Simultaneous Determination of Six Adenyl Purines in Human Plasma by High-Performance Liquid Chromatography With Fluorescence Derivatization

J Chromatogr B Biomed Sci Appl. 2001 Aug 25;760(1):159-63. doi: 10.1016/s0378-4347(01)00265-1.

Abstract

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A column with mobile phase consisting of 0.05 M citric acid-0.1 M dipotassium hydrogen phosphate (pH 4.0)-methanol (97+3). The detection limits were 100-1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2-22.2 pmol/ml), ADP (5.5-22.2 pmol/ml), AMP (0.8-3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.

MeSH terms

  • Calibration
  • Chromatography, High Pressure Liquid / methods*
  • Fluorescent Dyes / chemistry*
  • Humans
  • Purines / blood*
  • Purines / chemistry
  • Sensitivity and Specificity

Substances

  • Fluorescent Dyes
  • Purines