Development and Validation of an LC/MS Method of Glatiramer and Its Sequential Amino Acids, at Pharmaceutical Product

Analytical method by liquid chromatography coupled to mass spectrometry (LC/MS) was developed and validated for the quantification of the polypeptide mixture of glatiramer acetate (GA) as an injectable solution, the method was linear, precise, accurate and capable of separating the polypeptide mixture consisting of four amino acids that make up the glatiramer (L-glutamic acid, L-alanine, L-tyrosine and L-lysine), using a Waters Acquity® UPLC BEH300 C18 1.7 µm column, 2.1 × 100 mm, a mobile phase composed of 0.2% formic acid and acetonitrile at a flow rate of 0.2 mL/min, a mass spectrometry detector (WATERS Acquity QDa®). As a result, the fragmented ions related to each molecule were obtained with the following values: 564 m/z for GA, 148 m/z for glutamic acid, 90 m/z for alanine, 182 m/z for tyrosine and 147 m/z for lysine; with retention times of 4.1 min, 11.2 min, 11.3 min, 12.1 min and 10.9 min respectively. The method shows linear and reproducible responses in a range of 128-192 µg/mL GA, with a correlation coefficient (r) of 0.997, recoveries of 99 ± 1.2% and an accuracy of 0.5%.