INTRODUCTION:

Nabumetone, 4-(6-methoxynaphthalen-2-yl) butan-2-one, is a nonsteroidal anti- inflammatory drug (NSAID) of naphtylalkanone class. The drug has proved to be effective in the treatment of rheumatoid arthritis, osteoarthritis and acute soft tissue injuries. Nabumetone is a prodrug which undergoes extensive first pass metabolism to 6- methoxy-2-naphthylacetic acid (6-MNA), the major circulating metabolite; 6- MNA is largely responsible for the therapeutic efficacy of nabumetone[1-3]. Paracetamol (PARA) is chemically N-(4-hydroxyphenyl) acetamide, It has analgesic and antipyretic activity [1, 4].

Combination of Nabumetone and Paracetamol is effective as these agents act through different analgesic mechanisms and act synergistically. The combination offers faster as well as prolonged relief from pain and inflammation [3].

A: Paracetamol B: Nabumetone

Figure 1: Structure of Nabumetone and Paracetamol [2, 4]

Many RP-HPLC methods [5-7] have been reported for the determination of Nabumetone and its metabolite in tablet dosage form and also in human plasma. Simultaneous estimation of Naproxen and Nabumetone was also reported by RP-HPLC in human plasma, human urine and in pharmaceutical. While spectrophometry,[9-14] HPLC,[15-25] LC-MS[26] and capillary electrophoresis [27] had been reported for determination of PCM. But no method is available for simultaneous estimation of Nabumetone and Paracetamol in tablet dosage form.

Therefore, it was the purpose of this research to develop a rapid, simple, sensitive, reliable, and validated analytical method for the measurement of both drugs, which will be the first for their simultaneous analysis in API and tablet dosage form. The present dual wavelength spectrophotometric method was validated following the ICH guidelines [28].

EXPERIMENTAL:

Chemicals and Reagents Used

The reference standard of Nabumetone and Paracetamol were obtained as gift samples from Ipca laboratory and Biodeal laboratory respectively. Methanol (AR Grade, S. D. Fine Chemicals Ltd., Mumbai, India) and Whatman filter paper no. 41 (Millipore, USA) were used in the study. Tablet dosage form used for estimation in dosage form was NILTIS-P manufactured by Ipca laboratory. Each tablet containing 500 mg of Nabumetone and 500 mg of Paracetamol.

Instrumentation

Double beam UV-visible spectrophotometer (Shimadzu, model 1601) attached to computer software UV-Probe 2.21 having two matched quartz cells with 1 cm light path. Analytical balance (Keroy Pvt. Ltd.), pH meter (Analab scientific instrument Ltd.).

Preparation of Standard Stock Solutions of Nabumetone and Paracetamol

Standard solution of NABU and PCM were prepared in methanol by dissolving 10 mg of each in separate 100 ml volumetric flask to get stock solution having concentration 100 µg/ml of NABU and 100 µg/ml of PCM. From these stock solutions, working standard solutions of both drugs containing 10 µg/ml was prepared by appropriate dilutions.

Selection of Analytical wavelength for Nabumetone and Paracetamol

For estimation of PCM, the two wavelengths were so selected which eliminate interference due to absorbance of NABU. Two wavelengths selected for estimation of PCM were 237.4 nm and 214.4 nm (Figure 3). The difference in absorbance at 237.4 nm and 214.4 nm was found to be zero for NABU. Nabumetone was directly estimated at wavelength of its maximum absorption where PCM showed no absorbance. For NABU, 332.6 nm was selected as the analytical wavelength (Figure 2).

Figure 2: Overlain Spectra of NABU and PCM (10µg/ml each) in Methanol for Selection of Wavelength for NABU

Figure 3: Overlain Spectra of NABU and PCM (10µg/ml each) in Methanol for Selection of Wavelength for PCM

Preparation of Binary mixtures

Suitable aliquots of standard stock solutions of NABU and PCM were mixed and diluted with methanol to obtain different binary mixture solutions containing NABU and PCM in 1:1 ratio (in equal concentrations). Concentrations of solution (binary mixture) in the range of 2-20 µg/ml were prepared for the calibration curve of the drugs (Figure 4).

Figure 4: Overlain Spectra of NABU and PCM in Methanol Preparation of Calibration Curve

For each drug, appropriate aliquots were pipettes out from standard stock solution into a series of ten 10 ml volumetric flasks. Volume was made up to the mark with methanol to get solutions of concentrations 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 µg/ml for NAU and PCM. Calibration curve for NABU (Figure 5) was constructed by plotting absorbance at 332.6 nm against its concentration and Calibration curve for PCM (Figure 6) was constructed by plotting absorbance difference at 237.4 nm and 214.4 nm against its concentration.

Equation for estimation of Nabumetone and Paracetamol from pharmaceutical dosage form

Concentration of NABU and PCM was calculated using the equation:

Abs_NABU = A + B * C_NABU … … … (1)

Abs_PCM = A + B * C_PCM … … … (2)

Where, C_NABU = Concentration of Nabumetone, C_PCM = Concentration of Paracetamol, Abs_NABU = Absorbance of Nabumetone at 332.6 nm, and Abs_PCM = Absorbance of Paracetamol at difference of (237.4 – 214.4) nm.

Analysis of Marketed formulation

Twenty tablets were weighed and crushed separately to fine powder. A quantity of powder equivalent to 10 mg of Nabumetone and 10 mg Paracetamol was weighed and transferred to 100 ml volumetric flask and mixed with methanol (70ml) and sonicated for 20 min. Allow solution to cool and then make up the volume with methanol. The solution was filtered through whatmann filter paper No. 41. The above solution (1ml) was transferred in 10 ml volumetric flask and diluted to mark with methanol to obtain final solution with Nabumetone (10 µg/ml) and Paracetamol (10 µg/ml). For this method, absorbances of the sample solution were recorded at 332.6 nm, 237.4 nm and 214.4 nm.

RESULTS AND DISCUSSION:

Optimization of Analytical Conditions

Owing to the high solubility of NABU and PCM in methanol and absence of shift in the absorbance maxima of NABU and PCM in it, methanol was the solvent of choice.

The overlain spectra of NABU and PCM at different concentrations revealed that for estimation of PCM, two wavelengths selected were 237.4 nm and 214.4 nm (Figure 3) which eliminate interference due to absorbance of NABU. The difference in absorbance at 237.4 nm and 214.4 nm was found to be zero for NABU. Nabumetone was directly estimated at wavelength of its maximum absorption where PCM showed no absorbance. For NABU, 332.6 nm was selected as the analytical wavelength (Figure 2).

Method Validation

Linearity:

Data for calibration curve and regression analysis are given in Table 1 and Table 2 which shown that the method shows good linearity in concentration range of 2-20 µg/ml for nabumetone and 4-20 µg/ml for paracetamol with correlation coefficient of 0.9993 and 0.9992 respectively (Figure 5 and 6). The following equations for straight line were obtained for NABU and PCM.

Table 1: Calibration Data for Nabumetone

Concentration of NAB U (µg/ml)	Absorbance at wavelength 332.6 nm	RSD
2	0.021	1.608
4	0.039	1.404
6	0.059	0.928
8	0.077	0.618
10	0.098	0.854
12	0.122	0.686
14	0.140	1.374
16	0.160	0.948
18	0.178	0.502
20	0.197	1.359

Figure 5: Calibration Curve of NABU

Figure 6: Calibration Curve of PCM

Linear equation for NABU at 332.6 nm:

y = 0.0099x - 0.0001, r² = 0.9993

Linear equation for PCM at (237.4 – 214.4) nm:

y = 0.1109x - 0.3399, r² = 0.9992

Table 2: Calibration Data for Paracetamol

Concentration of PCM (µg/ml)	Absorbance at wavelength		Absorbance Difference at (237.4 - 214.4) nm	RSD
Concentration of PCM (µg/ml)	214.4 nm	237.4 nm	Absorbance Difference at (237.4 - 214.4) nm	RSD
2	0.395	0.409	0.014	1.597
4	0.704	0.807	0.104	1.720
6	1.018	1.361	0.343	0.530
8	1.185	1.749	0.564	0.486
10	1.416	2.165	0.745	0.528
12	1.551	2.534	0.976	0.466
14	1.602	2.793	1.191	0.209
16	1.651	3.101	1.451	0.329
18	1.682	3.335	1.655	0.168
20	1.701	3.598	1.893	1.310

Precision

The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples. It provides an indication of random error results and was expressed as coefficient of variation.

a) Repeatability

Standard binary mixture solutions containing NABU and PCM (2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg/ml) in equal concentration were prepared and absorbance was measured at 332.6 nm, 237.4 nm and 214.4 nm taking the methanol as the blank for each drug. The absorbance of same concentration solution was measured 3 times and RSD was calculated.

Table 3: Intraday Precision for NABU

Concentration of NABU (µg/ml)	Absorbance	Mean Absorbance	± SD (n=3)	RSD
8	0.078	0.078	0.001	1.282
	0.077
	0.079
10	0.098	0.098	0.001	0.587
	0.098
	0.099
12	0.121	0.120	0.002	0.959
	0.119
	0.121
Mean RSD: 0.942

b) Intra and inter day precision

Variation of results within the same day (intraday), variation of results between days (inter day) were analyzed. Intraday precision was determined by analyzing NABU and PCM for 3 times in the same day at 332.6 nm, 237.4 nm and 214.4 nm (Table 3 and 4). Inter day precision was determined by analyzing both drugs daily for three days (Table 5 and 6).

Table 4: Inter day Precision for NABU

Concentration of NABU (µg/ml)	Experiment Day	Mean Absorbance	± SD (n=3)	RSD
8	DAY 1	0.078	0.001	1.282
	DAY 2	0.076	0.002	1.950
	DAY 3	0.079	0.001	0.743
10	DAY 1	0.098	0.001	0.587
	DAY 2	0.097	0.002	1.039
	DAY 3	0.098	0.001	1.174
12	DAY 1	0.120	0.002	0.959
	DAY 2	0.119	0.002	1.465
	DAY 3	0.121	0.003	0.836
Mean RSD 1.121

Table 5: Intraday Precision for PCM

Concentration of PCM (µg/ml)	Absorbance	Mean Absorbance	± SD (n=3)	RSD
8	0.564	0.564	0.004	0.799
	0.560
	0.569
10	0.749	0.749	0.005	0.602
	0.744
	0.753
12	0.983	0.985	0.011	1.086
	0.976
	0.997
Mean RSD 0.828

Table 6: Inter day Precision for PCM

Concentration of PCM (µg/ml)	Experiment Day	Mean Absorbance	± SD (n=3)	RSD
8	DAY 1	0.564	0.005	0.799
	DAY 2	0.562	0.010	1.934
	DAY 3	0.563	0.001	0.182
10	DAY 1	0.749	0.005	0.602
	DAY 2	0.731	0.004	0.480
	DAY 3	0.742	0.009	1.178
12	DAY 1	0.985	0.011	1.086
	DAY 2	0.983	0.006	0.613
	DAY 3	0.984	0.005	0.480
Mean RSD 0.815

c) Reproducibility

Reproducibility test was determined between two analysts and instruments. The value of RSD was to be found below 1 showed reproducible of developed spectrophotometric method. The values obtained were evaluated using t- test to verify their reproducibility (Table 7).

Accuracy

To ascertain the accuracy of proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). The method showed % recovery in range of 98.62% to 101.80% whereas 98.50% to 100.49% for Paracetamol. The results indicate that developed method is accurate enough for routine use (Table 8).

Limit of Detection and Limit of Quantification

Calibration curve was repeated 5 times and standard deviation (SD) of intercepts (response) was calculated. Then LOD and LOQ were measured by using mathematical expressions by using equations:

Limit of Detection (LOD): 3.3 × σ/S,

Limit of Quantification (LOQ): 10 × σ /S

Where, σ = The Standard deviation of the response, S = Slope of calibration curve.

LOD and LOQ were found to be 0.30 µg/ml and 0.92 µg/ml for nabumetone and 0.41 µg/ml and 1.24 µg/ml for paracetamol, respectively.

Results of analysis of Marketed formulation

The results of analysis in terms of % label claim was found to be 99.51% ± 0.13 for Nabumetone and 98.83% ± 0.06 for Paracetamol for a formulation analyzed (Table 9).

Table 9: Analysis of Marketed Formulation

Tablet Dosage form	Label claim (mg)		Amount found (mg)		% Assay* ± SD
Tablet Dosage form	NABU	PCM	NABU	PCM	NABU	PCM
Nilitis P	500.0	500.0	497.6	494.1	99.51 ± 0.13	98.83 ± 0.06

* Average of three determination

CONCLUSION:

The proposed method for determination of Nabumetone and Paracetamol is simple, accurate, precise, feasible, sensitive as well as selective and suitable for routine analysis in laboratories. High % recovery (in developed methods) shows that the method is free from the interference of excipients used in the formulation. It could be applied to the determination of different tablet dosage forms

ACKNOWLEDGEMENT:

The authors are grateful to Ipca Laboratory and Biodeal laboratory, for providing gift samples of Nabumetone and Paracetamol, respectively.

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Received on 02.11.2012 Accepted on 21.11.2012

Asian J. Pharm. Ana. 2(4): Oct. - Dec. 2012; Page 122-127

Variable factor	Condition	*Drugs Concentration (µg/mL) (Mean ± SD)**		‘t’ Stat (t )	‘t’ Critical (ttab)	Interference
		NABU	PCM
Different analyst	Analyst-1	9.94 ± 0.01	9.87 ± 0.01	0.72	2.87	No Significance Difference
	Analyst-2	9.96 ± 0.02	9.82 ± 0.01
Different instrument	Shimadzu	9.96 ± 0.01	9.89 ± 0.01	1.24	2.91
	Elico	9.93 ± 0.02	9.88 ± 0.02

Sample Concentration (µg/ml)		Amount of Standard added (µg/ml)		Amount recovered (µg/ml)		*% Recovery ± SD**
NABU	PCM	NABU	PCM	NABU	PCM	NABU	PCM
10.0	10.0	8.0	8.0	17.8	18.1	98.62 ± 0.12	100.47 ± 0.06
10.0	10.0	10.0	10.0	20.2	20.1	101.21 ± 0.27	100.49 ± 0.09
10.0	10.0	12.0	12.0	22.3	21.7	101.81 ± 0.73	98.50 ± 0.04