Simultaneous determination of rosuvastatin and fenofibric acid in human plasma by LC-MS/MS with electrospray ionization: assay development, validation and application to a clinical study.

摘要

A simple, sensitive and specific LC-MS/MS method for simultaneous determination of rosuvastatin (RST) and fenofibric acid (FFA) was developed and validated with 500 microL human plasma using carbamazepine as an internal standard (IS). The assay procedure involved a simple one-step liquid/liquid extraction of RST and FFA and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto X-Terra MS C-18 column (4.6 mm x 50 mm, 5.0 microm). Separation of RST, FFA and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (45:55, v/v) at a flow rate of 0.40 ml/min. The API-3000LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Positive ion acquisition chromatographic run was used in the present method. Nominal retention times of RST, FFA and IS were 2.35, 4.70 and 2.32 min, respectively. Absolute recovery of RST, FFA and IS was 74, 61 and 69%, respectively. The lower limit of quantification (LLOQ) of RST and FFA was 1.00 ng/ml and 0.50 microg/ml, respectively. Response function was established for the range of concentrations 1.00-50.0 ng/ml and 0.50-20.0 microg/ml for RST and FFA, respectively, with a coefficient of determination (r2) of 0.999 for both the compounds. The inter- and intra-day precision in the measurement of RST quality control (QC) samples 5, 15, 400 and 800 ng/ml, were in the range 8.93-9.37% relative standard deviation (R.S.D.) and 1.74-16.1% R.S.D., respectively. Similarly, the inter- and intra-day precision in the measurement of FFA quality control (QC) samples 0.5, 1.5, 8.0 and 15.0 microg/ml, were in the range 9.78-11.6% relative standard deviation (R.S.D.) and 0.22-17.4% R.S.D., respectively. Accuracy in the measurement of QC samples for RST and FFA were in the range 88.1-108 and 87-115%, respectively, of the nominal values. RST and FFA were stable in the battery of stability studies, viz., bench-top, auto-sampler and freeze/thaw cycles. Stability of RST and FFA was established for 1 month at -80 degrees C. The application of the assay to a clinical study confirmed the utility of the assay.