INTRODUCTION:

Balsalzide(BSZ)^1,2 a new drug, which is chemically (E)-5[[-4-[[(2-carboxyethyl)amino] carbonyl]phenyl]azo]2-hydroxybenzoic acid, disodium salt, dihdrate. BSZ is a prodrug delivered intact to the colons by bacterial azo-reduction to release equimolar quantities of mesalamine (5-amion salicylic acid) an active portion of the molecule & 4-amino benzoyl – (beta) – alanine³. Literature survey revealed that a HPLC⁴ method with fluorescence detection was reported for the estimation of BSZ in plasma. But there are no methods reported for the estimation of BSZ in bulk drugs and in formulation by UV-Visible Spectrophotometry and derivative spectroscopy, hence, it was aimed to develop the simple, precise, accurate and economical methods for the estimation of BSZ inbulk and in formulation by uv visible spectrophotometry and derivative spectroscopy.

MATERIALS:

Shimadzu UV-1700 UV-Visible Spectrophotometer with 1 cm matched quartz cells was used for spectral and absorbance measurements. BSZ was a gift sample from Krebs Pharma, Chennai. The commercially available capsules formulation was procured from the local market. All the Chemicals used were of AR grade.

METHODS:

Simple UV Spectroscopic Method^5,6(method A)

This method is based on the measurements of absorbance of BSZ at its l max was found to be 358 nm. Stock solution of BSZ (0.5 – 5 ml of 200 mg/ml) were transferred into 50ml volumetric flask and made upto mark with distilled water. The absorbances of resulting solutions were measured at 358 nm using water as blank. Calibration curve was plotted by using concentration versus absorbance.

Simple colorimetric method⁷(method B)

This method is based on the colour formation when it was dissolved in alkaline media. When BSZ is dissolved in 1 M sodium hydroxide at pH 12, a dark red coloured chromogen was formed and it has given the absorption maximum at 456 nm. Stock solution of BSZ (0.5-5ml of 200 mg/ml) were transferred into 50 ml volumetric flask and made upto the mark with 1 M sodium hydroxide. The absorbance of dark red coloured chromogen was measured at 456 nm using 1 M sodium hydroxide as reagent blank. The calibration graph was constructed.

Derivative Spectroscopy method^8,9(method C)

The term derivative spectroscopy refers to a technique in which the rate of change of spectral intensity with wavelength is the slope of the spectrum is measured. It represents an elegant way of resolving overlapping spectra and has been successfully used for the determination of drugs alone or in mixture. Stock solution of BSZ (2.5 –12.5 ml of 100 mg/ml) were prepared with distilled water. The solution was scanned in UV-region of 200-500 nm, the spectra was derivatized to first order and measured at 383 nm. The different concentrations from stock solutions were prepared and absorbance were measured was linear with concentration in the range of 5-25mg/ml.

Table 1: Optical characteristics

Parameters	Method A	Method B	Method C
λmax (nm)	358	456	383
Beer’s law limit (µg/ml)	2-20	2-20	5-25
Molar absorptivity (mol/l/cm)	2.5855× 10⁴	2.5637× 10⁴	0.5753× 10⁴
Regression equation (Y=a+bx)
Slope (b)	0.0583	0.06928	0.00129
Intercept (a)	0.0056	0.0617	0.00022
Correlation coefficient (r)	0.9999	0.9999	0.9992
Relative standard deviation (n=6)	0.00163	0.0083	0.0640
Sandell’s sensitivity µg/cm2/0.001AU)	0.017075	0.0144	0.7732
LOD (µg/ml)	0.1015	0.1949	0.8851
LOQ (µg/ml)	0.3075	0.5905	2.6821

(Y=a+bx, where, x is concentration in µg/ml and Y is absorption units, a and b are intercept and slope, respectively)

Analysis of Formulation:

Sample preparation:

Capsules containing BSZ 750mg (Balacol, Torrent pharmaceutical Ltd., India) was taken for analysis, 20 capsule were weighed and average net weight was found out, powdered and equivalent to 50mg of BSZ was weighed accurately and dissolved in distilled water for method A and C and 1 M sodium hydroxide for method B by sonication for 3 minutes.

Assay procedure:

The volumetric flask were made upto volume to 50 ml with respective solvents and prepared concentration was 200mg/ml for method A and B and 100mg/ml for method C.the solutions were made upto the volume with appropriate solvents, then the solutions were centrifuged at 3500rpm for 15 minutes .the clear supernatant liquids were taken and The nominal concentration were prepared from calibration graph and the absorbance of the formulation were measured as per the above methods . To ensure the precision of the method, the repeated analysis of formulation were carried out for six times and the amount were calculated.

RESULTS AND DISCUSSION:

Three simple precise and accurate methods were developed for the estimation of BSZ in bulk and in formulations, Method A is uv simple spectroscopic method, Method B is the simple colorimetric method and Method C is derivative spectroscopic method. The linearity of the methods were found to be 2-20 mg/ml for method A and B and 5-25mg/ml for method C. The optical characteristics such as correlation co-efficient, slope, intercept, molar obsorptivity, sandel’s sensitivity LOD and LOQ were calculated and are shown in Table 1.

The correlation co-efficient was found to be 0.9999, 0.9999 and 0.9992 for method A, method B and method C, respectively . The nominal concentrations of the test solutions for each method was prepared, the absorbance were measured and the amount of BSZ in capsules formulation was found to be 742.95 mg ± 0.186, 745.28 mg ± 0.41 and 745.43 mg ± 1.003 for method A, Method B, and Method C, respectively; (Table 2) . To study the precision of the method the analysis of formulation was carried out for six times. The %RSD values were found to be 0.186 for method A , 0.413 for method B and 1.009 for method C Hence the precision of the methods were confirmed. Further, the precision was confirmed by intermediate precision. The analysis of formulation was carried out for three times in the same day and on three suceesive days. The %RSD value for interday and intraday analysis of formulation was found to be less than 2% and are shown in table 3The accuracy of method was confirmed by recovery studies¹⁰. A known amount of standard drug material was added with pre-analysed formulation in different levels. The amount of drug recovered was calculated and the percentage recovery was found to be in the range of 99.86% - 100.04% for method A, 98.22 – 100.39% for method B and 98.76% to 101.35% for method C. The procedure was repeated for 6 times for each concentration and the % RSD values were calculated. The low%RSD values ensure the accuracy of the method. The results of recovery studies are shown in table 4.

CONCLUSION:

Three simple, precise and accurate methods were developed for the estimation of BSZ in bulk drug and in formulations. The good precision ensures the reliability of the method. From the recovery studies it was found that there are no interference due to excipients used in formulation. Hence these three methods can be effectively used for the routine analysis of BSZ in bulk and in capsule formulation.

ACKNOWLEDGEMENT:

The authors are thankful to Arul thiru Amma, Thirumati amma, ACMEC TRUST and Dr. T. vetrichelvan, principAL, Adhiparashakthi College of Pharmacy for their kind help and providing all necessary facilities.

Table : 4. Recovery studies

Methods	Amount present (µg/ml)	Amount added* (µg/ml)	Amount found* (µg/ml)	Amount recovered* (µg/ml)	Percentage recovery* %	S.D	% RSD	S.E
A	5.28	5.08	10.36	5.07	99.80	0.243	0.242	0.099
	5.28	7.62	12.90	7.61	99.86	0.159	0.160	0.064
	5.28	10.16	15.44	10.15	99.90	0.208	0.209	0.084
B	5.27	5.00	10.27	5.01	100.20	0.528	0.528	0.215
	5.27	7.51	12.78	7.42	98.80	0.417	0.422	0.170
	5.27	10.01	15.28	9.93	99.20	0.432	0.435	0.176
C	7.34	7.41	14.75	7.34	99.06	0.726	0.723	0.296
	7.34	11.11	18.45	11.25	101.26	0.551	0.549	0.228
	7.34	14.81	22.15	15.15	102.03	0.835	0.831	0.340

* Mean ± SD of six observations

REFERENCE:

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Received on 02.09.2008 Modified on 08.09.2008

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008; Page 472-474

Methods	Label amount mg /capsule)	*Amount found(mg/capsule)**	%Label Claim*	SD	% RSD	SE
A	750	742.92	99.06	0.186	0.188	0.076
B	750	745.25	99.36	0.410	0.413	0.167
C	750	745.43	99.29	1.003	1.009	0.409

Methods	Amount found		% RSD
	Intraday*	Intraday*	Intraday*	Intraday*
A	742.95 mg	742.88 mg	0.046	0.294
B	746.18 mg	744.68 mg	0.563	0.244
C	741.20 mg	746.02 mg	0.309	1.109