Development of HPLC-CAD method for simultaneous quantification of nine related substances in ursodeoxycholic acid and identification of two unknown impurities by HPLC-Q-TOF-MS.

Ursodeoxycholic acid has gained increasing attention due to its recent discovery of the preventive effect on SARS-CoV-2 infection. Ursodeoxycholic acid has been included in various pharmacopoeias as an old drug, and the latest European Pharmacopoeia lists nine potential related substances (impurities A∼I). However, existing methods in pharmacopoeias and literature can only quantify up to five of these impurities simultaneously, and the sensitivity is inadequate, as the impurities are isomers or cholic acid analogues lacking chromophores. Herein, a novel gradient RP-HPLC method coupled to charged aerosol detection (CAD) was developed and validated for the simultaneous separation and quantification of the nine impurities in ursodeoxycholic acid. The method proved sensitive and allowed the quantification of the impurities as low as 0.02 %. Relative correction factors of the nine impurities were all within the range of 0.8-1.2 in the gradient mode by optimizing chromatographic conditions and CAD parameters. In addition, this RP-HPLC method is fully compatible with LC-MS due to the volatile additives and high percentage of the organic phase, which can be directly used for the identification of impurities. The newly developed HPLC-CAD method was successfully applied to commercial bulk drug samples, and two unknown impurities were identified by HPLC-Q-TOF-MS. The effect of CAD parameters on the linearity and correction factors was also discussed in this study. Overall, the established HPLC-CAD method can improve the methods in current pharmacopoeias and literature and contributes to understanding the impurity profile for process improvement.

Favipiravir (SARS-CoV-2) degradation impurities: Identification and route of degradation mechanism in the finished solid dosage form using LC/LC-MS method.

Favipiravir finished dosage was approved for emergency use in many countries to treat SARS-CoV-2 patients. A specific, accurate, linear, robust, simple, and stability-indicating HPLC method was developed and validated for the determination of degradation impurities present in favipiravir film-coated tablets. The separation of all impurities was achieved from the stationary phase (Inert sustain AQ-C18, 250 × 4.6 mm, 5-µm particle) and mobile phase. Mobile phase A contained KH2 PO4 buffer (pH 2.5 ± 0.05) and acetonitrile in the ratio of 98:2 (v/v), and mobile phase B contained water and acetonitrile in the ratio of 50:50 (v/v). The chromatographic conditions were optimized as follows: flow rate, 0.7 mL/min; UV detection, 210 nm; injection volume, 20 µL; and column temperature, 33°C. The proposed method was validated per the current International Conference on Harmonization Q2 (R1) guidelines. The recovery study and linearity ranges were established from the limit of quantification to 150% optimal concentrations. The method validation results were found to be between 98.6 and 106.2% for recovery and r2  = 0.9995-0.9999 for linearity of all identified impurities. The method precision results were achieved below 5% of relative standard deviation. Forced degradation studies were performed in chemical and physical stress conditions. The compound was sensitive to chemical stress conditions. During the study, the analyte degraded and converted to unknown degradation impurities, and its molecular mass was found using the LC-MS technique and established degradation pathways supported by reaction of mechanism. The developed method was found to be suitable for routine analysis of research and development and quality control.