Abstract
Objective: The present work aimed to develop a simple, rapid, specific and precise liquid chromatography-tandem mass spectrophotometric (LC–MS/MS) validated method for quantification of pomalidomide and internal standard (ISTD) Fluconazole in human plasma.Methods: 50 µl of 0.1% formic acid was added to plasma samples prior to liquid-liquid extraction (LLE) using 2.5 ml of ethyl acetate. Chromatographic separation was achieved on Xterra, RP18, 5 µ (50 x 4.6 mm) column using a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 12:88, v/v as the mobile phase. The flow rate was 0.50 ml/min. The LC eluent was split, and approximately 0.1 ml/min was introduced into Tandem mass spectrometer using turbo Ion Spray interface at 325 °C. Quantitation was performed by transitions of m/z 260.1 precursor ion to the m/z 148.8 for pomalidomide and m/z 307.1/238.0 for fluconazole.Results: The concentrations of nine working standards showed linearity between 9.998 to 1009.650 ng/ml (r2 ≥ 0.9968). Chromatographic separation was achieved within 2 min. The average extraction recoveries of three quality control concentrations were 53.86% for pomalidomide and were within the acceptance limits. The coefficient of variation was ≤15% for intra-and inter-batch assays. The %CV of ruggedness ranges 1.32 to 4.03. The % stability of short term and long term stock solution stability studies was found to be 99.01% and 98.49% respectively.Conclusion: The results obtained for specificity, linearity, accuracy, precision, ruggedness and stability studies were within limits. Thus the validated economical method was applied for pharmacokinetic studies of pomalidomide.
Highlights
Pomalidomide chemically 4-amino-2-(2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione, the newest immune-modulatory drugs (IMiD), was designed to be more potent and less toxic than thalidomide and lenalidomide [1,2]
Chromatographic separation was performed on Xterra, RP18, 5 μ (50 x 4.6 mm), analytical column and the mobile phase was a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 12:88, v/v
The response at the retention time of pomalidomide was less than 20% of LLOQ response, and at the retention time of internal standard (IS), the response was less than 5% of mean IS response in LLOQ
Summary
Pomalidomide chemically 4-amino-2-(2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione, the newest immune-modulatory drugs (IMiD), was designed to be more potent and less toxic than thalidomide and lenalidomide [1,2]. It is used for the treatment of relapsed and refractory multiple myeloma. As there is no literature on stability data of pomalidomide in human plasma, this study performed assay validations, according to the FDA guidelines [9]. While this method with validation details were economical and applied for pharmacokinetic studies of pomalidomide
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