Determination of Endogenous All-Trans-Retinoic Acid , 13-CIS-Retinoic Acid and 4-OXO-13-CIS-Retinoic Acid in Human Plasma by LC-MS-MS
Authors
Christopher J.L. Buggé, Jerry Mikolaichik, Anders Ljungqvist, and Michael P. Sullivan
Purpose
All-trans-retinoic acid, 13-cis-retinoic acid and 4-oxo-13-cis-retinoic acid are endogenous retinoids. The first two compounds have topical activity for dermatological disorders; we present an accurate and sensitive bioanalytical method to determine possible systemic exposure following such treatment.
Methods
Human heparinized plasma (0.5 mL) was fortified with internal standard (acitretin) and extracted with ethyl acetate/hexane 9:1. The organic layer was transferred, evaporated and dissolved in mobile phase (acetonitrile/75 mM ammonium acetate 93:7). An aliquot of the extract was injected onto a YMC ODS-AQ column (150 x 4.6 mm) and analyzed by SCIEX API-III LC-MS-MS in negative ion APCI in MRM mode. The run time was 15 min at a flow rate of 0.8 mL/min. Calibration standards and quality control (QC) samples were prepared by fortifying plasma that had been previously treated to remove endogenous retinoids.
Results
Baseline resolution of the isomers was obtained. The lower limit of quantitation of the assay was established at 0.50 ng/mL for all three analytes. Over the three days of validation, the accuracy and precision of the QC samples at low, medium and high levels were found to be acceptable (85-115% and <15% CV, respectively) for all three compounds. Stability studies (including 6h room temperature, 3-cycle freeze/thaw and 48h extract) demonstrated that the analytes were sufficiently stable provided that all operations were done under yellow lighting. Data from the analysis of plasma collected from 20 non-fasted male and female healthy volunteers are presented and discussed. Mean concentrations of endogenous all-trans-retinoic acid, 13-cis-retinoic acid and 4-oxo-13-cis-retinoic acid were 1.69 +/-0.36, 1.095 +/-0.22 and 1.75 +/-0.60 ng/mL, respectively. Little or no 4-oxo-all-trans-retinoic acid was seen (less than 0.1 ng/mL).
Conclusions
These results demonstrated an accurate and sensitive method for the determination of endogenous concentrations of these retinoids in human plasma.