Validation of a High-Performance Liquid Chromatography method for the determination of vitamin A, vitamin D3, vitamin E and benzyl alcohol in a veterinary oily injectable solution

• Main Authors: Maria Neagu, Georgiana Soceanu, Ana Caterina Bucur
• Format: Article
• Language: English
• Published: Romanian National Association of the Veterinary Products Manufacturers 2015-06-01
• Series: Medicamentul Veterinar
• Subjects: vitamin A; vitamin D3; vitamin E; validation; HPLC
• Online Access: http://www.veterinarypharmacon.com/docs/1539-2015_VD_ART_9.EN.pdf

A new simple, rapid, accurate and precise high – performance liquid chromatography (HPLC) method for determination of vitamin A, vitamin D3, vitamin E and benzyl alcohol in oily injectable solution was developed and validated. The method can be used for the detection and quantification of known and unknown impurities and degradants in the drug substance during routine analysis and also for stability studies in view of its capability to separate degradation products. The method was validated for accuracy, precision, specificity, robustness and quantification limits according to ICH Guidelines. The estimation of vitamin A, vitamin D3, vitamin E and benzyl alcohol was done by Waters HPLC system manager using gradient pump system. The chromatographic conditions comprised a reverse-phased C18 column (5 µm particle size, 250 mm×4.6 mm i.d.) with a mobile phase consisting of tetrahydrofurane, acetonitrile and water in gradient elution. The flow rate was 0.8 ml/min and 2.0 ml/min. Standard curves were linear over the concentration range of 16.50 µg/ml to 11.00 mg/ml for vitamin A, 10.05 µg/ml to 6.70 mg/ml for vitamin E, 0.075 µg/ml to 0.050 mg/ml for vitamin D3 and 1.25 mg/ml to 5.00 mg/ml for benzylalcohol. Statistical analyses proved the method was precise, reproducible, selective, specific and accurate for analysis of vitamin A, vitamin D3, vitamin E, benzyl alcohol and impurities.