A simple and sensitive LC-MS/MS method for simultaneous determination of temsirolimus and its major metabolite in human whole blood

摘要

A simple, fast and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of the concentrations of temsirolimus and its major metabolite, sirolimus, in human whole blood. The blood sample (100 μL) after adding temsirolimus-d7 and sirolimusd3 internal standards was precipitated with 0.200 mL of methanol/0.300 Mzinc sulfate (70/30, v/v), then analyzed by a Shimatzu LC system coupled to a Sciex API-5000 mass spectrometer. The chromatographic separationwas carried out on aBDSHypersilC8 column (50 × 3.0 mm, 5 μm) at 50 °C with amobile phase composed ofmethanol/water/formic acid (72/28/0.1) (v/v/v) containing 2.50 mM ammonium acetate. Mass spectrometric detection was performed using electrospray positive ionization with multiple reaction monitoring mode. Thismethodwas validated from0.250 to 100 ng mL~(-1) for temsirolimus and 0.100 to 40.0 ng mL~(-1) for sirolimus. The lower limits of quantitation were 0.25 ng mL~(-1) for temsirolimus and 0.1 ng mL~(-1) for sirolimus. The intra-day and inter-day precisions (CV %) of spiked quality control (QC) sampleswere less than 10.4 and 9.6 %, respectively.The accuracies as determined by the relative error for QC samples were less than 12.1 % for intra-day and 7.3 % for inter-day. No significant matrix effect was observed. This method has been successfully applied to analyze clinical pharmacokinetic study samples. The assay reproducibility was also demonstrated by using incurred samples.