Bioanalysis of Endogenous Isotretinoin in Altered and Unaltered Human Plasma by Liquid Chromatography-Tandem Mass...

Bioanalytical method development and validation of endogenous Isotretinoin with Isotretinoin D5 as internal standard was done as per current regulatory guidelines. The method is simple, rugged and sensitive enough to estimate endogenous Isotretinoin using the chromatography-tandem mass spectrometry technique. An alternative approach has been adopted for quantitative analysis of endogenous Isotretinoin in human plasma. Isotretinoin free matrix (surrogate matrix) was prepared and further used for the development and validation of Isotretinoin. The method was validated in altered and unaltered plasma. The chromatographic optimization was done with column (ACE C18, 100 × 4.6 mm I.D. 5 μm particle size), using a mobile phase containing 1 mM ammonium acetate, pH 3.0 as a solvent A and solvent B (1 mM ammonium acetate (pH 3.0) with acetonitrile in a ratio of 10:90). A flow rate was set at 0.75 mL/min in a binary gradient mode. The analyte was recovered by liquid-liquid extraction method with diethyl ether as an extraction solvent. Multi-reaction monitoring mode in negative polarity was implemented for the quantification of endogenous Isotretinoin in plasma. The calibration curve of Isotretinoin was linear (r2 > 0.9992) over the concentration range of 0.5-1000 ng/mL. The intra-day precision was found in a range of 2.0-3.9% CV for altered samples and 0.9-3.7% CV for unaltered samples. The inter-day precision was found 2.6-6.1% CV for altered samples and 1.3-3.8% CV for unaltered samples. The average recovery of the extraction procedure was found 64.6% for altered samples and 62.2% for unaltered samples.