Development and validation of liquid chromatography-tandem mass spectrometry method for the estimation of a potential genotoxic impurity 2-(2-chloroethoxy)ethanol in hydroxyzine.

2-(2-Chloroethoxy)ethanol (CEE) belongs to the so-called cohort of concerns which were classified as highly potent mutagenic carcinogens by the World Health Organization. It is widely used in the synthesis of the essential anti-histamine drug hydroxyzine. In addition, it is used as a primary solvent in dyes, nitrocellulose, paints, inks and resins. Owing to its potential genotoxicity, an efficient liquid chromatography-tandem mass spectrometry method was developed for the quantitative estimation of CEE traces in an active pharmaceutical ingredients and in tablet dosage forms of hydroxyzine-free base. The chromatographic separation was achieved on a C18 column using a gradient elution mode with a binary solvent system (ammonium formate and methanol). Mass detection was performed for CEE using a positive mode with selected ion monitoring technique at m/z value of [M + NH4 ]+ . The developed method was validated as per the International Conference on Harmonizaiton guidelines. The quantitation limit, linearity and recoveries were found to be 0.56 ppm, 0.56-7.49 ppm (r2 > 0.9985) and 93.6-99.3%, respectively. The proposed method was highly compatible and was used effectively to estimate CEE traces in different stages of drug synthesis and in tablet dosage forms of hydroxyzine for routine and stability testing.

Stability-indicating liquid chromatography method development and validation for impurity profiling of montelukast sodium in bulk drug and tablet dosage form.

Montelukast sodium (MLS) is a leukotriene receptor antagonist drug used in the treatment of asthma, bronchospasm, allergic rhinitis and urticaria. A reversed-phase high performance liquid chromatography method was developed to separate, identify and quantitative determination of MLS and its eight known organic impurities in tablet dosage form using a C18 column and mobile phases consisting of a gradient mixture of pH 2.5 phosphate buffer and acetonitrile. The stability-indicating character of the developed method was proven using stress testing (1 m HCl at 80°C/30 min, 1 m NaOH at 80°C/30 min, H2 O at 80°C/30 min, 3% H2 O2 at 25°C/1 min, dry heat at 105°C/10 h and UV-vis light/4 days) and was validated for specificity, quantitation limit, linearity, precision, accuracy and robustness. For MLS and its eight known impurities, the quantitation limits, linearity and recoveries were 0.015-0.03 µg/ml, correlation coefficient > 0.997 (R2 > 0.995) and 85.5-107.0%, respectively. The developed chromatographic method is suitable for impurity profiling and also for assay determination of MLS in bulk drugs and pharmaceutical formulations. The mass values (m/z) of newly formed degradation products (DP1 and DP2) of montelukast sodium were identified using liquid chromatography-mass spectrometry.

Stability Indicating LC Method Development for Hydroxychloroquine Sulfate Impurities as Available for Treatment of COVID-19 and Evaluation of Risk Assessment Prior to Method Validation by Quality by Design Approach.

A quality by design-based stability indicating HPLC method has been developed for hydroxychloroquine sulfate impurities. The optimized HPLC method can detect and quantify the hydroxychloroquine sulfate and related organic impurities in pharmaceutical solid oral dosage forms. Nowadays, for the quantification of impurities in drug products demands more comprehensive way of analytical method development. The quality by design approach allows the assessment of different analytical parameters and their effects with minimum number of experiments. A highly sensitive and stability indicating RP-HPLC method was developed and evaluated the risk assessment prior to method validation. The chromatographic separation was achieved with X-terra phenyl column (250 × 4.6 mm, 5 µm) using phosphate buffer (0.3 M and pH 2.5). The gradient method flow rate was 1.5 mL min-1 and UV detection was made at 220 nm. The calibration curve of hydroxychloroquine sulfate and related impurities were linear from LOQ to 150% and correlation coefficient was found more than 0.999. The precision and intermediate precision % RSD values were found less than 2.0. In all forced degradation conditions, the purity angle of HCQ was found less than purity threshold. The optimized method found to be specific, accurate, rugged, and robust for determination of hydroxychloroquine sulfate impurities in the solid oral dosage forms. Finally, the method was applied successfully in quality control lab for stability analysis.

QbD-based development of an extraction procedure for simultaneous quantification of telmisartan, amlodipine besylate and chlorthalidone in combination complex matrix formulation.

The main objective of this study was to establish an efficient extraction procedure for the estimation of telmisartan, amlodipine and chlorthalidone from their combination in sample matrix using an analytical quality by design approach. Initial screening studies were performed for optimization of a suitable diluent to extract active components from sample matrix. Further, the same study was extended for the identification of critical method attributes and the factors affecting the analytical target profile. This study also explains the rugged and robust quantitative determination of combinations drugs with a shorter run time. The design of experimental studies confirms that the current center point parameters are well suited to recoveries. The chromatographic separation was achieved with an X-Terra RP8, 150 × 4.6 mm, 3.5 µm column with an isocratic mobile phase (mixture of 20 mm aqueous ammonium acetate and acetonitrile). To demonstrate the stability-indicating nature of the optimized method, forced degradation studies were conducted and proved. The optimized method was validated according to International Conference on Harmonization guidelines.

Development and validation of a generic RP-HPLC PDA method for the simultaneous separation and quantification of active ingredients in cold and cough medicines.

A novel generic reverse phase high performance liquid chromatography (RP-HPLC) method is developed and validated for simultaneous determination of seven pharmaceutically active ingredients, namely, acetaminophen, dextromethorphan, doxylamine, phenylephrine, guaifenesin, caffeine and aspirin. All seven ingredients were quantified in soft gel, syrup and tablet formulations of the over-the-counter US-marketed products, as per the guidelines of the International Conference on Harmonization. The separation was achieved in a 16 min run time on an Agilent Zorbax Phenyl column using a gradient method with two mobile phases. Mobile phase A was 0.15% trifluoro acetic acid in purified water and while mobile phase B was a mixture of acetonitrile and methanol (750:250 v/v) with 0.02% trifluoro acetic acid. The flow rate was 1.0 mL min-1 and injection volume was 10 µL. Detection was performed at 280 nm using a photodiode array detector. As part of the method validation, specificity, linearity, precision and recovery parameters were verified. The concentration and area relationships were linear (R2 > 0.999), over the concentration ranges 20-120 µg mL-1 for acetaminophen, 75-450 µg mL-1 for dextromethorphan, 31.25-187.5 µg mL-1 for doxylamine, 25-150 µg mL-1 for phenylephrine, 25-150 µg mL-1 for aspirin, 6.5-39 µg mL-1 for caffeine and 12-72 µg mL-1 for guaifenesin. The relative standard deviations for precision and intermediate precision were <1.5%. The proposed RP-HPLC generic method is applicable for routine analysis of cold and cough over-the-counter products.

Stability-indicating HPLC method for simultaneous quantification of 14 impurities in excedrin tablet formulations and identification of new impurity by LC-MS in accelerated stability studies.

We developed novel stability-indicating HPLC method for simultaneous estimation of 14 impurities in excedrin tablet, a formulation with a combination of acetaminophen, aspirin, and caffeine. In addition, a new impurity that was generated through degradation of aspirin at high temperatures during the accelerated stability conditions was positively identified and confirmed, using liquid chromatography-mass spectrometry technique. The HPLC method was optimized using the Inertsustain C18 , 250 × 4.6 mm, 5.0 µm column, employing simple gradient method. Forced degradation studies were performed under acidic, basic, oxidative and thermal conditions to prove the scope and stability-indicating the nature of the method. The optimized method was validated as per the International Conference on Harmonization guidelines. The HPLC method showed linearity from LOQ concentration to 21 µg mL-1 . Precision and intermediate precision values were <5% RSD. The validated HPLC method is currently applied for the routine testing of excedrin tablet formulations in quality control laboratories.