1. INTRODUCTION:

Drotaverine HCl is 1-[(3, 4-dietoxyphenyl) methylene]-6, 7-diethoxy 1,2, 3, 4-tetrohydroisoquinoline HCl, is an isoquinoline HCl, is an isoquinoline derivative used as an antispasmodic agent which acts by inhibiting phosphodiesterase IV enzyme, specific for smooth muscles spasm and pain mainly labour pain.[1] The Drotaverine HCl was determined either individually by electrochemical methods [2], spectrophotometric methods [3] and HPLC methods [4-5].

Nimesulide is 4-Nitro-2-phenoxy-methanesulfonanilide.It is non steroidal anti-inflammatory drug. It is used in chronic arthritis (such as rheumatoid arthritis and osteoarthritis); surgery and post-traumatic acute pain and inflammation; otorhinolaryngological inflammation resulting in pain; dysmenorrheal; upper respiratory tract infection symptoms such as fever treatment. Literature survey revealed that Nimesulide alone or in combination with other drugs is reported to be estimated by A survey of the literature revealed that only a few UV-visible spectrophotometric [6], liquid chromatographic methods [7].

2. EXPERIMENTAL:

2. 1. Apparatus

A Shimadzu UV 2450 PC (Japan) double beam UV–Vis spectrophotometer, with 1 cm quartz cuvettes, a fixed slit width (2 nm) connected to an HCL-PC computer loaded with UV Probe 2.21software was equipped with a HP printer was used for all the absorbance measurements and treatment of data.

2.2 Material and Reagents:

Drotaverine HCl and Nimesulide reference standards were kindly supplied by Emcure Pharmaceuticals, Pune and Kirti Pharmachem, Sinnar, respectively. Methanol (HPLC grade), were purchased from Fischer Scientific (India).

2. 3. Pharmaceutical preparation:

A commercial pharmaceutical preparation (NOBEL SPAS® tablet Mankind Pharm. Ltd, batch no: NTS-416) was assayed. Its declared content was as follows: Drotaverine HCl, 40.0 mg; Nimesulide, 100.0 mg/ tablet.

2.4 Stock solutions

Stock solutions was prepared by dissolving 50 mg of drotaverine HCl and Nimesulide seperately in 50 ml of methanol to get concentration of 1mg/ml. Ten ml of stock solutions where futher diluted to 100 ml with methanol to get working standard of concentration 100µg/ml of each drug.

2.5 Sample preparation

Accurately weighed 10 tablets and powdered. The powder equivalent to 100 mg of Nimesulide was transferred to 100ml volumetric flask and made the volume to mark with methanol. This mixture was sonicated in ultrasonicator for 30minute and filtered through Whatman filter paper No. 41. Transferred 5 ml of the filtrate into a 50 ml volumetric flask and made the volume to mark with methanol.

2.6 Procedures:

2.6.1. Calibration for Q Absorbance ratio method.

Aliquots of standard solution equivalent to (5–25µg/ml) drotaverine HCl and aliquots of standard solution equivalent to (5–25µg/ml) nimesulide were transferred into two series of 10 ml volumetric flasks and the volume was completed with methanol. For method I a calibration curve for nimesulide and drotaverine HCl was obtained by measuring the absorbance at the 278nm and 304nm respectively. The calibration curves were constructed and the regression equations were recorded. (Table .1)

3. RESULTS AND DISCUSSION:

3.1. Absorbance ratio method

The absorbance method uses ratio of absorbance at two selected wavelengths, one at isoabsorptive point and other being the λ max of one of the two compounds. The zero-order spectra for the two drugs showed marked overlapping as shown in Fig. 1.

Absorbance Ratio Method:

The proposed methods have been applied to commercial tablets and was no evidence of interference from the excipients and the results obtained . The concentration of two drugs in mixture was calculated by using following equation. (Table 2,3)

C_NIM = Qm-Qy × A₁………………………(1)

Qx-Qy ax₁

C_DR = Qm-Qx × A₂……………………….(2)

Qy-Qx ax₁

Qm= A₂/A₁

Qy=ax₂/ax₁

3.3. Quantification, accuracy and precision of the proposed methods

Accuracy was measured by calculating mean percentage recovery. It was determined at 80,100 and 120% level. (Table 4,5). Precision of repeatability and reproducibility (intraday and inter day) were measured for three concentrations of each drug on 3 days and the standard deviations, RSD and standard error were calculated (Table 6,7).

Table 1. Statistical parameters of the calibration graphs (n=5) for Nimesulide and Drotaverine HCl by absorption ratio method

Parameter	Absorption Ratio Method
Parameter	Nimesulide	Drotaverine HCl
Beer’s law limit	5-25µg/ml	5-25µg/ml
Slope	0.0224	0.0339
Intercept	0.01319	-0.00302
Correlation coefficient.	0.9997	0.9998
Limit of detection (LOD)	1.17 µg/ml	0.58 µg/ml
Limit of quantitation (LOQ)	3.57µg/ml	1.8 µg/ml

Table. 2.: Absorptivity values of Nimesulide and Drotaverine Hydrochloride

Components	Absorptivity at 278nm	Absorptivity at 304nm
Nimesulide (x)	23.57 (ax₁)	26.41 (ax₂)
Drotaverine Hydrochloride (y)	21.83 (ay₁)	25.68 (ay₂)

Table 3: Analysis marketed formulation of Nimesulide and Drotaverine Hydrochloride

Sr. No	Label claimed (mg/tab)		Amount found (mg/tab)		% Estimated
Sr. No	Nim	DR	Nim	DR	Nim	DR
1	100	40	101.5	39	101.5	97.50
2	100	40	100.5	39.5	100.5	98.75
3	100	40	99.60	39.4	99.60	98.50
	Mean				100.5	98.25
	±S.D.				0.95	0.66
	%R.S.D				0.94	0.67
	±S.E.				0.54	0.38

Fig. 1 Overlay spectra of 25µg/ml Nimesulide and 25µg/ml Drotaverine HCl 278 nm (isoabsorptive point) and 304 nm (λ max of Drotaverine HCl )

Table 4: Recovery of marketed formulations. (n=3) of Nimesulide and Drotaverine Hydrochloride

Parameter	Nimesulide			Drotaverine hydrochloride
%Level Recovery	Recovery (%)	%RSD	SEM	Recovery (%)	%RSD	SEM
80	102	0.80	0.47	99.83	1.48	0.85
100	100.1	0.52	0.30	97.08	1.92	1.08
120	102.5	0.28	0.16	97.78	0.40	0.23

Table 5 Limit of Detection and Quantitation of Nimesulide and Drotaverine Hydrochloride

Parameter	Nimesulide	Drotaverine HCl
Limit of detection (LOD)	1.17 µg/ml	0.58 µg/ml
Limit of quantitation (LOQ)	3.57µg/ml	1.8 µg/ml

Table 6 Intraday precision of Nimesulide and Drotaverine Hydrochloride

Sr. No.	Parameter	Drotaverine HCl	Nimesulide
1	Standard Deviation	0.002	0.001
2	% RSD	1.45	0.69
3	Standard Error	0.15	0.09

Table 7 Interday precision of Nimesulide and Drotaverine Hydrochloride

Sr. No.	Parameter	Drotaverine HCl	Nimesulide
1	Standard Deviation	0.0019	0.0037
2	% RSD	0.58	0.95
3	Standard Error	0.13	0.26