Simultaneous Estimation of Paracetamol and Zaltoprofen in Pharmaceutical Dosage Form by HPTLC

 

KB Premakumari1, AR Mahesh1*, V Murugan1, V Ezhilarasan2

1Department of Pharmaceutical Chemistry, College of Pharmaceutical Sciences, Dayananda Sagar University, Bengaluru.

2Lotus Labs, Vasanth Nagar, Bengaluru.

*Corresponding Author E-mail: mahesh-sps@dsu.edu.in

 

ABSTRACT:

Objective: A method for HPTLC was developed and validated for simultaneous estimation of Paracetamol and Zaltoprofen in a pharmaceutical dosage form. Methods: The Chromatographic separation was achieved on TLC aluminum plates precoated with silica gel G60 F254 as the stationary phase. The mobile phase consisted of Hexane: Chloroform: Methanol in the ratio of 3.5:5.5:1v/v/v. Results: The method was found to be linear in the concentration range of 0.8-4µg/spot for PCM and 0.2-1 µg/spot for ZLT and the correlation coefficient were found to be 0.996 for PCM and 0.998 for ZLT. The %RSD value obtained for intraday precision were 0.237 and 0.377 and for inter-day precision were 0.259 and 0.766 respectively, the low % RSD indicates that proposed method is precise as per ICH guidelines. The accuracy of the method was determined and the mean recovery of PCM was 99.73-100.15% and ZLT was 99.57-100.18%. LOD and LOQ values were found to be 0.034 µg/spot and 0.105 µg/spot for PCM, 0.006µg/spot and 0.018 µg/spot for ZLT. Conclusion: The method reported is accurate, precise, specific, robust and linear for the estimation of Paracetamol and Zaltoprofen in Pharmaceutical dosage form.

 

KEYWORDS: Paracetamol, Zaltoprofen, HPTLC, Chromatography, HPTLC.

 

 


INTRODUCTION:

Paracetamol (PCM), N-(4-hydroxyphenyl) acetamide (Figure 1) is a non-opiate, non-salicylate, centrally and peripherally acting analgesic agent generally used in combination with many non-steroidal anti-inflammatory drugs. Zaltoprofen, 2-(10, 11-dihydro-10-oxodibenzo [b, f] thiepin-2- yl) propionic acid (Figure 2) is a potent non-steroidal anti-inflammatory drug (NSAID). It has powerful anti-inflammatory and analgesic effects on inflammatory pain, which preferentially inhibits COX-2 activity. It inhibits bradykinin-induced pain responses without blocking the receptors.1,2 In the present investigation, an attempt has been made to develop simple, sensitive, specific, economic HPTLC method for the simultaneous determination of PCM and ZLT in combined tablet dosage forms.

 

 

 

Fig. 1; Structure of PCM

 

Fig. 2; Structure of ZLT

 

MATERIALS AND METHODS:

Materials:

Hexane, Chloroform and Methanol were procured from merck chemicals. Paracetamol and Zaltoprofen were collected as a gift sample from Aeon formulations Pvt. Limited, Pondicherry, India. Zott tablets (Label claim: Paracetamol 325mg and Zaltoprofen 80mg) manufactured by Aeon formulations, Pondicherry were procured from local market.

Instrumentation:

A Camag HPTLC system (Switzerland) with Camag Linomat V sample applicator, Camag TLC Scanner 3, Camag Plate heater, Camag twin-trough glass chamber, Precoated plates (20cm X 20cm) with 200μm layer thickness, UV lamp (190-400nm), Camagwin CATS software, Hamilton syringe100μl and Sartorius Analytical balance were used for the study.

 

Chromatographic conditions:

The experiment was performed on silica gel 60F 254 aluminium sheets (20 x 20 cm) as stationary phase, using mobile phase comprised Hexane: Chloroform: Methanol in the ratio of 3.5:5.5:1v/v/v. The solutions were applied on TLC plate in the form of bands of 6 mm width under a stream of nitrogen gas using a Camag Linomat V automatic sample applicator, space between two bands were 10 mm. Ascending development to 80 mm was performed in 10 cm x 10 cm Camag twin trough glass chamber saturated with the mobile phase for 30 min at room temperature. The developed TLC plate was air dried and then scanned between 200 to 400 nm using Camag TLC scanner 3 using Win CATS software. Both components showed good response at 254nm.

 

Preparation of standard solution:

PCM (40mg) and ZLT (10mg) were weighed accurately, transferred to 10ml volumetric flask, dissolved and diluted with methanol to get 4000μg/ml and 1000μg/ml . Different volumes of mixed standard solution (0.2μl, 0.4μl, 0.6μl, 0.8μl and 1μl) were spotted on the TLC plate to obtain the concentrations of 0.8µg, 1.6µg, 2.4µg, 3.2µg and 4µg/spot for PCM and 0.2µg, 0.4µg, 0.6µg, 0.8µg and 1µg/spot for ZLT respectively.

 

Preparation of sample solution:

From the formulation of Zott (325mg PCM and 80mg ZLT), 10mg equivalent was weighed and taken in 10ml volumetric flask and the volume was adjusted up to the mark with methanol. 04μl of the sample solution was spotted on the TLC plate to obtain the concentrations of 1.6μg/spot for PCM and 0.4μg/spot for ZLT.2

 

Validation of the proposed method:

The Proposed method was validated according to the International Conference on Harmonization (ICH) guidelines.

 

Linearity (Calibration curve):

Calibration curves were plotted over the concentration range of 0.8-4μg/spot and 0.2-1μg/spot for PCM and ZLT, respectively. Accurately measured mixed standard solutions of PCM and ZLT were applied to the TLC plate. The TLC plate was developed and photometrically analysed as described under chromatographic separation. The calibration curve was prepared by plotting peak area versus concentration (μg/spot) corresponding to each spot. Each reading was an average of five determinations.4,5

 

Accuracy (% Recovery):

The accuracy of the method was determined by calculating recoveries of PCM and ZLT by the standard addition method. Known amounts of standard solutions of PCM and ZLT was added at 50, 100 and 150 % level to pre-quantified sample solution of PCM and ZLT respectively. The amount of PCM and ZLT was estimated by applying obtained values to the respective regression line equations.4,5

 

Method Precision (% Repeatability):

The precision of the instrument was checked by repeatedly injecting (n= 6) solutions of PCM and ZLT without changing the parameters of the proposed method. The results were reported in terms of relative standard deviation (% RSD).4,5

 

Intermediate Precision (Reproducibility):

The intraday and inter-day precision of the proposed method was determined by estimating the corresponding responses 3 times on the same day and on 3 different days over a period of one week for different concentration of standard solution of PCM and ZLT for the proposed method. The results were reported in terms of relative standard deviation (% RSD).4,5

 

Limit of detection (LOD) and limit of quantification (LOQ):

LOD and the LOQ of the drug were calculated using the following equations as per international Conference on Harmonization (ICH) guidelines.6,7,8

 

LOD = 3.3 X σ/S

LOQ = 10 X σ/S

Where = Standard deviation of the response

S = Slope of calibration curve

 

Specificity:

The specificity of the method was ascertained by analysing standard drugs and the sample. The spots for PCM and ZLT in the samples were confirmed by comparing the Rf and spectra of the spots with that of the standards.9,10,11, 12

 

Analysis of the marketed formulations:

Five microlitres of sample solution from formulation was applied separately on TLC plate, developed and scanned as described in chromatographic separation. The amount of PCM and ZLT present in the sample solution was determined by fitting area values of peak corresponding to PCM and ZLT into the respective calibration curve.9,10,11,12

RESULTS AND DISCUSSION:

The TLC procedure was optimized with a view to develop an assay method for the simultaneous estimation of PCM and ZLT. The standard solutions of both the drugs were spotted on the TLC plates and run in different solvent systems. The mobile phase consisting of Hexane: Chloroform: Methanol (3.5:5.5:1v/v/v) gave sharp and symmetrical peaks with the Rf values of 0.12 ± 0.002 and 0.30 ± 0.006 for PCM and ZLT, respectively. Well defined spots were obtained when the chamber was saturated with mobile phase for 30 min at room temperature (27 ±30şC). A combined densitogram of mixed standards and 3-D chromatogram showing peaks of PCM and ZLT in different concentrations at 254 nm are depicted in (Figure 3 and Figure 4), respectively. The proposed HPTLC method was validated in terms of linearity, precision, accuracy, LOD, LOQ and specificity. The calibration plot was found to be linear over the concentration range 0.8-4μg/spot for PCM and 0.2 to 1 μg/spot for ZLT, respectively with a correlation coefficient of 0.996 for PCM and 0.998 ZLT, respectively (Table 1 and Figure 3). LOD for PCM and ZLT were found to be 0.034μg/spot and 0.006 μg/spot, respectively. LOQ for PCM and ZLT were found to be 0.105μg/spot and 0.018μg/spot, respectively indicate the sensitivity of the method. The low % RSD values of intraday (0.259 for PCM and 0.766 for ZLT) and inter day (0.125 for PCM and 0.755 for ZLT) precision reveals that the proposed method is precise (Table 2 and 3). To study the accuracy of the method, recovery studies were performed. The percent average recoveries obtained were 99.73-100.15% and 99.47-100.18% for PCM and ZLT, respectively indicating that the proposed HPTLC method is highly accurate (Table 4). The proposed validated method was successfully applied to determine PCM and ZLT in tablet dosage forms. The percent average assay was found to be 99.90 ± 0.38 and be 99.88 ± 0.26 for PCM and ZLT, respectively (Table 5 and Figure.4). The low values of standard deviation indicate the suitability of this method for routine analysis of PCM and ZLT in pharmaceutical dosage forms. To confirm the specificity of the proposed method, the solution of formulation was spotted on TLC plate, developed and scanned. It was observed that the excipients present in the formulation did not interfere with the sample peak. The validation parameters are presented in Table 6.

 

Table.1: Linearity of PCM and ZLT

Sl. No

Concentration of PCM (µg/spot)

Concentration of LT

(µg/spot)

PCM

ZLT

1

0.8

0.2

3495

2018.9

2

1.6

0.4

6310.5

3906.2

3

2.4

0.6

9019.5

5416.3

4

3.2

0.8

11617.4

7333.8

5

4.0

1

14556

9089.6

 


 

Fig. 3: Linearity of PCM and ZLT

 


Table. 2: Intraday precision

Replicate

Concentration of PCM (µg/spot)

Concentration of ZLT (µg/spot)

PCM

ZLT

1

2.4

0.6

9013.4

5426.2

2

2.4

0.6

9055.1

5421.7

3

2.4

0.6

9014.4

5329.4

4

2.4

0.6

9068.3

5443.3

5

2.4

0.6

9037.6

5418.7

6

2.4

0.6

9059.2

5433.3

Mean

9041.33

5412.1

Standard deviation

23.475

41.464

%RSD

0.259

0.766

Table. 3: Interday precision

Replicate

Concentration of PCM (µg/spot)

Concentration of ZLT

(µg/spot)

PCM

ZLT

1

2.4

0.6

9035.0

5428.4

2

2.4

0.6

9067.7

5367.3

3

2.4

0.6

9055.5

5456.8

4

2.4

0.6

9045.0

5428.2

5

2.4

0.6

9056.7

5356.8

6

2.4

0.6

9057.4

5439.8

Mean

9052.883

5412.883

Standard deviation

11.342

40.869

%RSD

0.125

0.755

 

Table.4: Recovery study from binary mixture

Sl. No

Conc. of Standard added (μg/spot)

Amount Recovered

*%Recovery ± SD

PCM

ZLT

PCM

ZLT

PCM

ZLT

1

0.8

0.2

0.9971

0.9980

99.73 ± 0.166

99.574 ± 0.219

2

1.6

0.4

1.0015

1.0050

99.85± 0.375

100.182 ± 0.345

3

2.4

0.6

0.9998

0.9984

100.15 ± 0.227

99.476 ± 0.324

*Average of three determinations

 

Fig. 4: Chromatogram of PCM and ZLT with corresponding Rf values at 254 nm. Stationary phase: 10 X 10 cm HPTLC silica gel 60F254 aluminium plates, Mobile phase: Hexane: Chloroform: Methanol (3.5:5.5:1, v/v/v), Detection:UV at 254nm.

 

Table. 5: Assay of Paracetamol and Zaltoprofen

Drug

Label Claim (mg)

Amount found (mg)

*%Label Claim ±S.D.

 

Zott

PCM

ZLT

PCM

ZLT

PCM

ZLT

325

80

324.69

79.91

99.90 ± 0.38

99.88 ± 0.26

*Average of three determinations

 

Table. 6: Summary of Validation parameters

Validation Parameters

Paracetamol

Zaltoprofen

Linearity

0.8-4µg/spot

0.2-1µg/spot

Correlation Coefficient

0.99

0.99

Accuracy

99.73 to 100.15%

99.47 to 100.18%

Precision(%RSD)

Less than 2%

Less than 2%

LOD

0.034

0.006

LOQ

0.105

0.018

Specificity

Specific

Specific

 

CONCLUSION:

The proposed HPTLC method for simultaneous estimation of PCM and ZLT in combined dosage forms was validated and found to be applicable for routine quantitative analysis of PCM and ZLT. The result of linearity, precision, accuracy and specificity were proved to be within the limits. The method provides selective quantitative of LOTE and GAT. Therefore, this method can be employed for the routine analysis for simultaneous estimation of LOTE and GAT in quality control of formulation.

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Received on 06.01.2019          Modified on 10.02.2019

Accepted on 16.03.2019        © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(5):2075-2078.

DOI: 10.5958/0974-360X.2019.00343.3