High-performance liquid chromatographic determination of insulin in rat and human plasma.
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.
Khaksa
G
G
Department of Pharmaceutics, College of Pharmaceutical Sciences, Manipal, Karnataka State, India.
Nalini
K
K
Bhat
M
M
Udupa
N
N
D000328:Adult; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D007328:Insulin / Q000097:blood*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
D007328:Insulin