Impurities Control In Argatroban

Argatroban was originally developed by Mitsubishi Pharma, which is a new type of thrombin inhibitor. It can reversibly link to thrombin active target. It can be used for anticoagulation therapy for acute ischemic cerebral infarction patients. Japan pharmacopoeia (JP Sixteenth Edition) includes argatroban in the monograph, however, other pharmacopoeias still do not include argatroban. According to JP, the impurities are controlled by two methods, used to relevant substance1and relevant substance2, respectively. Relevant substance1is controlled by HPLC method, and the limit for individual impurity is less than0.10%. Relevant substance2is controlled by TLC which is a limit control method and the limit for individual impurity is less than0.3%. According to the ICH guideline IMPURITIES IN NEW DRUG SUBSTANCES Q3A(R2), sufficient clinical safety data should be available, if the limit of an impurity is more than0.10%. In order to assure the safety of drugs, the limit of impurity should be normally less than0.10%.The objective of this dissertation is to develop a quantitative analytical method to control the limit of the relevant substance2less than0.10%.The relevant substance2reference standards can not be purchased in the maket. These two impurities were obtained by synthesis and purification in our laboratory. The structures of argatroban impurities F and G were confirmed through HPLC, MS and NMR spectra. Argatroban impurities standard F and G were obtained for analytical method development.Normal phase HPLC method was investigated firstly. Waters e2695HPLC, Waters sunfire TM prep silica4.6×250mm,5μm column was used, mobile phase is to mix80volumes of n-hexane,20volumes of ethanol,0.1volume of trifluoroacetic acid and0.1volume of diethylamine, mix well. Flow rate is1.0mL/min, detection wavelength is254nm, column temperature is35℃, injection volume is20μL. In the method, impurity F, impurity G and argatrogan elute in this order:firstly, impurity F; secondly impurity G; and finally, argatroban. The resolution of impurity G and argatroban is more than1.5, the theoretical plate based on argatroban is not less than3000, the sensitivity of impurity F is0.04%, the sensitivity of impurity G is0.10%. The sensitivity of impurity G is not good enough.Then, reversed phase HPLC method was investigated. Agilent1261HPLC, YMC J’sphere ODS-H80,250*4.6mm,4μm column was used.Mobile phase A:1%acetic acid solution (adjust pH to4.0with ammonia solution, add1.0mL methanol to prevent growth of bacteria). Mobile phase B:acetonitrile. Isocratic elution:mobile phase A:mobile phase B=70:30. Flow rate is1.5mL/min, detection wavelength is254nm, column temperature is40℃, injection volume is50 μL. In the method, impurity G, argatroban, and impurity F elute in this order:firstly, impurity G; secondly argatroban; and finally impurity F. Impurity F has a pair of enantiomers, thus exibit two peaks in the chromatogram.The resolution of impurity G and argatroban is more than1.0, the sensitivity of impurity F is0.020%, the sensitivity of impurity G is0.020%. Reversed phase HPLC method was finally selected as the control method. The limit of relevant substance2is0.10%, which can reduce the clinical risk.A comprehensive analytical method validation was performed to give the evidence that the reversed phase HPLC method is suitable for relevant substance2control. The items of validation include specificity, limit of quantitation, solution stability, precision, robustness, linearity&relative response factor and accuracy. The limit of quantitation of impurity F, impurity G and argatroban are0.02%,0.02%and0.02%, respectively. The method has good repeatability. The RSD of intermediate precision of impurity F and G are6.5%and9.2%, respectively. The method has a good intermediate precision. The correlation coefficient (R) from the linear regression analysis of impurity F, impurity G and argatroban is more than0.99, so the method has good linearity. The relative response factor of impurity F is0.57, the relative response factor of impurity G is1.31. They all need to be taken into account for calculation. The recovery of impurity F and impurity G is more than70%, so the method has good accuracy for impurity F and impurity G.A reversed HPLC method was developed to substitute TLC method used in JP. A comprehensive analytical method validation demonstrates that this method is suitable for the control of argatroban relevant substance2.The limit of impurity is less than0.10%.