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.