Advanced HPLC Method Development, Validation and Force Degradation Study for Simultaneous Analysis of Allantoin, Hydroquinone and Tretenoin in Melasma Topical Cream Formation

Abstract

A simple, sensitive, rapid, precise and accurate RP-HPLC method has been developed for simultaneous estimation of hydroquinone, allantoin and tretinoin from cream formulation. Chromatographic separation was achieved on a reversed-phase Inertsil C18 (4.6 mm) I.D.× 250 mm, 5 µm) column using a mobile phase consisting of buffer (pH 3.5) 0.05 M potassium dihydrogen ortho phosphate-methanol (70:30% V/V) at a flow rate 1 mL min -1 and UV detection at λmax 223 nm. The method showed linearity with correlation coefficient of hydroquinone, allantoin and tretenoin being 0.999, 0.999 and 0.999 over the range of 100-300 µg mL-1, 50-150 µg mL-1 and 0.625-1.875 µg mL-1, respectively. The mean recoveries were found to be 99.00 to 101.00 % for all the components. The method was validated as per the ICH guidelines. The method was stable and specific and when sample was stressed under different conditions like acid, base, oxidative, thermal and photolytic, no interference of degradants were observed.