FEATURE CLINICAL CHEMISTRY 037


INTRODUCTION Ibuprofen (IBP) is chemically known as (±)-(R, S)-2- (4-isobutylphenyl)-propionic acid or α-methyl-4- (2-methylpropyl)-benzene acetic acid (see figure 1). It is a chiral 2-arylpropionic acid (2-APA) derivative non-steroidal anti-inflammatory drug (NSAID). IBP is widely accepted as the best tolerated NSAID; exhibiting good analgesic, anti-inflammatory, and antipyretic activity with the safest gastrointestinal profile. IBP is mainly marketed as a racemic


mixture, although anti-inflammatory activity, as determined by the in vitro inhibition of prostaglandin synthesis, resides almost exclusively in the S (+)-enantiomer. Clinically, IBP is extensively indicated to treat arthritis and is considered a promising drug because of its antiatherosclerotic property. It has also been used orally in the management of various


musculoskeletal disorders, pericarditis, and pain. Currently, there is renewed interest in the use of the enantiomers of IBP in the treatment of a variety of cancers including prostate and colon cancer. In the aim of overcoming the patient’s


most inconvenient nicotinic acid cutaneous flushing, IBP 2-hydroxyethyl ester - nicotinic acid codrug and IBP 2-hydroxyethyl ester have been synthesized (see figure 1). In vitro chemical and enzymatic stability of the aforementioned compounds were determined using HPLC analysis. Variety of HPLC procedures for IBP analysis and its derivatives have been reported. Many from the


previously developed methods for analysis of IBP and its related compounds employed the usage of gradient HPLC method or a complex tertiary mobile phase to accommodate


the differences in retention times between the parent drug and the prodrug, thus incapability of simultaneous analysis along with long retention times reaching 20 min. Some of the reported isocratic methods for determination of IBP and its derivatives utilized mobile phases consisting of large proportions of organic solvents, up to 70%, to elute the analytes within reasonable retention times; rendering them cost ineffective methods for routine use. In addition, few of the reported methods for analysis of IBP plasma samples are based on the usage of tedious extraction procedures requiring large sample volumes and multiple evaporations along with tertiary mobile phases to obtain reliable results. Herein, a simpler, faster, economic, and multi-task method is


repor ted in which the amounts of IBP (final parent drug) and the IBP 2-hydroxyethyl ester and the codrug were determined simultaneously in the same run. Fur thermore, it is an isocratic method not requiring any ratio adjustment of the relatively simple binary mobile phase to compensate the differences in retention times between IBP, its ester, and codrug.


MATERIAL AND REAGENTS Codrug of IBP 2-hydroxyethyl ester - nicotinic acid and IBP 2-hydroxyethyl ester have been synthesized at our laboratories in


Jordan University of Science and Technology (Irbid, Jordan) (figure 1). Reagents used were of analytical grade and solvents were HPLC- grade, Aldrich Chemical Company (Missouri, USA), ACROS Chemicals (Geel, Belgium), and Scharlau Chemical (Barcelona, Spain). IBP was kindly provided by the Jordanian Pharmaceutical Manufacturing (Naour, Jordan). Water used in the HPLC procedure was deionized.


APPARATUS AND CHROMATOGRAPHIC CONDITIONS The analytical HPLC system, which consists of SPD-M 10AVP diode array detector, SIL-10ADVP auto injector, DGV-14AVP degasser, and CTO-10ASVP column oven, was connected to a computer furnished with the appropriate software (Shimadzu, USA). The chromatographic separation was carried out under isocratic reversed phase conditions on Purospher®


at a column oven of 27°C±1. The injection volume was 30µL and the detection wavelength was 226 nm. The mobile phase was a mixture of acetonitrile and 0.02 M phosphate buffer pH 6 (55:45 v/v) and the flow rate was 2 mL min-1


and two of the parent drugs (IBP 2-hydroxyethyl ester and IBP) were detected with different retention times. This method was applied for determination of IBP and its reported ester and codrug in aqueous solutions, human plasma, and rat liver homogenate.


SAMPLE PREPARATION The standard solutions for each compound were obtained from the serial dilution of its 400 µg mL-1


of 40:60 v/v isopropanol HPLC-grade and 0.02 M phosphate buffer pH 6. The stock solution was prepared by dissolving 40 mg of each compound in 100 mL of the former solution mixture. The biological standard samples of each compound in human


plasma and rat liver homogenate were both prepared in a similar manner. Human plasma samples were prepared by first diluting pooled human plasma to (80% v/v) by usage of 0.05 M phosphate buffer of pH 7.4. A stock solution of 8 mg mL-1


in methanol was


prepared for each compound. The plasma standard solutions for each compound were obtained by the addition of 150 µL from the 8 mg mL-1


in an initial concentration of 400 µg mL-1 µg mL-1


25 µg mL-1


standard solution with 80% human plasma, 100 µg mL-1 standard solutions for each compound were obtained.


stock solution to 2.85 mL of 80% human plasma; resulting . By serial dilution of 400


and


Plasma proteins were then precipitated by usage of methanol in a ratio of 3:1 and centrifugation for 5 min at 4000 rpm for each diluted standard. The clear supernatant layer was analyzed for the three intended compounds by HPLC. Rat liver homogenate was prepared by homogenizing 2 g of rat liver pieces with 10 mL phosphate buffer saline (0.1 M, pH 7.4) for 5 min at 30,000 rpm in ice bath using a Teflon homogenizer to obtain (20% w/v) rat liver homogenate. The rat liver homogenate was centrifuged for 20 min at 5000 rpm and 4°C, and then the supernatant was collected and stored at -20°C until used. The same method adopted for preparation of compounds’ standard solutions in human plasma was used for rat liver homogenate.


VALIDATION CRITERIA Linearity and Sensitivity Two sets of calibration curves for IBP, IBP 2-hydroxyethyl ester, and codrug were constructed in three different days by injecting samples of the standard solutions’ for each compound containing concentrations ranging from 0.195 - 400 µg mL-1


. The area under the curve for each


compound was determined and plotted versus the concentration (µg mL-1


). The linearity was evaluated by linear regression analysis.  www.lifesciencesmagazines.com


RP-18C column (125x4 mm, 5 µm) (Merck, Germany)


. In the same chromatographic run, the codrug,


stock solution with solution mixture


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