/ MS have been reported to detect myocardial infarction (Kiernan, Nedelkov, & Nelson, 2006b). The combination of affinity chromatography with top-down analysis provided unequivocal identification of synthetic insulin analogs and metabolic products derived from human insulin and its artificial counterparts in plasma (Thevis, Thomas, & Schanzer, 2008). A novel affinity purification of human ceruloplasmin was achieved with an acharan sulfate stationary phase .…”

Section: Affinity Chromatographymentioning

confidence: 99%

Mass spectrometry of peptides and proteins from human blood

Zhu

Bowden

Zhang

et al. 2010

Mass Spectrometry Reviews

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It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post-translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post-translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ∼1 ng/mL.

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Section: Affinity Chromatographymentioning

confidence: 99%

Mass spectrometry of peptides and proteins from human blood

Zhu

Bowden

Zhang

et al. 2010

Mass Spectrometry Reviews

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“…Five determinations were performed over a period of 6 months with a CV of 3%. QC controls (8,16,40, and 80 IU/mL, or 48 -480 pmol/L) were made by spiking Human HypoOpticlear stripped serum to the target concentrations and then storing them in aliquots at Ϫ80°C. The newly made insulin QC controls and calibrators were assessed by comparison to NIBSC reference material.…”

Section: Standardsmentioning

confidence: 99%

“…The insulin A (molecular weight, 2377 Da) and B chains (molecular weight, 3431 Da) can be separated using suitable reducing agents. The insulin B chain has been studied extensively; however, a quantitative assay for the peptide has not been developed (12,16,17 ). Our aim was to develop an assay for quantification of total insulin concentrations in human serum via quantifying the B chain after reduction and liberation from the intact molecule.…”

mentioning

confidence: 99%

Quantitative Insulin Analysis Using Liquid Chromatography–Tandem Mass Spectrometry in a High-Throughput Clinical Laboratory

et al. 2013

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BACKGROUND Circulating insulin concentrations reflect the amount of endogenous insulin produced by the pancreas and can be monitored to check for insulin resistance. Insulin is commonly measured using immunochemiluminometric assays (ICMA). Unfortunately, differing crossreactivities of the various ICMA antibodies have led to variability in assay results. In contrast, liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based approaches can provide a highly specific alternative to immunoassays. METHODS Insulin was extracted from patient serum and reduced to liberate the insulin B chain. Subsequent resolution of the peptide was achieved by LC coupled to triple-quadrupole MS. Selected-reaction monitoring of B-chain transitions was used for quantification. Recombinant human insulin was used as a calibrator and was compared against the National Institute for Biological Standards and Control (NIBSC) reference standard. Bovine insulin and a stable isotopic-labeled (13C/15N) human insulin B chain were used and compared as internal standards. RESULTS The LC-MS/MS assay described herein has been validated according to CLIA guidelines with a limit of detection of 1.8 μIU/mL (10.8 pmol/L) and a limit of quantitation of 3 μIU/mL (18.0 pmol/L). A correlation between the LC-MS/MS assay and a US Food and Drug Administration-approved ICMA was completed for patient samples and the resulting Deming regression revealed good agreement. A reference interval for the assay was established. CONCLUSIONS A simple, high-throughput, quantitative LC-MS/MS insulin assay traceable to the NIBSC standard has been successfully developed and validated.

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“…These methods are immunoassay [15][16][17][18], high performance liquid chromatography [19][20][21][22][23][24][25], and mass spectrometry [26][27][28][29][30]. According to the pharmacopeial method for the determination of insulin lispro, the mobile phase is constituted by phosphate buffer solution 0.1mol/L (pH 2.3) and acetonitrile.…”

Section: Introductionmentioning

confidence: 99%

Development of a New HPLC Method for the Determination of Lispro and Glargine Insulin Analogues in Pharmaceutical Preparations

Moreno¹,

Lucchesi²,

Dib³

et al. 2018

JAPLR

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A rapid, accurate and sensitive HPLC method has been developed and validated for the quantitative determination of lispro and glargine insulin analogues in pharmaceutical preparations. Pharmacopeial method for the determination of insulin lispro employed mobile phase constituted by phosphate buffer solution 0.1mol/L (pH 2.3) and acetonitrile (74 + 26, v/v) and flow rate of 0.8mL/ min. Retention time was estimated to be 25 minutes and the temperature of the column maintained to 40ºC. No pharmacopeial method was found to the determination of insulin glargine. For this reason, the aim of this study was to develop and validate a new chromatographic method for the determination of lispro and glargine insulin analogues in pharmaceutical preparations. Solutions were prepared using the mobile phase (methanol-water, 70:30) as solvent and filtered through a 0.2 µm membrane. Aliquots of 20µL were injected into the HPLC. The method validation parameters yielded good results and included range, linearity, precision, accuracy, specificity, and recovery. The calibration curves for lispro and glargine insulin analogues were linear from 0.1 to 3.5UI/ mL, with correlation coefficients of 0.9990 and 0.9995, respectively. The interday and intraday precisions (relative standard deviation) were less than 1%. The accuracy was studied and the recovery test indicated mean absolute of 100.99% and 98.76% for lispro and glargine insulin analogues, respectively. The results obtained by HPLC method were calculated by analysis of variance (ANOVA). We concluded that the HPLC method proposed is satisfactory for the quantification of lispro and glargine insulin analogues in pharmaceutical preparations. Keywords: Development of a New HPLC Method for the Determination of Lispro and Glargine Insulin Analogues in Pharmaceutical Preparations 30Copyright: ©2018 Moreno et al.the sample or the stationary phase by means of ion pairing. It should be noted that a change in buffer could result in a change in selectivity [32,33].In this work, the parameters developed for the determination of insulin analogues did not employed buffer, and the mobile phase (methanol/water, 70:30) used is simple and advantageous; the most quality control applications can be carried out with methanol/water as a mobile phase.For this reason, the purpose of the present work was to describe the development and validation of the simple and accurate HPLC method for the analysis of lispro and glargine insulin analogues in pharmaceutical preparations. Materials and Methods SamplesInsulin lispro (lot9074) and glargine insulin (lot73A) reference solutions with assigned purity of 100UI/mL as well the pharmaceutical preparations (injections) were generous donated by Eli Lilly (São Paulo, Brazil) and Sanofi-Aventis Ltda (São Paulo, Brazil). Insulin lispro injection was claimed to contain 100UI/mL of drug and glycerol, metacresol, sodium phosphate, zinc oxide and water as excipient. Insulin glargine injection was claimed to contain 100UI/mL of drug and glycerol, cresol, hydrochlor...

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Mass spectrometric determination of insulins and their degradation products in sports drug testing

Cited by 96 publications

References 93 publications

Mass spectrometry of peptides and proteins from human blood

Mass spectrometry of peptides and proteins from human blood

Quantitative Insulin Analysis Using Liquid Chromatography–Tandem Mass Spectrometry in a High-Throughput Clinical Laboratory

Development of a New HPLC Method for the Determination of Lispro and Glargine Insulin Analogues in Pharmaceutical Preparations

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