Bioanalytical Assay
Plasma concentrations of lisinopril and amlodipine were measured by an established and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method at Suzhou Haike Pharmaceutical Technology Co., Ltd (Suzhou, Jiangsu Province, China). For the analysis of lisinopril, plasma samples were pretreated by liquid liquid extraction with isopropanol: ethyl acetate(1:2,V/V); LSN-d5 was used as internal standard; 5mM ammoniumacetate aqueous solution, 0.01% formic acid and methanol were used as mobile phase; chromatographic separation was performed on Atlantis-dC18 column(Waters, Massachusetts, USA) and the analytes were detected using Triple Quad TM 5500 tandem mass spectrometer(Sciex, Canada) in positive ion mode, with ion sprayin multiple reaction monitoring mode.The lower limit of quantification was 0.500ng/mL and the assay dynamic range was 0.500-100ng/mL. The analytes in matrix were stable when stored at -20℃ for 26 days, at -80℃ for 169 days and after four freeze-thaw cycles.
Amlodipine plasma concentrations were determinedusing a liquid chromatography unit(Shimadzu,LC-30AD,Japan) and a mass spectrometer(Sciex, Triple Quad TM 6500 plus, Canada). Under multiple reaction monitoring, LC-MS/MS system adopts positive ionization mode.Forthe analysis of amlodipine, the lower limit of quantification was 0.050ng/mL and the assay dynamic range was 0.050-10.0ng/mL.The analytes in matrix were stable when stored at -20℃ for 91days, at -80℃ for 207 days and after four freeze-thaw cycles.
Data collection and analysis was performed with Analyst 1.6.3 software (Sciex, Canada) and Watson LIMS(Thermo, USA). Calibration curves were constructed using linear regression equation obtained by the weighted (W=1/X2) least square method fitting for both analytes.Quantitation of qualitycontrol and clinical samples were also performed by theAnalyst software using the same mathematical algorithm asthat used in the calibration of standard curves.