Simultaneous determination of endogenous retinoic acid isomers and retinol in human plasma by isocratic normal-phase HPLC with ultraviolet detection.
We describe a rapid and simple procedure for the simultaneous quantitation of endogenous 13-cis-retinoic acid, all-trans-retinoic acid, and retinol by isocratic normal-phase HPLC with ultraviolet detection, in 0.5 mL of human plasma. A silica adsorption column was eluted with n-hexane:2-propanol:acetic acid (200:0.7:0.135 by vol) at 0.9 mL/min, and the effluent monitored at 350 nm. The arotinoid ethylsulfonic acid Ro 15-1570 was used as the internal standard. High sensitivity, allowing quantitation of physiological concentrations, was achieved, particularly for the retinoic acid isomers. The detection limits were 0.5 microgram/L in plasma for both 13-cis- and trans-retinoic acid, and 10 micrograms/L for retinol. The CVs for between-day determinations of the lowest quality-control concentration (n = 12) were 4.8% for 13-cis-retinoic acid, 3.4% for trans-retinoic acid, and 3.0% for retinol. The mean (+/- SD) concentrations of 13-cis-retinoic acid (1.79 +/- 0.56 microgram/L), trans-retinoic acid (1.35 +/- 0.42 microgram/L), and retinol (533 +/- 58 micrograms/L) measured in plasma from 22 healthy volunteers agreed well with those previously reported.
Meyer
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Laboratory of Medical Biochemistry and of Clinical Analysis, Faculty of Pharmaceutical Sciences, University of Ghent, Belgium.
Lambert
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WE
De Leenheer
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AP
D000328:Adult; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D006801:Humans; D008297:Male; D011786:Quality Control; D012016:Reference Values; D012680:Sensitivity and Specificity; D014212:Tretinoin / Q000097:blood*; D014801:Vitamin A / Q000097:blood*
D014801:Vitamin A; D014212:Tretinoin