DETERMINATION OF A POORLY SOLUBLE DRUG, IBUPROFEN IN RAT PLASMA BY A SIMPLE HPLC ANALYSIS AND ITS APPLICATION IN PHARMACOKINETIC STUDY

DETERMINATION OF A POORLY SOLUBLE DRUG, IBUPROFEN IN RAT PLASMA BY A SIMPLE HPLC ANALYSIS AND ITS APPLICATION IN PHARMACOKINETIC STUDY

A. Nurfazreen¹, B. E. Tommy Julianto¹ and A. H. Khuriah*^{1, 2}

Department of Pharmaceutics, Faculty of Pharmacy, UiTM, 42300, Bandar Puncak Alam, Selangor ¹, Brain and Neurosciences Communities of Research ², UiTM, 40450 Shah Alam, Selangor, Malaysia.

ABSTRACT: In this study, a reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for the quantification of ibuprofen in rat plasma. The mobile phase consisted of a mixture of acetonitrile and water that was adjusted to pH 2.5 using orthophosphoric acid (70:30). The flow rate was set at 0.5 ml/min and effluent was monitored by using UV detector at a wavelength of 223 nm with retention time of 6.1 min. The chromatographic separation was carried out using a C18 (150 mm × 4.6 mm i.d.) column. The proposed method was validated based on linearity, accuracy and precision. The linearity of ibuprofen was in the range of 0.39-50 µg/ml with mean correlation coefficient of 0.999. The percentage mean recovery was found to be at 99.16%, while the coefficients of variation of within-day and between-day measurements were all found to be less than 5%. The limit of quantification (LOQ) and limit of detection (LOD) of the method were 0.296µg/ml and 0.098µg/ml, respectively. The method was further engaged to identify the pharmacokinetic profiles of ibuprofen nanoemulsion and ibuprofen oil solution after oral administration with AUC value of 6670.10 ± 283.83µg/ml∙hr and 3060.32 ± 169.93µg/ml∙hr, respectively.

HPLC, Ibuprofen, Pharmacokinetic profiles, Oral administration, Bioavailability

INTRODUCTION: Ibuprofen, a phenyl propionic acid derivative, is a non-steroidal anti-inflammatory drug (NSAID) ¹. It is widely used in the treatment of rheumatoid arthritis, osteoarthritis and joint pain ^{1, 15}. Ibuprofen is available as a white crystalline powder with a molecular weight of 206.27, pKa (COOH) = 4.41 ^{1, 2}. Ibuprofen inhibits prostaglandin biosynthesis by blocking the enzyme cyclooxygenase, which converts arachidonic acid to prostaglandin ¹⁶. Ibuprofen is practically insoluble in water and has shown incomplete absorption in the GI tract ^{3, 23}.

Thus, ibuprofen has been formulated into inert lipid vehicles such as microemulsions, nanoemulsions, self-emulsifying formulations and liposomes in order to increase the bioavailability of this poorly soluble drug ⁴.

Based on previous studies conducted on various approaches in analysing ibuprofen, high performance liquid chromatography (HPLC) was found to be the most suitable method to determine the concentration of the drug in different types of samples. Recent studies stated that HPLC with UV-Vis detector at a wavelength of 223 nm has successfully determined the concentrations of ibuprofen in nanoemulsion formulations with a retention time of 7.45 min ¹². Meanwhile, another study has developed a method for the analysis of ibuprofen and paracetamol using reversed phase HPLC with UV detector at a wavelength of 230 nm with a retention time of 6.96 and 3.04 min respectively ¹³. The high sensitivity, accuracy, precision, linearity and stability of HPLC method in the analysis of ibuprofen have made it a widely used method for the determination of ibuprofen concentrations in nanoemulsion formulations.

Nanoemulsion is a heterogeneous system which comprised of oil, surfactant, cosurfactant and an aqueous phase ^{5, 19}. Developing nanoemulsions as vehicles for carrying active pharmaceutical ingredients is emerging as a promising approach to deliver drug to the targeted site of action ⁶. Nanoemulsion with an approximate droplet diameter of about 20-200 nm has the potential to increase the aqueous solubility of poorly water-soluble drugs ^{5, 30}. They are kinetically stable without any flocculation or coalescence after long-term storage because of the nanometer-sized droplets ^{9, 20}.

Several advantages of nanoemulsions include smaller droplet size with a larger surface area, increasing dissolution rate and solubility and enhancing mucosal permeability ^{7, 20}. In nanoemulsions, the oil droplets act as a reservoir for hydrophobic drugs ^{8, 10}. The most widely used oil molecules are saturated and unsaturated fatty acids, fatty acid esters, soybean oils as well as olive oils ^{8, 10}.

This study was conducted with the purpose of developing a simple reversed-phase high performance liquid chromatography (RP-HPLC) equipped with a photodiode array detector in order to determine the concentration of ibuprofen in rat plasma. Oral administration was also carried out in this study to identify and compare the pharmacokinetic profiles of ibuprofen nanoemulsion and ibuprofen oil solution respectively.

 MATERIALS AND METHODS:

Materials

Ibuprofen [2-(4-isobutylphenyl)-propionic acid] was purchased from Sigma-Aldrich (UK). HPLC grade acetonitrile and orthophosphoric acid were obtained from Merck (Darmstadt, Germany). Distilled water was purified prior to use using ELGA Water Purification System R15 supplied with pump and tank (Elga Water System, UK). Olive oil, glycerol, propyl paraben and methyl paraben were supplied by Zulat Pharmacy (Malaysia). Laurate Sucrose Monoester (SME) (L-1695) was obtained from Ryoto Mitsubishi-Kagaku (Japan). All other chemicals and solvents were of analytical grade.

 Preparation of standard solution

The stock solution of ibuprofen was prepared in acetonitrile at a concentration of 1 mg/ml. Working solutions of ibuprofen with concentrations in the range of 0.39 – 50μg/ml were obtained by diluting the stock solution with acetonitrile. Serial dilutions were prepared with concentration of 50μg/ml, 25μg/ml, 12.50μg/ml, 6.25μg/ml, 3.13μg/ml, 1.56μg/ml, 0.78μg/ml and 0.39μg/ml respectively.

Plasma was spiked with ibuprofen by mixing 0.1 ml of ibuprofen stock solution into 0.9 ml of plasma to yield final ibuprofen concentration of 100μg/ml. The standard solutions (n=6) were prepared with known amount of ibuprofen ranging from 0.39 – 50 μg/ml.

Plasma samples were treated with deproteinizing agent (DA) consisting of acetonitrile: propanol (1:1), at the ratio of 2:1 (DA: sample). The mixture was vortex for 1 min and centrifuged at 10,000 rpm for 10 min at room temperature. A volume of 50 µl of the supernatant was filtered and injected into HPLC system prior to analysis.

 Instrumental and chromatographic conditions

The HPLC analysis was performed using Waters ACQUITY UPLC^® system (Waters Corp., MS, USA), equipped with a binary solvent delivery system and autosampler. Ibuprofen was detected using Photodiode Array (PDA) detector at 223 nm ¹⁸. The chromatographic separation was carried out using Phenomenex reversed phase C18 column, Jupiter 5μ C18 with particle size of 5 μm (150 mm x 4.6 mm).

The filtered and degassed mobile phase comprised of a mixture of acetonitrile and water which was adjusted to pH 2.5 with concentrated orthophosphoric acid at a ratio of 70:30 ¹⁸. The analysis involved using isocratic elution at a flow rate of 0.5 ml/min. Each wash cycle consisted of 200μl of strong solvent (90% acetonitrile) and 600μl of weak solvent (50% acetonitrile).

Method validation

Linearity

Six linearity curves were analyzed; each calibration curve consisted of 8 concentrations that were prepared from stock solution over the range of 0.39 – 50μg/ml. Peak area was plotted against concentration and the regression lines were calculated by the least-square method ¹⁸.

 Accuracy and precision

Accuracy was defined as the closeness of the measured value obtained by the analytical method to the true value ¹⁶. Accuracy could be assessed by calculating the recovery of known amounts of analyte.

In the present study, recovery experiments were performed by spiking known amounts of ibuprofen in blank rat plasma over a range of 0.39 to 50 µg/ml. Samples were prepared at eight concentration levels. For each level, the drug content was determined in triplicates (n=3). The mean accuracy should be within 15% of the true value.

Meanwhile, precision refers to the reproducibility of multiple measurements of a homogenous sample ¹⁶. Precision could be measured as repeatability (within-day) and intermediate precision (between-day) ¹⁶. Within-day validation included the evaluation of precision and accuracy of six replicates of standard solution which were analysed on the same day. Between-day validation was performed by evaluating the precision and accuracy of same standard solutions on six consecutive days.

The precision and accuracy of all sample concentrations must not exceed 15% of the coefficient of variation (CV %) from the theoretical value ¹⁴.

 Detection and Quantitation Limits

The limit of detection (LOD) is described as the lowest concentration of the analyte in the sample that can be detected above baseline noise; typically, three times the noise level ¹⁶. Limit of quantification (LOQ) is the lowest concentration of analyte that can be quantitatively determined by a peak height with a signal-to-noise ratio higher than 10 ¹⁶. LOD and LOQ were calculated based on the formula recommended by ICH as follow:

LOD=3.3σ/S               LOQ=10σ/S

Where σ is the standard deviation of y-intercepts of regression lines and