Spectrophotometric Methods for Determination of Olsalazine Sodium

 

S.M. Malipatil*, Mogal Dipali and Bharath S. Athanikar

Department of Pharmaceutical Analysis, H.K.E.S's College of Pharmacy, Gulbarga-585105,  Karnataka (India).

Corresponding author: smmalipatil@gmail.com

 

ABSTRACT:

Three simple, sensitive, selective and accurate spectroscopic methods (A, B and C) have been developed for quantitative estimation of olsalazine sodium in bulk drug and pharmaceutical formulation (capsules). Method A and B is based on the reduction of ferric ions to ferrous ion by olsalazine sodium, which further in presence of 1,10-Phenanthroline, 2,2'-Bipyridyl produce blood red coloured chromogen with absorption maximum at 510 nm and 518 nm respectively. Method C is based on the direct colorimetric measurements of olsalazine sodium with 0.1N Sodium hydroxide. It gives yellowish-orange coloured chromogen with absorption maximum at 465nm. Method A, B and C obey Beer’s law in concentration range of 1-5 µg/ml, 5-25 µg/ml and 1-5 µg/ml respectively. Interference studies were conducted and it was found that the common excipients usually present in the dosage forms do not interfere in the proposed methods. The optical characteristics, regression analysis data, and precision of the methods were calculated. The proposed methods were found to be accurate, reproducible and consistent. It was successfully applied for the analysis of marketed formulation and could be effectively used for the routine analysis of formulations.

 

KEYWORDS: Olsalazine Sodium, double distilled water, 1,10-Phenanthroline, 2,2'-Bipyridyl, 0.1N Sodium hydroxide (0.1N NaOH).

 


 

INTRODUCTION:

Olsalazine1-3 (Fig.-1) is chemically 3,3'dicarboxy-4,4'-dihydroxy-azobenzene. Olsalazine is the azo-linked dimer of 5-amino salisylic acid (5-ASA). Olsalazine is a pro-drug which is used in the treatment of inflammatory bowel disease. Olsalazine is converted into mesalamine by the bacteria present in the colon, which is the therapeutically active agent used in the treatment of ulcerative colitis4. Mucosal production of arachidonic acid (AA) metabolites, both through the cyclooxygenase pathways i.e. prostanoids, and through lipoxygenase pathways, i.e. leukotrienes (LTs) and hydroxyecosatetraenoic acids (HETEs) is increased in patients with chronic inflammatory bowel disease and it is possible that mesalamine diminishes inflammation by  blocking cyclooxygenase and inhibiting prostaglandin (PG) production in the colon5. Literature survey reveals that there are some methods reported for determination of Olsalazine sodium in biological sample by HPLC6,7. Separation and determination of Olsalazine sodium and its impurities by HPLC8,9, capillary GC10 with Headspace, Spectrophotometric study11, different pulse voltametry12, electrochemical detection13, voltametric detection14 and electro-spray ionization mass spectroscopy15.

 

The objective of the present study was to develop simple, precise, accurate and economic analytical methods with the better detection range for estimation of olsalazine sodium in bulk drug and pharmaceutical formulations.

 

MATERIALS AND METHODS

Instrument:

A double beam Shimadzu UV-Visible spectrophotometer 1800, 1 cm matched glass cells were used to measure the absorbance of the resulting solution.

 

Materials:

Pure form was obtained from Emcure Ltd., Pune, India as a gift sample, double distilled water and 0.1N NaOH were used as a solvent in method A, B and C respectively. Two commercial brands of capsules having 250 and 500 mg strengths were purchased from local pharmacy.

 

Standard drug solutions:

Method A and Method B:

About 100 mg of Olsalazine sodium was accurately weighed and dissolved in double distilled Water, the total volume was made up to 100 ml with double distilled water. The final concentration was made to 1000 µg/ml (1 ml=1000 µg). From this solution 10ml of sample was transferred in to separate 100ml volumetric flask and the volume was made up to the mark with double distilled water the concentration found to be (100 µg/ml).

 

Fig.1

 

Method C:

Accurately about 100 mg of the pure drug was weighed and dissolved in 0.1N NaOH in 100 ml volumetric flask up to the mark to give standard stock solution (1000 μg/ml) and from this solution 10 ml of sample was transferred in to separate 100 ml volumetric flask and make up the volume up to mark with 0.1N NaOH to get concentration 100 µg/ml.

 

Calibration curves of Olsalazine Sodium:

Method A:

Fresh aliquots of Olsalazine sodium ranging from 0.1-0.5 ml (1ml-100 µg/ml) were transferred into a series of 10 ml volumetric flasks to provide final concentration range of 1 to 5 μg/ml. To each flask 1ml of ferric chloride (0.2%) solution and 0.5ml of alcoholic 1,10 phenanthroline (0.5%) were added and heated at 60şC for 20 minutes. The solutions were cooled to room temperature and made up to the mark with double distilled water.

 

For the selection of analytical wavelength, 3 μg/ml working standard solution was prepared and this solution was scanned in the spectrum mode from 800 nm to 400 nm. The absorbance of blood red colored chromogen was measured at 510 nm against the reagent blank (Fig. 2). The colored species was stable for 4 hours. The amount of Olsalazine sodium present in the sample solution was computed from its calibration curve (graph 1).

 

Fig 2: Visible spectrum of Olsalazine sodium by using 1,10-Phenanthroline (3 μg/ml)

 

Graph 1: Calibration curve of Olsalazine sodium with 1,10-Phenanthroline

 

Method B:

Fresh aliquots of Olsalazine sodium ranging from 0.5–2.5 ml (1 ml-100 µg/ml) were transferred into a series of 10 ml volumetric flasks to provide final concentration range of 5 to 25 μg/ml. To each flask 1ml of ferric chloride (0.2%) solution and 1 ml of alcoholic 2,2' Bipyridyl (0.1%) were added and heated at 600C for 10 minutes. The solutions were cooled to room temperature and made up to the mark with double distilled water.

 

For the selection of analytical wavelength, 15 μg/ml working standard solution was prepared and this solution was scanned in the spectrum mode from 800 nm to 400 nm. The absorbance of blood red colored chromogen was measured at 518 nm against the reagent blank (Fig. 3). The color species was stable for 4 hours. The amount of Olsalazine sodium present in the sample solution was computed from its calibration curve (graph 2).

 

Fig 3: Visible spectrum of Olsalazine sodium by using 2,2'-Bipyridyl (15 μg/ml)

 

Graph 2: Calibration curve of Olsalazine sodium with 2,2'-Bipyridyl

 

Method C:

Fresh aliquots of Olsalazine sodium ranging from 0.1-0.5ml (1ml-100µg/ml) were transferred into a series of 10ml volumetric flasks to provide final concentration range of 1 to 5 μg/ml. Made up the volume up to the mark with 0.1N NaOH set aside for 15 minutes. at room temperature.

 

For the selection of analytical wavelength, 3 μg/ml working standard solution was prepared and this solution was scanned in the spectrum mode from 800 nm to 400nm. The absorbance of yellowish-orange colored chromogen was measured at 465 nm against 0.1N NaOH reagent as blank (Fig.4). The colored species was stable for 2 hours. A calibration curve was plotted by taking the absorbance against the concentration of standard stock solutions (Graph 3). By using the calibration curve, the concentration of the sample solution can be determined.

 

Fig 4: Visible spectrum of Olsalazine sodium by using NaOH        (3 μg/ml)

 

Graph 3: Calibration curve of Olsalazine sodium with NaOH

 

Sample analysis of capsules formulation:

Method A and Method B:

For method A and B the estimation of Olsalazine sodium in capsule dosage form 10 capsules were weighed. Capsule powder equivalent to 100 mg of Olsalazine sodium was weighed, dissolved and further dilution was carried out with quantity sufficient of double distilled water. This was then filtered through the Whatmann filter paper No. 41 to get the stock solution of concentration 100 µg/ml. Further this stock solution was suitably diluted to get 3 μg/ml and 15 μg/ml these samples were analysed using proposed method A and B respectively.

 

Method C:

For method C, the estimation of Olsalazine Sodium in capsule dosage form 10 capsules were weighed. Capsule powder equivalent to 100 mg of Olsalazine sodium was weighed and dissolved in 0.1N NaOH and further dilution was carried out with quantity sufficient of 0.1N NaOH. This was then filtered through the Whatmann filter paper No. 41 to get the stock solution of concentration 100 µg/ml. Further this stock solution was suitably diluted to get 3 μg/ml and these samples were analysed using proposed methods.

 

UV reference method:

Accurately about 100 mg of the pure drug was weighed and dissolved in double distilled water in 100 ml volumetric flask up to the mark to give standard stock solution (1000 μg/ml) and from this solution 10 ml of sample was transferred in to separate 100 ml volumetric flask and make up the volume up to the mark with double distilled water to get concentration 100 µg/ml as a second stock. From resulting stock solution were 0.6 ml pipetted out and transferred in to 10 ml volumetric flask diluted with double distilled water up to the mark to get final concentration 6μg/ml of solution. The absorbance of the solution was measured at 366 nm. The content of the drug was calculated from the Beer-Lambert plot.

 

ANALYTICAL VALIDATION:

Linearity Range:

The statistical parameters and regression equations which were calculated from the calibration curves along with the standard error of the slope and intercept. Regression analysis indicated a linear relationship between absorbance and concentration.

 

To establish linearity of the proposed method, six separate series of solutions of the drug           (1-5 μg/ml, 5-25μg/ml and 1-5 μg/ml) were prepared from the stock solution and analysed.

 

Sensitivity:

The LOD and LOQ of the Olsalazine sodium by proposed methods were determined by using calibration standards. LOD and LOQ were calculated as follows:

LOD = 3.3σ/S

LOQ =10σ/S

Where, S is the slope of the calibration curve

σ is standard deviation of the y-intercept of regression equation (Table 1).

 

Specificity and Selectivity:

The comparison of standard spectra and spectra from capsule solutions shows that the wavelengths of maximum absorbance and maxima/minima did not change. It was concluded that the other excipients like starch, gelatin, talc, magnesium sterate, lactose, and steric acid present in the capsules forms did not interfere, with quantitation of Olsalazine Sodium in capsules by developed methods.

 

Accuracy:

Accuracy was assessed as the standard deviation and percentage relative standard deviation studies were found to be satisfactory (Table 2). To give additional support to accuracy of the developed assay method, standard addition method was done. The percent recovery of the added pure drug was calculated as,

% Recovery = [(Cv- Cu) / Ca] X 100

Where  Cv is the total drug concentration measured after standard addition

Cu is the drug concentration in the formulation

Cais the drug concentration added to the formulation (Table 2).

 

Precision:

Repeatability was determined by using different levels of drug concentrations (same concentration levels taken in accuracy study), prepared from independent stock solution and analysed (N=6) (Table 4). Inter-day and intra-day variation and instrument variation (shimadzu-1800) were taken to determine intermediate precision of the proposed methods (N=6). The % relative standard deviation of the predicted concentrations from the regression

 

RESULTS AND DISCUSSION:

Olsalazine Sodium exhibits reducing property due to the presence of hydroxyl moiety enabled the use of its oxidation reaction followed by complex formation of drug with 1,10-phenanthroline, 2,2' Bipyridyl in presence of ferric chloride to produced blood red coloured chromogen  exhibiting absorption maxima at 510 nm and 518 nm respectively. The method C was based on direct colorimetric estimation of Olsalazine sodium in 0.1N NaOH to produced yellowish-orange coloured chromogen exhibiting absorption maxima at 465 nm The optical characteristics such as Beer’s law limits, absorption maxima,  molar absorptivity,  sandell’s  sensitivity  are represented in Table 1.

 

Table-1: Optical characteristics and linearity data

Parameters

Method A

Method B

Method C

λ max (nm)

510

518

465

Beer’s Law limit (µg/ml)

1-5

5-25

1-5

Molar absorptivity

(L mol-1 cm­-1)

6.962 x 104

0.869 x 104

7.224 x 104

Sandell’s sensitivity

(µg cm-2/ 0.001 abs units)

0.0192

0.0132

0.0190

Regression equation (Y*)

Slope (b)

Intercept (a)

Correlation coefficient (r2)

 

0.153

0.007

0.9999

 

0.0229

0.0199

0.9989

 

0.1977

0.0451

0.9979

LOD (µg/ml-1)

0.0215

0.1436

0.0235

LOQ (µg/ml-1)

0.0652

0.4351

0.0713

% RSD

0.219

0.0010

0.724

Range of errors**

Confidence limits with 0.05 level

Confidence limits with 0.01 level

 

±0.00105

 

±0.00156

 

±0.001057

 

±0.001564

 

±0.001491

 

±0.000006

*Y = bC +a, Y is the absorbance unit and C is the concentration in µg/ml.

**Eight measurements


Table 2:Results of standard addition method

Method

Concentration of drug in formulation (μg/ml)

Concentration of pure drug added (μg/ml)

% level of pure drug added

Total concentration of drug found (μg/ml)

% Analytical recovery ± SD

Method A

3

2.4

80

5.39

99.58 ± 0.03

3

3.0

100

6.02

100.83 ± 0.11

3

3.6

120

6.59

99.86 ± 0.001

Method B

10

8.0

80

17.97

99.58 ± 0.12

10

10

100

20.16

101.66 ± 0.03

10

12

120

21.90

99.16 ± 0.006

Method C

3

2.4

80

5.46

101.2 ± 0.14

3

3.0

100

5.97

98.66 ± 0.04

3

3.6

120

6.69

101.5 ± 0.06

(Each value is a result of three separate determinations)

% Recovery = [(Cv- Cu) / Ca] X 100

Where, Cv is the total drug concentration measured after standard addition

Cu is the drug concentration in the formulation; Ca is the drug concentration added to the formulation


Table 3: Evaluation of Olsalazine Sodium in pharmaceutical dosage forms (capsules)

Sample

 

Labelled amount

(mg)

Amount obtained (mg)

Percentage recovery*

Proposed method

A

Proposed method

B

Proposed method

C

Reference method

(UV method)

Proposed method

A

Proposed method

B

Proposed method

C

C1

250

249.2

249.5

249.3

99.96

98.68

99.80

99.72

C2

500

497.8

498.7

500.0

99.94

99.56

99.74

100.02

*Mean and standard deviation of six determinations.

C1 and C2 are tablets from U.C.B. pharma and Pfizer Pharmaceuticals.

 

Table 4: Results of interday and intraday precision studies

Concentration (in μg/ml)

Inter-day repeatability

% RSD (N=6)

Intra-day repeatability % RSD (N=6)

Inter-instrument repeatability % RSD (N=6)

Day 1

Day 2

Day 3

Instrument-I

Instrument-II

Method A

1

0.16

0.14

0.14

0.15

0.19

0.18

3

0.21

0.24

0.22

0.23

0.24

0.24

5

0.28

0.25

0.27

0.27

0.22

0.23

Method B

5

0.009

0.006

0.008

0.007

0.007

0.006

 

10

0.002

0.004

0.001

0.002

0.002

0.001

25

0.004

0.005

0.003

0.005

0.004

0.004

Method C

1

0.63

0.65

0.64

0.66

0.68

0.66

3

0.72

0.75

0.70

0.74

0.71

0.74

5

0.50

0.52

0.51

0.55

0.57

0.58

Instrument-I     : Shimadzu UV-Visible spectrophotometer 1700.; Instrument-II   : Shimadzu UV-Visible spectrophotometer 1800.

 

 


The regression analysis using the method of least squares was made for the slope (b), intercept (a) and correlation coefficient (r2) obtained from different concentrations and the results are summarized in table 1. The percent relative standard deviation and percent range of errors (0.05 and 0.01 level of confidence limits) were calculated for two methods and results are given in table 1.

 

The values obtained for the determination of Olsalazine sodium in capsules by proposed and UV methods are compared in table 3. The percentage recoveries are given in table 2.

 

CONCLUSION:

In this study, colorimetric methods were developed and validated according to ICH guidelines. Olsalazine sodium can be directly determined in capsule, in presence of excipients by using spectrophotometric methods. The apparatus and reagents used are accessible in simple laboratories.

 

The results indicate that the methods reported here are found to be simple, sensitive, accurate, precise, and economical and can be used in the determination of Olsalazine from pharmaceutical dosage forms in a routine manner.

 

ACKNOWLEDGEMENT:

The authors are grateful to the Principal of H.K.E. Society’s Matoshree Taradevi Rampure Institute of Pharmaceutical Sciences for providing excellent research facilities in analysis department. We are also thankful to Emcure Ltd., Pune for providing pure sample of Olsalazine sodium as gift sample.

 

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Received on 05.09.2011          Modified on 21.10.2011

Accepted on 06.11.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 4(12): Dec. 2011; Page 1852-1856