Development and validation of rapid ultra high performance liquid chromatography with tandem mass spectroscopic method for the quantification of N-nitrosodimethyl amine and N-nitrosodiethyl amine in sitagliptin and metformin hydrochloride immediate and extended-release formulations.

A simple, effective LC-MS based method is developed and validated to determine N-nitrosodimethylamine and N-nitrosodiethylamine in pharmaceutical formulations of Sitagliptin and Metformin hydrochloride combination dosage forms. Atlantis T3 (100 × 3 mm, 3 µm) column, eluent-A (0.1% formic acid in water), and eluent-B (0.1% formic acid in methanol) were used to achieve chromatographic separation. A gradient program time (min)/%B: 0.01/3, 2/3, 4/55, 5/55, 5.5/90, 6.0/90, 6.5/3, and 7/3, and column flow rate: 0.75 mL/min was employed. The column oven and auto sample cooler temperatures were 40°C and 10°C, respectively. Atmospheric Pressure Ionisation positive mode with corona discharge potential as 4.0 V, drying gas (N2 ) flow as 110 mL/min, and nebulizer gas (N2 ) flow as 350 mL/min. Employing PerkinElmer triple quadrupole mass spectrometer, QSight 200 series, the source temperature was 450°C, and hot surface-induced desolvation temperature was 250°C. Under optimized conditions, diluent-1 and diluent-2 offered better recovery and improved peak shapes. The required method sensitivity of nitrosodimethylamine (LOQ 0.74 ng/mL) and nitrosodiethylamine (LOQ 0.37 ng/mL) for the nitrosamine impurities were achieved using an optimized test concentration of Metformin hydrochloride at 45.7 mg/mL.

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.

A validated stability-indicating reversed-phase-HPLC method for dipyridamole in the presence of degradation products and its process-related impurities in pharmaceutical dosage forms.

In this study, we developed and validated a method to determine dipyridamole-related impurities in pharmaceutical dosage forms using the reversed-phase-HPLC technique. All impurities were separated on a YMC pack C8 (150 mm × 4.6 mm, 3.0 µm) analytical column using a suitable mobile phase. Mobile phase A was 10 mM concentration of phosphate buffer (pH adjusted to 4.7 by adding diluted orthophosphoric acid) and mobile phase B was buffer:acetonitrile:methanol (at the ratio of 30:40:30 v/v). The optimized chromatographic conditions used in the experiment were as follows: flow rate, 1.0 mL/min; injection volume, 10 µL and column temperature, 35°C. Chromatographic detection was performed at 295 nm. The stressed samples were analyzed for degradation under acidic, basic, peroxide, water hydrolysis, and physical degradation conditions. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines, and found to be specific, linear, accurate and have a robust stability-indicating nature. The method showed excellent linearity from limit of quantification (LOQ) to 150% level of concentrations for all impurities. The correlation coefficient (r2 ) for all impurities was between 0.995 and 0.999. The recovery study was performed from LOQ to 150% level concentrations, with mean recovery values between 92.9% and 103.2%, respectively. The developed method can be used to determine dipyridamole and its relative impurities. The degradation and validated study results indicate its stability-indicating nature. Therefore, the method can be used in pharmaceutical research and development and quality control departments.

Determination of progesterone (steroid drug) in the semi-solid dosage form (vaginal gel) using a stability-indicating method by RP-HPLC/PDA detector.

A simple stability-indicating method was developed and validated for the determination of progesterone (a steroid drug) in the semi-solid dosage form. All the impurities were separated from the main compound with a simple stationary phase (Eclipse XDB, C8, 150 × 4.6 mm, 5 µm). The mobile phase A contained phosphate buffer and acetonitrile in the ratio of 90:10, v/v, and mobile phase B contained purified water and acetonitrile in the ratio of 10:90, v/v. The optimized chromatographic conditions were as follows: flow rate, 1.0 mL min-1 ; UV detection, 241 nm; injection volume, 10 µL; and the column temperature, 30°C. The method was validated as per the current ICH Q2 guidelines. The recovery study and linearity ranges were established from 50 to 300% optimal concentrations. The method validation results were found between 98 and 102% for accuracy and r2  = 0.999 for linearity. Forced degradation in hydrolytic, oxidative, thermolytic, and photostability conditions was performed, and the stability indicating nature of the method was proved. Based on the validation and forced degradation results, the current method was found to be specific, precise, accurate, linear, robust, and stability-indicating method. The developed method was cost effective and easy to handle for quality control analysis.

Low-level determination of 4-chlorobutyl-(S)-[4-chloro-2-(4-cyclopropyl-1,1,1-trifluoro-2-hydroxy-but-3yn-2-yl)phenyl] carbamate (4-CTHC) in efavirenz drug substance by LC-MS.

A rapid, simple, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS) test method was developed and validated for the trace level determination of 4-chlorobutyl-(S)-[4-chloro-2-(4-cyclopropyl-1,1,1-trifluoro-2-hydroxy-but-3yn-2-yl)phenyl] carbamate (4-CTHC). 4-CTHC is a potential genotoxic impurity in efavirenz drug substance and the acceptable level is 2.5 µg ml-1 with respect to analyte concentration according to ICH M7(R1) Multidisciplinary Guidelines M7(R1). The LC-MS/MS analysis of 4-CTHC impurity was carried out on a Kinetex C18 (150 × 4.6 mm, 5.0 µm) column. In this test procedure, the mobile phase was prepared with buffer (0.1% formic acid in water) and acetonitrile in the ratio of 1:3 (v/v). The method set flow rate was 0.4 ml min-1 . The method was developed with a short run time of <10 min. Selective ion monitoring acquisition mode and negative polarity electrospray ionization mode were used as an MS method to quantify genotoxic impurities at 422.25 Da. The method showed linearity in a range of 0.64-3.71 µg ml-1 with a correlation coefficient of 0.9992. The RSD for intra-day and inter-day precision was found to be <5%. The method's accuracy was in the range of 106.5-112.4% for the genotoxic impurity of 4-CTHC. The procedure was validated as per the current ICH Q2 (R1) guidelines and proved suitable for stability testing in the quality control laboratory for pharmaceutical preparations.