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157 | P a g e International Standard Serial Number (ISSN): 2319-8141

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International Journal of Universal Pharmacy and Bio Sciences 3(3): May-June 2014

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES IMPACT FACTOR 1.89***

ICV 5.13*** Pharmaceutical Sciences RESEARCH ARTICLE……!!!

UV SPECTROPHOTOMETRIC METHOD FOR THE SIMULTANEOUS

ESTIMATION OF SERRATIOPEPTIDASE AND NIMESULIDEIN

THEIR BULK AND COMBINED DOSAGE FORM

Ashish Sharma D*.,Prajapati Laxman M., Joshi Amit K., Kharodiya Mahammadali L.

Rajpurohit Prashant T., Patel Dhaval., Ramani Sandip.

Department of Quality Assurance, Shri B. M. Shah College of Pharmaceutical Education and

Research, Modasa-383315, Gujarat, India.

KEYWORDS:

Serratiopeptidase,

Nimesuldie , UV

spectrophotometry,

Distilled water.

For Correspondence:

Ashish Sharma*

Address:

Shri B. M. Shah College

of Pharmaceutical

Education and Research,

Modasa-383 315,

Gujarat, India.

Mob No.: 9725669755

Email:

[email protected]

m

ABSTRACT

A simple, rapid, economical, precise and accurate UV

spectrophotometric method for simultaneous estimation of

Nimesulide and Serratiopeptidase in bulk drug and combined dosage

form (Tablet Dosage form) has been developed. The method

employed solving of simultaneous equation based on

measurement of absorbance at two wavelengths, 276.8 nm and

389.2 nm, λmaxof Serratiopeptidase and Nimesulide respectively.

For Spectrophotometric method Linearity for Serratiopeptidase and

Nimesuldie were in the range of 50-300 µg/ml and 200-700 and the

Correlation coefficient (r2) 0.996 and 0.998 respectively. The %

recovery for Serratiopeptidase and Nimesuldie was found to be

99.3% to 101.1% and 99.76 % to 100.22%.



INTRODUCTION:

(SER) is chemically 5-Methyl-N-(4 (trifluoromethyl) phenyl)isoxazole-4-carboxamide Serrapeptase

(Fig 1)[1,2]

. Serratiopeptidase is a proteolytic enzyme isolated from the micro-organism Serratia E15

that has fibrinolytic, anti-edemic(prevents swelling and fluid retention),and anti-inflammatory

activity. (Fig 2)[1,2]

. Nimesulide is a relatively COX-2 selective, non-steroidal anti-inflammatory

drug (NSAID) with analgesic and antipyretic properties.

NEULITE-S is a fixed dose combination (FDC) of Nimesulide and Serratiopeptidase. Nimesulide is

chemically 4-Nitro-2-phenoxymethane sulphonanilide and Serratiopeptidase is proteolytic enzyme.

Various painful inflammatory conditions including those associated with osteoarthritis,

postoperative trauma, sports injuries, bronchitis, sinusitis etc are improved with Nimesulide.

Nimesulide is a potent and time tested NSAID and its spectrum of activity is improved by

Serratiopeptidase, it can be said that Nimesulide and Serratiopeptidase show synergistic potential

which is one of the most important factor in deciding the feasibility. Hypersensitivity to Nimesulide

and Serratiopeptidase. NEULITE-S is contraindicated in patients of active peptic ulcer disease,

moderate to severe hepatic impairment, severe renal failure and with blood coagulation disorders.

Fig. 1 : Chemical structures of the Serratiopeptidase

Fig. 2 : Chemical structures of the Nimesulide



MATERIALS AND METHODS:

Chemicals and solvents:

Serratiopeptidase (Alichem Laboratories pvt. Ltd. Ahmedabad.)

Nimesulide(Megh Pharmaceuticals pvt. Ltd, Modasa.)

Glass ware (Beaker, Pipette, volumetric flask)

Neulite S Tablet (Serratiopeptidase 15 mg and Nimesulide 100 mg)

(Indica Laboratories Pvt.Ltd. Ahmedabad)

Distilled water

Instruments:

Double beam UV-visible spectrophotometer ( Shimadzu 1601)

having two matched quartz cells with 1 cm light path

Digital analytical balance ( Shimadzu ATX 224)

Spectrophotometric condition:

Mode: Scan

Scan speed: Intelli scan

Wavelength range: 200 - 500 nm

Absorbance scale: -0.002 - 2.00 A

Initial base line correction: Distilled Water

Preparation of standard solution in Distilled Water

(A)Serratiopeptidase standard stock solution: (1000 μg/ml)

A 100 mg of SER was weighed and transferred to a 100 ml volumetric flask. Mix with

half quantity of distilled Water and volume was made up to the mark.

(B)Nimesuldie standard stock solution: (1000 μg/ml)

A 100 mg of NIM was weighed and transferred to a 100 ml volumetric flask. Mix with

half quantity of distilled Water and volume was made up to the mark.

Preparation of tablet mixture

A total of 20 tablets (15 mg SER and 100 mg NIM) were accurately weighed and powdered in a

mortar. The powder equivalent to the claimed amount was accurately weighed. The weighed

amount was quantitatively transferred to volumetric flask and dissolved in small quantity of

distilled water with vigorous followed by sonication for 30 minutes. The content was then

diluted up to 100 ml using distilled water.

Procedure for determination of wavelength for measurement

Simultaneous EquationMethod

The aliquots of SER and NIM stock solution were taken and diluted with the distilled water



individually, such that the final concentration of SER and NIM was 300 µg/ml and 700 µg/ml

respectively. The solution were scanned over the range of 200-500 nm using UV-visible

Spectrophotometer and spectrum was recorded. The wavelength ((λ1, λ2) at which maximum

absorbance was obtained was considered as λmax of the drug. These two wavelengths were used

to record absorbance using method shown in figure.

Preparation of calibration curve

A calibration curve was plotted over a concentration range of 50-300 µg/ml for SER and 200-

700 µg/ml for NIM individually. Accurately measured working standard solution of SER (0.5,

1.0, 1.5, 2.0, 2.5 and 3.0 ml) and working standard solution of NIM (2.0, 3.0, 4.0, 5.0, 6.0 and

7.0 ml ) were transferred to a set 10 ml volumetric flask individually, and diluted with distilled

water. The absorbance of each solution was measured at selected wavelengths. Calibration curves

were constructed by plotting absorbance versus concentration value for SER and NIM using UV-

Visible Spectrophotometer. The calibration curve is shown in figure for method. The results of

absorbance are shown in Table for simultaneous equation method.

Estimation of the SER and NIM using simultaneous equation method

The formula for SER and NIM was developed by adopting following proposed equation for the

purpose. Absorptivity of SER and NIM was determined of SER and NIM was determined and the

values are used to constitute the equation.

Simultaneous equation method: If a sample contains two absorbing drugs (X and Y) each of

which absorbs at the λ max of the other, it may be possible to determine both drugs by the

technique of simultaneous equations (Vierodt's method).

A2ay1 – A1ay2

Cx =------------------------------------

ax2ay1 – ax1ay2

A1ax2 – A2ax1

Cy =------------------------------------

ax2ay1 – ax1ay2

a) The absorptivity of X at λ 1 and λ 2, ax1 and ax2 respectively.

b) The absorptivity of Y at λ 1 and λ 2, ay1 and ay2 respectively.

c) The absorbances of the diluted sample at λ 1 and λ 2, A1 and A2 respectively.



Validation of proposed method

Parameters to be considered for the validation of method are:

Linearity and Range

The linearity response was determined by analyzing 6 independent levels of calibration curve

in the range of 50-300 µg/ml for SER and 200-700 µg/ml for NIM respectively (n = 3). The

calibration curve of absorbance vs. respective concentration was plotted and correlation

coefficient (r2) and regression line equations for SER and NIM were calculated.

Precision

Repeatability

Aliquots of 0.5 ml of working standard solution of SER (100 μg/ml) were transferred to a series of

10 ml volumetric flask. Aliquots of 3.0 ml of working standard solution of NIM (100 μg/ml) were

respectively transferred to the same above series of 10 ml volumetric flask. The volume was

adjusted up to mark with distilled water to get 50 μg/ml solution of SER and 300 μg/ml solution of

NIM. The absorbance of solutions were measured spectrophotometrically six times and relative

standard deviation (% R.S.D) was calculated.

Intraday precision

Aliquots of 0.5, 1.0 and 1.5 ml of working standard solution of SER (1000 μg/ml) were transferred

to a series of 10 ml volumetric flask. Aliquots of 2.0, 3.0 and 4.0 ml of working standard solution of

NIM (1000 μg/ml) were respectively transferred to the same above series of 10 ml volumetric

flask. The volume was adjusted up to mark with distilled water to get 50-150 μg/ml solution of

SER and 200-400 μg/ml solution of NIM. The absorbance of solutions were measured

spectrophotometrically three times on same day and relative standard deviation (%R.S.D) was

calculated.

Inter-day precision

Aliquots of 0.5, 1.0 and 1.5 ml of working standard solution of SER (100 μg/ml) were transferred to

a series of 10 ml volumetric flask. Aliquots of 2.0, 3.0 and 4.0 ml of working standard solution of

NIM (100 μg/ml) were respectively transferred to the same above series of 10 ml volumetric

flask. The volume was adjusted up to mark with distilled water to get 50-150 μg/ml solution of

SER and 200-400 μg/ml solution of NIM. The absorbance of solutions were measured

spectrophotometrically three times in three different days and relative standard deviation (%R.S.D)

was calculated.



Accuracy

Procedure

I) Preparation of Sample Solution for SER

Tablet mixture X: SER (150 μg/ml) + NIM (1000 μg/ml)

Standard solution Y: SER (1000 μg/ml)

Table : Step for accuracy measurement for SER

Sr. No Step 1 Step 2

Total Ser Conc.

(µg/ml)

1. Take 3.33 ml of solution X Add dis. Water to make up to 10 ml 50

2. Take 3.33 ml of solution X + 0.4 ml of solution Y

Add dis. Water to make up to 10 ml 90





II) Preparation of sample solution for NIM:

Synthetic mixture X: NIM (1000 μg/ml) + SER (150 μg/ml)

Standard solution Y: NIM (1000 μg/ml)

Table : Steps for accuracy measurement for NIM

Sr.

No

Step 1 Step 2 Total Nim

Conc

(µg/ml)

1. Take 3.0 ml of solution X Add dis. Water to make up to 10 ml 300







Each solution was scanned between 200 to 500 nm against dis. water as a blank. Absorbance of

solutions were measured at selected wavelengths for SER and NIM. The amount of SER and

NIM was calculated at each level (80%, 100%, and 120%) and % recoveries were computed.



Limit of detection

From the linearity curve equation, the standard deviation (SD) of the intercepts (response) was

calculated. Then LOD was measured by using mathematical expressions given in section.

The limit of detection (LOD) of the drug was calculated by using the following equations designated

by International Conference on Harmonization (ICH) guideline:

LOD = 3.3 Χσ/S

Where, σ = the standard deviation of the response

S = slope of the calibration curve.

Limit of quantification

From the linearity curve equation, the standard deviation (SD) of the intercepts (response) was

calculated. Then LOQ was measured by using mathematical expressions given in section.

The limit of quantification (LOQ) of the drug was calculated by using the following equations

designated by International Conference on Harmonization (ICH) guideline:

LOQ = 10 Χσ/S

Where, σ = the standard deviation of the response

S = slope of the calibration curve.

Simultaneous estimation of SER and NIM in tablet mixture

Simultaneous Equation Method

The tablet mixture of SER and NIM was prepared in ratio of 15:100. Twenty tablets containing

SER and NIM in ratio of 15:100 mg respectively was weighed and crushed to fine powder. Powder

equivalent to SER and was weighed and transferred in to 100 ml volumetric flask and dissolved in

few ml of dis. water, sonicated for 15 min and the volume was made up to the mark with dis. Water

and obtained a solution containing 150 µg/ml of SER and 1000 µg/ml of . This solution was filtered

through the whatmann filter paper no. 41 and filtrate was collected. Residues were washed with

dis water. The filtrate and washing were combined and volume of solution was made up to

100 ml with dis water. Absorbance of the resulting solution was measured at 276.8 nm and

389.2 nm against dis water as a reagent blank.



RESULTS AND DISCUSSION

Selection of Wavelength

UV spectrum of SER and NIM was recorded using UV-Visible Spectrophotometer, individually in the

range of 200-500 nm. The recorded spectrum of SER and NIM are shown in figure and figure

respectively being the wavelength at which drug solution possessed maximum absorbance 276.8 nm for

SER and 389.2 nm for NIM.

Figure : Overlain Spectra (SER 50 µg/ml and NIM 300 µg/ml)

Figure : Overlain spectra of SER showing linearity



Figure : Overlain spectra of NIM showing linearity

Preparation of Calibration Curve

Calibration curve of SER and NIM was developed individually by preparing different concentration of

SER and NIM measuring the absorbance at two selected wavelengths 276.8 nm and 389.2 nm. The

higher value of the regression coefficient confirmed the adherence to beer’s law and to establish

linearity range to be suggestive in setting the concentration of test solution. The selected concentration

and absorbance of the solution is noted in tables and at both wavelength along with calibration curve in

figure

Figure : spectra of SER for calibration curve



Figure : spectra of NIM for calibration curve

Simultaneous estimation of SER and NIM

Procedure: As discussed in methodology

SERRATIOPEPTIDASE

Table : Absorbance of SER at 276.8 and 389.2 nm

Concentrations

(µg/ml)

Absorbance at 276.8 Absorbance at 389.2

50 0.0389 0.0114

100 0.0658 0.0181

150 0.0983 0.0260

200 0.1296 0.0320

250 0.1603 0.0392

300 0.1973 0.0452



Calibration curve of SER at 276.8


NIMESULIDE

Table : Absorbance of NIM at 389.2 and 276.8 nm

Concentrations

(µg/ml)

Absorbance at 389.2 Absorbance at 276.8

200 0.0599 0.0471

300 0.1083 0.0717

450 0.1497 0.0956

500 0.2019 0.1188

650 0.2543 0.1420

700 0.3065 0.1637

y = 0.0006x + 0.0108R² = 0.998

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0 100 200 300 400 500 600 700 800

y = 0.0001x + 0.0048R² = 0.998

0

0.01

0.02

0.03

0.04

0.05

0 50 100 150 200 250 300 350



Calibration curve of NIM at 389.2


Table : Statistical data for SER & NIM by Spectrophotometric method

Parameters SER (at 276.8 nm) NIM (at 389.2 nm)

Linear Range (µg/ml) 50-300 200-700

Correlation coefficient (r2) 0.996 0.998

Slope 0.0006 0.0005

Intercept 0.0108 0.0415

Standard deviation of slope 0.000054 0.000057

Standard deviation of intercept 0.00015 0.0002

Limit of Detection (μg/ml) 15.4 19.1

Limit of Quantitation (μg/ml) 46.6 58

y = 0.0005x - 0.0415R² = 0.998

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0 100 200 300 400 500 600 700 800

y = 0.0002x + 0.0014R² = 0.999

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0 100 200 300 400 500 600 700 800



Validation of Proposed method

Linearity and range

Linearity for SER

Table: Absorbance of SER at selected wavelength 276.8 nm

Concentrations

(µg/ml)

Absorbance ± S.D. RSD

50 0.0389 ±0.0038 9.76

100 0.0658 ±0.0010 1.51

150 0.0983 ±0.0028 2.84

200 0.1296 ±0.0052 4.01

250 0.1603 ±0.0032 1.99

300 0.1973±0.0013 0.65


Range 50-300 µg/ml

y = 0.0006x + 0.0108R² = 0.998

0

0.05

0.1

0.15

0.2

0.25

0 50 100 150 200 250 300 350



Linearity for NIMESULDIE

Table : Absorbance of NIM at selected wavelength 389.2 nm

Concentrations

(µg/ml)

Absorbance ± S.D. % RSD

200 0.0599 ±0.0020 3.33

300 0.1083 ±0.0041 3.78

450 0.1497 ±0.0029 1.93

500 0.2019 ±0.0021 1.04

650 0.2543 ±0.0037 1.45

700 0.3065±0.0021 0.68


PRECISION:

REPEATBILITY

Table : Repeatability of sample application data for SER & NIM

n = 6 determination

Result: The % RSD is < 2 for SER & NIM which indicate that the method is precise.

y = 0.0005x - 0.0415R² = 0.998

0

0.05

0.1

0.15

0.2

0 100 200 300 400 500 600 700 800

SER (50 μg/ml) NIM (300 μg/ml)

Inj No. Area

1 0.0356 0.1101

2 0.0375 0.1065

3 0.0351 0.1123

4 0.0365 0.1021

5 0.0401 0.1085

6 0.0345 0.1073

Mean. 0.0365 0.1076

Std. Dev. 0.00020 0.00034

% RSD 0.54 0.31



Intra and inter day precision

Intra-day precision was determined by analyzing SER & NIM for three times in the same day.

Inter-day precision was determined by analyzing SER & NIM daily for Three days.

Table : Precision data for SER

Conc. (µg/ml) Intra-day (n=3) % RSD Inter-day (n=3) % RSD

50 0.0315±0.00019 0.31

0.0412±0.00013 0.60

100 0.0598±0.00024 0.40 0.0632±0.00033 0.52

150 0.1012±0.00044 0.31 0.1102±0.00035 0.43

Table : Precision data for NIM

Conc. (µg/ml) Intra-day (n=3) % RSD Inter-day (n=3) % RSD

200 0.0618±0.00021 0.20 0.0629±0.00013 0.33

300 0.1123±0.00011 0.26 0.1172±0.00031 0.97

400 0.1513±0.00041 0.29 0.1501±0.00045 1.97

Result: The % RSD is within 0.20 – 1.97 for SER &NIM which indicate that the method is precise.

ACCURACY (% RECOVERY STUDY)

To study the accuracy 20 Tablets were taken and analysis of the same was carried out. Recovery studies

were carried out by addition of standard drug to the sample at 3 different concentration levels taking in

to consideration percentage purity of added bulk drug samples.

Table : Determination of Accuracy (Tablet)

Amt. of sample

taken(μg/ml)

Amt. of drug std added

(μg/ml)

Amt. recovered % Recovery

SER NIM SER NIM SER NIM SER NIM

50 300 0 0 - - - -

50 300 40 240 89.4 541.2 99.3% 100.22%

50 300 50 300 101.1 598.6 101.1% 99.76%

50 300 60 360 110.6 659.1 100.54% 99.86%

Result: The % recovery is within limit (98.0 – 102.0 %) so the method is accurate.



APPLICATION OF DEVELOPED METHOD TO PHARMACEUTICAL FORMULATION

The proposed validated method was successfully applied to determination of SER &NIM in its tablet

dosage form.

Figure : Spectrum of Tablet formulation

Table : Assay Results of Marketed Dosage Forms

Formulation Actual concentration of drug

(µg/ml)

Amount obtained of

drug (µg/ml)

%

drug

SER NIM SER

NIM SER NIM

Tablet 15 100 16.64 100.65 97.94 98.74

Table : Summary of validation Parameters of UV Spectrophotometry

Parameters SER NIM

Linear Range (µg/ml) 50-300 200-700

Limit of Detection (μg/ml) 15.4 19.1

Limit of Quantitation (μg/ml) 46.6 58

Precision

( %RSD)

Repeatability 0.54 0.31

Intra day 0.40 0.26

Inter day 0.52 0.97

Accuracy ( % Recovery) 99.3-101.1 99.76-100.22

% Assay 97.94 98.74



ACKNOWLEDGEMENT:

Firstly, I offer my adoration to God Almighty and my ideal Parents who created me, gave me the

strength and courage to complete my dissertation and gave me the opportunity to thank all those

people through whom his grace was delivered to me. A successful outcome in any research endeavor

attributes itself to the selfless guidance of the mentor.

I would also like to thank MEGH PHARMACEUTICALS, Modasa and ALICHEM LABORATORIES,

Ahmedabad for providing me Nimesulide and Serratiopeptidase A.P.I. gift sample and Mukeshbhai for

providing me Neulite S tablet.

CONCLUSION:

The proposedUV spectrophotometric method was simple, precise, accurate, economic and rapid for the

determination of SER and NIM in bulk drug and in tablet dosage form with no interference of the

common additives and excipients.

So, it can be successfully adopted for routine quality control analysis of SER and NIM in combined

dosage form without any interference from common excipients and impurities.

The developed method was validated for the various parameters as ICH guideline.

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