Method validation

Lasmiditan Hemisuccinate (LDT) is an innovative medication designed for the treatment of acute migraines, operating by activating 5-HT1F receptors found within the central nervous system. The synthesis of LDT necessitates the use of various solvents. According to regulatory standards, assessing the levels of residual solvents in drug substances is imperative in API (Active Pharmaceutical Ingredients). Present study concentrates on the evaluation and validation of a method for quantifying residual solvents like methanol, acetone, isopropyl alcohol, dichloromethane, methyl ter. butyl ether, n-hexane, ethyl acetate, n-heptane in LDT. Analysis was conducted using a DB-1 capillary column, measuring 60 meters in length with an internal diameter of 0.32 mm and a film thickness of 5 µ. The oven temperature was set to 65°C for 12 minutes, followed by a ramping rate of 15°C per minute to reach 85°C, where it was held for 5 minutes. Subsequently, a second ramping phase increased the temperature at a rate of 10°C per minute until it reached a final temperature of 220°C, which was maintained for 5 minutes. The injector temperature was maintained at 200°C and nitrogen was utilized as the carrier gas with 1-methyl-2-pyrrolidinone (NMP) diluent serving as the sample solvent. A suitable sensitive and robust HSGC-FID method is developed for quantitation of residual solvents with flame ionization (FID) detector. The evaluated method can applied to analyse of solvents present in a various range of APIs, intermediates.

This study presents the analytical assay method development and validation of Itraconazole in an ointment dosage formulation by using reverse phase HPLC. The assay method development was carried out by using C18 column. In the beginning, the study was carried on Itraconazole API by taking USP method as a reference. By changing few chromatographic parameters, a symmetrical peak and a satisfactory result could able to achieve. Changes done as per USP chapter “allowable adjustment to United States Pharmacopeia method”.  The aim was to achieve an advanced chromatographic conditions on HPLC system with C18 column (150 × 4.6 mm, 5µm particle size) using mobile phase composed of acetonitrile and neutral buffer. The separation was achieved at different gradient flows for alternative methods.  The ideal wavelength was calibrated by using PDA detector and the same wavelength was used for UV- visible detector. The assay method developed was also validated with full agreement with present regulatory guidelines by applying well developed analytical method validation techniques and means which includes the parameters such as linearity, accuracy, method precision, specificity with force degradation, robustness, solution stability, system suitability. The method shows linearity over a concentration range of 10µg/ml to 250µg/ml with r²=0.999 and thus this represents the method is efficient to provide good detector response. The lower limit of detection and quantification was achieved by carrying out the studies like LOD and LOQ and the results were found to be 10µg/ml and 5µg/ml respectively.

A simple, precise, novel stability-indicating method was developed and validated for the estimation of Bendamustine in pharmaceutical forms. The Assay method was developed by using a detector set at 235nm and reverse phase c18 column (Inertsil ODS-2,150mmx4.6mm, 5 μm).The mobile phase consisted of 0.1%Trifluoroacetic acid: Acetonitrile 70:30 (v/v) in isocratic flow rate at 1.5mL/min for 10minutes.Sample cooler temperature and Column oven temperature were monitored in the method at 5°c and 25°c Respectively. The retention time of bendamustine was 4.8min.The proposed method was achieved and validated with respect to precision, specificity, accuracy, linearity, range, Analyte Stability, Filter variability, Robustness. The %Recovery of Bendamustine at Each level in formulations was found to be in a range of 98-102%. The linearity of the assay was established in the range of 5.053 to 75.793 µg/mL with correlation coefficient (R2) > 0.9999. The limits of detection and quantification were 0.03 and 0.05mg/mL, respectively. Specificity of the assay demonstrates no interference from and degrading products formed by alkaline, acidic, oxidative, UV light and high temperature (Thermal) conditions. This method showed more accurate, rugged and reliable determination of Bendamustine for drug stability assay in pharmaceutical studies.

In the present paper, the authors described a novel Liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of montelukast in human plasma using montelukast d6 as internal standard (IS). After solid phase extraction (SPE), the analyte and the IS were chromatographed on a C18 columns using a isocratic mobile phase composed of acetonitrile–5mM ammonium acetate (80:20, v/v) pumped at a flow rate of 0.8mL/min. The proposed method was validated in the range of 5.01–599.91 ng/mL as per the US FDA guidelines. Precision and accuracy results were calculated using five successful calibration curves. Analyte stability in true samples and in plasma samples under different conditions were established and results met the acceptance criteria. The chromatographic run time was set at 3 min, which makes the proposed method is high through put. The method was successfully applied to a pharmacokinetic study in healthy South Indian male subjects under fasting condition.

The authors proposed a simple, rapid and sensitive liquid chromatography / tandem mass spectrometry assay method for the determination of nateglinide in human plasma using carbamazepine as internal standard (IS). Analyte and the IS were extracted from the human plasma via liquid-liquid extraction using ethyl acetate. The chromatographic separation was achieved on a C18 column by using a mixture of 0.1% formic acid buffer –acetonitrile buffer (20:80, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The calibration curve obtained was linear (r2 ³ 0.99) over the concentration range of 10.0–10005 ng/mL. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The proposed method was found to be applicable to pharmacokinetic studies.

A simple, rapid, precise, and economical spectrophotometric method has been developed for quantitative analysis of Rilpivirine hydrochloride (RILH) in manufactured tablet formulations. The initial stock solution of RILH was prepared in dimethyl formamide: acetonitrile solvent and subsequent dilution were done in acetonitrile. The standard solution of RILH in acetonitrile showed absorption maxima at 281.6 nm. The drug obeyed Beer–Lambert’s law in the concentration range of 1–16 μg/mL with coefficient of correlation (R2) was 0.9999. It showed coefficient of variation below 2 % in intra-run and inter-run precision.  The results of analysis have been validated as per ICH guidelines. The method can be adopted in routine analysis of RILH in bulk and tablet dosage form and it involves relatively low cost solvents and no complex extraction techniques.