Simultaneous Estimation of Aceclofenac and ...

Research J. Pharm. and Tech. 3(4): Oct.-Dec. 2010

ISSN 0974-3618

www.rjptonline.org

RESEARCH ARTICLE

Simultaneous Estimation of Aceclofenac and Paracetamol in Bulk and Combined Tablet Dosage Form and From Biological Fluid by Planar Chromatography Poonam P. Patil1, Mahesh M. Deshpande1, Veena S. Kasture2* and Seema A. Gosavi2 1

2

Department of Pharmaceutical chemistry, MGV’S College of Pharmacy, Panchvati, Nashik-03 Sanjivani College of Pharmaceutical Edu. and Research, Kopargaon, Dist: Ahmednagar, (M. S.), India, 423603 *Corresponding Author E-mail: [email protected]

ABSTRACT: A simple, accurate, reproducible, rapid and precise high-performance thin-layer chromatographic method has been established and validated for simultaneous determination of Aceclofenac and Paracetamol in bulk and combined tablet dosage form and from biological fluids. Both the drugs were separated on aluminum plates precoated with silica gel 60 F254; Toluene: n- Butanol (7:3v/v) was used as mobile phase. Quantitative analysis was performed by densitometric scanning at 263 nm. The RF values of aceclofenac and paracetamol were 0.14 and 0.43, respectively. The calibration plot was linear over the range of 200–1200 ng/spot for aceclofenac and paracetamol, with correlation coefficients, r, 0.9979 and 0.9995, respectively. Recovery of aceclofenac and paracetamol was 100.14–101.80 and 100.04–100.32%, respectively. The suitability of densitometric TLC for quantitative analysis of these compounds was proved by validation in accordance with the requirements of ICH guidelines. The recovery of aceclofenac and paracetamol from biological fluids (albumin and plasma) by solvent - solvent extraction using ethyl acetate: acetonitrile (3:1v/v) was 8990% and 90-98% respectively.

KEYWORDS: Aceclofenac, Paracetamol, HPTLC, biological fluid. No published reports were found on the HPTLC determination of aceclofenac and paracetamol in combined dosage forms by Solvent - Solvent extraction method in biological fluid by RP-HPTLC. The objective of this work was to develop a simple, economical, reproducible, and rapid method for analysis of these drugs in their binary drug formulations.

NTRODUCTION: Aceclofenac (ACF), [o-(2, 6-dichloroanilino) phenyl] acetate glycolic acid ester has anti-inflammatory and analgesic properties1. ACF inhibits synthesis of the inflammatory cytokines interleukin-1b and tumour necrosis factor, and inhibits production of prostaglandin E2. ACF is used for relief of pain and inflammation in osteoarthritis, rheumatoid arthritis and ankylosing spondylitis 2. Paracetamol (PCM), 4-hydroxyacetanilide is a commonly used nonnarcotic, analgesic, antipyretic agent with weak anti-inflammatory activity. PCM blocks prostaglandin synthesis by the cyclooxygenase pathway and inhibits thromboxane A2 formation in platelets, reducing platelet aggregation. PCM is selective inhibitor of COX-33-5. The chemical structures of drugs are shown in Figure 1. Literature survey revealed various analytical methods, such as spectrophotometric, spectrofluorimetric, HPLC, and stability-indicating HPTLC methods for analysis of aceclofenac and paracetamol in a combined tablet dosage form has recently been reported 6-34.

Received on 09.05.2010 Accepted on 12.06.2010

2. EXPERIMENTAL: 2.1 Reagents and Chemicals: All chemicals and reagents used were of HPLC grade and supplied by S.D. Fine Chemicals, India. Aceclofenac and paracetamol reference standards were provided as gift samples by Ipca laboratory (Mumbai, India) and Cadila pharmaceutical (Ahemadabad, India) respectively. Commercial tablets (ZERODOL-P; Ipca laboratory Ltd,), labelled to contain aceclofenac 100 mg and paracetamol 500 mg, were obtained locally. 2.2 Preparation of Standard Solutions: Aceclofenac (10 mg) and paracetamol (10 mg) were weighed separately and dissolved in 100 mL methanol to furnish solutions of concentration 100 μg /mL for each drug.

Modified on 27.05.2010 © RJPT All right reserved

2.2.1 Preparation of plasma: Plasma purchased from blood bank was used for preparation of biological samples of the drugs.

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 470-473

1

Research J. Pharm. and Tech. 3(4): Oct.-Dec. 2010

O

3.1. Calibration and Linearity The standard solutions were prepared by dilution of the stock solution with methanol to reach a concentration range 100 μg/mL (10-60 µL) for ACF and PCM respectively. These standard solutions were spotted on the TLC plate to obtain final concentration 200–1200 ng/spot for ACF and PCM respectively. Each concentration was spotted three times on the TLC plate. The plate was developed on previously described mobile phase. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs. QC sample solutions were prepared at concentrations of 100 and 300 ng/spot.

COOH

O NH Cl

Cl

Aceclofenac

OH

3.2 Procedure for Analysis of Tablet Formulation Twenty tablets were weighed accurately and powdered. Powder equivalent to 500 mg paracetamol and 100 mg aceclofenac was taken and transferred to a 100 mL volumetric flask containing approximately 25 mL methanol. The mixture was sonicated for 10 min and final volume was made up to the mark with methanol. The solution was NHCOCH3 filtered through a 0.45 micron membrane filter paper and 1 Paracetamol mL of the filtrate was diluted to 10 mL applied to an Figure1: The chemical structures of ACF and PCM HPTLC plate to furnish 100 ng/spot for aceclofenac and 500 ng/spot for paracetamol. After chromatographic 2.2.2 Preparation of albumin matrix development the peak areas of the bands were measured at Taken 0.4 gm albumin dissolved in 10mL of distilled water 263 nm and the amount of each drug in each tablet was determined from the respective calibration plot. The 2.2.3 Preparation of biological samples analytical procedure was repeated six times for the An accurately weighed quantity of ACE (100mg) and PCM homogenous powder sample. The results are as shown in (100mg) was dissolved in methanol in 100 mL volumetric Table 1. flask and volume was made up to mark with methanol (1000 g/mL), from this 0.1, 0.2, 0.3mL was spiked with Table 1. Results of the Estimation of ACE and PCM in tablet 0.8 mL of plasma in centrifuge tube, each in triplicate. formulation 2.3 Chromatographic Conditions Chromatography was performed on 20 cm x 10 cm aluminium backed silica gel 60 F254 TLC plates (E.Merck, Darmstadt, Germany) stored in a desiccators. Spotting was done by means of Hamilton microsyringe (Switzerland), mounted on a Linomat V applicator (Camag, Muttenz Switzerland). Ascending development of the plate, migration distance 70 mm, (distance to the lower edge was =10 mm) was performed at 25±2°C with Toluene: nButanol (7:3 v/v) as mobile phase in a Camag chamber previously saturated with 20 mL of solvent mixture for 20 minutes. Samples were applied as 6 mm wide bands at a spraying rate of 15µLs-1; and the distance between the bands was 13 mm. The average development time was 20 minutes. After development the plate was dried at 500C in an oven for 5 minutes. Densitometric scanning was performed with a Camag TLC scanner III equipped with Wincats Software Version (1.4.2, Camag) at 263 nm using Deuterium light source. The slit dimensions were 6.00 Х 0.45 mm.

Labeled Claim (mg) Amount found (%) ±SD (n = 5) % RSD

ACE 100

PCM 500

101.10 0.1569

100.03 0.00546

0.1551

0.00544

3.3 Precision Precision was studied as intra-day and inter-day variation. Intra-day variation was determined by analysis of three different amounts, 600, 800, and 1000 ng/spot of ACF and PCM, three times a day. Inter-day precision was assessed by analysis of the same amounts of the drugs on three different days over a period of a week. 3.4 Accuracy Recovery of the drugs at different levels in the formulation was studied by overapplication of 50%, 100%, and 150% of the standard drug solution of ACF and PCM, and the mixtures were re-analysed by the proposed method. At each level three analyses were performed.

3.5 Sensitivity The sensitivity of measurement of ACF and PCM, was 3. Validation of the Method estimated as the limit of detection (LOD) and limit of The method was validated in accordance with ICH, and quantitation (LOQ). The LOD and LOQ were calculated by 35-40 FDA guidelines for bioanalytical method development . using the formula, LOD = 3.3 × σ/S and LOQ = 10 × σ/S, where σ is residual standard deviation of regression line and S is slope of corresponding regression line.

2

Research J. Pharm. and Tech. 3(4): Oct.-Dec. 2010 Table 2. Intra-day and Inter-day precision of ACF and PCM Intra-day precision Amount applied Amount found %RSD Drug (ng/spot) (ng/spot)±S.D. n=3 600 598.77±5.18 0.86 ACF 800 797.82±7.60 0.95 1000 998.13±5.27 0.52 600 598.42±6.51 1.08 PCM 800 799.89±4.57 0.57 1000 999.62±3.45 0.34

Inter-day precision Amount applied (ng/spot)±S.D 600 800 1000 600 800 1000

Amount (ng/spot)±S.D 598.38±5.61 798.21±7.19 998.52±5.94 598.85±6.10 801.45±5.66 998.84±2.46

found

%RSD n=3 0.93 0.90 0.59 1.01 0.70 0.24

chromatographic plate. The plate was developed using solvent system comprising of toluene: n- Butanol (7:3 v/v) in twin trough chamber to a distance of 70mm. The plate was then removed from chamber, air dried and scanned at 263 nm.

3.6 Specificity Peak purity for ACF and PCM, was tested by comparing spectra acquired at the start (S), apex (A), and end (E) of the peaks obtained from the drugs (Figure 2).

3.9.2. QC Samples For determination of stability of drugs in biological fluids, QC samples at three concentrations of 100, 200, 300 ng/spot for ACF and PCM respectively were used. 3.9.2.1. Matrix factor study The presence of unmonitored, co-eluting compounds from matrix may affect the detection of analytes this phenomenon is commonly known as matrix effect. 3.9.2.2. Benchtop stability Replicate QC samples in matrix at a minimum of two concentrations were analysed after keeping them at ambient temperature for 4 to 24 hours to cover at least the duration of time it takes to extract the samples. Figure 2. Peak purity spectras of ACF and PCM, scanned at the peak-start, peak-apex and peak-end positions of the spot (correlation > 0.99)

3.9.2.3. Freeze thaw stability QC samples in matrix at a minimum of 2 concentrations (low and high QC concentrations) were frozen overnight, at 3.7 Ruggedness o The ruggedness of the method was evaluated by two ambient temperature and thawed (-20 c), when completely thawed, the sample were frozen again at the same different analysts. temperature for 12 to 24 hrs. This freeze-thaw cycle is repeated three times. After third cycle, the samples were 3.8 Repeatability For repeatability 2.0 μL of drug solution was used for analysed. developing of TLC plate and the procedure was repeated Table 3: Results of recovery study five times. Level Amount Amount Drug

3.9 Stability of drugs in biological fluids 3.9.1. Extraction of drugs from biological fluids (albumin and plasma matrix): 0.1 mL of 1000 µg/mL of ACF and PCM was spiked with 8 mL of albumin or plasma solution in centrifuge tube. The solutions were kept aside for 2hrs till Cmax and Tmax was reached, and then extracted with 4.4mL extracting solvent (ethyl acetate: acetonitrile in ratio 3:1v/v) in each centrifuge tube. The solutions were mixed using Remi Cyclo mixer for 2 minute. Then centrifuge for 5 min each by using at 5000 rpm. Immiscible layer of extracting solvent was separated with the help of syringe. Its average volume was 3.7 to 3.8 mL measured. The solvent evaporated and the residue was diluted with 1mL methanol and quickly transferred to rubber stopper vials (4mL). The three different concentrations 100,200,300 µg/mL were applied on the

ACF

PCM

% 50 100 150 50 100 150

added [ng] 25 50 75 125 250 375

recovered [ng] n = 3 25.45 50.07 75.37 125.31 250.80 375.15

% Recovery

%RSD

101.80 100.14 100.50 100.25 100.32 100.04

0.34 0.27 0.18 0.27 0.26 0.49

4. RESULTS AND DISCUSSION: 4.1 Optimization of the Method Initially, toluene, methanol and ethyl acetate in different ratios were tried as mobile phase. Ultimately mobile phase consisting of Toulene: n- Butanol (7:3, v/v) gave good resolution and sharp and symmetrical peaks of RF 0.14 (ACF) and 0.43 (PCM). No tailing was observed when plates were scanned at 263 nm (Figure 3).

3

Research J. Pharm. and Tech. 3(4): Oct.-Dec. 2010 Table 4. Extraction efficiency in albumin matrix: Amount of Matrix taken (gm/ml) Solvent used 0.4 0.4 0.4 0.4

EA:ACT (3.5:0.5) EA:ACT (3.0:1.0) TOU:ACT (3.5:0.5) TOU: MEOH (3.5:0.5)

Amount of drug taken (ng/spot) ACE PCM 100 100 100 100 100 100 100 100

Table 5. Extraction efficiency in Plasma matrix: Amount of matrix Amount of drug taken (ng/spot taken(µl) ACE PCM 800 100 100 800 200 200 800 300 300

Rf values ACE 0.14 0.14 0.15 0.14

PCM 0.43 0.43 0.43 0.43

Rf values ACE 0.15 0.15 0.15

PCM 0.43 0.45 0.44

Extraction efficiency (%Recovery± SD) n = 3 ACE PCM 64.40 ± 0.15 91.6 ± 0.01 90.01± 0.01 98.02 ± 0.01 60.0± 0.14 20.16 ± 0.23 82.20± 0.01 15.63 ± 0.21

Extraction efficiency (%Recovery± SD) n = 3 ACE PCM 89.56±0.41 91.26±1.05 90.12±0.44 90.63±0.95 90.45±0.51 92.68±0.68

Table 6. Matrix factor study: Conc. of drug spiked (ng/spot)

Drug

100 300 100 300

ACF PCM

Table 7. Benchtop stability study: Sample condition Amount of matrix taken(µl) 800 800 800 800

Fresh Store Fresh Store

Table 8. Freeze thaw stability study Sample condition Matrix conc. (µl) Fresh Store Fresh Store

800 800 800 800

Matrix factor Mean n=3

%RSD

0.740 0.865 0.822 0.951

1.896 0.578 1.423 1.215



To recover the drugs from biological fluids several solvent systems were tried ethyl acetate: acetonitril (3:1v/v) was the better solvent system. Well-defined spots were obtained when plate was activated at 110°C for 5 min. and the chamber was saturated with the mobile phase for 20 min at room temperature.

Retention factor ACE PCM 0.14 0.45 0.15 0.45 0.14 0.44 0.15 0.45

% Recovery± SD* n = 3 ACE PCM 91.94±0.84 92.76±0.96 87.48±0.49 85.05±1.32 95.08±1.10 94.96±1.02 94.01±1.60 90.76±1.44

% Recovery± SD n=3 ACE PCM 91.29±0.689 92.11±0.9645 84.09±0.4523 80.55±1.452 94.93±1.1024 94.51±1.125 88.66±1.80 81.48±1.427

4.2.3. Determination of LOD and LOQ To determine the limits of detection and quantitation, concentrations in the lower part of the linear range of the calibration plot were used. LOD and LOQ were, respectively, 0.0133 and 0.0403 µg mL-1 for ACF and 0.638 and 1.933 µg mL-1 for PAR. This indicates the sensitivity of the method is adequate.

4.2 Validation 4.2.1. Linearity Response to ACF and PCM was a linear function of amount in the concentration ranges 200–1200 ng per band, respectively. The respective linear regression equations for ACF and PCM were Y = 632.89X + 527.51 and Y = 1055.8X+ 212.2, with correlation coefficients, r, 0.9979 and 0.9995, respectively, where Y is peak area and X is amount chromatographed. Six replicate readings were obtained at each concentration.

4.2.4. Accuracy Results from recovery experiments are listed in Table 3. RSD was