Simple RP-HPLC method for determination of triple drug combination of valsartan, amlodipine and hydrochlorothiazide in human plasma

A simple RP-HPLC method for the quantification of valsartan (VAL), amlodipine (AML) and hydrochlorothiazide (HCT) in human plasma was developed and validated. VAL, AML and HCT were resolved using a Gemini C18 column and mobile phase gradient starting from 20 % acetonitrile and 80 % 10 mmol L-1 ammonium formate (V/V, pH 3.5 ± 0.2, by formic acid) to 70 % acetonitrile and 30 % 10 mmol L-1 ammonium formate, over 20 minutes, with a flow rate of 1 mL min-1. The samples were purified by protein precipitation and extraction. Telmisartan was used as internal standard. The method was validated according to USFDA and EMEA guidelines with good reproducibility and linear responses R = 0.9985 (VAL), 0.9964 (AML), and 0.9971 (HCT). RSDs of intra- and inter-day precision ranged 2.2-8.1 and 4.6-11.7 %, respectively, for all three drugs. Mean extraction recoveries of three QCs for the triple drug combination were 76.5 (VAL), 72.0 (AML) and 73.0 (HCT) % for human plasma. Although the LC-MS/MS method is more sensitive than HPLC, HPLC is still suitable for preliminary pharmacokinetic study. The experiments performed demostrated that simultaneous determination of all components of the triple drug combination in human plasma can be done by this method. Proposed method can be also used for guidance to the LC-MS/MS method. U ovom radu opisana je i validirana jednostavna RP-HPLC metoda za određivanje valsartana (VAL), amlodipina (AML) i hidroklorotiazida (HCT) u humanoj plazmi. VAL, AML i HCT su razlučeni na koloni Gemini C18. Početni sastav mobilne faze bio je acetonitril (20 %) i 10 mmol L-1 otopina amonijevog formijata (80 %, V/V, pH podešen na 3,5 ± 0,2 pomoću mravlje kiseline), a nakon 20 minuta 70 % acetonitrila i 30 % 10 mmol L-1 amonijevog formijata, uz protok od 1 mL min-1. Uzorci su pročišćeni taloženjem proteina i ekstrakcijom. Telmisartan je upotrebljen kao unutarnji standard. Metoda je validirana prema uputama USFDA i EMEA uz dobro reproducibilnost i linearnost: R = 0.9985 (VAL), 0.9964 (AML), and 0.9971 (HCT). Ponovljivost i intermedijarna preciznost bile su u rasponu 2,2-8,1, odnosno 4,6-11,7 % za sve tri ljekovite tvari. Srednji povrat ekstrakcije iz humane plazme za ovu kombinaciju lijekova iznosila je 76,5 (VAL), 72,0 (AML) i 73,0 (HCT) %. Iako je LC-MS/MS metoda osjetljivija od HPLC metode, HPLC je prihvatljiva za preliminarna farmakokinetička ispitivanja. Provedeni pokusi pokazuju da se predlož enom metodom mogu simultano odrediti sastavnice trostruke kombinaci