Development and validation of chiral HPLC method for quantitative determination of hydrazine analogue in Argatroban monohydrate

Abstract

Abstract Argatroban is an anticoagulant which is used as direct thrombin inhibitor. It consists of hydrazine analogue, which is probably similar to Argatroban. The single HPLC method is already reported in to United States Pharmacopeia (USP). In that method, Argatroban peak eluted as R-Argatroban and S-Argatroban while hydrazine analogue eluted as P1 and P2 peaks. The P2 peak of Hydrazine analogue is eluted as retention time of R-Argatroban peak. Therefore it was difficult to determine the accurate concentration of Hydrazine analogue in to Argatroban. The existing method is longer run time up to 102 min. The present HPLC method shows appropriate resolution between hydrazine analogue and Argatroban. The total run time reduces up to 40 min, which is a ∼2.5 time lesser than reported in literature. In present method hydrazine analogue and Argatroban are eluted to be 9.0 min and 12.5 min respectively. The effective chromatographic separations are achieved on YMC chiralart cellulose-SC, 250×4.6 mm, 5μ column and normal phase with linear elution. The mobile phase was uniform mixture of methanol, acetonitrile, dichloromethane, trifluoroacetic acid, triethylamine with ratio of 30:50:920:3:4 and delivered flow rate 0.7 mL/min. Analysis is performed on UV/PDA detector with detection wavelength is adjusted at 259 nm. The injection volume is 5μL and column oven temperature is 35°C. The present method is accurate and precise as well as linear in the range of LOQ to 150 % level. The finalized concentration of hydrazine analogue for limit of detection and quantitation is 0.003 and 0.010%. The correlation coefficient of hydrazine analogue is 0.99947. The recovery for ranged between 100.00 to 103.29%. In robustness study for of mobile phase flow rate and column oven temperature, no significant changes are observed in system suitability parameters and result. This method has been developed and validated using the ICH guideline Q2(R1).The new method is sensitive, precise, accurate, and rapid; it also qualifies all the criteria of linearity, stability, as well as robustness.