Development and Validation of RP-HPLC Method for Simultaneous Estimation of Ibuprofen and Chlorzoxazone in Synthetic Mixture

ABOUT AUTHORS
Anil C. Patel*, Dr Paresh U. Patel , Rinku B. Patel
Department of Quality Assurance, S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University, Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India.
*anilpatel002@gmail.com

ABSTRACT
A novel, precise, accurate and rapid isocratic reversed-phase high performance liquid chromatographic/ultraviolet (RP-HPLC/UV) method was developed, optimized and validated for  simultaneous determination of Ibuprofen and Chlorzoxazone. The method showed adequate separation for Ibuprofen and Chlorzoxazone and best resolution was achieved with ACE 5 C18 column (150 mm × 4.6 mm i.d, 5 μm particle size) using Acetonitrile-Phosphate buffer pH 3.5 - Methenol (20:20:60, v/v; pH adjusted to 3.5 with O-phosphoric acid and TEA(Tetra ethyl amine) as a mobile phase at a flow rate of 0.7 ml/min and wavelength of 221 nm. The calibration curves were linear over the concentration ranges of 2-30 μg/ml for Ibuprofen and Chlorzoxazone. The limit of detection (LOD) and limit of quantification (LOQ) for Ibuprofen were 0.96 and 2.92 μg/ml while for Chlorzoxazone were 0.69 and 2.09 μg/ml, respectively. All the analytes were separated in less than 6.0 min. The proposed method could be applied for routinelaboratory analysis of Ibuprofen and Chlorzoxazone in pharmaceutical dosage form. Methods were validated statistically and recovery studies were carried out. The proposed methods have been applied successfully to the analysis of cited drug either in pure form or in synthetic mixture of both drugs with good accuracy and precision. The method herein described can be employed for quality control and routine analysis of drugs in pharmaceutical formulations.

REFERENCE ID: PHARMATUTOR-ART-1729

INTRODUCTION
Ibuprofen(IBU) is chemically (RS)-2-(4-(2-methylpropyl) phenyl) propanic  acid  (Figure 1) is a well known Nonsteroidal anti-inflammatory drug ¹. It is official in Indian Pharmacopoeia (IP)² , British Pharmacopoeia (BP)³, United states Pharmacopeia (USP)⁴, Japanese Pharmacopoeia (JP)⁵. IP and JP describe Aqueous titration method for its estimation, while BP , IP ,USP describes liquid chromatography method for its estimation.   Literature survey reveals   UV^6-7, HPLC^8-10, HPTLC¹¹ methods  for estimation of IBU alone. Literature survey also reveals UV^12-15, HPLC^16-18 , HPTLC¹⁹ spectrophotometry  methods for determination of IBU with other drugs in combination. Chlorzoxazone (CHL) is chemically 5-chloro-3H-benzooxazol-2-one(Figure 2). Chlorzoxazone (CHL) is official in United states Pharmacopeia (USP)²⁰. USP  describes UV and  liquid chromatography method for its estimation. Literature survey reveals Flourimetry²¹, HPLC^22-25 methods  for determination of CHL alone. Literature survey also reveals UV^26-28 ,  HPLC^29-31, HPTLC³² methods  for the determination of CHL with other drugs combination. The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of IBU and CHL in their combined synthetic mixture or dosage forms. Literature survey  reveal only simultaneous method³³ for IBU and CHL in synthetic mixture or combined dosage forms. The present communication describes simple, sensitive, rapid, accurate, precise and cost effective chromatographic method.

EXPERIMENTS

Apparatus
- RP-HPLC instrument equipped with a UV-Visible detector and a photodiode array detector, (Shimadzu, LC-2010C_HT, Japan,),auto sampler, ACE 5 C18 column (150 mm × 4.6 mm i.d, 5 μm particle size)and LC-solution software were used.
- Analytical balance (Sartorius CP224S, Germany)
- Triple distillation unit consisting of borosilicate glass
- Digital pH meter (LI 712 pH analyzer, Elico Ltd., Ahemedabad)
- Ultra sonic cleaner (Frontline FS 4, Mumbai, India)

Reagents and materials
IBU and CHL bulk powder was kindly gifted by Acme Pharmaceuticals Ltd. Ahmedabad,  Gujarat, India. Methanol (AR Grade, S. D. Fine Chemicals Ltd., Mumbai, India), Acetonitrile (AR Grade, S. D. Fine Chemicals Ltd., Mumbai, India) and Whatman filter paper no. 41 (Millipore, USA) were used in the study.

Preparation of standard stock solutions
An accurately weighed standard IBU and CHL powder (10 mg) were weighed and transferred to 100 ml separate volumetric flasks and dissolved in methanol. The flasks were shaken and volumes were made up to mark with methanol to give a solution containing 100 μg/ml of each IBU and CHL.

Preparation of Working Standard Solutions
An aliquot of stock solution 1 ml of standard stock solution was transferred in 10ml volumetric flask and adjusted up to mark with methanol having concentration (10µg/ml).

Preparation of Synthetic mixture
Ibuprofen (40 mg) and Chlorzoxazone (50 mg) were taken and then both the drug were mixed with, Starch, Lactose, Magnesium Stearate, DCP and Talc. Total 200 mg of mixture was prepared and it was used.

Chromatographic Condition
Stationary phase: ACE 5 C₁₈ column (150 mm x 4.6 mm i.d., 5 µm particle size) was used at ambient temperature.
Mobile Phase:  Acetonitrile: Phosphate buffer pH 3.5: Methanol [20:20:60, v/v/v]
Flow rate: 0.7 ml/min
Injection volume: 20 µL
Detection: The elution was monitored at 221 nm using PDA detector.

Method development
A satisfactory separation and good peak symmetry for IBU and CHL was obtained with a mobile phase Acetonitrile: Phosphate buffer pH 3.5: Methanol [20:20:60, v/v/v] at a flow rate of 0.7 ml/min to get better reproducibility and repeatability (Figure 3).Overlain UV spectrum showed that both drugs showed good absorbance at 221 nm, hence the wavelength of 221 nm was selected for quantification of IBU and CHL (Figure 4).

Method Validation

Calibration Curve (Linearity)
Calibration curves were constructed by plotting peak areas Vs concentrations of IBU and CHL and the regression equations were calculated. The calibration curves were plotted over the concentration range 2-30 µg/ml for IBU and 2- 30µg/ml for CHL. Accurately measured standard working solutions of IBU (0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 ml) and CHL (0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 ml) from100 µg/ml of stock solution were transferred to a series of 10ml of volumetric flasks and diluted to the mark with methanol.  20 µL of each solution were injected under the operating chromatographic conditions described above

Accuracy (% Recovery)
The accuracy of the method was determined by calculating recovery of IBU and CHL by the standard addition method. Known amounts of standard solutions of IBU were added to pre quantified sample solutions of CHL. Known amounts of standard solutions of CHL were added to pre quantified sample solutions of IBU. The amounts of IBU and CHL were estimated by applying obtained values to the regression equation of the calibration curve.

Method Precision (% Repeatability)
The precision of the Method was checked by repeatedly injecting six sample solutions of IBU (20 μg/ml) and CHL (20 μg/ml) under the same chromatographic condition and measurements of peak area, retention time and tailing factor. Percentage relative standard deviation (RSD) or % coefficient of variation (CV) should not be more than 2.

Intermediate Precision (Reproducibility)
The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of sample solutions of IBU (10, 20, 25 μg /ml) and CHL (10, 20, 25 μg /ml). The results were reported in terms of relative standard deviation (RSD).

Limit of Detection and Limit of Quantification
LOD and the LOQ of the drug were calculated using the following equations as per International Conference on Harmonization (ICH)⁽³⁴⁾ guidelines.

LOD = 3.3 × σ/S

LOQ = 10 × σ/s

Where
σ= the standard deviation of the response
S = Slope of calibration curve.

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ANALYSIS OF IBU AND CHL IN SYNTHETIC MIXTURE:-
The response of the sample solution was measured at 221 nm under the chromatographic condition mentioned above for the quantification of IBU and CHL. The amounts of IBU and CHL present in sample solution were determined by applying values of the peak area to the regression equations of the calibration curve.

RESULTS AND DISCUSSION:-

Linearity
Linear correlation was obtained between peak area Vs concentrations of IBU and CHL in the concentration of 2-30 µg/ml for IBU and 2-30 μg/ml for CHL. Regression parameters are mentioned in Table and the calibration curves of these two drugs at 221 nm are shown in Fig 5 and 6.

Method Precision (% Repeatability)
The RSD values for IBU and CHL were found to be 1.95 and 0.34 %, respectively . The RSD values were found to be <2 %, which indicates that the proposed method is repeatable.

Intermediate Precision (Reproducibility)
The low RSD values of interday and intraday for IBU and CHL, respectively, reveal that the proposed method is precise .

LOD and LOQ
LOD values for IBU and CHL were found to be  0.96  µg/mland 2.92 µg/ml, respectively and LOQ values for IBU and CHL were found to be 0.69µg /ml and 2.09µg/ml, respectively (Table 5). These data show that the proposed method is sensitive for the determination of IBU and CHL.

Accuracy
The recovery experiment was performed by the standard addition method. The recoveries obtained were 98.57 ± 0.49% and 99.09 ± 0.85 % for IBU and CHL, respectively (Table 3). The low value of standard deviation indicates that the proposed method is accurate. Results of recovery studies are shown in Table 3.

Assay of the synthetic mixture
The proposed validated method was successfully applied to determine IBU and CHL in their synthetic mixture. The result obtained for IBU and CHL was comparable with the corresponding labeled amounts (Figure 7) (Table 4)

Table 1 System Suitability Parameters of Chromatogram for IBU and CHL

                             Table 2 Recovery Data for the proposed Method

Table 3 Analysis of IBU and CHL (n = 6)

Table 4  Regression Analysis Data and Summary of Validation Parameter for the proposed Method

CONCLUSION:-
In this proposed method the linearity is observed in the concentration range of 2 to 30 µg/ml and 2 to 30 µg/mlwith co-efficient of correlation, (r²) = 0.997 and (r²) = 0.997 for IBU and CHL, respectively at 221 nm. The result of the analysis of pharmaceutical formulation by the proposed method is highly reproducible and reliable and it is in good agreement with the label claim of the drug. The method can be used for the routine analysis of the IBU and CHL in synthetic mixture without any interference of excipients. 

ACKNOWLEDGEMENT
The authors are thankful to Acme Pharmaceutical Ltd., Ahmedabad, India for providing gift sample of IBU and CHL for carry out the research work.

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