Analvtical Method Of Impurities In Cvclophosphamide Active Pharmaceutical Ingredient

In the modern pharmaceutical industry, drug development and manufacturing, impurity problems appeared during raw material preparation, sample production and storage. Since the impurities and their corressponding by-products may have potential damages to human being, identification of trace impurities and by-products is extremely important. There are stringent requirements for the identification of drugs purity and main medical mediates in Pharmacopoeia of many countries.Cyclophosphamide is an alkyl agent, used as the clinical immune inhibitor to cure different tumors. Some impurity and by-product are produced during the production and storage of cyclophosphamide. The degradation product A (Bis(2-chloroethyl)amine hydrochloride) is highly toxicity to human being. According to USP Pharmacopeial Forum, the impurity should be separated and identified before going to the market. This experiment used three independent methods, ie., USP PF (Thin-layer chromatography), High performance liquid chromatography and Ion chromatography to separate and identify various impurities in cyclophosphamide. The main results were as following:1. In the sensitivity test, limit concentration of impurity1and degradation products A of cyclophosphamide in various concentrations were undetectable by using the thin-layer chromatography method as recommended by USP. While, the spot of cyclophosphamide standard sample appeared during the determination. Based on the results obtained above, it is clear that the USP PF is not suitable for the determination of degradation product A. In the detection limit test, limit of dection of degradation impurity B-D is0.03%, which is less than limit of0.06%. The method show good specificity, thus it is suitable for limit determination of degradation impurity B-D in cyclophsphamide. The method show good specificity, thus it is suitable for limit determination of degradation impurity B-D in cyclophsphamide.2. HPLC-UV method was established to quantivative determine of the degradation impurities B, impurity1:Using a Dalian Split Hypersil ODS-2column (250×4.6mm×5μ m), the mobile phase consist of water and acetonitrile degassed mixture (77/23), the flow rate was1.0ml/min with UV detection set at195nm; the column temperature was25℃; inject volume was20μL. The linear range for the content of three impurities was1.75～47.43μg/ml (R>0.996, n=6), the recovery of accuracy test was in the range of94to 110%, its relative standard deviation of precision does not exceed5.0%, and the lowest detection limit of the method is up to0.08μg/ml.3. Ion exchange chromatography-TCD method was established to measure quantivative determine of degradation impurity A in cyclophosphamide:Using a Lonpac CS12A column (250×4.0mm Analytical), the mobile phase was methane sulfonic acid, the flow rate was1.0ml/min with TCD detector; the column temperature was25℃; inject volume was25μL. The detection and quantification limit is0.54and1.80μg/ml, respectively. The linear range for the content of degradation impurity A was LOQ to24.04μg/ml (R>0.999, n=6), the average recovery of in the concentration level of LOQ to24.04μg/ml was in the range of76to103%, the relative standard deviation does not exceed5.0%. The method show good specificity, precision and accuracy.