HPLC

Artesunate (1) (ART), also called as dihydroartemisinin-12-α-succinate, it is a semisynthetic peroxide-bridged sesquiterpene lactone compound derived from Artemisinin, the bioactive component of the Chinese medicinal herb called Artemisia annua.  An accurate, simple and precise HPLC method was developed and validated for forced degradation studies of drug Artesunate .The column was used C18 column (150X4.6mmX5µm ) column  by isocratic elution. The mobile phase composition consisted of Acetonitrile Water and trifluoroacetic acid (TFA) in the ratio of 55:45:0.1 v/v. The analysis was performed at a 1ml/min flow rate. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and lower limit of quantification (LOQ) were determined according to International Conference for Harmonization ICH Q2 (R1) guidelines.

In this review articles, the development, formulation, and manufacture of drugs, analytical method development & validation play a critical role. Methods are developed for ensuring purity, identity, potency, and performance of pharmaceutical products. Methods should be applied to the extent that they are sufficient for their intended purpose. Throughout the life cycle of a drug product and substance, a range of activities are associated with developing and validating methods. An objective of method validation is to prove that the procedure can be used as intended. Once the method is developed, validation is performed. Different national and international committees have defined the parameters for method validation. The International Conference on Harmonization attempted to harmonize pharmaceutical applications. In accordance with the ICH, other organizations define Linearity, Selectivity/Specificity, Range, Accuracy, Precision (repeatability, intermediate precision, and reproducibility), Limit of quantitation, Limit of detection, Ruggedness, and Robustness.

The quantitative analysis of bulk materials, drug formulations, drug products, impurities, and biological products containing pharmaceuticals and their metabolites is challenging in the field of pharmaceutical research. It is also complicated to choose the best method. Pharmacokinetic studies frequently make use of quantitative or qualitative studies of a drug and its metabolite. Developing a generic product with quantitative equivalence, which increases regulatory flexibility, seems to be the ultimate aim. Knowing the exact components of reference items and their concentrations is extremely helpful when developing generic formulations. The quantitative composition of the dosage forms is kept secret by the innovators. In such a situation, the quantitative formula of the dosage form is being decoded by generic manufacturers through reverse engineering. To quantify them, we need reliable, non-destructive analytical tools. In this article, we covered excipient quantification techniques, analytical data reports, challenges, and applications.

Reverse phase high performance liquid chromatographic method for the estimation degradation impurities of Mebeverine HCl in bulk drug is illustrated. The method consists of; Mobile phase B: Acetonitrile and Mobile phase A: 0.051 M Phosphate buffer (pH-4.5) in gradient pump mode. A linear response was observed in the range of 0.8 to 48 µg/ml with a regression coefficient of 0.999 for both Mebeverine HCl and Veratric acid. The force degradation study was performed to determine the degradation pathways which also proves the specificity of the method. The method is cost effective as well as sensitive. Validation parameters were carried out as per the guidelines of International Conference for Harmonization.

A simple Reverse phase liquid chromatographic method has been developed and subsequently validated for estimation of lansoprazole in tablet dosage form. The separation was carried out using a mobile phase consisting of Methanol and 0.1% OPA (Ortho Phosphoric Acid) in the ratio of 70:30. The column used was C18 and 250 mm length with flow rate of 1.2 ml / min using UV detection at 285nm. The described method was linear over a concentration range of 10-50 μg/ml for the assay of Lansoprazole. The retention time of Lansoprazole was found to be 6.6 min, and all the results of analysis were validated statistically. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of Lansoprazole in tablet dosage form and in its pharmaceutical dosage forms.

A rapid liquid chromatographic tandem mass spectrometry (LC-MS/MS) assay for the measurement of metoclopramide level in human plasma was developed and validated. One ml plasma samples containing metoclopramide and 0.25 µg of loratadine as internal standard (IS) were extracted with 5 ml tert-butyl methyl ether and reconstituted in 80 µl of acetonitrile. Analysis was performed on reversed phase Atlantis dC18 column using a mobile phase of 0.4% formic acid (pH=3.0 ± 0.05) and acetonitrile (20:80, v:v) delivered at a flow rate of 0.25 ml/minute. Analytes were quantified multiple reaction monitoring in positive ion mode with transition mass to charge ratio (m/z) of 299.8→226.9 and 383.4→337.2 for metoclopramide and IS, respectively. Retention times of metoclopramide and IS were around 1.4 and 2.1 minutes, respectively. No significant matrix effect was observed on metoclopramide and IS peaks. Detection limit of metoclopramide in plasma was 0.3 ng/ml. Relationship between metoclopramide level and peak area ratio of metoclopramide / IS was linear (R2 ³ 0.9964) in the range of 0.5–100 ng/ml and inter-day coefficient of variations (CV) and absolute bias were ≤ 12.0% and ≤ 6.0%, respectively. Mean extraction recoveries for metoclopramide and the IS were 91% and 88%, respectively. The method was applied to assess stability of metoclopramide under various conditions generally encountered in the clinical laboratory. Stability of metoclopramide was ≥ 94% and ≥ 95% after 24 hours at room temperature or 48 hours at -20 ºC, respectively, in processed samples and 100% and ≥ 99% after 24 hours at room temperature or 12 weeks at -20 ºC, respectively, in unprocessed samples.

A gradient reversed phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for the determination for related substances of Dapagliflozin drug substance. Chromatographic separation of Dapagliflozin from its process and degradation related substances was achieved on YMC Pack Pro C18, 250mm × 4.6mm 5m i.e A stainless steel column 250 mm long, 4.6 mm internal diameter filled with Octadecyl silane chemically bonded to porous silica particles of 5 mm diameter maintained column oven temperature at 25°C. Orthophosphoric acid buffer is mobile phase A and acetonitrile is mobile phase B. Wavelength for UV detection: 225nm, flow rate: 0.8 ml/min and Injection volume: 20µl. The developed method suitability was checked and validated as per ICH guidelines for specificity, linearity, accuracy, precision, limit of quantification, limit of detection robustness and ruggedness experiments. Dapagliflozin drug substance was subjected to stress conditions of thermal, hydrolysis, humidity, peroxide and photolytic to observe the degradation products. Limit of detection of each RS is less than 0.008%w/w indicating that the developed method is highly sensitive. The experiment results are given in detailed in this research article.

A rapid liquid chromatographic tandem mass spectrometry (LC-MS/MS) assay for the measurement of loratadine level in human plasma was developed and validated. One ml plasma samples containing loratadine and 0.18 µg of metoclopramide as (internal standard, IS) were extracted with 5 ml tert-butyl methyl ether and reconstituted with 80 µl of acetonitrile. Analysis was performed using a reversed phase Atlantis dC18 column and a mobile phase consisting of 0.4% formic acid and acetonitrile (20:80, v:v) and delivered at a flow rate of 0.25 ml/min. The eluents were monitored using electrospray ionization in the positive ion mode with transition mass to charge ratio (m/z) at 383.4→337.2 and 299.8→226.9 for loratadine and IS, respectively. The retention times of the IS and loratadine were around 1.53 and 2.33 min, respectively. Mean matrix effect was measured as -11.4% for loratadine and -14.4% for the IS. Detection limit of loratadine in plasma was 0.3 ng/ml. The relationship between loratadine concentration in plasma and the peak area ratio of loratadine / IS was linear (R2 ³ 0.9945) in the range of 0.5–100 ng/ml, and the intra- and inter-day coefficient of variations (CV) were ≤ 11.3%. Mean extraction recoveries for loratadine and the IS were 87% and 91% respectively, whereas accuracy (relative recovery) ranged from 99% to 111% quality control samples and from 93% to 105%  using back- calculated concentrations. The method was applied to assess the stability of loratadine under various conditions generally encountered in the clinical laboratory. Stability for processed samples (24 hours at room temperature, 48 hours -20 ºC) and unprocessed samples (24 hours at room temperature, 12 weeks -20 ºC) was ≥ 94%. Key words: Loratadine, Metoclopramide, Human plasma, HPLC  

A simple and reproducible method was developed for carbimazole by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Carbimazole was separated on C18 column [4.6x250mm, particle size 5μm] at the UV detection of 291nm. Methanol and OPA (0.1%) was used as a mobile phase with various ratios and flow rates, eventually 80:20 v/v Methanol and OPA (0.1%) was being set with the flow rate of 0.7mL/min. The statistical validation parameters such as linearity, accuracy, precision, inter-day and intra-day variation were checked, further the limit of detection and limit of quantification of carbimazole concentrations were found to be within the limits. Recovery and assay studies of carbimazole were within 99 to 102% indicating that the proposed method can be adoptable for quality control analysis of carbimazole.

A simple, specific, accurate and precise reverse phase high performance liquid chromatographic method was developed and validated for the estimation of Venlafaxine hydrochloride in pure and Pharmaceutical dosage form. Kromasil C18 column having 150 mm x 4.6 mm internal diameter, 5 µm particle sizes in isocratic mode with mobile phase containing mixture of methanol and water in the ratio of 65:35 v/v was used. The flow rate was 1.0 ml/min and effluents were monitored at 225 nm. The retention time for Venlafaxine was 2.424 min. The method was validated for linearity, accuracy, precision, specificity, limit of detection, limit of quantification and robustness. Limit of detection and limit of quantification were found 2.97µg/ml and 9.92 µg/ml respectively and recovery of Venlafaxine from tablet formulation was found 100.4 %. The proposed method was successfully applied for the quantitative determination of Venlafaxine in tablet dosage form.