Development and validation of a LC-MS/MS method for simultaneous quantification of Ivabradine and Metoprolol in rat plasma.

The combination of Ivabradine (IVA) and Metoprolol (MET) was approved by US-FDA for symptomatic treatment of chronic stable angina pectoris. Hence, a potential analytical method that can simultaneously quantify these two drugs is required. In view of this, a novel and fully validated LC-ESI-MS/MS method has been established for the quantification of IVA and MET in rat plasma. Analytes and their deuterated analogues were quantitatively extracted from rat plasma by protein precipitation technique. The analytes were separated using acetonitrile-water consisting 0.1% orthophosphoric acid buffer (30:70 v/v) as a mobile phase with a flow-rate of 1.0 mL/min and 5 min run time on Waters, X-Bridge-C18 (150× 4.6 mm, 3.5 μm) analytical column. The multiple reaction monitoring transitions, m/z 638.14 → 124.22 for IVA, 498.33 → 110.59 for MET; 644.37 → 130.41 for IVA-D6 and 504.46 → 116.28 for MET-D6 were chosen to achieve high selectivity in the analysis. The method exhibited great improvement in sensitivity and good linearity over the concentration range of 0.1-1.5 ng/mL for IVA, 1.0-15.0 ng/mL for MET, with satisfactory precision and accuracy according to USFDA guidelines. Accuracy was within 99.71-100.3% and 99.9-100.31% for IVA and MET. The intra- and inter-day precision ranged between 0.048 and 12.68% and 0.1-2.66% CV for IVA and MET respectively. Further, the results of the pharmacokinetic parameters including Cmax, tmax, AUC0-t, AUC0-∞ and t1/2 values of drugs indicated that the method is useful for successful quantification of the drugs in rat plasma. The developed method is significant and is useful for simultaneous quantification of IVA and MET.