INTRODUCTION:

Ibuprofen¹ (IBP) is chemically (RS)-2-(4-(2-methylpropyl) phenyl) propionic acid, it is come under category of Non-steroidal anti-inflammatory drug (NSAID). Ibuprofen is an inhibitor of the enzyme cyclooxygenase (COX), which converts arachidonic acid to prostaglandin H2 (PGH2). PGH2, in turn, is converted by other enzymes to several other prostaglandins (which are mediators of pain, inflammation, and fever) and to thromboxane A2 (which stimulates platelet aggregation, leading to the formation of blood clots).

Methocarbamol¹ (MCB) is chemically 2-hydroxy-3-(2-methoxyphenoxy) propyl carbamate, it is a centrally acting skeletal muscle relaxant which reduces the skeletal muscle tone by a selective action on the cerebral spinal axis, without altering consciousness.

Basu D et al². have suggested a method using high performance liquid chromatography for the determination of ibuprofen and paracetamol in tablets. Zarapkar S.S. et al³.have reported, Reverse phase HPLC determination of ibuprofen, paracetamol and methocarbamol in tablets. Erk N et al⁴.published a method of Simultaneous determination of paracetamol and methocarbamol in tablets by ratio spectra derivative spectrophotometry.

Literature survey reveals that no stability indicative analytic method for the estimation of Ibuprofen and Methocarbamol combination of drugs. Hence, there is a need to develop and validate as an stability indicating analytical method for the estimation of Ibuprofen and Methocarbamol.

EXPERIMENTAL:

Apparatus:

A Shimadzu HPLC with Class-VP version 6.12 SP1 software, UV-Visible Detector (UV/PDA), Pump (LC-10AT) and (LC-10ATvp), UV-Visible double beam Spectrophotometer (Shimadzu), Electronic Balance (Sartorius CP-25D), Ultra Sonicator (Crest 257D ), PH Analyzer (Mettler toledo), Milli Pore (Merck).

Materials and Reagents⁵ :

IBP and MCB were kindly gifted from Granules India Limited. Chromatographic grade: methanol, acetonitrile, water. Analytical grade: phosphoric acid, tetrahydrofuran AR, Potassium dihydrogen orthophosphate, hydrogen peroxide.

Selection of wavelength by uv-spectroscopy^6:

The Maximum absorbance of IBP and MCB were found to be 254 nm and 274 nm respectively. From the UV Visible spectrophotometric results, Simultaneous estimation of two spectra shows maximum absorbance at 220nm (Fig: 1)

Fig 1: Selection of wavelength by uv-spectroscopy

Preparation of 0.1M phosphate buffer :

13.6g of potassium dihydrogen phosphate was dissolved in 1000ml of water and the contents were dissolved completely.

Preparation of 0.1M phosphoric acid solution:

65ml of concentrated phosphoric acid was transferred into a 1000ml beaker and the volume was making up with water.

Preparation of mobile phase:

A mixture of 0.1M potassium dihydrogen phosphate (pH 3.5 adjusted with orthophosphoric acid), methanol, acetonitrile (20:25:55) and 0.3ml of tetrahydrofuran was added, mixed. It was filtered through 0.45µ nylon filter and degassed.

Preparation of standard solution:

Accurately weighed quantity of about 62.5mg of methocarbamol WS and 25.0mg of ibuprofen were added into a 50 ml volumetric flask and dissolved under sonication by 30ml of diluent, the volume was made by diluent and mixed well. 5ml of the resulting solution was transferred into a 25ml volumetric flask and the volume was made by mobile phase and mixed, the solution was filtered through 0.45u nylon filter. Final concentration of the resulting solution was 0.25mg/ml of MCB and 0.1mg/ml of IBP.

Assay sample preparation:

20 caplets were taken and made into fine powder. Powder equivalent to 62.5mg of MCB and powder equivalent to 25mg of IBP were taken into two separate 50ml volumetric flasks and dissolved under sonication by 30ml of diluent, the volume was made by diluent and mixed well.5ml of the resulting solutions was transferred into a 25ml volumetric flasks and the volume was made by mobile phase and mixed, the solution was filtered through 0.45µ nylon filter. Final concentration of the resulting solutions was 0.25mg/ml of MCB and 0.1mg/ml of IBP.

Assay of pharmaceutical formulation:

Separate and filtered portions of equal volume of (about 20µl) standard preparation and assay preparations were injected into the chromatograph and the chromatogram was recorded and the peak responses of the major peak were measured. The quantity of % of MCB and IBP in the portion of caplets was calculated by using the following formula.

=AT/AS*WS/50*5/25*100/WT*100/5*P/100*AV/LC*100

Where,

AS: Average peak area due to standard preparation

AT: Peak area due to assay preparation

WS: Weight of methocarbamol / ibuprofen WS in mg

WT: Weight of sample in assay preparation

P: Purity of methocarbamol / ibuprofen WS

AV: Average weight of caplets in mg

LC: Labeled claim of methocarbamol/ ibuprofen

RESULTS AND DISCUSSION:

Development Of Method^7,8:

The main objective of the present work is to develop and validate a suitable high precision and accurate analytical method for the simultaneous estimation of Ibuprofen and Methocarbamol in caplet dosage form by reverse phase high performance liquid chromatography (RP-HPLC). Different columns were tested and finally Inertsil ODS-3V C18 Column (150 mm x 4.6mm x 5 µm) was selected. Mobile phase compositions 0.1M phosphate buffer: methanol: acetonitrile: tetrahydrofuran in the ratio of 20:25:55:0.3 is decided as the mobile phase which gave good resolution between IBP and MCB within a period of 12 min .The flow rate was 1ml/min, Volume of injection was 20ul.The detection was carried out at a wave length of 220nm.

Table 1: Linearity data for Ibuprofen:

Table 2: Linearity data for Methocarbamol:

Validation of the method⁹:

Specificity of the method is performed by injecting placebo and standards separately the results shows that there is no interference of IBP and MCB peaks due to excipients (Fig 2, 3).

The linearity and range of the method was established by measuring the responses of the standard preparations of seven different preparations of seven different concentrations of IBP and MCB i.e. 25%, 50%, 75, %100, %125, %150 and 175% (tables 1,2). The correlation coefficient (r²) values for IBP and MCB were found to be 1, 0.999 respectively (Fig: 4, 5).

The accuracy of the method was established from recovery experiments. The recovery was performed by adding IBP and MCB to the placebo in the range of 50 % to 150% of test concentration i.e. 0.1mg/ml of IBP and 0.25mg/ml of MCB, the analysis was measured at five concentration levels i.e. 50%, 75%, 100%, 125%, and 150% and expressed as %RSD for both the drugs separately. The percentage mean recovery of IBP and MCB in the presence of placebo is 100.61% and 99.23% respectively (Tables3, 4).

Table 3: Recovery of Ibuprofen working standard in presence of placebo:

Table 4: Recovery of Methocarbamol working standard in presence of placebo

Table 5: Data for Ibuprofen precision

Table 6: Data for Methocarbamol precision

Fig: 2 Placebo for specificity

Fig 3: Standard solution for specificity

Fig 4: Linearity graph for Ibuprofen

Fig 5: Linearity graph for Methocarbamol

Fig 6: Acid stress

Fig 7: Alkali stress

Fig 8: peroxide stress

Fig 9: Thermal stress

Table7: Data for intermediate precision

Parameters	Ibuprofen		Methocarbamol
Parameters	Analyst –I	Analyst -II	Analyst -I	Analyst –II
No. of samples	6	6	6	6
Mean assay (%)	103.5	102.9	103.3	103.9
%RSD	0.60	0.65	0.48	0.65

Table 8: % of RSD and % of assay of robustness study of Ibuprofen

S.No.	Parameters	Low		High
S.No.	Parameters	Assay%	%RSD	Assay %	%RSD
1	Flow rate	105.2	1.78	105.2	1.59
2	Wavelength	107.6	0.17	107.3	0.23
3	Mobile phase	103.1	0.80	102.3	0.81

Table 9: % of RSD and % of assay of robustness study of Methocarbamol

S.No	Parameters	Low		High
S.No	Parameters	Assay%	%RSD	Assay %	%RSD
1	Flow rate	103.6	0.49	103.9	0.59
2	Wavelength	104.6	1.81	103.6	1.87
3	Mobile phase	101.9	0.39	102.2	0.21

Precision:

Repeatability: The repeatability of the method was established by estimating the assay for six different sample preparations of the same batch. The relative standard deviation of 6 determinations of ibuprofen was found to be 0.6% and for methocarbamol found to be 0.48%., which is well within the acceptance criteria of 2.0% (Tables 5, 6).

Intermediate precision (Ruggedness): The intermediate precision of the method was established by estimating the assay of ibuprofen and methocarbamol caplets for six different sample preparations of the same batch by different analysts using a different HPLC with similar column on a different day. The difference in the assay of IBP and MCB caplets between two analysts was found to be less than 2.0% of the absolute assay value (Table 7).

Table 10: Degradation studies of ibuprofen and methocarbamol

Conditions	Sample weight (mg)	Peak Area (Ibuprofen)	Peak Area (Methocarbamol)
Sample Control	851.2	6040407	10911457
Acid Degradation	848.3	41898376	80021792
Alkali Degradation	846.8	44035380	81800585
Thermal Degradation	849.2	43276449	70240812

Table 11: Solution stability of ibuprofen

Time intervals	Sample area	Standard area	Assay%
0-Hour	6581352	6409462	102.6
	6585349
2^nd-Hour	6591453	6414125	102.7
	6594092
4^th-Hour	6597954	641786	102.8
	6598094
8^th-Hour	6606655	6430020	103.0
	6606531
12^th-Hour	6614203	6439772	103.1
	6614173
16^th-Hour	6621623	6446239	103.2
	6621791
20^th-Hour	6626398	6458215	103.3
	6625086
24^th-hour	6630154	6460532	104.3
	6631086
Average	6609125	6434529	103.0
SD	16289	19801	0.2623
%RSD	0.25	0.31	0.25

Robustness:

Due to small changes in flow rate, mobile phase composition and wavelength the assay results are not very much affected .Hence, the method proved as robust. Flow rates used 0.9ml/min, 1.1ml/min; wave lengths used 217nm, 223nm, Mobile phase compositions 20:27:53:0.2 and 20:23:57:0.4. The results are shown in Tables 8, 9.

Limit of detection:

The detection limit (DL) may be expressed as:

DL = 3.3 s/S

Where,

s = the standard deviation of y-intercepts of regression lines.

S = the slope of the calibration curve.

LOD for ibuprofen = 198.4 ppm.

LOD for methocarbamol = 478.4 ppm.

Limit of quantitation:

The quantitation limit (QL) may be expressed as:

QL = 10 s/S

Where,

s = the standard deviation of the response.

S = the slope of the calibration curve.

LOQ for ibuprofen = 62.33 ppm.

LOQ for methocarbamol = 153.44 ppm

Table 12: solution stability of Methocarbamol

Time intervals	Sample area	Standard area	Assay%
0-Hour	11088490	10628715	103.8
	11100511
2^nd-Hour	11114053	10643057	104.0
	11115202
4^th-Hour	11124061	10649132	104.1
	11124337
8^th-Hour	11144102	10682549	104.3
	11147788
12^th-Hour	11155975	10696558	104.4
	11158900
16^th-Hour	11152053	10655998	104.3
	11150706
20^th-Hour	11157446	10701393	104.4
	1152738
24^th-hour	11136637	10691810	104.2
	11138010
Average	11135063	10672402	104.2
SD	21667	27773	0.2084
%RSD	0.19	0.26	0.20

Forced degradation:

The forced degradation of placebo and formulation was carried out as per ICH guidelines (ICH, Q2B) in acid, base, oxidation and water. The acid, base, oxidation and water stress studies were carried out by refluxing sample flasks at 60^oC for 1 hour with 20ml 1N Hcl, 1N NaOH, 5% H₂O₂ and at 105⁰c for 24hours respectively. The drug and formulation were found to be stable under all the stress conditions. All the stress conditions with purity angle, purity threshold and purity flag results are reported in Fig 6 to 9 and Table 10.The areas of both the peaks were increased a lot assuming the formation of chromophores at the particular wavelength.

Stability of analytical solutions:

The %RSD for area of ibuprofen at periodic time intervals of standard and sample preparations is found to be 0.31 and 0.25 respectively. And the %RSD for assay of ibuprofen was found to be 0.25. The %RSD for area of methocarbamol at periodic time intervals of standard and sample preparations is found to be 0.26 and 0.19 respectively. And the %RSD for assay of methocarbamol was found to be 0.20. (Table 11,12).

CONCLUSION:

From the above experimental data results and parameters it was concluded that, the chromatographic method developed for the simultaneous estimation of ibuprofen and methocarbamol was found to be simple, precise, accurate and cost effective and it can be effectively applied for routine analysis in research institutions, quality control department in industries, approved testing laboratories, bio-pharmaceutical and bio-equivalence studies and in clinical pharmacokinetic studies in near future.

REFERENCES:

1. United States Pharmacopoeia, Asian Edn., United Pharmacopoeial Convention, Inc., Rockville, 2005, 1266, 2610.

2. Basu D, Mahalanabis K.K, Roy B., Application of least squares method in matrix form: simultaneous determination of ibuprofen and paracetamol in tablets. J Pharm Biomed Anal. 1998, 16: 809-812.

3. Zarapkar S.S., Hulkar U.P., Bhandari N.P., Reverse phase HPLC determination of ibuprofen, paracetamol and methocarbamol in tablets, Indian Drugs, 1999, 36(11).

4. Erk N, Ozkan Y, Banolu E, Ozkan SA, Sentürk Z. Simultaneous determination of paracetamol and methocarbamol in tablets by ratio spectra derivative spectrophotometry and LC. J Pharm Biomed Anal.2001, 24: 469-475.

5. Perry, J. A. Glunz, L. G. Szczerba, T. J. Hocson, V. S.; Reagents For Ion Pair Reversed-Phase HPLC; American Laboratory 1984, 16(10), 114—119.

6. Hobart H. Willard, Lynne L. Merritt, J. J. A. Dean and A. S. Frank, Instrumental Method of Analysis, 5^th ed., CBS Publishers and Distributors, New Delhi, 1986, 3.

7. E. Michael, I. S. Schartz and Krull, Analytical Method Development and Validation, Interpharm Publishers, 2004, 25-46.

8. Lloyd R. Snyder, Joseph J. Kirkland, Practical HPLC Method Development, 2^nd edition, 1997, 600-620.

9. International Conference on Harmonization, Draft Guideline on Validation of Analytical Procedures, Definitions and Terminology. Federal Register 1995, 1260.

Received on 04.12.2012 Modified on 15.12.2012

Research J. Pharm. and Tech. 6(2): Feb. 2013; Page 178-183