Development and Validation of Absorption Correction Method for Simultaneous Estimation of Paracetamol and Nimesulide in Bulk and Combined Tablet Dosage Form

 

Dnyaneshwari D. Waichal*, Vrushali S. Tambe

Dept. of Quality Assurance Technique PES’s Modern College of Pharmacy (For Ladies), Moshi, Pune-412 105, Maharashtra, India

*Corresponding Author E-mail: dkmbsp@gmail.com

 

ABSTRACT:

The present paper describes simple, accurate, rapid, precise and sensitive UV spectrophotometric absorption correction method for the simultaneous determination of paracetamol and nimesulide in combined tablet dosage form. This method is more simple than reported methods and is based on use of 0.1 N NaOH as a solvent. The wavelengths selected for the analysis were 257 nm and 393 nm. Beer’s law was obeyed in the concentration range of 4-18 µg/ml and 10-30 µg/ml for paracetamol and nimesulide respectively. The mean percentage drug content for paracetamol and nimesilide were found to be 100.03% w/w and 101% w/w respectively. The % RSD value was found to be less than 2 which shows the precision of method. The high recovery and low coefficients of variation conforms it’s suitability for the routine quality control analysis of paracetamol and nimesulide in pure and tablet dosage forms.

 

KEYWORDS: Paracetamol, Nimesulide, Spectrophotometric absorption correction method.

 

 


INTRODUCTION:

Paracetamol (PARA Fig 1a) is N-(4 – hydroxyphenyl) acetamide, a para-aminophenol derivative, with analgesic, antipyretic properties and weak anti-inflammatory activity. The log P value of PARA is 0.31. It is insoluble in water, very soluble in ethanol and it’s pka value is 9.5. It is official in Indian Pharmacopoeia[1] and British Pharmacopoeia [2,3]B.P.

 

The I.P. and B.P. both suggest titrimetric and UV spectrophotometric assay method for PARA in bulk and tablet formulations. Nimesulide (NIME Fig 1b) chemically is [N-(4-nitro-2-phenoxyphenyl)] methanesulfonamide. It is non steroidal antiinflammatory drug with good analgesic and antirheumatic properties. The log P value of NIME is 2.7. It is soluble in ethanol, DMSO and DMF and sparingly soluble in aqueous buffer. It has pka value of 6.46. Combination of PARA and NIME is available in tablet dosage form. Some HPLC[4, 5] and spectrophotometric[6,7,8] methods have been reported in literature for it’s estimation. UV and HPLC methods have been reported in literature for determination of PARA and NIME combination[4-12]. There are two UV spectrophotometric methods for simultaneous determination of these drugs in tablet dosage form. The reported methods are simultaneous equation method and Q ratio method, yet absorption correction method is not reported in literature. Hence, the objective of the present work is to develop and validate new analytical method for simultaneous determination of PARA and NIME in tablet dosage form by absorption correction method.

 

 

Fig. 1(a) Structure of PARA

 

 

Fig.1(b) Structure of NIME

 

MATERIALS AND METHODS:

APPRATUS AND EQUIPMENT:

A Shimadzu UV–visible spectrophotometer (UV-1800, Shimadzu) was used for all absorbance measurements with 1 cm paired quartz cell.

 

REAGENTS AND CHEMICALS:

Pharmaceutical grade PARA and NIME were supplied as a gift sample from Medico Remedies Pvt Ltd, Mumbai, and Alicon Pharmaceutical Pvt Ltd, Mumbai, respectively. Sodium hydroxide used in analysis was of Analytical grade. It was purchased from Research-Lab Fine Chem Industries, Mumbai, India.

 

PREPARATION OF STANDARD STOCK SOLUTION: (100µg/mL):

An accurately weighed quantity of 10 mg PARA was transferred to 100 mL volumetric flask, dissolved in 0.1N sodium hydroxide and sonicated for 15 min, volume was then made to the mark with 0.1N sodium hydroxide. An accurately weighed quantity of 10 mg NIME was transferred to 100 mL volumetric flask, dissolved in 0.1N sodium hydroxide and sonicated for 15 min, volume was then made up to the mark with 0.1N sodium hydroxide.

 

SELECTION OF WAVELENGTH FOR ANALYSIS:

The standard stock solution was diluted to obtain a solution containing 10 μg/ml of PARA and 10 μg/ml of NIME separately. These diluted solutions were scanned in the range 200-400 nm separately. The wavelength selected for analysis were 393 (ʎmax of NIME) and 257 nm (ʎmax of PARA). As at 393 nm, there was no interference due to absorption of PARA, absorbance correction method was successfully developed. PARA showed ʎmax at 257 nm while it does not show any absorbance at 393 nm. The absorbance value at 393 nm is due to NIME only in the combined mixture of both drugs.

 

DETERMINATION OF ABSORPTIVITY VALUE:

The stock solutions of both the drugs were further diluted separately with 0.1 N sodium hydroxide to get a series of standard solutions in the concentration range of 4-18 μg/ml for PARA and 10-30 μg/ml for NIME. The absorbance were measured at the selected wavelengths and absorptivities for both the drugs at selected wavelengths were determined by plotting the calibration curve.

 

ASSAY OF TABLET FORMULATION:

Twenty tablets were weighed and average weight was determined. The tablets were triturated to a fine powder. An accurately weighed quantity of powder (54.2 mg) equivalent to 32.5 mg of PARA and 10 mg of NIME was transferred in to 100 ml volumetric flask and added 50 ml of 0.1 N sodium hydroxide to dissolve the actives. The solution was sonicated for 15 minutes, then made to the volume up to the mark and filtered through Whatmann filter paper No. 41. From the clear solution, 4.0 ml of the filtrate to 10 ml was diluted with 0.1 N sodium hydroxide to obtain solution containing 13 µg/ml of PARA and 4 µg/ml of NIME theoretically. The absorbance of sample solution was measured at the selected wavelengths. The content of PARA and NIME in sample solution of tablet was calculated using formula given below,

 

CNIME = A393 /axNIME393                                                                                     (I)

CPARA = A257– axNIME257 × CNIME / axPARA257                        (II)

 

Where, A393 and A257 are absorbance of sample solution at 393 nm and 257 nm, respectively.

AxPARA, absorptivity coefficients of PARA at 257 nm.

AxNIME, absorptivity coefficients of NIME at 257 nm and 393 nm, respectively.

CPARA and CNIME are concentrations of PARA and NIME, respectively in mixture.

 

METHOD VALIDATION:

The method was validated according to ICH Q2B guidelines for validation of analytical procedures in order to determine the linearity, precision, accuracy, robustness, LOD and LOQ of the method.

 

 

 

LINEARITY:

The calibration curves were plotted over a concentration range of 4-18 μg/ml for PARA and 10-30 μg/ml NIME. Accurately measured standard stock solutions of each PARA (0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 ml) and NIME (1, 1.5, 2, 2.5, 3) were transferred to a series of 10 ml volumetric flask separately and diluted up to the mark with 0.1 N sodium hydroxide. The absorbances of solution were then measured at 257 nm and 393 nm. The calibration curves were constructed by plotting absorbance versus concentration and the regression equations were calculated (Figure 2,3,4)

 

PRECISION:

It is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings. It was determined by studying repeatability, intra-day and intermediate precision of the method.

 

INTERMEDIATE PRECISION:

Stock solution containing 32.5 μg/ml PARA and 10 μg/ml of NIME was analyzed on 3 different days by three different analyst. The % RSD of assay value was calculated.

 

INTRADAY PRECISION:

Stock solutions containing 32.5 μg/ml PARA and 10 μg/ml of NIME was analyzed three times on the same day. The % RSD of assay value was calculated.

 

REPEATABILITY:

The precision of the method was checked by repeated (n=6) measurement of absorbance of solution containing PARA (13 µg/ml) and NIME (4 µg/ml) at 257nm and 393nm without changing the method parameter. The % RSD of response was calculated.

 

ACCURACY:

The validity and reliability of proposed methods were assessed by recovery study (standard addition method). Reference standard of each drug was added at three different concentrations levels (80, 100 and 120%) to prequantified sample solution of PARA (32.5µg/ml) and NIME (10µg/ml) tablet dosage form. Then the method described under assay section was followed. The amount of both drugs recovered was calculated. The value for recovery (%) was found to be 99.79% to 102.5% w/w for PARA and NIME respectively. These result revealed that the drug can be successfully recovered in the assay process.

 

LIMIT OF DETECTION AND LIMIT OF QUANTITATION:

LOD and LOQ of the drug were calculated using the following equations designated by International Conference on Harmonization (ICH) guideline:

LOD= 3.3σ/S

LOQ=10σ/S

Where,

σ = Standard deviation of the response,

S = Slope of calibration curve.

 

ROBUSTNESS:

To evaluate the robustness of the developed method, small deliberate variations in the optimized method parameters were done. The effect of change in wavelength were studied. The method was found to be unaffected by small changes like ± 2 nm change in wavelength (Table 4). The absorbance of test solution was measured at 255, 259, 391 and 395nm.

 

 

Table 1. Regression analysis data and summary of validation parameters for the proposed method.

PARAMETERS

ABSORBANCE CORRECTION METHOD

PARA

NIME

Wavelength (ʎmax)

257 nm

393 nm

Linearity (µg/ml)

4 -18

10 – 30

Slope

0.06

0.0487

Intercept

0.237

0.066

Correlation coeficient

0.991

0.997

Assay of formulation(%w/w)

100.03

101

Repeatability % RSD

0.25

0.55

Intermediate Precision % RSD

0.28

1.19

Intra day Precision % RSD

0.55

1.83

Accuracy (%w/w) Recovery

99.79 – 100.17% w/w

100 – 102.5% w/w

LOD (µg/ml)

1.49

1.23

LOQ (µg/ml)

4.51

3.73

 

 

 

 

Figure 2. Calibration curve of PARA at 257 nm

 

 

Figure 3. Calibration curve of NIME at 257 nm

 

 

 

Figure 4. Calibration curve of NIME at 393 nm

 

 

Figure 5. UV spectra of Paracetamol

 

 

Figure 6. UV spectra of Nimesulide

 

 

Figure 7. Overlain spectra of PARA and NIME

 

 

Figure 8. UV spectra of test solution

 


Table 2. Precision data of intermediate and intraday assay

Sample No.

% Assay of PARA

% Assay of NIME

Intermediate

Intraday

Intermediate

Intraday

1

100.38

100.76

100

99.5

2

99.46

100

98

100.5

3

100.23

101.38

98.5

98

Mean

100.02

100.71

98.83

99.33

± SD

0.281

0.556

1.19

1.82

% RSD

0.28%

0.55%

1.20%

1.83%

 


Table 3. Recovery studies

Drug

Amount Present (µg/ml)

Actual Amount Added (µg/ml)

Conc. after dilution (µg/ml)

Amount found (µg/ml)

Amount recovered (µg/ml)

% Recovered

± SD

% RSD

PARA

32.5

26

11.7

44.2

11.72

100.17

0.213

0.212

32.5

32.5

13

45.5

13.02

100.15

32.5

39

14.3

46.8

14.27

99.79

NIME

10

8

3.6

13.6

3.69

102.5

1.322

1.30

10

10

4

14

4.02

100.5

10

12

4.4

14.4

4.41

100

 


Table 7. Robustness study data at different wavelength

Concentration (µg/ml)

Wavelength variation (nm)

Absorbance

% Assay (w/w)

40

255

0.849

100.07

40

257

0.850

100.76

40

259

0.851

100.30

40

391

0.193

99.25

40

393

0.194

99.5

40

395

0.194

99.58

 

RESULT AND DISCUSSION:

VALIDATION OF PROPOSED METHOD [13]

The proposed method has been validated for the simultaneous determination of PARA and NIME in tablet dosage form using following parameters.

 

LINEARITY:

Linear correlation was obtained between absorbance versus concentrations in the range of 4-18 µg/ml and 10-30 µg/ml for PARA and NIME respectively for the proposed method. Regression parameters and is linearity data mentioned in table 1.

 

PRECISION:

INTERMEDIATE PRECISION:

The low % RSD values of intermediate and intraday assay for PARA and NIME, reveals the proposed method is precise (Table 2).

 

REPEATABILITY:

The % RSD values for PARA and NIME were found to be 0.25 and 0.55. The low values of % RSD indicate the proposed method is repeatable.

 

ACCURACY:

The recovery experiment was performed by the standard addition method. The recoveries were 99.79 – 100.17 % w/w and 100 -102.5 %w/w for PARA and NIME, respectively (Table 1). Results of recovery studies are shown in Table 3.

LOD AND LOQ:

LOD for PARA was found to be 1.49 µg/ml at 257nm and LOD for NIME was found to be 1.74 µg/ml at 257 nm and 1.23 µg/ml at 393 nm respectively. LOQ for PARA was found to be 4.51 µg/ml at 257nm and of NIME was found to be 4.69 µg/ml at 257 nm and 3.73 µg/ml at 393 nm respectively (Table 1). This data show that method is sensitive for the determination of PARA and NIME.

 

ROBUSTNESS:

The minor variation in detection wavelength did not significantly altered % assay value. The % assay value was found to be in the range of 99.79% -100.17w/w. Hence the method is found to be robust.

 

CONCLUSION:

The developed method was found to be simple, rapid, economical, precise, accurate and robust. The proposed  method use 0.1 N NaOH. Hence proposed method is ecofriendly and cost effective. Hence, the proposed method could be effectively applied for the routine analysis of PARA and NIME in bulk and in combined tablet dosage form.

 

ACKNOWLEDGEMENT:

The authors are thankful to Progressive Education Society’s Modern College of Pharmacy, Moshi, Pune for providing all the necessary facilities required for the research work. The authors are also thankful to all the teaching and non-teaching staff and the colleagues for their constant support. Hearty thanks to our parents.

 

REFERENCES:

1.     Indian Pharmacopoeis Vol II, New Delhi, The controller of Publications.Govt of India, 1996,554.

2.     British National Formulary (BNF 41) British medical association: London. 2001: 464.

3.     British Pharmacopoeia, Vol II, London. 1998, p.1854.

4.     Prasanna Reddy. Battu, Simultaneous RP-HPLC Determination of Nimesulide and Paracetamol in Tablets, International Journal of PharmTech Research, Vol.1, July-Sept 2009 pp 514-516.

5.     Deepali Gharge, Simultaneous Estimation of Nimesulide and Paracetamol in Solid Dosage Form by Rp-Hplc Method, International Journal of PharmTech Research, Vol.2, April-June 2010, pp 1330-1333.

6.     Rupali Kirtawade, Simultaneous UV Spectrophotometric Method for Estimation of Paracetamol and Nimesulide in Tablet Dosage Form, International Journal of ChemTech Research, Vol.2, April-June 2010, pp 818-821.

7.     Satyabrata Sahu, Simultaneous Spectrophotometric Estimation of Nimesulide and Paracetamol in Liquid Dosage Form, Journal of Pharmacy Research Vol.3.Issue 8, August 2010 pp 1973-1975.

8.     Magda Mohammed El-Henawee, Spectrophotometric Determination of Nimesulide in Pure and in Pharmaceutical Formulations using Ion-Associates Complex Formation, Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry. 2, 2014, 240- 248.

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10.  EL-Saharty, Y.S, Refaat, M. and EI-khateeb, Drug. Develop. Ind. Pharm, 2002, 28,91.

11.  Zawilla.N.H, Mohamad,M.A., EI-ousy, N.M.,and ELMoghazy Aly. J.Pharma. Biomed. Anal., 2002, 27,243.

12.  Hinz,B., Auge.D., Rau, T., Rietbrck, S., Brune, K. and Werner, U. Biomed Chromatogram, 2003, 17,268.

13.  International Conference on Harmonization (ICH), Q2R1, Validation of Analytical Methods: Methodology, Yokohama, Japan, (2005)

 

 

 

 

 

 

Received on 06.12.2017          Accepted on 18.02.2018        

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(1): 33-38.

DOI:    10.5958/2231-5675.2018.00006.6