Simultaneous analysis of retinol, all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction.
A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.
Teerlink
T
T
Department of Clinical Chemistry, Free University Hospital, Amsterdam, Netherlands.
Copper
M P
MP
Klaassen
I
I
Braakhuis
B J
BJ
D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D006801:Humans; D015474:Isotretinoin / Q000097:blood*; D012995:Solubility; D014212:Tretinoin / Q000031:analogs & derivatives* / Q000097:blood* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014801:Vitamin A / Q000097:blood*
D014801:Vitamin A; C002202:4-oxoretinoic acid; D014212:Tretinoin; D015474:Isotretinoin