Photochemically-Induced Fluorescence Properties and Determination of Flufenamic Acid, a Non-Steroidal Anti-Inflammatory Drug, in Urine and Pharmaceutical Preparation

Turk J Chem 25 (2001) , 165 – 171. c TÜBİTAK Photochemically-Induced Fluorescence Properties and Determination of Flufenamic Acid, a Non-Steroidal Anti-Inflammatory Drug, in Urine and Pharmaceutical Preparation∗ Leila BETTAIEB and Jean-Jacques AARON† Institut de Topologie et de Dynamique des Systèmes de l’Université Paris 7-Denis Diderot, associé au CNRS, UPRES-A 7086, 1, rue Guy de la Brosse, 75005 Paris-FRANCE Received 09.10.2000 The photochemically-induced fluorescence (PIF) properties of flufenamic acid (FF), a non-steroidal anti-inflammatory drug (NSAID) were investigated in acidic (pH 1.0) aqueous solutions at room temperature. An optimization procedure, including the effects of UV irradiation time, pH and solvent, was established for the determination of FF. A linear logarithmic calibration plot was obtained over a wide concentration range of four orders of magnitude. A low limit of detection of 0.14 ng/mL was found. The relative standard deviation (RSD) was 6.6% . The PIF method was applied to the quantitative analysis of FF in urine and in a pharmaceutical preparation with satisfactory recovery values. Key Words: Photochemically-induced fluorescence; flufenamic acid; urine analysis; pharmaceutical preparation. Introduction Flufenamic acid (FF) (Scheme 1) belongs to a family of important nonsteroidal anti-inflammatory drugs (NSAID) with a diphenylamine structure that are widely used in the treatment of rheumatoid arthritis, other musculo-skeletal disorders and post-trauma inflammation. Several analytical methods, such as HPLC1,2 GCMS3 , and UV-visible spectrophotometry4 and spectrofluorimetry5 generally based on the formation of metal complexes, have been reported for the determination of FF and other structurally-related NSAID. Voltammetric, spectrofluorimetric and UV spectrophotometric techniques have also been proposed for FF analysis, using the cyclization reaction of FF with sulfuric acid to produce the corresponding acridone derivative6 . The analytical usefulness of direct native fluorescence of FF is still a subject of controversy7,8 . Recently, it has been shown that diphenylamine derivatives can undergo photocyclization and yield stable photoproducts under UV irradiation9. Therefore, we decided to apply the photochemically-induced fluorescence (PIF) ∗ This paper has beed presented at MBCAC III (3rd Mediterranean Basin Conference on Analytical Chemistry) 4-9 June, 2000 Antalya-Turkey † Corresponding author. 165 Photochemically-Induced Fluorescence Properties and..., L. BETTAIEB, J.-J. AARON approach10,11 to the quantitative analysis of FF. Indeed, photochemical derivatization has the advantage of being a simple, clean and efficient analytical technique, providing strongly fluorescent photoproducts from non-fluorescent or weakly fluorescent analytes12 . O C OH H N C F3 Flufenamic acid C1 4 H10 F3 N O2 In this work, we investigated the PIF properties of FF in various media. Under UV irradiation, this compound exhibited in an acidic aqueous solution, an intense fluorescence emission due to its phototransformation into stable, fluorescent photoproducts. We applied the PIF method to the determination of FF in urine and in a pharmaceutical preparation. Experimental Apparatus Fluorescence and PIF spectra and intensity measurements were measured on a Kontron (Zürich, Switzerland) SFM 25 spectrofluorimeter, using a quartz cuvette (10 mm optical pathlength). An Osram (Germany) 200W high-pressure mercury arc lamp powered with a Spotlight power supply and located in a Schoeffel Instruments GmbH light-box was utilized for the photochemical studies. Chemicals Flufenamic acid was purchased from Sigma and used as received. Deionized water and analytical grade solvents (dioxane, ethanol, acetonitrile and dimethyl sulfoxide) were utilized for the preparation of solutions. The pH buffers were obtained from Merck. HClO4 was purchased from Aldrich (spectroscopic grade). The pharmaceutical preparation Movilisin (Sanky Pharma, GmbH, Germany) was obtained in a commercial pharmacy. Procedures Determination of FF in standard solutions Aliquots of FF samples, prepared in the optimal conditions and containing FF concentrations of 0.3-2,800 ng/mL, were introduced into a 1 cm quartz cuvette and then irradiated for the optimum irradiation time (defined as the irradiation time corresponding to the maximum PIF signal) at room temperature with the UV high-pressure mercury arc lamp. Then, the cuvette was transferred to the spectrofluorimeter and the fluorescence intensities were measured at the wavelengths of the excitation and emission maxima of the uncorrected PIF spectra. The procedure used for photochemical-fluorimetric measurements was the same as one previously utilized10,11. Afterwards, a direct calibration curve was drawn. 166 Photochemically-Induced Fluorescence Properties and..., L. BETTAIEB, J.-J. AARON Determination of FF in urine and pharmaceutical preparation For these applications, the classical standard addition procedure was chosen. In the case of FF determination in urine, a 20 µL volume of a 10−3 M FF aqueous solution was placed in a 10 mL flask and completed to the mark with a filtrated human urine sample. Then, 40 µL volumes of the resulting solution were put in 10 mL flasks and completed to the mark with FF aqueous standard solutions of increasing concentrations ; in these conditions, the original urine sample was diluted 250 times. For pharmaceutical preparation, 1 mL of Movilisin r (containing 30 mg of FF) was placed in a 100 mL flask and completed to the mark with water; a 100 µL volume of this solution was introduced into a 10 mL flask and completed to the mark with water. A constant volume (100 µL) of the diluted pharmaceutical sample was spiked with FF acidic aqueous standard solutions of increasing concentrations in 10 mL flasks. Photochemical-fluorimetric measurements were performed on these solutions. Results and Discussion PIF properties of FF in aqueous media FF (10−5 M, 2,800 ng/mL) displayed no significant fluorescence at room temperature in an acidic aqueous medium (0.1M HClO4 ), but upon UV irradiation of FF for 4 min in the same solution, a very strong fluorescence band appeared, with an emission maximum at 424 nm and a shoulder at 444 nm (Figure 1). The corresponding excitation spectrum showed two peaks at 256 and 390 nm. The same emission spectrum was obtained upon excitation at both wavelengths, which indicates that these excitation bands correspond to two electronic transitions of the same fluorescing molecule (Figure 1, curves a and b). This behaviour suggests that FF undergoes a photocyclization reaction, yielding strongly fluorescent photoproduct(s), in agreement with recent literature results9 . 390 0,8 0,7 424 444 EX 0,6 EM IF R.U. 0,5 0,4 0,3 256 0,2 a 4 min b 0,1 0,0 -0,1 0 min 200 240 280 320 360 400 440 480 520 WAVELENGTH(nm) Figure 1. PIF excitation (EX) and emission (EM) spectra of flufe (...truncated)