A RP-HPLC method for the simultaneous estimation of paracetamol and nabumetone in API and in tablet dosage form

C.K.Oza^1*, Ranju Nijhawan¹, M. K. Pandya², A. J. Vyas³, A. I. Patel³

^1.Xylopia, Ahmadabad, India.

^2.R.K University, Rajkot, India.

_3.B. K. Mody Govt. Pharmacy College, Rajkot, India.

*Corresponding Author E-mail: chiragkoza@gmail.com

ABSTRACT:

A reverse phase high performance liquid chromatography method was developed for the simultaneous estimation of Nabumetone and Paracetamol. The separation was achieved by octadecyl column (C18) and Acetonitrile: water (75:25 v/v) at pH 6.5 using ortho phosphoric acid as eluent, at a flow rate of 1.0 ml/min. UV detection was carried out at 239.0 nm. The retention time of NABU and PCM was found to be 4.9 and 2.7 min respectively. The method has been validated for linearity, accuracy and precision. Linearity for Nabumetone and Paracetamol were in the range of 2-20 µg/ml for both drugs. The correlation coefficients were better than 0.999 and 0.999 for NABU and PCM respectively. The lower limit of detection (LOD) for NABU and PCM were found to be 0.27 and 0.31, respectively. The lower limit of quantification (LOQ) for NABU and PCM were found to be 0.81 and 0.93 respectively. The % recovery obtained for Nabumetone and Paracetamol were 99.66% to 101.00 % and 99.68% to 100.66 % respectively. The developed method was found to be accurate, precise, selective and rapid for simultaneous estimation of Nabumetone and Paracetamol in tablets.

KEY WORDS: Reverse phase high performance liquid chromatography (RP-HPLC), Nabumetone, Paracetamol, ortho phosphoric acid.

INTRODUCTION:

Nabumetone, 4-(6-methoxynaphthalen-2-yl) butan-2-one (fig.-1), 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 (fig.-1), it has analgesic and antipyretic activity^[1,4].

A: Paracetamol B: Nabumetone

Figure 1: Structure of Nabumetone and Paracetamol^{[2, 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].

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,^[8-14] 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 by RP-HPLC.

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 RP-HPLC method was validated following the ICH guidelines.^[28]

Figure 2: Calibration curves of Nabumetone

Figure 3: Calibration curves of and Paracetamol

EXPERIMENTAL:

Chemicals and Reagents Used

The reference standard of Nabumetone and Paracetamol were obtained as gift samples from Ipca laboratory and Biodeal laboratory respectively. All chemicals used such as acetonitrile, ortho-phosphoric acid and water were of HPLC grade of Merck Limited. 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

High performance liquid chromatography including JASCO PU-2080 Plus pump equipped with universal injector (Rheodyne) with injection volume 20 μL, JASCO UV-2075 Plus UV-visible detector. Analytical balance (Keroy Pvt. Ltd.), pH meter (Analab scientific instrument Ltd.), Ultra sonicator (Trans-O-Sonic Ltd.) were used.

Chromatographic Conditions

The separation was achieved by octadecyl column (C₁₈) and Acetonitrile: water (75:25 v/v) at pH 6.5 using ortho phosphoric acid as eluent, at a flow rate of 1.0 ml/min. UV detection was carried out at 239.0 nm. The retention time of NABU and PCM was found to be 4.9 and 2.7 min respectively. The injection volume of standard and sample solutions was 20µl.

Preparation of Standard And Sample Solution

Weigh accurately and transfer about 10 mg of nabumetone and 10 mg of paracetamol in a 100 ml volumetric flask separately. Dissolve and dilute to volume with acetonitrile. (Nabumetone 100 µg/ml and Paracetamol 100 µg/ml). From these stock solutions, working standard solutions of both drugs containing 10 µg/ml were prepared by appropriate dilutions with mobile phase.

Preparation of Calibration Curve

Different binary mixture solutions of NABU and PCM were chromatographed in 8 minute time period and the peak area of these solutions were measured at 4.9 and 2.7 minute corresponding to the retention time of NABU and PCM respectively. Calibration curves for both the drugs were plotted separately. The method shows good linearity in the range of 2-20 µg/mL for the two drugs. The results are given in Figure 2 and 3.

RESULTS AND DISCUSSION:

Optimization of Analytical Conditions

Different columns containing octyl, octadecyl, phenyl and base deactivated silane stationary phase were tried for separation and resolution. The Inertsil base deactivated silane column became more advantageous over the other columns. Individual drug solution was injected into column, both elution pattern and resolution parameters studied as a function of pH, as a function of mobile phase component and their ratio. To develop a suitable LC method for estimation of nabumetone and paracetamol in formulations, different mobile phases were employed to achieve the best separation. The selected and optimized mobile phase was acetonitrile: water (75:25 v/v) at pH 6.5 using ortho phosphoric acid as eluent, at a flow rate of 1.0 ml/min. UV detection was carried out at 239.0 nm (Figure 4: Overlain Spectra of NABU and PCM showing 239 nm as wavelength selected for RP-HPLC determination). The retention time of NABU and PCM was found to be 4.9 and 2.7 min respectively. Run time was 8 min. Here the peaks were separated and showed better resolution, theoretical plate count and asymmetry were also found good for Nabumtone and Paracetamol. The typical chromatogram of drugs assayed is shown in Figure 5 and Overlain Chromatograph of Nabumetone and Paracetamol calibration is shown in Figure 6.

Table 1: RESULTS OF SYSTEM SUITABILITY STUDY

System Suitability Parameters	Proposed Method
System Suitability Parameters	NABU	PCM
Retention time* (RT) (min)	4.98 ± 0.02	2.76 ± 0.02
Relative retention time* (min)	2.22 ± 0.01	-
Tailing factor*	1.46 ± 0.03	1.43 ± 0.02
Theoretical plates (N)	6453	4453
Capacity factor (k`)	0.80
Resolution* (R_s)	10.75 ± 0.10

METHOD VALIDATION

System Suitability

The system suitability of the method was studied to determine the reproducibility of the chromatographic system and column performance was acceptable for the intended analytical application. Four parameters i.e. precision of peak area of five replicate injections,retention time of eluted drugs, number of theoretical plates, asymmetry factor and resolution between two peak of analytes were evaluated. The results are shown in Table 1.

TABLE 2: RESULTS OF LINEARITY STUDY

Nabumetone		Paracetamol
Concentration of (µg/ml)	Area	Concentration of (µg/ml)	Area
2	568221	2	352562
4	865784	4	541229
6	1174060	6	775710
8	1471732	8	968143
10	1729662	10	1152930
12	2068244	12	1392570
14	2371662	14	1586804
16	2687302	16	1811079
18	2983746	18	1989649
20	3250969	20	2186501

Linearity

The Linearity of analytical method is its ability to obtain test results, which are directly proportional to the concentration of analyte in the test sample .The linearity of the assay method, was established by injecting test samples in the range of 2-20 µg/ml for Nabumetone and also for Paracetamol. Each solution was injected twice into HPLC and the average area at each concentration was calculated. The regression analysis was carried out from graph of peak area Vs Concentration; correlation co-efficient and Y- Intercept of plot was also evaluated.

Table 3: RESULTS OF ACCURACY STUDY

Sample Conc. (µg/ml)		Amount of Std. 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	17.9		17.9		99.66 ±0.29	99.68 ± 0.03
10.0	10.0	10.0	10.0	19.9		20.1		99.78 ±0.04	100.66 ± 0.15
10.0	10.0	12.0	12.0	22.2		21.9		101.00 ±0.06	99.72 ± 0.10

*Average of five experiments

Figure 6: Overlain Chromatograph of Nabumetone and Paracetamol

The results are shown in Table 2. Linear regression equation and correlation coefficient was found as

NABU: y = 150227 x + 264637, r² = 0.999

PCM: y = 102778 x +145156, r² = 0.999

Where ‘y’ is area of peak and ‘X’ is the concentration of drug solution, respectively.

Accuracy

The accuracy study was performed by spiking placebo with known quantity of API. The accuracy of test method was demonstrated by preparing recovery samples at the level of 80%, 100%, and 120% of target concentration. The recovery samples were prepared in triplicate at each level. The above samples were injected and the percentage recovery for amount added, were estimated. The precision of recovery at each level was determined by computing the relative standard deviation of triplicate recovery results. The result for accuracy is shown in Table 3, indicating good accuracy of the method for simultaneous determination of two drugs.

Precision

Precision was determined by two ways; by System precision and Intermediate precision. System precision was demonstrated by making five replicate injections of standard solution. The peak area of analyte for replicate injections was recorded. The %RSD for the analyte peak area of these replicate injections was evaluated. The intermediate precision of test method was demonstrated by carrying out precision study at three concentration level as 80 %, 100%, 120% (i.e 8, 10, 12 µg/ml). Intermediate precision study includes intra-day and inter-day analysis. The result summary of intermediate precision is shown in Table 4A and 4B.

Limit of Detection And Limit of Quantitation

Limit of detection and limit of quantitation was established based on the residual standard deviation method. LOD and LOQ were found to be 0.26 µg/ml and 0.81 µg/ml for nabumetone and 0.31 µg/ml and 0.93 µg/ml for paracetamol, respectively.

Table 4: A- Results of Intermediate Precision for Nabumetone

Conc. (µg/ml)	Intra-day (n=3)		RSD	Inter-day (n=3)		RSD
Conc. (µg/ml)	Mean Peak Area	± SD	RSD	Mean Peak Area	± SD	RSD
8	1471543	4418	0.436	1471092	5673	0.386
10	1726816	3577	0.207	1726997	3711	0.525
12	2067876	5961	0.922	2066802	4941	0.739

B- Results of Intermediate Precision for Paracetmol

Conc. (µg/ml)	Intra-day (n=3)		RSD	Inter-day (n=3)		RSD
Conc. (µg/ml)	Mean Peak Area	± SD	RSD	Mean Peak Area	± SD	RSD
8	968750	1875	0.194	968699	1897	0.196
10	1152926	4801	0.850	1152579	6621	1.095
12	1392477	6721	0.842	1394793	4581	1.045

TABLE 5: ANALYSIS OF MARKETED FORMULATION

Formulations	Label claim (mg)		Amount found (mg)		*% Assay ± SD**
Formulations	NABU	PCM	NABU	PCM	NABU	PCM
Nilitis P	500.0	500.0	496.8	498.6	99.36 ± 0.39	99.72 ± 0.26

* Average of five determination

Analysis of Marketed Formulation

Twenty tablets were weighed and crushed to fine powder. A quantity of powder equivalent to 10 mg of each drug were weighed and transferred to 100 ml volumetric flask. 70 ml of acetonitrile was added and sonicated for 20 min. Allow the solution to cool, and then make up the volume with acetonitrile. The solution was filtered through whatmann filter paper No. 41. The filtrate of 10 ml was taken and diluted to 100 ml with mobile phase in another 100 ml volumetric flask to obtain final concentration of 10 µg/ml for each drug. Results are shown in Table 5.

CONCLUSION:

The data demonstrate that the ion pair chromatographic method we have developed showed acceptable linearity, specificity, accuracy, precision and robustness in the concentration range of 2-20 µg/ml for Nabumetone and Paracetamol, as per the requirement of ICH guidelines. The method described is rapid since chromatographic run time is 8 min. The limit of quantification value for Nabumetone and Paracetamol are observed to be 0.81 and 0.93 µg/ml, respectively. The proposed method is precise, accurate, and robust and does not suffer from any interference from other excipients. In conclusion, the proposed method could be routinely used for the analysis of Nabumetone and Paracetamol in pharmaceutical dosage form.

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 03.08.2012 Modified on 22.08.2012

Asian J. Research Chem. 5(9): September, 2012; Page 1118-1122