After studying molecular structure and solubility, the method development was started according to hydrophobic interaction and polarity of hydrocortisone and clotrimazole. The process development was carried out at 1.0 mL per min flow rate, and the chromatographic temperature (column temperature) was 40 °C. In the start, a mobile phase consisting of acetonitrile and buffer was used with a 20-μL injection volume of a 0.2-mg/mL concentration separately for hydrocortisone and clotrimazole. The peaks which were obtained are symmetrical, so there was no necessity for method optimization of unusual chromatograms, i.e., asymmetrical peaks, overlapping. In order to reduce the peaks’ retention time and to make the method cost-effective only play with the composition of the mobile phase.
Similarly, it was observed that different parameters like column type and pH also have different effects on HYD and CL. System suitability parameters were tested according to USP.
The working standard solution of HYD and CL was injected separately into HPLC to determine their retention time, and also chromatographic purity and UV spectrum. Then, the working standard solution of a mixture of HYD and CL was injected, and their retention time, chromatographic purity, and UV spectrum were confirmed. For this purpose, we injected six standard injections, and their RSD, tailing factor, theoretical plates, and resolution were calculated. System suitability parameters confirmed that the given chromatographic conditions were good for the method development and validation.
The mixture of working standard solutions, placebo of cream, and blank was injected separately, and chromatograms are obtained; no extra peak was observed. The peaks which are obtained are of HYD and CL in the cream formulation. Linearity solutions were prepared by quantitative dilutions of the stock solution of hydrocortisone and clotrimazole. The correlation coefficient (r) should not be less than 0.999, is considered as acceptable, and was obtained for both HYD and CL.
Accuracy was tested by spiking known quantities of HYD and CL in the placebo of cream. The accuracy samples were prepared at three-level concentrations over a range of 75%, 100%, and 125%. The obtained recovery result should be between 98 and 102%, and their %age RSD should not be more than 2 (RSD ≤ 2%). There are three stages of precision: intra-assay precision(repeatability), reproducibility, and intermediate precision . Repeatability of the suggested analytical method was carried out by making six samples and injecting each of them three times in HPLC of the selected cream formulation. All the results which were obtained met the requirements RSD ≤ 2% and are given in Table 2. Intermediate precision was performed on the same sample on different days by two different analyses. The value of RSD is not more than 2% for both HYD and CL.
The concentration interval for HYD and CL over which acceptable linearity, precision, and accuracy were obtained is given in Table 2. Robustness was performed regarding wavelength, oven temperature, and flow rate. The results which were obtained are within acceptable limits that are 98–102%, and their RSD is 0.18% for HYD and 0.34% for CL.
The LOD of an analytical method is the lowest quantity of analyte in a given sample which can be easily detected but not quantified to the given value. The LOQ of an analytical method is the lowest amount of analyte in a given sample which can be easily quantified with appropriate accuracy and precision.
Initially, physical appearance of experimental cream was off-white and homogeneous and chromatograms of initial testing were given in Fig. 3. The samples were tested after 3 months at 50 °C stress condition, and it was observed that the physical appearance of a sample remains almost the same and little degradation of HYD and CL was observed. The degradation of HYD and CL was also observed and tested after 3 and 6 months, no change in physical appearance observed, and little degradation of HYD and CL was observed (Fig. 4). These conditions are not as much drastic as 50 °C. The placebo sample was also tested in both conditions, and it also shows no change in physical appearance and almost the same results were obtained as at initial conditions.
Sunlight stress on experimental topical cream shows that little degradation of HYD and CL was observed, and a physical appearance of cream also remains nearly unchanged. The developed and validated stability indicating method was also applied for the detection of HYD and CL assay in three topical creams from the market: Clozox H and Gytrim labeled on HYD and CL content. These creams are the same in composition but are different with respect to excipient composition, and their texture and physical appearance also vary a bit.