Simultaneous HPTLC Determination of Nabumetone and Paracetamol in Combined Tablet Dosage Form

Um novo método de cromatografia em camada delgada de alta eficiência (HPTLC) para determinação simultânea de nebumetona e paracetamol em forma combinada em comprimido foi desenvolvido e validado. As separações foram efetuadas em placas de alumínio prerrecobertas com sílica gel 60 F254 utilizando tolueno:propanol-2:ácido acético (8:2:0.1, v/v/v) como fase móvel. A determinação quantitativa das bandas foi feita por varredura densitométrica a 236 nm. Os fatores de retenção calculados para nebumetona e paracetamol foram 0,78 ± 0,03 e 0,32 ± 0,03, respectivamente. O método foi validado com relação à linearidade, exatidão, precisão e robustez. A curva de calibração mostrou-se linear dentro do intervalo de 50-250 ng por banda de ambas as drogas. O método tem sido aplicado com sucesso nas análises das drogas em formulações farmacêuticas. As porcentagens de precisão de ensaio (média ± desvio padrão) foram 99,89 ± 1,15 para nabumetona e 101,10 ± 0,71 para paracetamol, ambos de comprimidos disponíveis comercialmente.


Introduction
Nabumetone (NAB), chemically, 4-(6-methoxy-2naphthyl)-2-butanone is a non-steroidal anti-inflammatory drug of the arylalkanoic acid family used to treat pain or inflammation caused by arthritis, 1 being official in British Pharmacopoeia 2 and United States Pharmacopeia 3 and Indian Pharmacopeia. 4Paracetamol (PAR), 4-hydroxyacetanilide is a widely-used analgesic and antipyretic drug. 51][12] Also colorimetric 13 and micellar stabilized room temperature phosphorescence quantitation 14 of NAB as single component, or in combinations with other drugs, has been reported.Analytical methods have been reported for the determination of PAR including: high-performance liquid chromatography-diode array (HPLC-DAD) with online post-column photochemical derivatization, 15 non-suppressed ion chromatography(IC), 16 UV-Visible spectrophotometry, 17 HPLC, [17][18][19][20] gas chromatographmass spectrometry (GC-MS) 21 as single component or in combinations with other drugs.The chemical structures of the drugs are given in Figure 1.
No reports were found for simultaneous determination of NAB and PAR in combined tablet dosage form by high performance thin layer chromatographic method (HPTLC).This paper describes simple, accurate, precise and sensitive Vol.22, No. 6, 2011   HPTLC method for simultaneous determination of NAB and PAR in combined tablet dosage form.The proposed HPTLC method can be used for analysis of ten or more formulations on a single plate and is a rapid and costeffective quality-control tool for routine simultaneous analysis of drugs.The technique is simpler and provides more flexibility than HPLC.The method was optimized and validated as per the International Conference on Harmonization (ICH) guidelines. 22

Reagents and chemicals
Authentic samples of NAB and PAR were obtained from Divi's Laboratories Ltd. (Hyderabad, India) and Cipla Ltd. (Pune, India), respectively, used as such without further purification.Brand of tablets Niltis P (Ipca laboratories Ltd., India), labeled to contain 500 mg of NAB and 500 mg of PAR were procured from the local market.Methanol, toluene, 2-propanol and acetic acid (all AR grade) were obtained from Sisco Research Laboratories (Mumbai, India).Silica gel 60 F 254 TLC plates (20 × 20 cm, layer thickness 0.2 mm, from Merck, Germany) were used as stationary phase.

Preparation of standard stock solutions
Standard stock solution of NAB and PAR was prepared by dissolving 10 mg of each drug in 10 mL of methanol separately to get concentration of 1 mg mL -1 from which 0.5 was further diluted to 10 mL to get stock solution of 50 ng µl -1 of each drug.

Chromatographic condition
The plates were prewashed with methanol and activated at 110 o C for 5 min, prior to chromatography.The slit with dimensions of 5 × 0.45 mm and scanning speed of 20 mm s -1 were employed.The linear ascending development was carried out in 10 × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) using toluene:2-propanol:acetic acid (8:2:0.1,v/v/v) as mobile phase, after saturation of the chamber with mobile phase vapor for 10 min.The development distance and time were 9 cm and 20 min, respectively.After, chromatography plates were dried in a current of air with the help of a hair dryer.A Camag HPTLC system containing Camag Linomat V semiautomatic sample applicator, Hamilton syringe (100 µl), Camag TLC Scanner-3 with winCATS software version 1.4.2 and Camag twin-trough chamber (10 × 10 cm) were used for the present study.The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 200 to 400 nm.

Selection of detection wavelength
After chromatographic development bands were scanned over the range of 200-400 nm (with a spectrum scan speed of 100 nm s -1 ) and the spectra were overlain.It was observed that both drugs showed considerable absorbance at 236 nm, which was selected as the wavelength for detection (Figure 2).

Preparation of calibration curves
The standard stock solutions of NAB and PAR (50 ng µL -1 each) were applied by overspotting on TLC plate in range of 1-5 mL with the help of CAMAG 100 mL sample syringe, using Linomat 5 sample applicator to get concentrations 50, 100, 150, 200 and 250 ng per band.The plate was developed and scanned under above established chromatographic conditions.Each standard in five replicates was analyzed and peak areas were recorded.Calibration curves of NAB and PAR were plotted separately of peak area vs. respective concentration of NAB and PAR.

Analysis of tablet formulation
Twenty tablets were weighed accurately and finely powdered.A quantity of powder equivalent to 10 mg of each NAB and PAR was weighed and transferred to a 10 mL volumetric flask containing approximately 7 mL of methanol, ultrasonicated for 5 min, and volume was made up to the mark with the methanol.The solution was filtered through Whatman 41 filter paper, and 0.5 mL of filtrate was further diluted to 10 mL with methanol.Then, 2 µL volume were were applied to a TLC plate to furnish final concentration of 100 ng per band for both NAB and PAR.After chromatographic development the peak areas of the bands were measured at 236 nm and the amount of each drug present in sample was estimated from the respective calibration curves.Procedure was repeated six times for the analysis of homogenous sample.

Recovery studies
To check the accuracy of the method, recovery studies were carried out by addition of standard drug solution to preanalyzed sample solution at three different levels, 50, 100 and 150%.Chromatogram was developed and the peak areas were noted.At each level of the amount, three determinations were carried out.

Precision
Set of three different concentrations in three replicates of mixed standard solutions of NAB and PAR were prepared.All the solutions were analyzed on the same day in order to record any intra-day variations in the results.For inter-day variation study of three different concentrations of the mixed standard solutions in linearity range were analyzed on three consecutive days.

Robustness studies
In the robustness study, the influence of small, deliberate variations of the analytical parameters on peak area of the drugs were examined.Factors varied were development distance (± 5%), time from application to development (0, 10, 20 and 30 min) and from development to scanning (0, 30, 60, and 90 min).One factor at a time was changed to study the effect.Robustness of the method was checked at a concentration level of 100 ng per band for both NAB and PAR.

Results and Discussion
Different mobile phases containing various ratios of toluene, methanol, acetic acid were examined (data not shown).Finally the mobile phase containing toluene:2-propanol:acetic acid (8:2:0.1,v/v/v) was selected as optimal for obtaining well defined and resolved peaks.The optimum wavelength for detection and quantitation used was 236 nm.The retention factors for NAB and PAR were found to be 0.78 ± 0.03 and 0.32 ± 0.03, respectively.Representative densitogram obtained from a mixed standard solution of NAB and PAR is shown in Figure 3.
Straight-line calibration graphs were obtained for NAB and PAR in the concentration range 50-250 ng per band for both the drugs with high correlation coefficient > 0.999 (Figure 4).For NAB, the percentage of recovery study ranged from 99.44 to 100.41% with % RSD values ranging from 0.85 to 1.28%.For PAR, the recovery results ranged from 99.41 to 100.77% with % RSD values ranging from 0.99 to 1.34%.The method was found to be accurate and precise, as indicated by recovery studies as recoveries were close to 100% and % RSD not more than 2. Results of recovery studies are reported in Table 1.Intra-day variation, as % RSD, was 0.78 for NAB and 0.92 for PAR.Inter-day variation, as % RSD was 1.64 for NAB and 1.43 for PAR, as presented in Table 2.
Robustness of the method checked after deliberate alterations of the analytical parameters showed that areas of peaks of interest remained unaffected by small changes of the operational parameters (% RSD < 2).The results are given in Table 3.The proposed method was also evaluated by the assay of commercially available tablets containing NAB and PAR.The % of assay (mean ± S.D.) was found to be 99.89 ± 1.15 for NAB and 101.10 ± 0.71 for PAR.

Conclusions
The validated HPTLC method employed proved to be simple, fast, accurate, precise and robust, thus can be used for routine analysis of NAB and PAR in combined tablet dosage form.

Figure 1 .
Figure 1.Chemical structures of NAB and PAR.

Table 1 .
Recovery studies of NAB and PAR a Average of three determinations.

Table 2 .
Percentages of recovery for intra-and inter-day variation studies

Table 3 .
Relative standard deviation of peak area obtained under robustness study

.
Calibration curves for NAB and PAR.