Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization.
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.
Katayama
M
M
Department of Functional Bioanalysis, Meiji Pharmaceutical University, Tokyo, Japan. mkatayam@my-pharm.ac.jp
Matsuda
Y
Y
Shimokawa
K
K
Tanabe
S
S
Kaneko
S
S
Hara
I
I
Sato
H
H
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D011687:Purines / Q000097:blood* / Q000737:chemistry; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D011687:Purines