Validation of a Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry Method for Determination of All-Trans Retinoic Acid in Human Plasma

A sensitive, reliable and specific LC-MS-MS method was developed and validated for the identification and quantitation of all-trans retinoic acid (ATRA) in human plasma. Acitretin was used as the internal standard (IS). After liquid-liquid extraction of 500 μL plasma with methyl tert-butyl ether (MTBE), ATRA and the IS were chromatographed on a HyPURITY C18 column (150 mm × 2.1 mm, 5 μm) with the column temperature set at 40 °C. The mobile phase was consisted of 40% phase A (MTBE–methanol–acetic acid, 50:50:0.5, v/v) and 60% phase B (water–methanol–acetic acid, 50:50:0.5, v/v) with a flow rate of 0.3 mL/min. The API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode via the positive electrospray ionization interface using the transition m/z 301.4 → 123.1 for ATRA and m/z 326.9 → 177.1 for IS, respectively. The calibration curve was linear over the range of 0.45–217.00 ng/mL (r ≥ 0.999) with a lower limit of quantitation (LLOQ) of 0.45 ng/mL. The intraand inter-day precisions values were below 8% relative standard deviation and the accuracy was from OPEN ACCESS Molecules 2014, 19 1190 98.98% to 106.19% in terms of relative error. The validated method was successfully applied in a bioequivalence study of ATRA in Chinese healthy volunteers.