Development and validation of analytical spectrophotometric and RP-HPLC methods for the simultaneous estimation of Hydroquinone, Hydrocortisone and Tretinoin ternary mixture in topical formulation

  • MARWA K. AL JAMAL Beirut Arab University, Faculty of Pharmacy, Department of Pharmaceutical
  • AZZA A. GAZY Beirut Arab University, Faculty of Pharmacy, Department of Pharmaceutical Technology


Direct spectrophotometric and RP-HPLC methods were developed, compared and validated for the simultaneous determination of Hydroquinone (HQ), Hydrocortisone (HC) and Tretinoin (TRT) ternary combination in laboratory made mixtures and pharmaceutical preparation. The developed spectrophotometric method characterised by it simplicity and time saving property. The proposed 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, where at this wavelength peaks were well resolved and eluted after 4.0, 8.2 and 19.7 minutes, for Tretinoin (TRT), Hydroquinone (HQ) and Hydrocortisone (HC), respectively.

The signals obtained (A or D1 value or peak areas) plotted against the concentration of each of the three components showed linear response in the concentration ranges of 10-50 µg/mL  for HQ, 2-10 µg/mL HC and 0.5-5 µg/mL TRT for the spectrophotometric method and in the concentration range of 2-10 µg/mL for HQ, 0.1-1 µg/mL for HC and 0.05-2 µg/mL for TRT for the RP-HPLC method with excellent linear regression values. Both proposed methods were applied for the determination of the active ingredients in the pharmaceutical formulation and the common excipients present in the formulation did not interfere in the analysis. Both methods were validated in terms of linearity, LOD, LOQ, accuracy, precision and selectivity. The results obtained from the analysis of the co-administered mixture by the proposed spectrophotometric method was statistically compared to those obtained by the applied RP-HPLC method.

Keywords: Hydroquinone, Hydrocortisone, Tretinoin, Ternary mixture, Spectrophotometry and RP-HPLC


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K. AL JAMAL, M., and A. A. GAZY. “Development and Validation of Analytical Spectrophotometric and RP-HPLC Methods for the Simultaneous Estimation of Hydroquinone, Hydrocortisone and Tretinoin Ternary Mixture in Topical Formulation”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 11, no. 11, Sept. 2019,
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