Asian J. Research Chem. 2(3): July-Sept.

, 2009
,

ISSN 0974-4169 www.ajrconline.org

RESEARCH ARTICLE

HPTLC estimation of Paracetamol, Diclofenac Sodium and Chlorzoxazone

in Tablet dosage form
Smita J Pawar*, Amol P Kale, Manoj P Amrutkar, Jyotsna J Jagade, Nikhil S Pore and
Ashok V Bhosale
PDEA's Seth Govind Raghunath Sable College of Pharmacy, Saswad, Pune-412301
*Corresponding Author E-mail: smitapawar4477@yahoo.co.in

ABSTRACT
A simple, rapid, reliable and accurate HPTLC method has been developed for the quantitative determination of
Paracetamol, Diclofenac Sodium and Chlorzoxazone in tablets. The drugs were extracted from (CIP-ZOX). Various
aliquots of this sample solution were spotted automatically by means of Camag (Muttenz; Switzerland) Linomat V
sample applicator on Merck HPTLC plates (0.2mm thickness) precoated with silica gel 60 F254 on aluminium sheet as
stationary phase prewashed with methanol using Chloroform: Methanol: Ammonia (20:5:0.2v/v/v) as mobile phase.
The spots were scanned at =254nm using Camag TLC scanner 3. The Rf values of Paracetamol, Diclofenac Sodium
and Chlorzoxazone were found to be 0.53, 0.27, and 0.68 respectively. Calibration curves were linear in range of
1000-5000ng per spot. The limit of detection (LOD) and quantitation (LOQ) for Paracetamol, Diclofenac sodium and
Chlorzoxazone were found to be 100, 100, 500 and 300, 300, 1500ng per spot respectively. The suitability of this
method for quantitative determination of compounds was proved by validation in accordance with requirements of
pharmaceutical regulatory standards. The proposed method is valid, simple, sensitive and accurate. Therefore this
method can be applied for routine analysis of these drugs in tablet formulation.

KEYWORDS: Paracetamol, Diclofenac sodium, Chlorzoxazone, HPTLC, Pharmaceutical dosage form

INTRODUCTION: Chlorzoxazone is 5-chloro-3H-benzooxazol-2-one.

Paracetamol is N-(4-hydroxyphenyl) acetamide; Chlorzoxazone is a centrally acting muscle relaxant used
commonly known as acetaminophen1,2. It is a widely-used to treat muscle spasm and the resulting pain or
as analgesic and antipyretic medication. Derived from discomfort1,4. These drugs are official in IP, BP, and USP.
coal tar, it is the active metabolite of phenacetin, but HPLC method has been reported for this combination5-7.
unlike phenacetin, paracetamol has not been shown to be
carcinogenic.It is available over-the-counter. It is No HPTLC method has been reported for this multidrug
commonly used for the relief of fever, headaches, and combination. In recent years HPTLC technique has been
other minor aches and pains3,4. In combination with non- improved to incorporate certain features automated
steroidal anti-inflammatory drugs (NSAIDs) or opioid sample application, controlled development environment,
analgesics, paracetamol is used also in the management of computer controlled densitometry etc.
more severe pain.
Validation of an analytical procedure is done using the
Diclofenc sodium is 2-(2-(2,6-dichlorophenylamino) guidelines suggested by the International Conference on
phenyl) acetic acid. is a non-steroidal anti-inflammatory Harmonization [ICH Topic Q2 (R1)].8,9 The validation of
drug (NSAID) taken to reduce inflammation and as an an analytical method development is an essential part
analgesic in conditions such as arthritis or acute injury1,2,4. after the method has been developed to check whether it
It can also be used to reduce menstrual pain, is suitable for different conditions and with varying
dysmenorrhea3,4. concentrations of the sample.11,12 The aim of the present
work is to develop and validate HPTLC method for
estimation of Paracetamol, Diclofenac sodium,
Chlorzoxazone in tablet dosage forms.

Received on 04.07.2009 Modified on 09.08.2009

Accepted on 17.08.2009 © AJRC All right reserved
Asian J. Research Chem. 2(3): July-Sept., 2009, page 306-309

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Table No.1 Results of Assay, LOD and LOQ values for Paracetamol, Diclofenac sodium and Chlorzoxazone in
pharmaceutical dosage forms
Brand Name Sr. No. Amount Label claimed (mg)
Paracetamol (500mg) Diclofenac Sodium (50mg) Chlorzoxazone (500mg)
1 500.12 50.31 499.93
2 499.06 49.67 500.21
CIP-ZOX 3 499.87 49.53 499.43
4 499.63 49.93 499.84
5 499.60 49.31 500.03
Mean assay 499.65 ± 0.60 49.75 ± 0.72 499.88 ± 0.40
R.S.D (%) 1.67 1.54 1.87
LOD 100 100 500
LOQ 300 300 1500

Table No 2: Results from precision evaluation

Drug Concentration (ng/spot) Intra-day precision ( % RSD, n=3) Interday precision (% RSD, n= 3 )
2000 0.252 0.563
Paracetamol 3000 0.793 0.931
4000 0.382 1.23
3000 0.573 0.592
Diclofenac 4000 0.984 0.783
sodium 5000 0.703 0.739
2000 1.04 0.963
Chlorzoxazone 3000 0.232 0.762
4000 0.653 0.547

Table No 3: Results from recovery studies

Label claim Initial amount Amount added Amount % recovered % RSD
Component (mg/tablet) (ng) (ng) recovered (ng)
3000 1500 1496 99.73 0.659
Paracetamol 500 3000 0 2993 99.76 0.874
3000 4500 4502 100.04 0.459
4000 2000 1994 99.70 1.65
Diclofenac 50 4000 0 3995 99.87 1.36
sodium 4000 6000 5999 99.98 1.05
3000 1500 1487 99.13 0.597
Chlorzoxazone 500 3000 0 2999 99.96 0.542
3000 4500 4504 100.08 1.466

Figure No.1 A typical chromatogram of Paracetamol, Diclofenac Sodium and Chlorzoxazone measured at 254nm. Mobile
phase- Chloroform: Methanol: Ammonia (20:5:0.2v/v/v).

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Figure No.2 Typical Absorption Spectra of Paracetamol, • Chromatography:

Diclofenac Sodium and Chlorzoxazone Chromatography was performed on 20cm X 10cm
aluminium backed silica gel 60 F254 HPTLC plates
(Merck, Dermatadt, Germany). The plates were
prewashed with methanol and dried in an oven at 500C for
5min. Samples were applied as 6mm bands spraying at a
rate of 15µL-1s by means of a Camag (Muttenz;
Switzerland) Linomat V sample applicator equipped with
a 100-µL syringe (Hamilton). The distance between the
bands was 10.0mm. Ascending development of the plate,
migration distance 80mm was performed at 25±20C with
Chloroform: Methanol: Ammonia (20:5:0.2v/v/v) as
mobile phase in a Camag twin trough chamber previously
saturated for 20min with paper. The average development
time was 20min. Densitometric scanning was then
performed with a Camag TLC scanner 3 equipped with
Wincats Software Version 1.4.4 at =254nm using
Deuterium light source, the slit dimensions were 5.00 X
0.45mm.

MATERIALS AND METHODS: RESULT AND DISCUSSION:

• Instrumantation: Validation of HPTLC method: 8-12
Camag (Muttenz; Switzerland) Linomat V applicator, • Linearity:
Camag twin trough TLC chamber, Camag TLC scanner 3, Amounts of standard solutions equivalent to 1000-5000
Camag Wincats software and Hamilton Syringe (100µl) ng/spot Paracetamol, Diclofenac sodium, Chlorzoxazone
was spotted on prewashed HPTLC plates. The plate was
• Materials and reagents: developed, dried and scanned as described above. The
Analytically pure gift samples of Paracetamol, Diclofenac calibration plot was constructed by plotting peak area
sodium, Chlorzoxazone were obtained from Hetero Labs Ltd. against the corresponding concentrations of all the drugs
Mumbai; India was used as working standard. Chloroform, (ng/spot) individually. The linearity of response for
Methanol and Ammonia of HPLC grade (Merck Chem.) to Paracetamol, Diclofenac sodium, Chlorzoxazone was
prepare mobile phase were used without further purification. assessed in the concentration range 1000-5000 ng/spot, in
terms of intercept and correlation coefficient values. The
• Standard stock solutions: calibration spots showed the correlation coefficients
Standard stock solutions containing 0.5mg/mL of all of the (r=0.9956, 0.9988, 0.9988), intercept were (2309, 2782,
Paracetamol, Diclofenac sodium and Chlorzoxazone were 1140) respectively over the concentration range studied
prepared by dissolving 5mg standard Paracetamol, with six replicate readings of each concentration.
Diclofenac sodium and Chlorzoxazone in 10ml methanol in
3 different flasks and are used as working standard solutions. • Sensitivity:
The sensitivity of measurement of Paracetamol,
• Sample preparation: Diclofenac sodium and Chlorzoxazone by the use
Twenty CIP-ZOX tablets manufactured by CIPLA proposed method was estimated in terms of Limit of
containing 500mg of Paracetamol, 50mg of Diclofenac Quantitation (LOQ) and the lowest concentration detected
sodium and 500mg of Chlorzoxazone were weighed and under the chromatographic conditions as the Limit of
powdered. An amount of powder equivalent to 500mg of Detection (LOD). The LOD and LOQ were calculated by
Paracetamol, 50mg of Diclofenac sodium and 500mg of the use of the equation LOD = 3 X N/B and LOQ = 10 X
Chlorzoxazone was transferred to 250ml calibrated N/B where N is the standard deviation of the peak areas
volumetric flask containing 100ml methanol sonicated for of the drugs taken as a measure of noise and B is the
15min. Then the resulting solution was filtered using slope of the corresponding calibration curve. The results
0.45µm filter. Take 1ml of solution from filtrate and of sensitivity are as shown in Table No.1.
dilute it for 10ml of methanol. Also take 4ml of solution
from filtrate and dilute it for 10ml of methanol. A sample • Assay and Precision evaluation:
solution (6µL containing 3000ng of Paracetamol and The amount of Paracetamol, Diclofenac sodium and
Chlorzoxazone; 1200ng of Diclofenac sodium) was Chlorzoxazone was applied in number of replicates of
spotted for assay of Paracetamol, Diclofenac sodium, both pharmaceutical preparations (n = 6) and the assay
Chlorzoxazone. results are reported in Table No.1. Precision studies were
performed using standard solution of Paracetamol,
Diclofenac sodium and Chlorzoxazone the concentration
of drugs covering the entire calibration range. The
precision of the method in terms of intra-day variation (%
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CV) was determined by analysing Paracetamol, Chlorzoxazone simultaneously in pharmaceutical
Diclofenac sodium and Chlorzoxazone standard drug formulation.
solution within the calibration range, three times on the
same day. Inter-day precision (% CV) was assessed by ACKNOWLEDGEMENT:
analysing Paracetamol, Diclofenac sodium and The authors are thankful to Hetero Labs Ltd. Mumbai,
Chlorzoxazone standard drug solution within the India for supplying the gift samples and to Anchrom Labs
calibration range on three different days over a period of Mumbai, India for providing technical support. We are
one week. The results of precision studies are as shown in also thankful to the Principal S.G.R.S College of
Table No.2. Pharmacy,. Saswad, Pune for providing necessary
facilities.
• Accuracy:
The accuracy of the method was determined by the use of
REFERENCES:
standard additions at three different levels i.e. multiple 1.
www.drugs.com
level recovery studies. Sample stock solution of tablet 2.
www.wikipedia.com.
formulation of 3000ng/spot of Paracetamol, 3.
Indian Drugs Review. A. Mediworld Publication; New
Chlorzoxazone and 4000ng/spot of Diclofenac sodium Delhi, (2006).
was prepared from tablet solution and spiked with 4. Tripathi K. D.; Essential of Medical Pharmacology;
amounts equivalent to 50, 100. 150 % of amounts of Jaypee Brothers Medical Publishers (P) Ltd.; New
drugs in the original solutions. When these solutions were Delhi, 5th Ed., (2003), 48-52.
analyzed the recoveries were found to be within the 5. Indian Pharmacopoeia Govt. of India. Ministry of
acceptable limits of 98-102%. The reports of recoveries Health and Family Welfare. Controller of Publication,
are as shown in the Table No.3. Vol. I, 244, (1996).
6. British Pharmacopoeia, Vol. III, HMSO, Cambridge,
• Specificity: International edition, , 2257, 2544, (2003).
th
The mobile phase designed for the method resolved the 7. United States Pharmacopoeia, 26 edition. US
drugs very efficiently as shown in Figure No.1 The Rf Pharmacopoeial Convention INC (NF-21), 18,441,
values of Paracetamol, Diclofenac Sodium and (2004)
8. ICH Q2A Harmonized Tripartite Guideline, Text on
Chlorzoxazone were found to be 0.53, 0.27 and 0.68
Validation of Analytical Procedures, IFPMA In:
respectively. The typical absorption spectrum for
Proceedings of International Conference of
Paracetamol, Diclofenac Sodium and Chlorzoxazone is Harmonization, Geneva, March (1994).
shown in Figure No.2. The peak purity was tested by 9. ICH Q2B Harmonized Tripartite Guideline, Text on
correlating the spectra’s of Paracetamol, Diclofenac Validation of Analytical Procedures Methodology,
Sodium and Chlorzoxazone at the peak start (S), peak Proceedings of International Conference of
apex (A) and peak end (E) positions. Correlation between Harmonization, Geneva, March (1996).
these spectra’s indicated purity of Paracetamol, 10. ICH Guidance on Analytical Methods, International
Diclofenac Sodium and Chlorzoxazone peak correlation r Convention on Quality for Pharmaceutical Industry,
(S, A) = 0.9996, 0.9994, 0.9999, and r (A, E) = 0.9997, Toronto, Canada, September, (2002).
0.9992, 0.9998. It can be thus concluded that no 11. Reviewer guidance, Validation of Chromatographic
impurities or degradation products were found with the methods,:7-29 (1994)
peaks of standard solution. 12. Sethi PD, High Performance Thin Layer Chromatography:
Quantitative analysis of pharmaceutical formulations, CBS
• System suitability: Publications, New Delhi,162-5, (1996)
According to USP system suitability tests are an integral
part of a chromatographic analysis and should be used to
verify that the resolution and reproducibility of the
chromatographic system is adequate for analysis. To
ascertain the effectiveness of the method developed in this
study system suitability tests were performed on freshly
prepared standard stock solutions of Paracetamol,
Diclofenac Sodium and Chlorzoxazone.

CONCLUSION:
The proposed HPTLC method provides simple, accurate
and reproducible, quantitative analysis for simultaneous
determination of Paracetamol, Diclofenac sodium,
Chlorzoxazone in tablet dosage form. The method was
validated as per ICH guidelines. Statistical test indicates
that the proposed HPTLC method reduced the duration of
analysis and appears to be equally suitable for routine
determination of Paracetamol, Diclofenac sodium,

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