1 to 207.2, for losartan acid was monitored from m/z 437.1 to 235.2, for amlodipine was monitored from m/z 409.3 to 238.0, and for IS was monitored from m/z 429.2 to 206.9. Method development A mobile phase consisting of methanol and 0.1% formic acid (85:15, v/v) was found suitable as the analytes were kinase inhibitor DAPT secretase protonated and well separated from endogenous components in this phase. Zorbax XDB-Phenyl column (75 mm �� 4.6 mm; 3.5 micron particle size; Agilent Technologies, USA) gave a good peak shape and response, even at LLOQ level, for all the analytes and IS. The mobile phase was operated at a flow rate of 1.0 mL/min. The retention time of losartan, losartan acid, amlodipine, and IS are low enough (1.3, 1.4, 1.7 and 1.5 min), allowing a small run time of 2.5 min.

Specificity and selectivity The specificity of the method was evaluated by injecting each analyte at the highest concentration in human plasma samples in the presence of other analytes. A typical chromatogram for the control human plasma (free of analyte and IS), human plasma spiked with IS, and human plasma spiked with analytes at LLOQ and IS is shown in Figures Figures22–44 (a�Cc). Results demonstrate the lack of chromatographic interference between each analyte and from endogenous components at the retention time of analyte and IS.

Figure 2 Typical MRM chromatograms of losartan (left panel) and internal standard [IS] (right panel) in (a) human blank plasma (b) human plasma spiked with IS (c) a LLOQ sample along with IS Figure 4 Typical MRM chromatograms of amlodipine (left panel) and internal standard [IS] (right panel) in (a) human blank plasma (b) human plasma spiked with IS (c) a LLOQ sample along with IS Figure 3 Typical MRM chromatograms of losartan acid (left panel) and internal standard [IS] (right panel) in (a) human blank plasma (b) human plasma spiked with IS (c) a LLOQ sample along with IS Sensitivity The lowest limit of reliable quantification for the analytes was set at the concentration of the LLOQ. The precision and accuracy at LLOQ concentration were found to be 5.31% and 103%; 5.36% and 109%; 6.88% and 105% for losartan, losartan acid, and amlodipine, respectively. Extraction efficiency Solid-phase extraction with HLB cartridge proved to be robust and provided the cleanest samples. The recoveries of analytes and IS were good and reproducible.

The mean overall recoveries (with the precision range) of losartan, losartan acid, amlodipine, and IS are summarized in Table 1. Table 1 Mean overall recoveries of losartan, losartan acid, amlodipine and IS Matrix effect No significant matrix effect was observed in all the six batches of human plasma for the analytes at LQC and HQC concentrations. The precision and accuracy for losartan, losartan acid, and amlodipine at LQC concentration were found to be 4.98% and 94.8%; 2.29% and Cilengitide 103%; 1.18% and 100%, respectively.