Chromatographic method development for the detection of mefenamic acid in human plasma using HPLC with UV-VIS detector

Abstract

Mefenamic acid (MA) [ 2-[(2,3-dimethylphenyl) amino]benzoic acid], is from group of nonsteroidal anti-inflammatory drugs (NSAIDs). Generic MA is widely used nowadays as it is expected to have same efficacy and strength as the brand drug. Bioequivalence study for generic formulation is prime requisite of generic drug registration. Bioequivalence study consists of clinical part and analytical part. Method development and subsequent validation of MA in human plasma using HPLC UV-vis detector for analytical part of bioequivalence study was the aim of this study. Two sample preparation technique was successfully developed and validated which were protein precipitation technique and liquid liquid extraction technique. Optimization of MA was performed with selecting several mobile phase, column, and selection of internal standard candidates. Acetonitrile and 2 % triethylamine buffer adjusted to pH 4.2 by using phosphoric acid with ratio (60:40 v/v) was selected as mobile phase. Agilent Zorbax Eclipse XDB C 18 column with 3.5 µm particle size (150 mm x 4.6 mm) was selected as the column for separation of MA compound. Diclofenac sodium was selected as internal standard. Retention time of MA and diclofenac sodium was found 5.4 and 3.8 minutes respectively. Protein precipitation technique was performed using 500 µl spiked plasma sample added with 100 µl of internal standard and 2 ml acetonitrile. For liquid liquid extraction technique relatively small volume of sample was used, 100 µl of spiked plasma with MA, was added with 100 µl diclofenac sodium and 100 µl 20 mM ammonium acetate buffer with pH 3.9. Dichloromethane was used as extracting solvent for MA and IS. Both of these sample preparation technique was validated according to CDER bioanalytical method validation guideline. Protein precipitation technique and liquid liquid extraction was validated for selectivity and showed no interference at retention time of MA and IS. Calibration plot was linear within the range of 250 – 5000 ng ml-1 with the coefficient of determination (r2) above 0.990 for both technique. Limit of detection was 40 ng ml-1 whereas limit of quantification was 250 ng ml-1. QC samples consists of LLOQ 250 ng ml-1, LQC 400 ng ml-1, MQC 2700 ng ml-1, and HQC 4500 ng ml-1. Accuracy and precision was performed for QC samples for intraday and interday. The range for intraday or within day accuracy was between 91.67 % to 110.84 %. The range for intraday and interday precision (CV) was between 1.45 % to 5.5 %. Recovery of MA was more than 80 %. Limit of detection for MA for liquid liquid extraction was 50 ng ml-1 whereas limit of quantification 250 ng ml-1. The range of accuracy for intraday and interday accuracy was within 95.11 % to 106.56 % whereas the range for intraday and interday precision (CV) was 1.47 % to 6.45 %. Recovery of MA was found more than 90 % for this technique. For the stability study for both liquid liquid extraction technique and protein precipitation technique, MA was found stable, for short term, freeze up to three cycle and thaw and long term stability the content of MA about 4 month found about 90 % for all QC samples. Thus two different chromatographic technique were developed and validated for MA in plasma which were efficient, reliable, economic and also fulfill the green chemistry approach. Both the methods are suitable for bioequivalence study of MA.