High-Performance Liquid Chromatographic Determination of Insulin in Rat and Human Plasma

✍ Scribed by G. Khaksa; K. Nalini; M. Bhat; N. Udupa

• Publisher: Elsevier Science
• Year: 1998
• Tongue: English
• Weight: 67 KB
• Volume: 260
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1998.2660

✦ Synopsis

A rapid and simple isocratic chromatographic procedure for the determination of insulin in human and rat plasma using reversed-phase (RP) high-performance liquid chromatography with an ultraviolet/visible detector is described. The method includes extraction of insulin from human and rat plasma into dichloromethane, followed by back-extraction into 0.05 N hydrochloric acid. The organic phase was evaporated under a stream of nitrogen. The aqueous phase was filtered and a 100 microliters aliquot was analyzed on a RP-C18 column eluted with a mobile phase consisting of a mixture of 74 vol of 0.2 M sodium sulfate anhydrous adjusted to pH 2.3 with phosphoric acid and 26 vol of acetonitrile. The flow rate was 1.2 ml/min and the wavelength was set at 214 nm. The calibration curve was linear over the range of 75-800 microIU/ml. The precision of the assay expressed as coefficients of variation was less than 6% over the entire concentration range. The recovery for insulin ranged from 79 to 81% from human and rat plasma, with coefficients of variation less than 6%. The intra- and interassay coefficients of variation were less than 5.7%. The limit of detection was 50 microIU/ml.