Purpose: The active molecule is a derivative of Pyruvic acid which is being investigated for treatment of psoriasis. Since it is a cytotoxic compound, the target concentration was set at a low level in the topical formulation. The API is unstable above pH 3 with a maximum half-life of approximately 30 hours at room temperature. Along with low dosing, instability, presence of acidic modifier in the formulation, very polar nature, and weak UV absorption spectra of Active Pharmaceutical Ingredient (API) present challenges for the development of a reliable analytical methodology. A HPLC method was developed to quantify the API in various formulation by resolving the above-mentioned issues.
Methods: It was a requirement for this method to be capable of determining levels of API at ~10 µg/mL. Due to poor UV absorption, achieving this low sensitivity was attempted with Charged Aerosol Detector (CAD) but these efforts were not successful. Therefore, reversed-phase chromatography (RP-HPLC) was attempted and several columns were screened including several aqueous columns, organic acid column, and HILIC column. These columns were evaluated to separate the acidic API in the formulations containing other acid modifiers such as acetic acid, citric acid, phosphoric acid, lactic acid, and benzoic acid. Most of the columns were selective to specific formulations, and satisfactory separations were not obtained except HILIC column which successfully separated API from other acids and excipients. API was detected using a gradient chromatographic separation at 202 nm wherein the mobile phases used consists of 5 mM Phosphate buffer (pH 2.6) and Acetonitrile. The run time is 27 min and the column and autosampler temperature was maintained at 35°C, and 8 °C, respectively.
A simple extraction procedure was developed for the formulation to meet the active concentration requirement of ~10 µg/mL. Using the developed extraction method and HPLC conditions, several formulations including acetic acid, and phosphate buffer were successfully analyzed.
Results: The retention time for API was found to be approximately 16 minutes. The API system suitability and peak characteristics met the USP/ICH requirements. The RSD for n=5 standard injections was ≤ 2.0%, the capacity factor was ≥ 6, and the tailing factor was 1.0. The purity profile was evaluated with a PDA detector and the λmax determined to be 202 nm. The peak was found to be pure. The calibration curves were found to be linear in the concentration range of 50% (~4 µg/mL) to 150% (~12 µg/mL) level. The correlation coefficient (R2) was found to be 0.999. The resolution between the API and acid modifier varied was acceptable for the assay determination.
The drug product extraction method was confirmed to be precise with % RSD of ~0.6%. Accuracy was established by spiking a known amount of API in a placebo formulation and the average recoveries were ~95%.
Conclusion: A HILIC chromatography under reversed-phase UV-LC condition was developed for quantification of acidic API in acidic formulations. The required low sensitivity was achieved and the API was successfully separated from the organic acids using a simple reverse-phase gradient elution program. The method has been determined to be linear, accurate, precise and specific. The methodology presented here can be applied to other formulations with different inorganic/organic acids, and buffers systems.