DEVELOPMENT AND VALIDATION OF METHODOLOGY FOR QUANTITATIVE ANALYSIS OF ENOXAPARIN SODIUM.

TF27

Samantha Sant’ Anna Marotta de Oliveira samantha@fcfrp.usp.br (1); Cristiane Masetto de Gaitani (1); Juliana Maldonado Marchetti (1). 

(1) Faculty of Pharmaceutical Sciences of Ribeirão Preto. University of São Paulo – USP.

Heparin is the most used anticoagulant worldwide for the treatment of deep vein thrombosis (DVT) and its major complications such as pulmonary embolism and chronic venous insufficiency. Enoxaparin sodium is a fractionated heparin (FH) obtained from unfractionated heparin (UH) (extracted from porcine intestinal mucosa) through benzylation followed by alkaline depolymerization. Its molecular weight is more homogeneous, ranging from 2,000 to 8,000 Da (average 4,500). Its anticoagulant effect is more predictable than UH with less risk of bleeding. Its therapy does not require constant laboratory monitoring for most patients. There is a great shortage of studies published about methodology of quantitative analysis of both intact FH and UF by HPLC. The analysis is complicated because it is a macromolecule sulphated, acidic, negatively charged and it has variable molecular weight. In this work, a method for the analysis of enoxaparin sodium based on turbidimetry was developed and validated. The methodology consists of a reaction of sulphated groups existing in the enoxaparin molecule with the quaternary ammonium groups of the cetylpyridinium chloride after heating at 37°C for one hour, resulting in an insoluble precipitate in water and turbidity of the reaction environment. This turbidity is directly proportional to concentration of enoxaparin present in the sample. The samples were analyzed using a spectrophotometer in several wavelengths to achieve greater sensitivity and range of application. The method was linear in the range 30.0 to 300.0 µg mL^-1, 10.0 to 100.0 µg mL^-1 and 5.0 to 75.0 µg mL^-1 with wavelengths 500, 340 and 290 nm, respectively. The linear correlation coefficient was higher than 0.99. The values found showed maximum deviation of 12% on the nominal value and precision intra and inter-assays showed coefficient of variation below 3% and 5% respectively. The proposed method was validated for linearity, specificity, precision, accuracy and robustness and it was shown to be useful for the analysis of quantitative enoxaparin sodium for the wavelengths studied.