Development and validation of analytical method by HPLC-DAD for determination of vasodilator active in pharmaceutical ophthalmic forms

Abstract

4-Aminobenzamidine dihydrochloride (4-AD) is one of the degradation products of diminazene aceturate, which have been demonstrating anti-glaucoma potential. As glaucoma is the second leading cause of blindness worldwide, it is relevant to study new therapeutic alternatives, for example, 4-AD carried in polymeric inserts of prolonged release. The present work aims to develop and validate an analytical method for the quantification of 4-AD. An HPLC with UV-Vis detector was used, using a wavelength of 290 ƞm and an ACE® C18 column (125x4.6 mm, 5 μm) mobile phase consisting of phosphate (pH 7.4 ) and triethylamine in the concentration of 0.03% (v / v), in isocratic mode and flow of 1.0 mL / min. . The retention time for 4-AD was 3.2 minutes. The method was developed and validated in accordance with the recommendations of the Resolution of the Collegiate Board (RDC) No. 166 of 2017 of ANVISA, of the Brazilian Pharmacopoeia 5th edition and of the ICH quality guides. The linearity range was established between the concentrations of 5 and 25 μg / mL (r2), the accuracy, repeatability, intermediate precision tests showed a relative standard deviation less than or equal to 5%, in addition the method was also considered selective, exact and robust, pH being its critical factor. Therefore, the HPLC analysis method is robust and can be used to quantify the substance 4-AD in pharmaceutical forms for ocular application.

Keywords

• hplc
• validation
• ophthalmic insert
• high performance liquid chromatography