Facile separation of four co-formulated ternary antihypertensive drug combinations with a customized elution protocol using supercritical fluid chromatography

Abstract A novel supercritical fluid chromatography (SFC) method is described to analyze four antihypertensive drug combinations containing a diuretic (hydrochlorothiazide, HCT), a calcium channel blocker (amlodipine, AML) and an angiotensin receptor blocker (olmesartan (OLM)/ telmisartan(TEL)/valsartan(VAL)/renin inhibitor (aliskiren, ALI) using a customized protocol. The effect of mobile phase, analytical column, temperature and pressure were suitably optimized. The best conditions for separation was achieved on DCPak P4VP (250 × 4.6 mm, 5 µm) column using CO2 and 0.1% formic acid in methanol by gradient elution for all drug combinations. The chromatographic separations were accomplished within 5.0 min at 125 bar back pressure and 40 °C column temperature. Detection and quantitation of analytes was performed at 230 and 250 nm using a photo diode array detector. The resolution factor Rs between the drugs ranged from 1.52-3.85 for different combinations. The study showed decrease in retention factors (k) of the drugs with increase in temperature and pressure. The van’t Hoff plots revealed enthalpy driven process for the separation of all drug combinations. The method was validated as per ICH guidelines for specificity, sensitivity, accuracy and precision and recovery. Method linearity was established from 10.0-300, 1.0-20/25, 2.00-100, 5.00-200, 2.00-100 and 5.00-200 µg/mL for ALI, AML, HCT, OLM, TEL and VAL, respectively. Application of the method was demonstrated by analyzing single pill combinations of ALI–AML–HCT, OLM–AML–HCT, TEL–AML–HCT and VAL–AML–HCT. The significance of the method for accuracy and precision was assessed using Student’s t-test and F-test.