Qualitative Determination of Synthetic Analogues of Insulin in Human Plasma by Immunoaffinity Purification and Liquid Chromatography−Tandem Mass Spectrometry for Doping Control Purposes

Thevis, Mario; Thomas, Andreas; Delahaut, Philippe; Bosseloir, and Alain; Schänzer, Wilhelm

doi:10.1021/ac050066i

Cited by 102 publications

(77 citation statements)

References 34 publications

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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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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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“…The XXXI Summer Olympic Games (August [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]2016) and the XV Paralympic Games (September 7-18, 2016) were held in Rio de Janeiro, marking the first time that these events were organised in South America. Doping control analyses have been conducted in Brazil since 1989, were incorporated into the International Olympic Committee (IOC) accreditation in 2002, and were absorbed by the World Anti-Doping Agency (WADA) accreditation system in 2005.…”

Section: Introduction and Facility Descriptionmentioning

confidence: 99%

Doping control analysis at the Rio 2016 Olympic and Paralympic Games

Pereira

Sardela

Padilha

et al. 2017

Drug Testing and Analysis

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This paper summarises the results obtained from the doping control analyses performed during the Summer XXXI Olympic Games (August 3-21, 2016) and the XV Paralympic Games (September 7-18, 2016). The analyses of all doping control samples were performed at the Brazilian Doping Control Laboratory (LBCD), a World Anti-Doping Agency (WADA)-accredited laboratory located in Rio de Janeiro, Brazil. A new facility at Rio de Janeiro Federal University (UFRJ) was built and fully operated by over 700 professionals, including Brazilian and international scientists, administrative staff, and volunteers. For the Olympic Games, 4913 samples were analysed. In 29 specimens, the presence of a prohibited substance was confirmed, resulting in adverse analytical findings (AAFs). For the Paralympic Games, 1687 samples were analysed, 12 of which were reported as AAFs. For both events, 82.8% of the samples were urine, and 17.2% were blood samples. In total, more than 31 000 analytical procedures were conducted. New WADA technical documents were fully implemented; consequently, state-of-the-art analytical toxicology instrumentation and strategies were applied during the Games, including different types of mass spectrometry (MS) analysers, peptide, and protein detection strategies, endogenous steroid profile measurements, and blood analysis. This enormous investment yielded one of the largest Olympic legacies in Brazil and South America.

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“…[2][3][4] From the five-fold charged molecule, the mass of the determined insulin can be accurately calculated, which matches that of insulin aspart. In addition, the product ions shown in the extracted ion chromatograms (739.4, 626.3, 248.1 and 219.1) unequivocally corroborate the identity of the substance.…”

Section: Case Reportmentioning

confidence: 81%