1. INTRODUCTION:

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 spondylitis². 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-3^3-5. 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.

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.

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.

2.2.1 Preparation of plasma:

Plasma purchased from blood bank was used for preparation of biological samples of the drugs.

Aceclofenac

Paracetamol

Figure1: The chemical structures of ACF and PCM

2.2.2 Preparation of albumin matrix

Taken 0.4 gm albumin dissolved in 10mL of distilled water

2.2.3 Preparation of biological samples

An accurately weighed quantity of ACE (100mg) and PCM (100mg) was dissolved in methanol in 100 mL volumetric flask and volume was made up to mark with methanol (1000 mg/mL), from this 0.1, 0.2, 0.3mL was spiked with 0.8 mL of plasma in centrifuge tube, each in triplicate.

2.3 Chromatographic Conditions

Chromatography was performed on 20 cm x 10 cm aluminium backed silica gel 60 F₂₅₄TLC 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: n- Butanol (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.

3. VALIDATION OF THE METHOD:

The method was validated in accordance with ICH, and FDA guidelines for bioanalytical method development^35-40.

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.

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 filtered through a 0.45 micron membrane filter paper and 1 mL of the filtrate was diluted to 10 mL applied to an HPTLC plate to furnish 100 ng/spot for aceclofenac and 500 ng/spot for paracetamol. After chromatographic development the peak areas of the bands were measured at 263 nm and the amount of each drug in each tablet was determined from the respective calibration plot. The analytical procedure was repeated six times for the homogenous powder sample. The results are as shown in Table 1.

Table 1. Results of the Estimation of ACE and PCM in tablet formulation

	ACE	PCM
Labeled Claim (mg)	100	500
Amount found (%) ±SD (n = 5)	101.10± 0.1569	100.03± 0.00546
% RSD	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 estimated as the limit of detection (LOD) and limit of quantitation (LOQ). The LOD and LOQ were calculated by 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.

Table 2. Intra-day and Inter-day precision of ACF and PCM

	Intra-day precision			Inter-day precision
Drug	Amount applied (ng/spot)	Amount found (ng/spot)±S.D.	%RSD n=3	Amount applied (ng/spot)±S.D	Amount found (ng/spot)±S.D	%RSD n=3
ACF	600	598.77±5.18	0.86	600	598.38±5.61	0.93
	800	797.82±7.60	0.95	800	798.21±7.19	0.90
	1000	998.13±5.27	0.52	1000	998.52±5.94	0.59
PCM	600	598.42±6.51	1.08	600	598.85±6.10	1.01
	800	799.89±4.57	0.57	800	801.45±5.66	0.70
	1000	999.62±3.45	0.34	1000	998.84±2.46	0.24

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).

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)

3.7 Ruggedness

The ruggedness of the method was evaluated by two different analysts.

3.8 Repeatability

For repeatability 2.0 μL of drug solution was used for developing of TLC plate and the procedure was repeated five times.

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 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.

3.9.2. 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.

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 ambient temperature and thawed (-20^oc), when completely thawed, the sample were frozen again at the same temperature for 12 to 24 hrs. This freeze-thaw cycle is repeated three times. After third cycle, the samples were analysed.

Table 3: Results of recovery study

Drug	Level %	Amount added [ng]	Amount recovered [ng] n = 3	% Recovery	%RSD
ACF	50	25	25.45	101.80	0.34
	100	50	50.07	100.14	0.27
	150	75	75.37	100.50	0.18
PCM	50	125	125.31	100.25	0.27
	100	250	250.80	100.32	0.26
	150	375	375.15	100.04	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 R_F 0.14 (ACF) and 0.43 (PCM). No tailing was observed when plates were scanned at 263 nm (Figure 3).

Table 4. Extraction efficiency in albumin matrix:

Amount of Matrix taken (gm/ml)	Solvent used	Amount of drug taken (ng/spot)		Rf values		Extraction efficiency (%Recovery± SD) n = 3
		ACE	PCM	ACE	PCM	ACE	PCM
0.4	EA:ACT (3.5:0.5)	100	100	0.14	0.43	64.40 ± 0.15	91.6 ± 0.01
0.4	EA:ACT (3.0:1.0)	100	100	0.14	0.43	90.01± 0.01	98.02 ± 0.01
0.4	TOU:ACT (3.5:0.5)	100	100	0.15	0.43	60.0± 0.14	20.16 ± 0.23
0.4	TOU: MEOH (3.5:0.5)	100	100	0.14	0.43	82.20± 0.01	15.63 ± 0.21

Table 5. Extraction efficiency in Plasma matrix:

Amount of matrix taken(µl)	Amount of drug taken (ng/spot		Rf values		Extraction efficiency (%Recovery± SD) n = 3
	ACE	PCM	ACE	PCM	ACE	PCM
800	100	100	0.15	0.43	89.56±0.41	91.26±1.05
800	200	200	0.15	0.45	90.12±0.44	90.63±0.95
800	300	300	0.15	0.44	90.45±0.51	92.68±0.68

Table 6. Matrix factor study:

Drug	Conc. of drug spiked (ng/spot)	Matrix factor Mean n=3	%RSD
ACF	100	0.740	1.896
ACF	300	0.865	0.578
PCM	100	0.822	1.423
PCM	300	0.951	1.215

Table 7. Benchtop stability study:

Sample condition	Amount of matrix taken(µl)	Amount of drug taken (ng/spot)		*% Recovery± SD n = 3**
		ACE	PCM	ACE	PCM
Fresh	800	100	100	91.94±0.84	92.76±0.96
Store	800	100	100	87.48±0.49	85.05±1.32
Fresh	800	300	300	95.08±1.10	94.96±1.02
Store	800	300	300	94.01±1.60	90.76±1.44

Table 8. Freeze thaw stability study

Sample condition	Matrix conc. (µl)	Amount of drug taken (ng/spot)		Retention factor		% Recovery± SD n=3
Sample condition	Matrix conc. (µl)	ACE	PCM	ACE	PCM	ACE	PCM
Fresh	800	100	100	0.14	0.45	91.29±0.689	92.11±0.9645
Store	800	100	100	0.15	0.45	84.09±0.4523	80.55±1.452
Fresh	800	300	300	0.14	0.44	94.93±1.1024	94.51±1.125
Store	800	300	300	0.15	0.45	88.66±1.80	81.48±1.427

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.

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.

4.2.2. Precision

Results from determination of intra-day and inter-day precision, by analysis of standard solutions covering the entire calibration range, are listed in Table 2.

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.

4.2.4. Accuracy

Results from recovery experiments are listed in Table 3. RSD was <2%, indicating the method was accurate.

4.2.5. Ruggedness

When the method was performed by two different analysts, recovery was 99.52 and 100.67% for ACF and 99.21 and 99.00% for PCM.

4.2.6. Repeatability

Repeatability of sample solution was assessed by application of 2.0 μL drug solution, containing 200 ng ACF, and 1000 ng PCM, five times to a TLC plate. RSD of peak area for ACF and PCM 0.83 and 0.92 respectively.

Figure 3. Typical HPTLC Chromatogram of ACF and PCM Standard Drug Solution, measured at 263 nm, mobile phase Toluene: n- Butanol (7:3 v/v)

4.3. Stability of drugs in biological fluids

The extraction efficiency of drugs from biological fluids (albumin and plasma matrix) using ethyl acetate: acetonitrile (3:1, v/v ), were shown in Table 4 and 5.

The stability of ACF and PCM was investigated in biological fluid samples. Each experiment was repeated three times and the mean concentration of ACF and PCM was calculated. The variability in matrix factors, as measured by the coefficient of variation (%RSD), should be less than 15%. The Results from recovery of drug samples from biological fluid are listed in Table 6, 7 and 8.

5. CONCLUSIONS:

In conclusion, a new, sensitive, and specific assay has been established for analysis of ACF and PCM in pharmaceutical formulation and biological fluids. The method validation results indicate the method is sufficiently accurate and precise for recovery studies of the drug.

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Received on 09.05.2010 Modified on 27.05.2010

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1194-1199