Development and Validation of Two Novel Chromatographic Methods: HPTLC and HPLC for Determination of Bromhexine Hydrochloride in Presence of Its Two Impurities.

Abstract

Two simple, sensitive and validated chromatographic methods were developed for quantitative determination of bromhexine hydrochloride (BHX) in presence of its major impurities, impurity B (IMB) and impurity C (IMC), as specified by British Pharmacopoeia. First method (I) was high-performance thin layer chromatography-densitometry at which the chromatographic separation was performed using silica gel plates and developing system consisted of hexane:acetone:ammonia solution (9:0.5:0.08, by volume) with ultraviolet scanning at 240nm and linearity was achieved in the ranges of 0.40-10.00, 0.20-2.00 and 0.20-2.00μg/band of BHX, IMB and IMC, respectively. Also, second chromatographic method (II) was high-performance liquid chromatography (HPLC) where the separation was carried out on C18 column at isocratic mode at flow rate 1.5mL/min. The mobile phase consisted of methanol:water (90:10, v/v) adjusted to pH2.5 with O-phosphoric acid and temperature was adjusted to 40°C. The scanning wavelength was 240nm. The chromatographic run time was 6min. Linearity of this method was achieved in the ranges of 4.00-40.00, 0.20-10.00 and 0.50-10.00μg/mL for BHX, IMB and IMC, respectively. The validation of these chromatographic methods was made according to International Conference on Harmonization guidelines. These methods were successfully applied for determination of BHX in its pharmaceutical formulation. Also, statistical comparison was attained between the developed methods and the reported HPLC method using Student's t-test and F-test, and the obtained results showed that there was not any significant difference between them concerning with accuracy and precision.