Abstract

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 μ1 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 μIU/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 μIU/ml.

Original languageEnglish
Pages (from-to)92-95
Number of pages4
JournalAnalytical Biochemistry
Volume260
Issue number1
DOIs
Publication statusPublished - 15-06-1998

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Plasma (human)
Rats
Insulin
Plasmas
Liquids
Hydrochloric Acid
Methylene Chloride
High performance liquid chromatography
Reverse-Phase Chromatography
Calibration
Limit of Detection
Assays
Nitrogen
High Pressure Liquid Chromatography
Flow rate
Detectors
Recovery
Wavelength

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "High-performance liquid chromatographic determination of insulin in rat and human plasma",
abstract = "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 μ1 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 μIU/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 μIU/ml.",
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High-performance liquid chromatographic determination of insulin in rat and human plasma. / Khaksa, G.; Nalini, K.; Bhat, M.; Udupa, N.

In: Analytical Biochemistry, Vol. 260, No. 1, 15.06.1998, p. 92-95.

Research output: Contribution to journalArticle

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AB - 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 μ1 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 μIU/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 μIU/ml.

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