Gradient High Performance Liquid Chromatography Method Development and Validation for Simultaneous Determination of Phenylephrine and Ibuprofen in Tablet Dosage Form

Purpose: To develop a gradient high performance liquid chromatography (HPLC) method for the simultaneous determination of phenylephrine (PHE) and ibuprofen (IBU) in solid dosage form. Methods: HPLC determination was carried out on an Agilent XDB C-18 column (4.6 x 150mm, 5 μ particle size) with a gradient mobile phase composed of 0.1 % orthophosphoric acid and acetonitrile at a ratio of: 0.01/95/5, 2.5/95/5, 6/10/90, 8/10/90, 8.1/95/5 and 13/95/5 for time (min)/0.1 % orthophosphoric acid (%)/acetonitrile (%) at a flow rate of 1.0 mL/min. Column temperature was maintained at 30 °C and detection was carried out using a photodiode array (PDA) detector at 210 nm. Validation parameters, including system suitability, linearity, precision, accuracy, specificity, limit of detection (LOD), limit of quantification (LOQ), stability of sample and standard stock solutions as well as robustness were obtained as per International Conference on Harmonization (ICH) guidelines. The proposed method was applied to the determination of phenylephrine and ibuprofen in commercial tablets. Results: Retention time for phenylephrine and ibuprofen were 2.7 and 8.4 min, respectively while % recovery was 99.42 and 99.80 %, respectively. The relative standard deviation (% RSD) for assay of the tablets was < 2 %. Conclusion: The method is fast, accurate, precise and sensitive, and hence it can be employed for routine quality control of tablets containing both drugs in quality control (QC) laboratories and pharmaceutical industry. Scopus, Embase, EBSCO,


INTRODUCTION
Phenylephrine (PHE) is chemically named as (R)-3-[-1-hydroxy-2-(methylamino) ethyl] phenol ( Figure 1A). It is a nasal decongestant which helps to relieve a blocked nose. It reduces the size of the blood vessels in the nose and sinuses thus enabling one to breathe more easily. It is also used as paroxysmal supraventricular tachycardia, mydriasis, and haemorrhoids [1]. Ibuprofen (IBU) is chemically named as (RS)-2-(4-(2-methylpropyl) phenyl) propanoic acid ( Figure 1B). It is used to relieve symptoms of a wide range of illnesses such as headaches, backache, pains, migraine, cold and flu symptoms and arthritis. Its effects are due to the inhibitory actions on cyclo-oxygenases, which are involved in the synthesis of prostaglandins.
Prostaglandins have an important role in the production of pain, inflammation and fever [2]. Various ultra violet (UV) and HPLC assay methods have been reported in the literature for the determination of phenylephrine [3][4][5][6] and ibuprofen [7][8][9][10][11] individually and in-combination with other drugs. According to the literature, there is no official method for the simultaneous determination of both drugs by reverse phase HPLC in combined tablet dosage forms. Hence, an attempt has been made to develop new method for simultaneous determination [12][13][14] and validation of phenylephrine and ibuprofen in a tablet formulation in accordance with ICH guidelines [15][16][17].

EXPERIMENTAL Instrumentation
Chromatography was performed with Water's 2695 HPLC systems provided with Hamilton syringe, auto sampler and 2996 photodiode array detector. All HPLC systems were equipped with a column compartment with temperature control and an on-line degasser. Data acquisition, analysis, and reporting were performed by Empower2 (Waters) chromatography software.

Reagents and chemicals
The reference samples of PHE and IBU were provided as gifts from Spectrum Pharma research solutions, Hyderabad. HPLC grade acetonitrile, HPLC grade methanol and all other chemicals were obtained from Merck chemical division, Mumbai. HPLC grade water obtained from Milli-Q water purification system was used throughout the study. Commercial tablets (ADVIL -dosage: PHE -10 mg and IBU -200 mg) were purchased from a local pharmacy.

Preparation of standard stock solution
Standard stock solutions were prepared by transferring 10 mg of phenylephrine and 200 mg of ibuprofen into a clean and dry 100 mL volumetric flask, to which 70 mL of diluent was added, sonicated for 5 min and volume made up to 100 mL with diluent to get stock solution.

Sample preparation
Twenty tablets were weighed and crushed into fine powder. An amount of the powder equivalent to the weight of five tablets was taken and dissolved in 1000 mL diluent, sonicated for 20 min and filtered through PVDF 0.45 µ filter. From the filtrate, 1 mL was pipetted into a 10 mL volumetric flask and the solution made up to the volume with the diluent.

System suitability test
To ensure that the resolution and reproducibility of the HPLC system was adequate for the analysis, a system suitability test was established. Data from six injections of 10 µL of the working standard solutions of PHE and IBU were used for the evaluation of the system suitability parameters like tailing factor, the number of theoretical plates, retention time and resolution factor.

Linearity
By appropriate aliquots of the standard PHE and IBU solutions with the mobile phase, six working solutions ranging between 5 -25 μg/mL of PHE and 100 -500 of IBU μg/mL were prepared. The linearity point of each experiment was performed in triplicate according to the optimized chromatographic conditions. The peak areas of the chromatograms were plotted against the concentration of PHE and IBU to obtain the calibration curve.

Accuracy
Recovery studies by the standard addition method were performed with a view to justify the accuracy of the proposed method. Previously analyzed samples of PHE and IBU to which known amounts of standard PHE and IBU corresponding to 50, 100 and 150 % of target concentration, were added. The accuracy was expressed as the percentage of analyte recovered by the proposed method.

Precision
Precision was determined as repeatability and intermediate precision (ruggedness), in accordance with ICH guidelines. The intra-day and inter-day precision were determined by analyzing the samples of PHE and IBU. Determinations were performed on the same day as well as well as on consequent days.

Limit of detection and the limit of quantification
Limit of detection (LOD) and limit of quantification (LOD) of PHE and IBU were determined by calibration curve method. Solutions of both PHE and IBU were prepared in linearity range and injected in triplicate. Average peak area of three analyses was plotted against concentration. LOD and LOQ were calculated by using following equations: LOD = (3.3 × Syx)/b, LOQ = (10.0 × Syx)/b, where Syx is residual variance due to regression and b is slope.

Robustness
The robustness of the method was performed by deliberately changing the chromatographic conditions. Organic strength was varied by ± 5 %, column temperature by ± 5 o C and flow rate by ± 0.1 mL.

Stability
The sample and standard solutions were injected at 0 h (control) and after 24 h (stability sample) at ambient room temperature. Stability was determined by determining RSD for sample and standard solutions.

Statistical analysis
Where applicable, results were expressed as mean ± SD. % RSD and data were analyzed statistically by using t-test with aid of Microsoft Excel-2007 software and data were considered significantly different at p ≤ 0.05.

Method development
Initially reverse phase liquid chromatography separation was tried using various ratios of methanol and water, acetonitrile and water as mobile phases, in which both the drugs did not responded properly, and the resolution was also poor. The organic content of mobile phase was also investigated to optimize the separation of both drugs.
To analyze both drugs, detection was tried at various wavelengths from 205 nm to 280 nm. Both PHE and IBU showed maximum absorption at a wavelength of 210 nm, which was selected as the detection wavelength for PDA detector. The retention times were found to about 2.7 min and 8.4 min for PHE and IBU, respectively. The chromatogram obtained was shown in the Figure  2.

System suitability
System suitability parameters such as number of theoretical plates, peak tailing, retention time and resolution factor were determined. The total run time required for the method is only 13 min for

Accuracy
To pre-analyzed sample solution, a definite concentration of standard drug (50, 100 and 150 % level) was added and recovery was studied. The percentage Mean recovery for PHE and IBU are 99.27 and 100.66 %, respectively and these results are within acceptable limit of 98-102%. The % RSD for PHE and IBU are 1.2 and 0.7, respectively and the percentage RSD for PHE and IBU is within limit of ≤ 2. Hence the proposed method is accurate and the results were summarized in Table 2.

Repeatability
Six replicates injections in same concentration were analyzed in the same day for repeatability and the % RSD for PHE and IBU were found to be 1.1 and 1.0, respectively and which is for PHE and IBU found to be within the acceptable limit of ≤2 and hence, the method is reproducible as presented in Table 3.

Intermediate precision
Six replicates injections in same concentration were analyzed on two different days with different analyst and column for verifying the variation in the precision and the % RSD for PHE and IBU was 0.3 and 1.3, respectively, and is within the acceptable limit of ≤ 2. The overall % RSD for PHE and IBU was found to be 0.8 and 1.1, respectively, and it is within the acceptable limit of ≤ 2 and hence, the method is reproducible on different days with different analyst and column and the results are as shown in Table 3.

Statistical Analysis of Precision Result:
Probability value (P) for PHE and IBU at 5% significance level was found to be 0.75 and 0.68, respectively, which are greater than 0.05 and hence no significant difference was observed in the precision results carried out for two consecutive days, and the results are shown in Table 4.

Robustness
The robustness was established by changing the flow rate, column temperature and composition of the mobile phase within allowable limits from actual chromatographic conditions. It was observed that there were no marked change in mean RT and RSD is within limit of ≤ 2 .The tailing factor, resolution factor and number of theoretical plates were found to be within     Table 5 while the results for IBU are shown in Table 6.  Table  7.

LOD and LOQ
LOD and LOQ for PHE were 0.03895 and 0.11803 μg/mL respectively, and LOD and LOQ for IBU were 0.338187 and 1.024809 μg/mL, respectively.

Results of method application to tablet
The content of PHE and IBU in the tablets was found by the proposed method and the results were shown in Table 8.

DISCUSSION
RP-HPLC method was developed and validated for the simultaneous determination of phenylephrine and ibuprofen in tablet dosage form. The resolution between two peaks is always more than 2. The system suitability tests revealed that numbers of theoretical plates were above 2000 and tailing factor is less than 2. PHE and IBU showed a linearity of response between5-25 µg/ml and 100-500 µg/ml. The mean peak area of the chromatograms was plotted against the concentration of PHE and IBU to obtain the calibration curve. Linearity was high as well as recovery of PHE and IBU, indicating high accuracy of the method. Repeatability and intermediate precision values were within the acceptable limits. This indicates that the method is precise. Specificity experiment shows that there is no interference or overlapping of the peaks of excipients or diluents with the main peaks of PHE and IBU. The lowest values of LOD and LOQ as obtained by the proposed method indicate that the method is sensitive. The stability studies indicate that both