Development of bioanalytical method for determination of intact human insulin from plasma using LC/MS/MS

Human insulin is a peptide hormone composed of 51 amino acids, and has a molecular weight of 5808 Da. It is a heterodimer of two peptide chains connected by disulphide bonds that is secreted by beta cells of pancreas, and is central to regulating carbohydrate and fat metabolism in the body. Animal form insulin, including porcine and bovine insulin, has been used clinically for the treatment of diabetes. However, biosynthetic human insulin is preferred because side reactions are generally less common. Quantitation of insulin from biological fluids is important to understand its pharmacokinetics. Traditionally, measurement of this peptide hormone is carried out using immunoassays such as ELISA or RIA. However, the major drawbacks of these techniques are cross reactivity, prolonged assay development times, reagent optimization, reagent procurement and matrix effects. LC/MS/MS technique which has been established as a gold standard for small molecule quantitation, is now emerging as a tool for quantitation of bio-therapeutics as well. In comparison to conventional LBAs’, LC/MS/MS based approach offers many advantages in terms of selectivity by MRM, improved throughput by multiplexed detection of targets, wide dynamic range, short development time lowering cost of analysis etc. However, few challenges still exist in quantitation of proteins using LC/MS/MS such as nonspecific binding, poor fragmentation etc. Different approaches of extraction, separation and detection of human insulin have been reported in the literature such as enrichment using immunocapture, SPE, quantitation of insulin B chain by reducing intact insulin, use of low-flow HPLC, 2D-LCMS etc. Here, simple SPE technique was used for sample pretreatment and LC/MS/MS method was developed for quantitation of intact human insulin in plasma using LCMS-8060, a triple quadrupole mass spectrometer from Shimadzu Corporation, Japan

Content Type:
Paper
Document Number:
PO-CON1747E
Product Type:
Liquid Chromatograph-Mass Spectrometry, Mass Spectrometry
Keywords:
insulin, amino acids, porcine, bovine, peptide hormone, SPE technique,low-flow HPLC, 2D-LCMS, Pharmaceutical, Life Science, Drug discovery, Development (Formulation, Scale-up, Method development), LCMS-8060
Language:
English
File Name:
apo21765.pdf
File Size:
550kb

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