DEVELOPMENT AND VALIDATION OF ANALYTICAL SPECTROPHOTOMETRIC AND RP-HPLC METHODS FOR THE SIMULTANEOUS ESTIMATION OF HYDROQUINONE, HYDROCORTISONE AND TRETINOIN TERNARY MIXTURE IN TOPICAL FORMULATION
Objective: Development and validation of spectrophotometric and RP-HPLC methods for the simultaneous determination of Hydroquinone (HQ), Hydrocortisone (HC) and Tretinoin (TRT) ternary combination in pharmaceutical preparation.
Methods: The proposed spectrophotometric method was able to determine TRT directly from its absorption spectrum at 362 nm, however, HQ and HC from their first derivative spectra at 284 nm and 252 nm, respectively, without any separation step. The RP-HPLC method was developed using a C18 Sunfire© waters column with a mobile phase composed of acetonitrile: phosphate buffer (adjusted to pH 6.1 using ortho-phosphoric acid) in the ratio of 30:70 %, v/v, respectively at a flow rate of 0.8 ml/min. Quantification was based on measuring peak areas at 260 nm.
Results: The spectrophotometric method was able to selectively quantify each of HQ, HC and TRT in the ranges of 10-50 µg/ml, 2-10 µg/ml and 0.5-5 µg/ml, respectively. The RP-HPLC method was able to produce well-resolved peaks after 3.0, 8.2 and 20.2 min, in the ranges of 2-10 µg/ml, 0.1-1 µg/ml and 0.05-2 µg/ml, for HQ, HC and TRT, respectively. The obtained A, D1 or peak areas values plotted against the concentration of each of the three components showed linear response in the stated ranges. Both methods were validated in terms of linearity, LOD, LOQ, precision, accuracy and selectivity.
Conclusion: Both developed proposed methods were applied for the determination of the active ingredients in the pharmaceutical formulation and the common excipients did not interfere in the analysis. The RP-HPLC method proved to be more sensitive when compared to the applied spectrophotometric method. However, the applied spectrophotometric methods, considered as green analytical chemistry, is a simple, time-saving method that requires minimal use of a hazardous solvent.
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