High performance liquid chromatography

HPLC is the dominant separation technique in modern pharmaceutical and biomedical analysis because it results in highly efficient separations and in most cases provides high detection sensitivity. Most of the drugs in multi component dosage forms can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision and ease of automation in this method. HPLC methods development and validation play important roles in new discovery, development, manufacture of pharmaceutical drugs and various other studies related to humans and animals. It is the most versatile, safest, dependable and fastest chromatographic technique for the quality control of drug components. This article was prepared with an aim to review different aspects of HPLC, such as principle, types, instrumentation, validation of HPLC and application. 

Process analytical technology (PAT) has been defined as a mechanism to design, analyze and control pharmaceutical manufacturing processes through measurement of critical process parameters which affect critical quality attributes. PAT checks the quality of raw material attributes both physically and chemically (i.e. at off-line, on-line, in-line). PAT involves a shift from testing the quality of building to the quality of products by testing at several intermediate steps. PAT saves a huge amount of time and money required for sampling and analysis of products. Pharmaceutical companies face many challenges and problems while implementing PAT into their new and pre-existing manufacturing processes. The potential for improved operational control and compliance resulting from continuous real time quality assurance was highlighted as a likely benefit that would result from PAT implementation. In this paper, we will start with brief PAT concepts, Introduction, PAT tools, Pat implementation and a review of their application in the wider pharmaceutical industry.

A simple reverse phase specific and selective high performance liquid chromatographic method for determination of Exemestane in Exemestane tablets has been developed and validated with Isocratic elution and UV detection. Chromatographic separation was achieved by using Hypersil, C-18, 150 X 4.6mm, 5µ column with a mixture of Acetonitrile and Purified water in the ratio of 35:65, filter and degas the mobile phase same is used as diluent. Detection was at 249 nm. By this method all known and unknown Impurity & Degradation products are well separated from Exemestane main peak.  Peak purity factor for Exemestane peak is not less than 99.0%. Both the Precision (System Precision, Method Precision, Intermediate Precision) and Linearity were within acceptable range. Response was a linear function between concentration and area of peak over the   range from 50% to 150% of assay concentration for Exemestane. It can be concluded that the Exemestane Peak is found to be degraded more in acid, alkali and peroxide stress condition. Exemestane Peak purity factor was more than 99.0% and all degradation product formed were well separated from Exemestane Peak. By this it was found that this method is robust and system suitability test was established and related parameters are recorded. This method is validated hence this method can be used for routine analysis of stability sample.

Clobazam is an antiepileptic drug. There have been very less number of analytical methods developed for estimation of Clobazam in pure bulk form and in dosage form. In the present research a simple, accurate, precise and cost effective High performance liquid chromatographic method for the estimation of Clobazam, in bulk and pharmaceutical dosage form was carried out. HPLC method was developed on a Symmetry C-8 (4.6×150mm), 3.5µm particle, reversed-phase column. The mobile phase was acetonitrile: phosphate buffer (0.05M, pH- 4.5), 60:40 (v/v) at a flow rate of 0.8ml/min. The eluate was monitored at 231 nm. The method was validated reaching satisfactory results for selectivity, precision and accuracy. The retention time of the drug was found to be 3.38min in the mobile phase, acetonitrile: 0.05M potassium dihydrogen orthophosphate buffer (pH-4.5) 40: 60 (v/v). A linear response was observed in the range of 20-60µg/ml with a regression coefficient of 0.999. Validation parameters were carried out as per the guidelines of International Conference for Harmonization (ICH). This method can be used in the industries for determination of Clobazam to analyze the quality of formulation without interference of the excipients.

Terbinafine hydrochloride (TFH), is a potent antifungal agent of the allylamine class with broad spectrum activity against yeasts, dimorphic fungi, molds, and dermatophytes. A new, simple, rapid, selective, precise, accurate, and stability indicating high performance liquid chromatographic method has been developed for the determination of terbinafine hydrochloride (TFH) in pharmaceuticals. The assay was performed using an Zorbax Eclips XDB C-18 (3.5 µm, 4.6 × 150 mm i.d.,) column at 30ºC temperature with UV-detection at 222 nm. A mobile phase consisting of buffer (1000 mL water, 2 mL triethylamine, pH 3.4 adjusted with trifluoroacetic acid.), isopropyl alcohol and methanol (40:12:48, v/v/v), was used in the assay at a flow rate of 1 mL min-1. The method was validated and system suitability parameters were investigated. An excellent linearity was obtained over the concentration range 1 - 80 μg mL-1 TFH with limits of detection (LOD) and quantification (LOQ) values of 0.3 and 1.0 mg mL-1, respectively. The proposed method were applied successfully to the determination of TFH in tablets. The results obtained were in good agreement with those obtained by a reference method, with high precision and without any detectable interference from tablets excipients. The validity and reliability of the proposed methods were further assessed by the recovery studies via a standard addition method. In addition, forced degradation of TFH was conducted in accordance with the ICH guidelines. Acidic, basic water hydrolysis, thermal stress, peroxide and photolytic degradation were used to assess the stability indicating power of the method. Slight degradation was observed during oxidative degradation and no degradation was observed under other stress conditions.

Ultra performance liquid chromatography (UPLC) system involves significant technological advances in particle size performance, system optimization, data processing, detector design and control. When all brought together, the specific achievements in each area have created a step-function progress in chromatographic performance. This new technique of analytical separation science uses the principles and practicality of HPLC with increasing the attributes of speed, sensitivity and resolution. Now a day’s pharmaceutical industries are in search of new ways to reduce cost and time for analysis of drugs. Analytical laboratories are not exception in this trend. Ultra high performance liquid chromatography (UPLC) with better resolution, assay sensitivity and high sample throughput allows a greater number of analysis to be performed in a shorter period of time and it also impart cost effective advantage over HPLC analysis. So that conventional assay was transferred and optimized for UPLC system. This review introduces the theory of UPLC, and involves some of the most advanced work in the field.