pharmaceutical formulation

In the last few decades, the hyphenated analytical techniques including liquid chromatography (LC) in combination with a mass spectrometer (MS) i.e., LC/MS, have made a major impact in pharmaceutical drug discovery and development. LC-MS hase been routinely used in pharmaceutical formulation development for drug substance and drug product characterization, molecular weight and fragmentation patterns determination, breakdown studies, and to identify impurities and degradation products. Recent advancements in LC-MS instrumentation, have allowed the technique to be implemented in several indications i.e., cancer, cardiovascular, respiratory, neurological, rare diseases etc. across preclinical (in vitro and in vivo) and clinical projects to evaluate pharmacokinetic and pharmacodynamic factors. LC-MS has overcome the sensitivity and dynamic range challenges to successfully identify and characterize drug molecules to help projects that use small molecules, biologics, and gene and cell therapy/editing platforms as drug modalities. This technique may provide several advantages over other analytical approaches including specificity, multiplexing, precision to quantify drug analyte or a biomarker in a variety of matrices like blood (plasma/serum), tissue, cerebrospinal fluid, urine, cells etc. In this review, we have highlighted LC-MS applications to study pharmaceutical formulations, pharmacokinetics and pharmacodynamics involving small molecule modality.

Two sensitive and reproducible methods are described for the quantitative determination of Glipizide and Metformin Hydrochloride in presence of its degradation products. The first method was based on high performance liquid chromatographic (LC) separation of the drug from its degradation products on the reversed phase, Cosmosil® column [C18 (5 mm, 4.6 x 150 mm, i.d.)] at ambient temperature using a buffer consisting of 10 mM potassium dihydrogen phosphate (pH adjusted to 2.5 with diluted o- phosphoric acid) and acetonitrile 50: 50 v/v as optimized mobile phase in a gradient program. The flow rate was 0.7 ml min-1 and quantitation was achieved UV determination at 225 nm based on peak area with linear calibration curves at concentration range 10-25 µg ml-1 and 20-50 µg ml-1 respectively. The second method was based on   high performance thin-layer chromatographic (HPTLC) separation followed by densitometric measurement of spots at 216 nm. The separation were carried out on Merck HPTLC aluminium sheets of silica gel 60 F 254 using Toluene: Methanol: Ethyl acetate: Formic acid in the ratio of (3:6:3:0.2, v/v/v/v), as mobile phase. This system was found to give compact spots for Glipizide and Metformin hydrochloride after double development (retention factor, RF 0.08 ± 0.02 and RF 0.74 ± 0.02 respectively). The second order polynomial regression analysis data was used forth regression line in the range of 200-1400 ng spot-1 and 200-1400 ng spot-1 respectively. Both the method has been successively applied to pharmaceutical formulation. No chromatographic interference from the tablet exicipients was found. Both the methods were validated in terms of precision, robustness, recovery, limits of detection and quantitation.

A simple, new, precise, accurate and reproducible RP-HPLC method for simultaneous estimation of mefenamic acid, ethamsylate and tranexamic acid in bulk and pharmaceutical formulations. Separation of mefenamic acid, ethamsylate and tranexamic acid was successfully achieved on a Kromasil C8 (250 mm x 4.6mm x 5µ ) in an isocratic mode utilizing Ammonium acetate buffer and methanol (60:40 v/v) at a flow rate of 1.0 mL/min. The method was validated according to ICH guidelines for linearity, sensitivity, accuracy, precision, specificity and robustness. The response was found to be linear in the drug concentration range of 25-75 mg/mL for mefenamic acid ,25-75 mg/mL for ethamsylate 50-150 mg/mL for tranexamic acid. The correlation coefficient was found to be 0.9997 for both the drugs. The limit of detection (LOD) was 0.158,0.2183 and 0.321 for  mefenamic acid, ethamsylate and tranexamic acid respectively. The limit of quantification (LOQ) was 0.527,0.7278 and 1.071 for mefenamic acid, ethamsylate and tranexamic acid respectively. The relative standard deviation (RSD) of six replicates is less than 2%. This HPLC method is applied successfully to the simultaneous quantitative analysis of mefenamic acid, ethamsylate and tranexamic acid in commercial tablets.

A simple, precise, accurate and reproducible RP-HPLC method for simultaneous estimation of clarithromycin, pantoprazole and Metronidazole in bulk and pharmaceutical formulations. Separation of clarithromycin, pantoprazole and Metronidazole was successfully achieved on a YMC Pack Pro C18 (250 mm x 4.6mm x 5µ) in an isocratic mode utilizing sodium dihydrogen phosphate buffer and methanol (65:35 v/v) at a flow rate of 1.0 mL/min. The method was validated according to ICH guidelines for linearity, sensitivity, accuracy, precision, specificity and robustness. The response was found to be linear in the drug concentration range of 31-93.75 mg/mL for clarithromycin and 5-15 mg/mL for pantoprazole and 50-150 mg/mL for Metronidazole. The correlation coefficient was found to be 0.999 for both the drugs. The limit of detection (LOD) was 0.228, 0.0309 and 0.743 for clarithromycin, pantoprazole and Metronidazole respectively. The limit of quantification (LOQ) was 0.758, 0.1030 and 2.475 for clarithromycin, pantoprazole and Metronidazole respectively. The relative standard deviation (RSD) of six replicates is less than 2%. This HPLC method is applied successfully to the simultaneous quantitative analysis of clarithromycin, pantoprazole and Metronidazole in commercial tablets.

A new simple, precise, accurate and reproducible RP-HPLC method for simultaneous estimation of gentamicin and clobetasol in bulk and pharmaceutical formulations. Separation of gentamicin and clobetasol was successfully achieved on a Zorbax C18 (150 mm x 4.6mm x 5µ ) in an isocratic mode utilizing disodium hydrogen phosphate buffer and methanol (60:40 v/v) at a flow rate of 1.0 mL/min. The method was validated according to ICH guidelines for linearity, sensitivity, accuracy, precision, specificity and robustness. The response was found to be linear in the drug concentration range of 0.1-0.30 mg/mL for gentamicin and 0.05–0.15 mg/mL for clobetasol. The correlation coefficient was found to be 0.9997 for both the drugs. The LOD and LOQ for gentamicin were found to be 0.3525 µg/mL and 1.1751 µg/mL respectively. The LOD and LOQ for clobetasol were found to be 0.1938 µg/mL and 0.6460 µg/mL respectively.  The proposed method was found to be good percentage recovery for gentamicin and clobetasol, which indicates that the proposed method is highly accurate. The specificity of the method shows good correlation between retention times of standard solution with the sample solution. Therefore, the proposed method specifically determines the analyte in the sample without interference from excipients of pharmaceutical dosage forms.