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About Authors:
Mona Karia*1, Bhargav Gohel2, Bina Thanki3, Darshan Madiya4, Shital Faldu5
1,3M.Pharm, Smt. R.D.Gardi B.Pharmacy College. Rajkot, Gujarat, India
2Q.A. Officer, Intas Pharmaceutical Ltd., Ahemdabad.
4Assistant Professor of Smt. R.D.Gardi B.Pharmacy College, Rajkot
5Principal  of Smt. R.D.Gardi B.Pharmacy College, Rajkot

An accurate, simple, precise and sensitive HPLC method with UV detection was developed and validated for simultaneous estimation of Etamsylate and Mefenamic acid in human plasma as per USFDA guidelines. Etamsylate and Mefenamic acid were spiked with human plasma and 5 % w/v Trichloro acetic acid used for protein precipitation in ration of 1:9 %v/v of human plasma. The column used was SUSEIDO C18 column (250mm x 25 mm, i.d, 5 µ). Analysis was carried out with Methanol: ACN (70:30 % v/v): Buffer (0.05M Ammonium acetate buffer) pH 6.0 (85:15,% v/v) as mobile phase at flow rate of 1.5 ml/min at 298 nm with UV detection. The retention time of Mefenamic acid observed was 1.98 min and for Etamsylate 3.85 min respectively. The method was validated over the range of 0.5-30 μg/ml for Etamsylate and 0.2-18 µg/ml for Mefenamic acid with coefficients of regression 0.9939 and 0.9914 for Etamsylate and Mefenamic acid respectively. LOD and LOQ were 0.17 ng/ml and 0.51 ng/ml for Etamsylate and 2.62 ng/ml and 7.95 ng/ml for Mefenamic acid.The mean % recovery for MEF and ETM were found to be 89.07% and 88.98%with % C.V. of 1.87% and 1.91% respectively. Inter-day as well intra-day replicate, gave % RSD below 4.44 and 2.63 for Etamsylate and 3.20 and 3.75 for Mefenamic acid respectively. This RP-HPLC method is sensitive, selective, accurate, precise and rapid for the simultaneous estimation of Etamsylate and Mefenamic acid in human plasma with limit of Quantification in nano-gram level was developed and validated as per USFDA guideline and can be applied for a bioequivalence study to compare relative bioavailability of drug.


Etamsylate [N-Ethylethanamine 2, 5-dihydroxybenzenesulphonat] is used commonly in combination with Mefenamic acid [2(2, 3-dimethyl-phenyl amino)] benzoic acid. Etamsylate is a haemostatic drug and Mefenamic acid is a non-steroidal anti-inflammatory drug used to treat pain, including menstrual pain. So, Etamsylate and mefenamic acid in combination are used in the prevention and treatment of capillary bleeding in menorrhea, after abortion, after tooth extraction.

Etamsylate is believed to work by increasing capillary endothelial resistance and promoting platelet adhesion. Plasma Cmax is found 1-15 µg/ml andPlasma tmax is found 3.7 hrs.Plasma protein binding is 92 % (mainly Albumin). Excretion (mainly kidney) is 72 % unchanged in urine.

Figure No.1: Chemical structure (A) Etamsylate (B) Mefenamic acid

Mefenamic acid binds the prostaglandin synthetase receptors COX-1 and COX-2, inhibiting the action of prostaglandin synthetase. Protein binding efficiency is found 90 % (mainly Albunmin)andPlasma Cmax is found 10-20 µg/ml. Plasma tmax is 2 hrs. Excretion (mainly kidney) is 52 % unchanged in urine [1-6].

Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, urine, is reliable and reproducible for the intended use.

The measurement of Etamsylate and Mefenamic acid concentration in plasma could be of help to clinicians in assessing compliance, maximizing response, or avoiding toxicity. Literature survey revealed that there are various methods have been reported for estimation of both drugs such as UV spectrophotometry, HPLC, GC individually. Literature survey also reveals that there is no HPLC method available for the determination of these analytes in combination from human plasma[8-17]. We develop simple, rapid, accurate, reproducible and economic RP-HPLC method for simultaneous determination of Etamsylate and Mefenamic acid from human plasma. The proposed methods were validated as per the USFDA bio-analytical method validation guidelines.


Solvents and Materials
Etamsylate and Mefenamic acid were supplied by Balaji Drugs, Surat (Gujarat, India). Acetonitrile, water and methanol were of HPLC grade and obtained from Ranbaxy ltd., (Mumbai). Ammonium acetate was obtained from Suvidhinath Lab, Rajkot (Gujarat, India). Drug free Healthy human plasma was obtained from Nathani Blood Bank, Rajkot.

Analysis by HPLC was performed using LC-100 pump combined with a LC-100 UV Detector. The column used was CAPCEL PAK C18 (SUSEIDO) (250 mm×4.6mm, particle size 5-µm). An UV-Visible double beam spectrophotometer (heλios Alpha, Model - V 7.09) having two matched quartz cells with 10 mm light path was used for wavelength detection. Weighing of materials were done on high precision balance (Contech, Model-CA34)

Chromatographic condition
Simultaneous estimation of bothe the drus was carried out by using Methanol:ACN(70:30 %v/v): Buffer (0.05M Ammonium acetate buffer) pH 6.0 (85:15, %v/v) as mobile phase with 1.5ml/min flow rate at detection wavelength 298 nm. Prior to injecting solutions, the column was saturated for at least 15 min with the mobile phase flowing through the system.

Protein precipitation procedure
The protein precipitation method was carried out by use of 5% w/v Trichloro Acetic Acid (TCA) solution in ratio of 9 (Spiked drugs in plasma):1 (5% w/v TCA solution). The amount of the both the drugs (ETM and MEF) added for spiking in plasma were such that the free drugs (ETM and MEF) meeting the Cmax level of both the drugs after de-proteinization and being centrifuged at 3000 rpm for 10 min. After centrifugation, supernatant plasma (protein free) was collected which meeting the Cmax level of Etamsylate and Mefenamic acid.

Preparation of Standard Stock Solution
Standard stock solution of pure drug containing 1000 μg/ml Etamsylate and 1000 μg/ml of Mefenamic acid prepared separately in methanol. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in methanol.

Procedure for Determination of Wavelength for Measurement
Appropriate required quantity from working standard stock solution of ETM and MEF were diluted with methanol to get a concentration of 10 μg/ml of ETM and MEF separately. Each solution was scanned between 200-400 nm against methanol as a reagent blank. Cross point was observed at 298 nm which was used as wavelength of detection in HPLC for simultaneous estimation of both the drugs.

Construct the chromatogram of the analytes, calculate the peak area and plot calibration curve peak area against concentration in µg/ml. Concentration of analyte is calculated using linear equation,

y = mx + c

Where, y = peak area of the analyte, m = slope of the calibration curve,
x = concentration of the analyte (µg/ml), c = intercept of the calibration curve


Bioanalytical method development
Different proportion of 0.05 M ammonium acetate buffer (pH 6.0) and mixture of methanol and acetonitrile as mobile phase were used for determination of both the drugs and found that 85:15 %v/v of 0.05 M ammonium acetate buffer (pH 6.0) : (mixture of methanol and acetonitrile, 70:30 % v/v) with 1.5 ml/min flow rate shows better sharp peak and very shorter retention time for both the drugs. Where as in the other proportion ratio of mobile phase and other flow rate, retention times of both drugs were becomes longer with tailing, fronting, broadening of peak.

Bioanalytical method validation

Calibration curve, Linearity and Range
ETM and MEF were spiked to the drug free human plasma and being subjected for deproteinization and samples are injected with 85:15 %v/v of 0.05 M ammonium acetate buffer (pH 6.0) : (mixture of methanol and acetonitrile, 70:30 % v/v) with 1.5 ml/min flow rate as mobile phase. The calibration plot (peak area versus concentration in µg/ml) was generated by replicate analysis (n = 6) at all concentration levels and the linear relationship was evaluated and R2 value was determined. The method was found to be linear in the range of 0.5-30 μg/ml with correlation coefficient R2=0.9939 for ETM and 0.2-18 µg/ml with correlation coefficient R2=0.9914 for MEF.

Figure No. 2: (A) Calibration curve of ETM and MEF.MEF, (B) Plotted calibration curve of MEF, (C) plotted calibration curve of ETM



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