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Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2009, 8, 22-37

Analytical Procedure for Determination of Cyclooxygenase-2 Inhibitors in Biological Fluids by High Performance Liquid Chromatography: A Review
Giuseppe Carlucci*
Universit degli Studi G. DAnnunzio Chieti-Pescara - Facolt di Farmacia - Dipartimento di Scienze del Farmaco via dei Vestini - 66100 Chieti Italy
Abstract: High-performance liquid chromatographic methods for the analysis of cyclooxygenase 2 inhibitors (COX-2) in biological fluids are reviewed. In particular, sample preparation and handling procedures, chromatographic conditions and detection methods are discussed. A summary of published high-performance liquid chromatographic assays for individual nabumetone, celecoxib, rofecoxib, nimesulide, etoricoxib, etodolac, deracoxib, lumiracoxib, valdecoxib, and meloxicam is included.

Keywords: Sample preparation, NSAIDs, COX-2 inhibitors, biological fluids, HPLC. 1. INTRODUCTION The anti-inflammatory, analgesic, and antipyretic drugs are a heterogeneous group of compounds, often chemically unrelated (although most of them are organic acids), which nevertheless share certain therapeutic actions and side effects. The prototype is aspirin, hence these compounds are often referred to as aspirin-like drugs; they also are frequently called non-steroidal anti-inflammatory drugs, or NSAIDs, an abbreviation that is used throughout this review to refer to these agents. There has been substantial progress in elucidating the mechanism of action of NSAIDs. Inhibition of cyclooxygenase (COX), the enzyme responsible for the biosynthesis of the prostaglandins and certain related autacoids, generally is thought to be a major facet of the mechanism of NSAIDs. Some of the shared properties of NSAIDs are considered first; the more important drugs are discussed in some detail. Since the principal therapeutic effects of NSAIDs derive from their ability to inhibit prostaglandin production, the enzymatic activities involved in prostaglandin synthesis are described here briefly. The mechanisms by which varying NSAIDs interfere with prostaglandin synthesis then are outlined. The first enzyme in the prostaglandin synthetic patway is prostaglandin endoperoxide synthase, or fatty acid cyclooxygenase. This enzyme converts arachidonic acid to the unstable intermediate s PGG2 and PGH2. It is now appreciated that there are two forms of cyclooxygenase, termed cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX-2) [1]. COX-1 is a constitutive isoform found in most normal cells and tissues, while COX-2 is induced in settings of inflammation by cytokines and inflammatory mediators [2]. However, COX-2 also is constitutively expressed in certain areas of kidney and brain [3, 4]. Importantly, COX-1, but not
*Address correspondence to this author at the Universit degli Studi G. DAnnunzio Chieti-Pescara - Facolt di Farmacia - Dipartimento di Scienze del Farmaco - via dei Vestini - 66100 Chieti Italy; E-mail: g.carlucci@unich.it 1871-5230/09 $55.00+.00

COX-2, is constitutively expressed in the stomach. This accounts for the markedly reduced occurrence of gastric toxicity with the use of selective inhibitors of COX-2. 2. NABUMETONE Nabumetone (1) or 4-(6-Methoxynaphthalenyl)-2-butanone, is a nonsteroidal anti-inflammatory drug of the acetic chemical class. Nabumetone is a prodrug that requires conversion to an active metabolite 6-methoxy-2-naphthyacetic acid (6-MNA) for its anti-inflammatory, analgesic, and antipyretic activity. Structures of nabumetone and its metabolite are shown in Fig. (1). Nabumetone is the only NSAID of the acetic acid class with a half-life long enough to support once daily administration. 6-MNA competitively inhibits both cyclooxygenase isoenzymes, COX-1 and COX-2, by blocking arachidonate binding resulting in analgesic, antipyretic, and anti-inflammatory pharmacologic effects [5-8]. The enzymes COX-1 and COX-2 catalyze the conversion of arachidonic acid to prostaglandin G2 (PGG2), the first step of the synthesis of prostaglandins and thromboxanes that are generated in rapid physiological responses [9, 10]. Nabumetone is used in the treatment of osteoarthritis, rheumatoid arthritis and other musculoskeletal disorders. Preliminary studies to quantify this drug in biological fluids involved radioassay scintillation counting of isotopes and gas chromatography

Fig. (1). Chemical structures of nabumetone and 6-methoxy-2naphtyacetic acid (6-MNA).

2009 Bentham Science Publishers Ltd.

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with flame ionization detection and reversed-phase highperformance liquid chromatography using violet or fluorescence detection [11-16]. 6-chloro-2-naphthylacetic acid [15],
-naphtol [22] or naproxen [27, 32] were used as internal standards for the determination in biological samples. Several high-performance liquid chromatographic methods have been published for the individual determination of nabumetone in biological fluids, serum and urine samples [17-23], however there have been no reports concerning the simultaneous determination of nabumetone and 6-MNA by isocratic HPLC. Direct injection of diluted urine [17, 19, 20] and liquid-liquid extraction as a sample clean-up procedure [14, 17, 19, 22] are frequently used for the naphthalene derivatives in biological fluids. For the liquid-liquid extrac-tion procedure, back extraction and/or some derivatization techniques induced to increase the sensitivity are often carried out to avoid interference substances [23-25]. Mikami et al. [15] describes a reversed-phase HPLC method for the simultaneous quantitation of nabumetone and its metabolite 6-MNA in human urine, using methyl p toluate as the internal standard. The ability of this method to distinguish intact nabumetone from its metabolite was demonstrated. Nabumetone and 6-MNA were extracted from the sample matrix using solidphase extraction by Bond-Elut Certify II cartridges containing reversed-phase and anion exchange functionalities for the analysis of these drugs in human urine with fluorimetric detection. Briefly, aliquots of 1.0 mL of diluted methanolic urine samples were applied to pre-conditioned Bond-Elut Certify II cartridges and slowly drawn through the cartridge. The cartridges were washed with 4.0 mL of water and dried for five min by aspiration with a moderate to strong vacuum. The analytes were eluted with 6 mL of n-hexane-ethyl acetate (1:1). These n-hexane -ethyl acetate (1:1) solutions were evaporated to dryness in a water bath at 40C. The residues were redissolved in 200 L of ethanol containing methyl-p toluate, and aliquots of 10 L were injected into the chromatograph. The assay of Nabumetone and 6-MNA and the internal standard utilized a fluorescence detector with excitation at 280 nm, and emission at 350 nm. The lower limits of quantification in human urine were 150 and 16 ng/mL for nabumetone and 6-MNA, respectively. Jang et al. [22] and Ray et al. [14] both described liquidliquid extractions, following acidification of drug-containing plasma. Jang et al., acidified using a combination of 0.1 M HCl and 0.5M citrate buffer (pH 3), while Ray et al. used 1.5 M HCl only. Both authors then extracted the analyte into an organic phase ether and n-hexane-ethyl acetate 50:50, respectively and evaporated to dryness under a gentle stream of nitrogen, after which the samples were reconstituted and injected into a normal - phase HPLC column. While Jang et al., detected fluorometrically with excitation at 284 nm and emission at 320 nm, Ray et al. made use of UV detection at 280 nm, fluorimetric detection having a higher sensitivity (0.1 g/mL). The extraction procedure described by AlMomani et al. [16] appears to be loosely based on the method described by Ray et al., while using UV detection at 270 nm. Jang and Al-Momani used approximately 1.0 mL of plasma or serum, while Ray et al. used 0.5 mL of plasma. De Jager et al. [25] described an HPLC assay for 6-MNA in

which samples are prepared by protein precipitation, using naproxen as internal standard. In this study the authors describe a procedure that is well suited for rapid sample processing, requiring only a small volume of plasma (200L). 6MNA was separated from the internal standard and endogenous plasma components using a LiChrospher 100 RP8 stainless-steel column, fitted with a guard column, dry filled with Perisorb RP18 pellicular packing. A simple and sensitive HPLC method for the determination of nabumetone in human plasma was developed by Kobyliska et al. [26]. The procedure used involves liquid-liquid extraction with ethyl acetate and reversed-phase chromatography with fluorimetrica detection with excitation at 230 nm and emission at 356 nm. Briefly, 0.5 mL of plasma, 25 L of an internal standard working solution and 0.2 mL of 0.01 M phosphate buffer pH 10, were added. Then, the plasma was briefly mixed with a vortex mixer and 3.0 mL of ethyl acetate was added. The solution was shaken for 5 min, and centrifuged at 1500 x g for 5 min. After freezing at 70C for 20 min, the organic layer was transferred to another glass tube and evaporated to dryness at 45C under a gentle stream of nitrogen. The sample residue was dissolved in 200 L freshly prepared mobile phase, centrifuged, transferred to autosampler vials and 40 L aliquot was injected onto the HPLC system for analysis. The limit of quantification was established as 0.313 ng/mL and the calibration curve was linear up to 20 ng/mL. The disposition of nabumetone after a single oral dose administration of this drug to humans and minipigs was investigated by Nobilis et al. [27, 28]. These authors developed a simple HPLC method for the simultaneous determination of nabumetone, 6-MNA and the other metabolites. Naproxen was chosen as the internal standard, both UV for higher concentrations and fluorescence detection for very low concentrations were used. The identity of the nabumetone metabolites in biological samples was confirmed using HPLC-MS experiments. Pharmacokinetics of nabumetone, 6-MNA and 6-HNA (6-hydroxy-2-naphthylacetic acid) in human plasma and minipig plasma was evaluated and compared. A selective, simple and sensitive heavy atom induced-room temperature phosphorimetric method for the determination of 6MNA in biological fluids was developed by Pulgarn et al. [29] and Ray et al. [30]. The phosphorescence signals are a consequence of intermolecular protection when analytes are, exclusively, in the presence of heavy atom salt and sodium sulfite as scavinger to minimize room temperature phosphorescence quenching. This technique enables us to determine analytes in complex matrices without the need for a tedious separation process. 3. CELECOXIB Celecoxib or 4-[5-(4-Methylphenyl)-3-(trifluoromethyl)1H-pyrazol-1-yl]benzenesulfonamide is a selective inhibitor of COX-2 for the treatment of rheumatoid arthritis and osteoarthritis [31-35]. COX-2 is induced at inflammation sites [31, 32] but is also found constitutively in brain, spinal cord, kidney and some other tissues [33-35]. Clinical studies indicate that celecoxib is metabolised in the liver via the oxidative pathway to the corresponding alcohol and carboxylic acid and is removed by renal excretion as a glucuronide metabolite [36]. The chemical structures of celecoxib, hydroxycelecoxib, and carboxycelecoxib are shown in Fig. (2). The clinical pharmacokinetics and pharmackodinamics of cele-

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Fig. (2). Chemical structures of Celecoxib, hydroxycelecoxib and carboxycelecoxib.

coxib have been reported [37]. Development of sensitive and specific analytical techniques for the determination of celecoxib in biological samples is highly required for clinical investigations and pharmacokinetic studies. Many chromatographic methods for the assay of celecoxib were reported. Woolf et al. [38] described a method for the determination of celecoxib in human plasma. The HPLC method included a column switching procedure, in which late eluting compounds were diverted to waste, to reduce run-time to 12 min. Briefly, 1.0 mL of plasma was pipetted into a 15 mL polypropylene conical tube. A 25 L aliquot of 10 g/mL working internal standard solution was pipetted into each of the tubes containing the samples and the previously prepared standards. Tubes containing samples received an additional aliquot of acetonitrile (50 L) to make these samples equivalent in organic content to the standards. The tubes were vortexed. Four 250 L aliquots of acetonitrile were added to each tube vortexing for 10 s after each addition. The tubes were capped and centrifuged for 10 min at 2500 g. The supernatant was decanted into a disposable glass culture tube containing 3 mL of 0.10 M, pH 3.0, sodium phosphate buffer and mixed. The buffered samples were poured into individual 10 mm/6mL 3M Empore C18 solid-phase cartridges positioned on a 20-place vacuum manifold equipped with stopcocks at each position. SPE cartridges for plasma were conditioned using sequential washes of 2 mL acetonitrile and 2

mL water. Upon transfer of the sample to conditioned cartridge, the buffered plasma was aspirated through the SPE cartridges using a vacuum pressure. The cartridges were rinsed with 1 mL of water and 1 mL of acetonitrile-water (25:75). The cartridges were allowed to aspirate to dryness after which they were removed from the manifold and suspended into 15 mL centrifuge tubes. The analytes were eluted from the SPE cartridges by drawing two 1 mL aliquots of acetonitrile through each cartridge using centrifugation. The tube containing the elution solvent was placed in a Zymark Turbovap LV evaporator, and the solvent was evaporated using nitrogen (50 C, 15 p.s.i., 30 min). The residue in the tube was reconstituted using 250 L of mobile phase and transferred to a low-volume conical glass vial prior to injection onto the HPLC. The samples were chromatographed under normal-phase HPLC conditions using a Nucleosil-NO2 column. Detection was accomplished using UV absorbance at 260 nm. The assay was linear in the concentration range of 25-2000 ng/mL. The limit of quantification for celecoxib was 25 ng/mL. Brutigam et al. [39] describes a liquid chromatography-tandem mass spectrometry for the quantification of celecoxib in human plasma and rat microdialysis samples using 4-[5-phenyl-3-(trifluoromethyl)1H-pyrazol-1-yl]benzesulfonamide as the internal standard. Celecoxib and the internal standard were extracted from plasma by solid-phase extraction with a C18 cartridges. Microdialysis samples were injected directly into the LC-MSMS system. Extraction was not necessary. Thereafter compounds were separated on a narrow bore RP-C18 column. Chromatographic separation of extracted plasma samples was performed in isocratic mode with a Nucleosil C18 column (30 x 2.0 mm I.D., 5m particle size and 100 pore size). The mobile phase consisted of a mixture of acetonitrile-water-ammonium hydroxide solution 25% (85:15:0.1); while for microdialysis samples a modified chromatographic procedure was used. Microdialysates were separated using a Nucleosil C18 column (70 x 2.0 mm I.D., 5m particle size and 100 pore size) with a mobile phase acetonitrile-waterammonium hydroxide solution 25% (65:35:0.1). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The limits of quantitation for celecoxib were in human plasma 0.25 ng/mL and in microdialysis samples 0.5 ng/mL, respectively. A robust, highly reliable and reproducible liquid chromatographic-mass spectrometric assay is developed for the determination of celecoxib in human plasma using sulindac as an internal standard by Abdel-Hamid et al. [40]. The method involved extraction of the analytes from plasma samples at pH 5 with ethyl acetate and evaporation of the organic layer. The reconstituted solution of the residue was injected onto a Shim Pack GLC-CN, C18 column and chromatographed with a mobile phase comprised of acetonitrile-1% acetic acid solution (4:1) at a flow-rate of 1.0 mL/min. The mass spectrometer was programmed in the positive single-ion monitoring mode to permit the detection and quantification of the molecular ions of celecoxib and sulindac at m/z 382 and 357, respectively. The limits of quantification and detection of celecoxib in plasma were determined by analysing plasma samples spiked with celecoxib at relatively low concentrations (20-50 ng/mL) using the developed LC-MS method under the described conditions. The limit of quantification for celecoxib in plasma was found to be 50 ng/mL (mean

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predicted concentration 41.04.5 ng/mL). This concentration yielded an RSD of 10.2% and an accuracy of 9.0% expressed as (% deviation) of the nominal concentration. On the other hand, the limit of detection for celecoxib in plasma was 20 ng/mL. A simple and sensitive method for the determination of celecoxib in human serum by HPLC with fluorescence detection was developed by Schnberger et al. [41]. Briefly, after protein precipitation with acetonitrile, samples were extracted with chloroform. Separation was achieved on a Prontosil C18 AQ column (150 x 3 mm I.D., 3 m particle size) at a flow-rate of 0.35 mL/min using water-acetonitrile (40:60) as the mobile phase. Aliquots of 10 L were used for HPLC analysis. A demethylated analogue of celecoxib was used as internal standard. Using fluorescence detection with excitation at 240 nm and emission at 380 nm, the limit of quantification was 12.5 ng/mL for a sample size of 0.5 mL of serum. The assay was linear in the concentration range of 12.5 1500 ng/mL and showed good accuracy and reproducibility. At all concentrations intra- and inter-assay variabilities were below 11% with a less than 9% error. The method was applied to the determination of celecoxib for pharmacokinetic studies in man. An innovative reversedphase high-performance liquid chromatographic method is validated for the simultaneous determination of celecoxib and rofecoxib in human plasma by Hamama et al. [42]. Briefly, plasma samples are extracted into an organic solvent (1-chlorobutane) and evaporated under an air flow. The chromatographic separation is achieved using a Zorbax SBCN 5 m analytical column operated at room temperature and mobile phase consisting of acetonitrile and water containing 0.1 M potassium dihydrogen orthophosphate buffer adjusted to pH 2.4 with 85% orthophosphoric acid (43:58). The 4-n-pentyl-phenylacetic acid was used as the internal standard. The assay of celecoxib and rofecoxib in human plasma utilized a ultraviolet detector set at 254 nm. The calibration curves for the two drugs were linear over the range 20 to 2000 g/mL for celecoxib and 10 to 500 g/mL for rofecoxib, respectively. The lower limit of quantification for the celecoxib and rofecoxib is 20 and 10 g/mL, respectively, using 1.0 mL of human plasma. Werner et al. [43] developed a method for the quantification of celecoxib in human plasma based on reversed-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionisation (APCI) mass spectrometry after liquidliquid extraction. The method is rapid, sensitive and highly selective. The limit of quantitation was 5 g/L. Rofecoxib was used as an internal standard. After validation, the method was used to study the pharmacokinetic profile of celecoxib following administration of a single oral dose (200 mg) in 12 healthy volunteers. A simultaneous determination of celecoxib, hydroxycelecoxib, and carboxycelecoxib in human plasma using gradient reversed-phase liquid chromatography with ultraviolet absorbance detection was developed by Strmer et al. [44]. Briefly, following a solid-phase extraction procedure, the samples were separated by gradient reversed-phase HPLC and quantified using UV detection at 254 nm. The method was linear over the concentration range 10-500 ng/mL. The achieved limits of quantification of 10 ng/mL for each analyte allowed the determination of the pharmacokinetic parameters of the analytes after administration of 100 mg celecoxib. A simplified solid-phase extraction procedure and liquid chromatographic determination of cele-

coxib in rat plasma was developed by Guermouche et al. [45]. In this work, a poly(divinylbenzene-co-N-divinylpirrolidone) Oasis HLB 60 mg SPE cartridge was used to directly extract celecoxib from rat serum without any supplementary step. The optimal chromatographic conditions for the celecoxib determination were investigated. After pilot investigations, ibuprofen was choose as the internal standard. Separations were carried out on a Novapak C18 column (150 x 4.0 mm I.D.) preceded by a C18 home-made guard column (20 x 4.0 mm) using a mobile phase of acetonitrile- acetate buffer 0.075 M (pH 5.0). The detection limit at a signal to noise ratio of 3 was 0.002 mg/L. For the limit of quantification 0.01 mg/L, was taken. Jalalizadeh et al. [46] describes a reversed-phase HPLC method for the quantitation of celecoxib in human plasma, using flutamide as the internal standard. The method is based on liquid-liquid extraction with chloroform. The residue after extraction was reconstituted in 100 L of mobile phase, mixed well and 60 L of the final clear solution was injected into HPLC system. The assay utilizes a Nucleosil CN column and UV spectrophotometer detection at 260 nm. The mobile phase consists of acetonitrile-water (60:40). The limit of quantification of the method, defined as the minimum concentration that could be measured with a CV<5% was found to be 10 ng/mL in 500 L of plasma sample. The limit of detection with a signal to noise ratio of 3:1 was 4 ng/mL in plasma. Zhang et al. [47] described a procedure for determination of celecoxib in human plasma and breast milk by HPLC. Briefly, acetonitrile 0.2 mL was added to 0.2 mL each of blank, standard, QC or the samples plasma or milk to precipitate the proteins. Each mixture was vortexed for 30 s, centrifuged at 15000 x g for 10 min, and 40 L of clear supernatant was injected into the HPLC system. The method did not include an internal standard. The chromatographic separation was achieved in an Aqua C18 5 m reversed-phase column, connected to an Aqua C18 (4 mm x 3.0 mm I.D.) guard column. The mobile phase used was a mixture of acetonitrile and 0.01 M phosphate buffer pH 3.5 (50:50) containing 0.1% triethylamine. The plasma and milk calibration curves of celecoxib were constructed by spiking drug-free human plasma or breast milk with celecoxib working standard solution at concentrations of 10 to 2000 g/L. The limit of quantification of celecoxib was around 10 g/L in both plasma and milk, at which the mean values were within 15% of the spiked values and the intra- and inter-day coefficients of variation were < 5%. Chow et al. [48] described a method for the determination of celecoxib in human plasma using a solid-phase extraction (BakerBond Octadecyl SPE cartridges, 100 mg) for sample preparation, followed by HPLC with ultraviolet detection set at 215 nm. The samples were chromatographed on a Nova Pak C8 column (150 x 3.9 mm; 5 m). Use of the analytical column was preceded by that of a direct-connect column prefilter. The limit of quantification of celecoxib was 40 ng/mL with 0.25 mL of plasma. A reversed-phase HPLC method was developed for the separation and simultaneous determination of two cyclooxygenase inhibitors, celecoxib and refecoxib, in addition to two well-known non-steroidal antiinflammatory drugs, sodium diclofenac and niflumic acid in human serum by Navas et al. [49]. Chromatographic separation was achieved using a Hypersil ODS (150 x 4.1mm I.D., 5 m) analytical column, connected to a C18 (5 x 4.1 mm, I.D.) precolumn, applying a gradient with acetonitrile and

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water, from 15 to 60% acetonitrile. The mobile phase containing 1.0% trifluoracetic acid as an organic modifier. Detection was made using a diode array detector (DAD). The analytical procedure in the serum pre-treatment consisting of extraction using a C18 solid-phase extraction cartridges. Briefly, 2.0 mL of human serum were placed into a 5.0 mL plastic tube, and the pH of the sample was adjusted with 6 mL of pH 6 phosphate buffer solution 0.1 M. After blending, solid phase extraction was performed using a C18 solid phase extraction 200 mg cartridge fitted with 3.0 mL reservoir. The cartridges were conditioned with 2.0 mL of methanol and 2.0 mL of pH 6 phosphate buffer solution 0.1M. The buffered serum samples were passed through the C18 cartridges by gravity. Before eluting the drugs with 3.0 mL of acetonitrile, a clean up procedure with 3.0 mL of water was performed and the cartridges were dried by passing air. The eluted drugs were collected in a glass tube. The eluate was evaporated to dryness with nitrogen. The residue was dissolved in 200 L of water-acetonitrile (50:50), and injected into the chromatograph for analysis. The limits of quantification for celecoxib and refecoxib were 1.8 mg/L and 2.5 mg/L, respectively. A simple and rapid HPLC method for determination of celecoxib in human plasma for application in pharmacokinetic studies was developed and validated by Zarghi et al. [50]. The assay enables the measurement of celecoxib for therapeutic drug monitoring with a minimum quantification limit of 10 ng/mL. The method involves a simple, one-step extraction procedure, and analytical recovery was 100.51.3%. Briefly, to 450 L of plasma in a glassstoppered 15 mL centrifuge tube were added 50 L of nefenamic acid as internal standard (5.0 g/mL), 500 L of acetonitrile and 100mg NaCl. After mixing, the mixture was centrifuged for 15 min at 8000 g. Then 20 L of supernatant was injected into the liquid chromatography. The separation was performed on Chromolith Performance (RP-18e, 100 x 4.6 mm) column. The assay of celecoxib in plasma utilized a UV absorbance detector set at 254 nm with a mobile phase consisting of acetonitrile, methanol and water (45:10.45) containing 0.2% acetic acid (pH 3.5). Under the chromatographic conditions described, celecoxib and the internal standard peaks were well resolved. Endogenous plasma components did not give any interfering peaks. Separation was performed on a reversed-phase monolithic column, which has lower separation impedence compared with the particulate packings, and therefore, it allows easy optimising chromatographic conditions to obtain desirable resolution in a shot time. Owing to the use of a monolithic column, much faster separations are possible as compared with traditional chromatographic columns packed with porous particles. Accordingly, the chromatographic elution step is undertaken in a short time (less than 6 min), with high resolution. 4. ROFECOXIB Rofecoxib or 4-[4-Methylsulfonyl-phenyl]-3-phenyl-2(5H)-furanone is a selective inhibitor of COX-2 for the treatment of rheumatoid arthritis and osteoarthritis. Its chemical structure is shown in Fig. (3). Details of its pharmacokinetics, therapeutic efficacy, and toxicity have been reviewed by Scott et al. [51] and Matheson et al. [52]. Recently its use has become more controversial due to cardiovascular side effects on prolonged use. A high-performance

Fig. (3). Chemical structure of Rofecoxib.

liquid chromatographic assay for the determination rofecoxib in human plasma utilizing liquid-liquid extraction for sample preparation followed by HPLC with post- column photochemical derivatization and fluorescence detection was developed by Woolf et al. [53]. Briefly, an Astec Beam Boost photochemical reactor equipped with a 254 lamp and a 10 m reaction coil (0.3 mm I.D.) was used. The assay utilizes a reversed-phase BDS-Hypersil C18 analytical column (100 x 4.6 mm I.D., 5 m) preceded by a threaded guard column (20 x 4 mm) packed with the same material used for the separation. In this case a mobile phase was a mixture of acetonitrile-water (35:65). The columns operated at ambient temperature (approximately 22C). The sample injection volume was 150 L. The fluorescence detector was set at an excitation wavelength of 250 nm and an emission wavelength of 375 nm. The limit of quantification of the assay, was 0.5 ng/mL. The use of HPLC-with post-column photochemical derivatization-fluorescence detection has been reported for the determination of various drugs in biological fluids [54-58]. In many cases the technique has been reported to be highly competitive with HPLC with tandem mass spectrometric detection in terms of speed of analysis, selectivity and sensitivity [59]. Chavez-Eng et al. [60] also developed an HPLC method with tandem mass spectrometric detection of rofecoxib in human plasma. The method is based on high-performance liquid chromatography with atmospheric pressure chemical ionisation tandem mass spectrometric (APCI-MS-MS) detection in negative ionisation mode using a heated nebulizer interface. Rofecoxib and the internal standard were isolated from basified plasma using liquid-liquid extraction. Cleaner extracts were obtained when methyl-tert-butyl ether (MBTE) was utilized as an extraction solvent and plasma was basified before extraction. The chromatographic separation was achieved in a YMC ODS AQ (100 x 3.0 mm I.D. 3 m) analytical column, connected to a YMC ODS AQ (20 x 3.0 mm I.D. precolumn). The mobile phase used was a mixture of acetonitrile-water (50:50). The assay was validated in the concentration range of 0.1 to 100 ng/mL of plasma. A high-throughput, semi-automated determination of Rofecoxib in human plasma and urine using solid-phase in the 96-well format and high-performance liquid chromatography with post-column photochemical derivatization-fluorescence detection was developed by Mattews et al. [61]. Isolation of Rofecoxib and the internal standard was achieved by solid-phase extraction (SPE) in the 96well format. A C8 SPE plate was used for the extraction of the drug from human plasma while a mixed mode (C8Cation) SPE plate was used to isolate the analytes from human urine. The analyte and internal standard were chroma-

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tographed on a Keystone Scientific Prism-RP guard column (20 x 4.6 mm I.D., 5 m) connected to a Prism-RP analytical column (150 x 4.6 mm I.D., 5 m) using a mobile phase consisting of 45% acetonitrile in 10 mM acetate buffer (pH 4). Analytes were found to form highly fluorescent products after exposure to UV light (254 nm). Thus, the analytes were detected by fluorescence (
ex = 260 nm,
em = 375 nm) following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 5 to 500 ng/mL for human plasma and urine yielded a linear response (R2 > 0.99) when a 1/y weighed linear regression model was employed. Injection volume was 35 L for both the human plasma and urine assays. Werner et al. [62] developed a selective and rapid liquid chromatography-mass spectrometry method for the quantification of Rofecoxib in pharmacokinetic studies with humans. The analytical technique is based on reversed-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionisation mass spectrometry. Briefly, an aliquot of plasma (1.0) mL, 100 L acetonitrile, and 75 L internal standard solution were wortex-mixed for 1 min. Afterwards, 1.0 mL 0.1M sodium acetate buffer (pH 5) and 4 mL dichloromethane-hexane (50:50) were added. The tubes were capped, agitated in an overhead shaker for 10 min and centrifuged at 4000 g for 10 min. The organic layer was transferred to another tube. The solvent was evaporated under a stream of nitrogen at 40C. The resulting residue was reconstituted in 140 L of mobile phase and aliquots of 100 L injected onto the column. HPLC was carried out isocratically at ambient temperature using a Nucleosil C8 column, and the eluent comprised of methanol- water (50:50) and 1% acetic acid at a flow-rate of 200 L/min, the outlet coupled to the mass spectrometers APCI source. The method has been validated over a linear range from 1 to 500 g/L using celecoxib as internal standard. After validation, the method was used to study the pharmacokinetic profile of rofecoxib in 12 healthy volunteers after administration of a single oral dose (12.5 mg). An improved procedure for the determination of rofecoxib in human plasma involving 96-well solid-phase extraction and fluorescence detection was developed by Mattews et al. [63]. The analyte and an internal standard were extracted from the plasma matrix using solid-phase extraction in the 96-well format with an Empore extraction plate. Briefly, a 96-well disk SPE plate C8-standard density, was conditioned by passing 0.3 mL of methanol followed by 0.6 mL of water through each of the wells. Low negative pressure was applied during plate conditioning to prevent drying of the wells. Aliquots of 0.75 mL of the diluted plasma solutions were transferred into the conditioned SPE plate. The solutions were draw through the plate wells using negative pressure. A 1.0 mL volume of 10% acetonitrile in water was passed through each of the extraction wells. The plate was then positioned on the top of an ELISA plate and the extraction plate/ELISA plate assembly was centrifuged for 5 min at 1500 rpm to removal residual solvent. The extraction plate was then placed on top of a 96-deep well plate. Each well in this plate contained 300 L of water. Acetonitrile 150 L was added to each of the extraction wells. The SPE disk plate/deep round well plate assembly was centrifuged for 5 min at 1500 rpm to elute the analytes. The resulting solutions in each of the wells of the plate were mixed. The plate was sealed with a 96-well storage mat and placed on the 96-well

plate compatible autosampler. The analytes are chromatographed on a Waters Symmetry C18 analytical column (50 x 4.6 mm I.D., 3,5 m) connected to a Water Symmetry C18 cartridge guard column with a mobile phase consisting of acetonitrile-water (35:65). Analyte detection was via fluorescence following post-column photochemical derivatization. The method was partially automated using a combination of a Packard Multi-Probe liquid handling system and a TomTec Quadra 96 workstation. The limit of quantification of the assay was 0.5 ng/mL in human plasma using 0.5 mL of plasma. Chavez-Eng et al. [64] describes a high-performance liquid chromatographic-tandem mass spectrometric evaluation and determination of stable isotope labeled analogs of rofecoxib in human plasma samples from oral bioavailability studies. The use of stable isotope lebeled compounds to study the pharmacokinetics of drugs has been reviewed in two review papers [65, 66]. In these bioavailability studies, the unlabeled and labelled drugs are administered simultaneously by a different route of administration, and plasma concentrations vs time profiles are used to calculate the necessary pharmacokinetic parameters to determine the oral bioavailability. To conduct these studies, validated quantitative methods are developed for the simultaneous determination of the labelled and an unlabeled drug. The labeled compounds have been employed to study absorption, distribution, metabolism, and excretion of drugs in animal and human subjects. Most commonly, stable isotope labelled analogs of drugs are utilized to determine absolute or relative bioavailability of a test compounds [67-71].The method is based on HPLC with atmospheric pressure chemical ionisation tandem mass spectrometry (APCI-MS-MS) detection in the negative ionisation mode using a heated nebulizer interface. Two different stable isotope labeled anaogs of rocecoxib were initially evaluated for their use as intravenous markers in the bioavailability study. [13CD3] rofecoxib was shown to be isotopically unstable in plasma and water containing solvent and an efficient deuterium exchange prevented its use in the study. On the other hand, the isotopic integrity of the subsequently synthesized [13C7] rofecoxib was maintained, as expected, in plasma and other solvent systems. The results of these experiments clearly demonstrated the need for the careful evaluation of the isotopic integrity of standards in the quantitative analysis of drugs in biological fluids. Briefly, after liquid-liquid extraction of rofecoxib, [13C7] rofecoxib and internal standard from plasma, the analytes were chromatographed on a YMC ODS AQ narrow-bore (100 x 3.0 mm I.D. 3 m) C18 analytical column connected to a YMC ODS AQ precolumn, with a mobile phase consisting of acetonitrile-water (1:1) at a flowrate of 0.5 mL/min. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in the selective reaction monitoring mode. The assay was validated in the concentration range of 0.1 to 100 ng/mL of plasma for both rofecoxib and [13C7] rofecoxib. 5. NIMESULIDE Nimesulide or 4-nitro-2-phenoxymethanesulfonanilide, is a non - steroidal anti-inflammatory drug (NSAID) that, at therapeutic doses, markedly inhibits cyclooxygenase (COX)2 with less effect on COX-1 [72]. Its chemical structure is shown in Fig. (4). Numerous studies have demonstrated the

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Fig. (4). Chemical structure of Nimesulide.

good antipyretic, analgesic and anti-inflammatory activities of nimesulide in a wide range of clinical conditions [73]. In the last few years it has been demonstrated that prostaglandins, which are synthesized from arachidonic acid by COX, play an important role in the modulation of spinal neuron activity and that NSAIDs have a direct action on central processing of peripheral information [74, 75]. Furthermore, in the light of epidemiological findings, some investigators have recently suggested a potential utility of NSAIDs for the treatment of different disorders such as cancer and dementia [76, 77]. In particular, the selective COX-2 inhibitors slow the progression of dementia in Alzheimer patients [78, 79]. For all these reasons, the opportunity to determine nimesulide in human plasma and other biological fluids is of crucial importance to better evaluate the effects of the drug and to understanding the pharmacokinetic-pharmacodynamic relationship in the inhibition of neuroinfiammation exerted by this drug. A rapid and sensitive method for determination of nimesulide in human plasma by high-performance liquid chromatography was developed by Khaksa et al. [80]. Briefly, the chromatographic system uses a reversed-phase Zorbax ODS ( 250 x 4.6 mm I.D., 5 m) analytical column, protected by a guard column ODS ( 100 x 4.6 mm I.D.), with UV detection at 230 nm the mobile phase consisted of phosphate buffer (5.5)-methanol-acetonitrile (50:20:30) at a flowrate of 1.4 mL/mim. The HPLC method described did not include an internal standard and required 1.0 mL of sample. Nimesulide was extracted in a single step into dichloromethane. The calibration curve was linear in the concentration range of 0.05 to 5.0 g/L, and the lower limit of detection was 30 ng/mL. Ptek et al. [81] developed a rapid and simple HPLC determination of nimesulide in human plasma. The method is based on protein precipitation with methanol and reversed-phase chromatography with spectrophotometric detection at 404 nm. The separation was performed on a Nucleosil 120-5 C18 (50 x 4.0 mm, I.D., 5 m) analytical column and the mobile phase consisted of acetonitrilemethanol-15 mM potassium dihydrogenphosphate buffer, pH 7.3 (30:5:65). Only 250 L of plasma are used for sample preparation and no internal standard is necessary. The calibration curve is linear up to 10000 ng/mL and the limit of quantifiction is 80 ng/mL. The assay was used for pharmacokinetic studies. A simultaneous determination of nimesulide and hydroxynimesulide in rat plasma, cerebrospinal fluid and brain by liquid chromatography using solid-phase extraction was developed by Ferrario et al. [82]. Briefly, plasma samples (250 L) and brain homogenates added with the right amount of internal standard are extracted on C18

disposable cartridges by solid-phase extraction (SPE) while cerebrospinal fluid samples are analysed without any extraction. The separation is performed at room temperature on a Waters Symmetry C18 (150 x 4.6 mm I.D., 3.5 m) column with acetonitrile-sodium citrate buffer pH 3.0 (53:47) as mobile phase, and detection at 240 nm. Th lower limits of quantitation for either nimesulide and hydroxynimesulide are 25 ng/mL for plasma, 20 ng/mL for cerebrospinal fluid and 25 ng/mL for brain tissue. Maltese et al. [83] also developed an HPLC method with ultraviolet detection (300 nm) for the analysis of nimesulide in rabbit aqueous humor. Briefly, a simple pre-treatment with acetonitrile was used to deproteinize aqueous humor samples. The samples were chromatographed on a C18 reversed-phase column (Ultracarb ODS, 150 x 4,6 mm I.D., 5 m). In this case the mobile phase was a mixture of acetonitrile-water containing 1% triethylamine (TEA) adjusted to pH 3.2 with orthophosphoric acid. The limit of quantification was 50 ng/mL. Other HPLC methods have been reported for the analysis of nimesulide in biological fluids [84-89]. 6. ETORICOXIB Etoricoxib or 5-chloro-6-methyl-3-[4-(methylsulphonyl) phenyl]-2,3-bipyridine is the newest addition to the group of non-steroidal anti-inflammatory drugs (NSAIDs) known as selective cyclooxygenase-2 inhibitors [90-93]. Its chemical structure is shown in Fig. (5). Being the latest molecule, very few HPLC methods are reported in the literature. Rose et al. [93] developed a LC-MS-MS with atmospheric pressure chemical ionization (APCI) using stable isotope of etoricoxib as an internal standard. The method was validated over the concentration range of 0.2 to 250 ng/mL. The validation of a liquid chromatography-tandem mass spectrometry method for the determination of the cyclooxygenase-2 inhibitors etoricoxib in human plasma with phenazone as internal standard is described by Brutigam et al. [94]. The plasma samples were extracted by solid-phase extraction using polymerbased cartridges (Oasis HLB, 1mL, 30 mg). Briefly, prior to the extraction procedure, an aliquot of 200 L was spiked with 20 L phenazone solution, vortexed and centrifuged at 12000 g. The cartridges were placed on a 16-place manifold equipped with stopcocks, conditioned with 1 x 1 mL methanol and equilibrated with 1 x 1 mL water. The prepared plasma samples were cautiously loaded onto the cartridges and washed with 1 x 1 mL water-methanol (95:5). The cartridges were dried under reduced pressure for 5 min. The analyte was eluted with 1 x 2 mL acetonitrile-ethyl acetate (50:50). The solvent was evaporated under reduced pressure at 45C with a centrifugal evaporator. The residue was re

Fig. (5). Chemical structure of Etoricoxib.

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constituted in 200 L mobile phase. Chromatography in isocratic mode, was carried out on a short, narrow bore RP C18 column (30 x 2.0 mm I.D. 5 m particle size and 100 pore size). The mobile phase consisted of acetonitrile-water (90:10). The injection volume was 15 L. Detection was achieved by a Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo ion spray source working in positive ion mode. The respective mass transitions used for quantification of etoricoxib and phenazone were m/z 359.2
280.2 and m/z 189.0
104.1. The analytical method was validated over the concentration range 0.2 to 200 ng/mL. The limit of quantification was 0.2 ng/mL. The method is applicable to pharmacokinetic studies in humans. A validate liquid chromatographic ultraviolet method for the quantitation of etoricoxib in human plasma using liquid-liquid extraction was developed by Ramakrishna et al. [95]. Briefly, 1 mL volume of plasma was transferred to a glass test tube, and the 50 L of internal standard (zaleplon, 50 g/mL) was spiked). Next a 5 mL aliquot of extraction solvent, diethyl ether/ dichloromethane (70:30) was added. The sample was vortexmixed for 5 min. The sample was then centrifuged for 3min at 800 g . The organic layer was quantitatively transferred to a 6 mL glass tube and evaporated to dryness at 40C under a stream of nitrogen. Then, the dried extract was reconstituted in 250 L of water-methanol (50:50) and a 100 L aliquot was injected into chromatographic system. The analyte and internal standard were separated using a isocratic mobile phase of water-acetonitrile (58:42) on reversed-phase Waters Symmetry C18 column (250 x 4.6 mm I.D., 5 m) at 30C temperature. Detection was set at a wavelength of 284 nm. A linear range of 5 to 2500 ng/mL was established. This limit of quantification was 5 ng/mL, with a relative standard deviation of less than 20%. 7. ETODOLAC Etodolac or 1,8-diethyl-1,3,4,9-tetrahydropyano[3,4-b] indole-1-acetic acid, is a non-steroidal anti-inflammatory drug which has been shown to be effective in the treatment of rheumatoid and osteoarthritis and a selective COX-2 inhibitors in a wide range of clinical relevant assays in direct comparisons with other NSAIDs [96, 97]. Its chemical structure is shown in Fig. (6). Details of its pharmacology have been reviewed by Balfour et al. [98]. Etodolac, is metabolized in humans by hydroxylation and acyl glucuronidation to yield the corresponding 1-O-glucuronides. The acylglucuronides of etodolac and one of its hydroxylated metabolites were determined by Berendes et al. [99] by HPLC using a RP18 column. In order to increase the lipophilicity of these metabolites the hydroxy groups were acetylated and the carbonilic functions were methylated. The S-etodolac-glucuronide is mainly excreted during the first 6-hours after

Fig. (6). Chemical structure of Etodolac.

administration of 400 mg of etodolac whereas the elimination of the R-glucuronide predominates at longer periods of time. The s-glucuronide of 7-hydroxy-etodolac was preferentially excreted during the period of time observed. An unknown metabolite of etodolac could be characterized by chemical and enzymatic hydrolysis and by MS and NMR. An evaluation of the stereoselective metabolism of the chiral drug etodolac by high-perormance liquid chromatography was proposed by Becker-Scharfenkamp et al. [100]. Briefly, the enantiomers of the racemic drug etodolac have been resolved by fractional crystallization of the diastereoisomeric salts with optically active 1-phenylethylamine and analized in urine using a simple HPLC method with a conventional reversed-column. An application of a stereospecific highperformance liquid chromatography assay to a pharmacokinetic study of etodolac enantiomers in humans was developed by Jamali et al. [101]. Briefly, following the addition of internal standard, ()-2-(4benzoylphenyl)butyric acid, the constituents were extracted from the specimen into a mixture of isooctane-isopropanol (95:5). The organic layer was evaporated and the drug and internal standard were sequentially derivatized with ethyl chloroformate and (-)-
phenyethylamine. The diastereoisomers thus formed were extracted and chromatographed on a normal-phase column, with a mobile phase consisting of hexane-ethyl acetateisopropanol (85:15:0.2). The etodolac diastereoisomers were separated with a resolution factor of 6.4 and detected at a wavelength of 280 nm. Excellent linear relationships were found between the peak area ratios (etodolac/internal standard and the plasma and the urine concentrations (0.2 20 mg/L). A direct high-performance liquid chromatography separation of etodolac enantiomers using chiral stationary phases was described by Caccamese [102]. Briefly, The enantionmers of the etodolac were separed without derivatization on Chiracel OD and Pirkle (R)-DNBPG colums. Enantiomeric purity can be determined in less than 10 min. Optimisation of separation was evaluated using various concentrations of 2-propanol (doped with TFA) in hexane as the mobile phase. Brocks et al. [103] also developed a stereoselective disposition of etodolac enantiomers in synovial fluids. Jin et al. [104] developed a stereoselctive RP-HPLC assay to determine the enantiomers of etodolac in human plasma. Briefly, chiral drug enantiomers were extracted from human plasma with liquid-liquid extraction. Then etodolac enantiomers reacted with (S)-(-)-
-(1-naphthyl)ethylamine) using 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBT) as coupling agents. The derivatized products were separated on an Agilent Zorbax C18 (250 x 4.6 mm I.D., 5 m) column with a mixture of methanol-0.01 M phosphate buffer (pH 4.5) (70:30) as mobile phase. The detection wavelength of UV detector was set at 278 nm. The assay was linear from 0.5 to 50 g/mL for each enantiomer. The limit of quantification of the method was 0.5 g/mL. Cosyns et al. [105] describes a sensitive high-performance liquid chromatographic method for the determination of etodolac in serum. The limit of detection was 0.2 g/mL. The specificity of control sera, sera spiked with etodolac congeners, and sera obtained from rats treated with a variety of other drugs. The isolation of a unknown metabolite of etodolac in urine and its identification a 5-hydroxy etodolac was developed by Strickmann et al. [106]. For the identification elecrospray ionisation mass

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spectrometry (ESI-MS) as well as 1NMR and 13C-NMR spectroscopy as been used. Lee et al. [107] also developed a simple and specific method using a one-step liquid-liquid extraction with butyl acetate followed by HPLC coupled with positive ion electrospray ionisation tandem mass spectrometry (ESI-MS/MS) detection for the determination of etodolac in human plasma, using indomethacin as an internal standard. Chromatographic separation was performed isocratically using a Capcellpak MGII C18 column ( 50 x 2.0 mm I.D., 3 m) with 65% acetonitrile and 35% water containing 10 mM ammonium formate (adjusted to pH 3.5 with formic acid). The calibration curve exhibited good linearity in the concentration range of 0.1 - 25.0 g/mL. The limit of quantification was 0.1 g/mL with a relative standard deviation of less than 15%. 8. DERACOXIB Deracoxib or 4-[3-(difluoromethyl)-5-(3-fluoro-4-methoxyphenyl)-1H-pyrazol-1-yl] benzene sulfonamide is a selective cyclooxygenase 2 inhibitor [108]. Its chemical structure is shown in Fig. (7). The determination of deracoxib in feline plasma samples using high-performance liquid chromatography was developed and validated by Cox et al. [109]. Briefly, previously frozen plasma samples were vortexed and 1.0 mL placed in a centrifuge tubes followed by 100 L of tolbutamide (internal standard, 200 g/mL) and 6 mL of isopropilic alcohol -chloroform (80:20). Samples were centrifuged at 1000 x g for 15 mim. Supernatant were transferred to a clean tube and the organic phase evaporated at 30C with nitrogen. Samples were reconstituted in 1.0 mL of mobile phase and a 100 L injection was analysed. The compounds were separated with an Atlantis D C18 (150 x 4.6 mm, I.D. 5 m) column with an Atlantis D C18 guard column. The mobile phase used was an isocratic mixture of 10 mM potassium phosphate buffer (pH 4.5) and acetonitrile (52:48). Ultraviolet detection is carried out at 252 nm. The procedure produced a linear curve over the concentration range 10 - 1500 ng/mL. The calibration curve had to have a correlation coefficient of 0.99 or better. The limit of quantification was 10 ng/mL. There are no other analytical method available for this drug in the literature.

that confers weakly acidic properties (pKa 4.7). Its chemical structure is shown in Fig. (8). The absorption, metabolism, disposition and mass balance of 14C-lumiracoxib were investigated in healthy male subjects after a single 400 mg oral dose by Mangold et al. [110]. Serial blood and complete urine and feces were collected for 168 h post dose. Lumiracoxib was rapidly adsorbed, achieving mean plasma concentrations > 1g/mL within 1 h of postdosing. Unchanged drug plasma accounted for 81 to 91 % of radioactivity up to 2.5 h postdose, suggesting a modest first-pass effect; unchanged drug was the major circulating component in plasma, accounting for approximately 43% of the AUC. The terminal half-life of lumiracoxib in plasma was 6.5 h. Major plasma metabolites were the 5-carboxy, 4-hydroxy, and 4hydroxy-5-carboxy derivatives. Excretion involved both renal (54.1%) and fecal (42.7/%) routes, and dose recovery was almost complete (96.8%). Lumiracoxib was extensively metabolised before excretion, with little unchanged drug in urine (3.3% of dose) or feces (2.2% of dose). The major metabolic patways of lumiracoxib is oxidation of the 5methyl group and hydroxylation of the dihaloaromatic ring. Glucuronic acid conjugates of lumiracoxib metabolites (and to a minor extend lumiracoxib itself). Were identified, although there was no evidence of cysteine, mercapturic acid or glutathione conjugates. A validated high-performance liquid chromatographic assay for determination of lumiracoxib in human plasma was developed by Cheremia et al. [111]. Briefly, samples were prepared by adding 500 L 1M hydrochloric acid and 0.1 mL internal standard stock solution (10 g/mL niflumic acid in methanol) to 1.0 mL plasma followed by the addition of 4 mL hexane-diethyl ether (70:30). The tubes were capped, agitated and centrifuged at 4000 rpm for 10 min. The organic layer was transferred into a glass tube and evaporated under a stream of nitrogen at room temperature. The residue was dissolved in 150 L of methanol-water (50:50). The reconstituted specimens were vortex, transferred to Eppendorf tubes and used for HPLC analysis. The injected volume of the samples was 100 L. The analyte was separated using a reversed-phase Nucleosil (120-3 C8) column protected by a C8 precolumn. The column was maintained at 30C. The mobile phase consisted of acetonitrile and 0.05% trichloracetic acid (35:65). Uv detection was set at 270 nm. Linearity of the calibration cures for lumiracoxib was achieved for concentrations between 10 and 10000 ng/mL. The correlation coefficients were greater than 0.997. The limit of quantification was determined to be 10.0 ng/mL of lumiracoxib in plasma.

Fig. (7). Chemical structure of Deracoxib.

9. LUMIRACOXIB Lumiracoxib or 2-[2-fluoro-6-chlorophenyl)amino]-5methyl-benzenacetic acid is a distinct cyclooxygenase-2 selective inhibitor, which has been developed for the treatment of osteoarthritis, rheumatoid arthritis, and acute pain, is chemically distinct from the other coxib in that it lacks a sulfur-containing moiety and possesses a carboxylic group
Fig. (8). Chemical structure of Lumiracoxib.

10. VALDECOXIB Vadecoxib or 4-(5-methyl-3-phenyl-4-isoxazolyl)benzenesulfonamide is a anti-inflammatory drug that is highly

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selective for inhibition of the inducible form of ciclooxygenase (COX-2) [112]. This drug has been approved for the treatment of rheumatoid arthritis, osteoarthritis and pain [113-115]. Valdecoxib is metabolized primarily by cytochrome P450 2C9 and 3A4 to the pharmacological active hydroxylated metabolite and the carboxylic acid metabolite in humans [116-118]. Chemical structures of valdecoxib, hydroxylated valdecoxib, and carboxylic acid valdecoxib are shown in Fig. (9). A simple, sensitive and specific automated SPE-LC-MS-MS assay was developed and validated for determination of valdecoxib, hydroxylated valdecoxib, and carboxylic acid valdecoxib metabolite in human urine by Zhang et al. [119]. Valdecoxib and its metabolites and an internal standard were extract on a C18 solid-phase extraction cartridges using a Zymark RapiTrace automation system. Briefly, frozen urine samples were thawed in a water bath in a microwave oven for 60 s on defrost cycle. Aliquots of 500 L were placed in disposable glass tubes and 500 L of internal standard solution (100 ng/mL) in 100 mM ammonium acetate buffer (pH 6.8) was added. The urine samples were vortexed and placed in the loading modules of a RapidTrace automatic SPE System. C18 SPE cartridges (100 mg, 1.0 mL) were conditioned with 2 mL of methanol and 2 mL of water. The urine samples were loaded onto the cartridges that were washed with 4 mL of water and eluted with 500 L of acetonitrile. The solvent was removed under a stream of nitrogen on a TurboVap at room temperature to obtain residues that were reconstituted in 100 L of the mobile phase of acetonitrile-water (50:50; pH 6.0) containing 10 mM 4methylmorpholine, and transferred into autosampler vials. Then 20 L of the reconstituted samples was injected onto the LC-MS-MS system for analyses. Enzymatic hydrolysis of glucuronide coniugate of hydroxylated valdecoxib was conducted by adding approximately 100 units of
glucuronidase in 0.5 mL of 0.2 M sodium acetate buffer (pH 5.0; 1:1) into 0.5 mL of human urine. The separation of valdecoxib, hydroxylated valdecoxib, carboxylic acid valdecoxib and the internal standard were carried out on a narrow-bore reversed-phase Prism RP HPLC column (50 x 2.0 mmI.D., 5 m) with a isocratic mobile phase consisting of acetonitrile-water (50:50, pH 6.0) containing 10 mM 4methylmorpholine. The column effluent was directly introduced into the mass spectrometer using electrospray ionization in negative mode. Standard curves were linear over the concentration range of 1 200 ng/mL for valdecoxib, and hydroxylated valdecoxib, and 2-200 ng/mL for carboxylic acid valdecoxib metabolite. The lower limit of quantitation for human urine was 1 ng/mL for both valdecoxib, hydroxylated valdecoxib. The LOQ for carboxylic acid valdecoxib metabolite was raised to 2 ng/mL at the concentration of 1 ng/mL did not meet the validation criteria (<20%). A LCMS-MS assay to measure simultaneous valdecoxib, and hydroxylated valdecoxib, in human plasma samples by Zhang et al. [120]. The procedure consisted of an automated C18 solid-phase extraction (SPE) of valdecoxib, hydroxylated valdecoxib and an internal standard from 0.4 mL of human plasma using a Zymark RapidTrace automation system. After extraction, the samples were injected onto a reversedphase Zorbaz XDB C8 HPLC column for separation. The analytes were detected by mass spectrometry using negative ion electrospray ionisation with MRM mode. A versatile and sensitive HPLC assay was developed by Ramakrishna et al.

Fig. (9). Chemical structures of Valdecoxib, hydroxylated valdecoxib, and carboxylic acid valdecoxib.

[121]. Measurements of human plasma valdecoxib concentrations were determined by HPLC with ultraviolet detection using rofecoxib as the internal standard. Briefly, a 1 mL volume of plasma was transferred to a 15 mL glass tube test, and the 50 L of internal standard was spiked. Next a 5 mL aliquot of extraction solvent, diethyl ether/dichloromethane (70:30) was added. The sample was vortex-mixed for 5 min, was then centrifuged for 5 min at 800 g. The organic layer was quantitatively transferred to a 6 mL glass tube and evaporated to dryness. Then, the dried extract was reconstituted in 200 L of water - methanol (80:20) and 100 L aliquot was injected into chromatographic system. The separation of compounds was made on a YMC ODS-AQ column (250 x 4.6 mm, I.D., 5 m). The mobile phase was a mixture of water-methanol (47:53). Detection was set at a wavelength of 210 nm. Werner et al. [122] utilized a LC-MS for the measurement of both etoricoxib and valdecoxib in human plasma. The analytical technique is based on reversed-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionisation mass spectrometry. Mass analysis was performed in the positive ion mode. An aliquot of plasma sample (1.0 mL), 100 L acetonitrile - water (50:50), and 100 L internal standard solution were vortexed for 1 mim. Afterwards, 1 mL 0.1 M sodium acetate buffer

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(pH 5) and 4 mL dichloromethane - hexane (50:50) were added. The tubes were agitated and centrifuged at 4000 g for 10 min. The organic layer was transferred into another tube. The solvent was evaporated and the residue reconstituted in 500 L of mobile phase. Aliquots of 20 L (etoricoxib) or 100 L (valdecoxib) injected onto the column. HPLC was carried out using a Nucleosil C8 column and an eluent comprising methanol - water (50:50) and 1% acetic acid. The limit of quantitation, was 10 and 5 g/mL, respectively. Nageswara Rao et al. [123] describes a reversed-phase HPLC method for the simultaneous separation and quantitation of COX-2 inhibitors (clecoxib, rofecoxib, valdecoxib, nimesulide and nabumetone) using 4-chloro-2-nitroaniline as internal standard, in pharmaceuticals and its application to biological fluids. The chromatographic analysis was performed on Intersil C18 ODS ( 250 x 4.6 mm I.D., 5 m) column with methanol and 0.05% glacial acetic acid (68:32) as mobile phase. A diode array detector was used. Another work for the simultaneous determination several analytes in plasma by HPLC with UV detection was developed by Pavan Kumar et al. [124]. Briefly, the method employed a simple liquidliquid extraction of the analytes and internal standard from human plasma (500 L) into acetonitrile. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-C18 column (250 x 4.6 mm I.D., 5 m). The chromatographic separation was achieved by gradient elution consisting of 0.05 M formic acid (pH 3)-acetonitrile-methanol-water. The eluate was monitored using an ultraviolet detector set at 235 nm. The standard curve for etoricoxib, valdecoxib and celecoxib, was linear (r2>0.999) in the concentration range 0.1 - 50 g/mL. 11. MELOXICAM Meloxicam or 4-hydroxy-2-methyl-N-(5-methyl-2thiazolyl)-2H-1,2benzothiazine-3-carboxamide 1,1-dioxide is a representative drug belonging to the oxicam derivatives which shown preferential inhibition of cyclooxygenase-2 and prostaglandin synthesis. It has definite activity in treating rheumatoid arthritis, osteoarthritis, and other joint diseases [125-128]. Its chemical structure is shown in Fig. (10). A number of analytical methods for the determination of meloxicam in biological samples were reported. They include the methods using high-performance liquid chromatography with UV detector (HPLC-UV) [129, 130], HPLC with diode array detector [1006] and LC-tandem mass spectrometry (LC-MS-MS) [132-134]. A non-extracting procedure for the determination of meloxicam in plasma by HPLC-diode array detection was developed by Medvedovici et al. [131]. Briefly, the sample preparation procedure was based on protein precipitation with a misture of methanol-acetonitrile, trifluoracetic acid and sodium sulfate solution. Piroxicam was used as internal standard. The stepwise gradient elution profile of the chromatographic method allows injection of a high volume of the sample (500 L) with the focusing of both analytes in a Chromolith Performance RP-18e column. The mobile phase constituents are methanol and aqueous 20 mM Na2HPO4 buffer solution brought to pH 6 with H3PO4 . The assay utilized a UV absorbance detector set at 356 nm. The limit of quantitation was around 30 ng/mL. Velpandian et al. [129] describes a new high-performance liquid chroma-

Fig. (10). Chemical structure of Meloxicam.

tographic estimation method of meloxicam in biological samples, using piroxicam as the internal standard. Acidified plasma samples were extracted with chloroform, evaporated to dryness, reconstituted in the mobile phase and then a volume of 10 L of the prepared sample was injected in the column. Lichrocart (125 x 4.0 mm, I.D. 5 m) was used for analytical separation. The mobile phase consisted of an aqueous solution of diammonium hydrogenorthophosphate (50 mM), methanol and acetonitrile in the ratio (50:40:10). The UV detection was achieved at 364 nm. The limit of detection was found to be 0.029 g/mL and the limit of quantification was found to be 0.1 g/mL. Dasanti et al. [130] also developed an HPLC method with UV detection for the analysis of meloxicam in human plasma. The analytical procedure in the plasma pre-treatment consists of extraction using perchloric acid and acetonitrile mixture. The supernatant was directly injected to the HPLC. The samples were chromatographed on a C18 reversed-phase column. In this case the mobile phase was a mixture of sodium acetate buffer (pH 3.3) 170 mM-acetonitrile (62:38). Detection was by UV detector at 355 nm. The response was linear over a range of 50 1500 ng/mL in human plasma. The limit of the method is 50 ng/mL. Meloxicam was quantified in human plasma after a single 15 mg oral dose of the drug was given to 26 healthy volunteers by Wisner et al. [132]. An Applied Biosystems Sciex API 2000 triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode using TurboIonSpray (TIS) in the positive ione mode, was used. Protein precipitation with acetonitrile was followed by C18 reversed-phase liquid chromatography and tandem mass spectrometry. Piroxicam was used as the internal standard. The mean recovery for meloxicam was 92% with a lower limit of quantification of 8.96 ng/mL. The work of Baeyens et al. [135] was therefore directed towards the determination of meloxicam in human plama by application of an alkyl-diol silica precolumn in a column switching system. The group of LiChrospher alkyl-diol silica (ADS) phases that make-up part of the unique family of restricted-access materials, have been developed as special packing used in the liquid chromatographic integrated sample processing of biofluids. An online elimination of the protein matrix is achieved with a quantitative recovery together with an on-column enrichment. In this work Baeyens, describes a hand-operated online switching high-performance liquid chromatographic system for the determination of meloxicam. Briefly, spiked plasma samples were introduced on the ADS precolumn using a 0.05 M phosphate buffer, pH 6.0. After washing with the buffer the ADS column was backflushed with the mobile phase 0.05 M phosphate buffer-30% acetonitrile-25 mM tbutylamine at a pH of 7.0, thus transferring the analyte to the analytical column. The eluent was monitored by a UV-

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detector set at 364 nm. Ji et al. [136] described a procedure for the determination of meloxicam and other drugs in human plasma by LC-MS-MS. Briefly, meloxicam and other drugs (piroxicam and tenoxicam) and isoxicam (internal standard) were extracted from human plasma with ethyl acetate at acidic pH and analysed on a Sunfire column with the mobile phase methanol-ammonium formiate (15 mM, pH 3.0) (60:40). The analytes were detected using a mass spectrometer, equipped with electrospray ion source. The instrument was set in multiple reaction monitoring (MRM) mode. The standard curve was linear (r = 1.000) over the concentration range of 50 to 200 ng/mL. The lower limit of quantification for meloxicam was 0.50 ng/mL using a 100 L plasma sample. A highly sensitive liquid chromatographytandem mass spectrometry method was developed to determine meloxicam at low concentration in human plasma by Yuan et al. [137]. After a simple sample preparation procedure by one-step protein precipitation with methanol, meloxicam and the internal standard piroxicam were chromatographed on a Zorbax SB C18 column. The mobile phase used consisted of acetonitrile-water-formic acid (80:20:0.2). detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionisation (ESI) source. The method had a lower limit of quantification of 0.10 ng/mL. The calibration curve was demonstrated to be linear over the concentration range of 0.10 to 50.0 ng/mL. The validate method was successfully applied on the determination of meloxicam in human plasma collected up to 180 h after a transdermal administration of 30 mg meloxicam for evaluation of the pharmacokinetics. Concentrations of meloxicam were analyzed by HPLC with ultraviolet detection by Bae et al. [138]. After extraction with ethyl ether, the chromatographic separation of meloxicam was carried out using a reversed-phase Sunfire C18 column (150 x 4.6 mm, I.D., 5 m) with a mobile phase of acetonitrile-20 mM potassium hydrogen phosphate (40:60) (pH 3.5) and UV detection at a wavelength of 355 nm. The lower limit of quantification was 10 ng/mL. The curve was linear within the concentration range, 10-2400 ng/mL. Igualada et al. [139] described a rapid method for the determination of NSAIDs drugs in animal tissue by liquid chromatography-mass spectrometry with ion-trap detector. Briefly, after chemical hydrolysis, an organic extraction from homogenised tissue was performed. Final extract was injected in a liquid chromatograph with an ion-trap mass spectrometer with electrospray interface. For quantitative purposes flunix-D3 was used as internal standard. The method was validated using fortified blank muscle from different animal species according to the European decision criteria. The decision limits (CC
) and detection capabilities (CC ) were determined and their values were at concentrations near to the maximum residue limits (MRL) for each substance. CONCLUSIONS A overview of the current state-of art analytical methods for determination of cyclooxygenase COX-2 inhibitors has been presented. The literature compilation has revealed that a variety of methods are available for the first generation of COX-2 inhibitors viz., nimesulide and meloxicam. For drugs like celecoxib, valdecoxib, lumiracoxib and etoricoxib only a limited number of methods were reported, while no methods

were reported for tirmacoxib and cimicoxib. This is because of the fact that selective COX-2 inhibitors were introduced in late nineties. Our analysis of the published data revealed that the HPLC was extensively used for estimation of COX-2 inhibitors in biological fluids. Many methods were published during the period of 1993-2008; several methods are based on HPLC, showing that it is the technique of choice for analysis of COX-2 inhibitors. Most of the workers used the reversed-phase mode with UV absorbance detection because this provided the best available reliability, repeatability, analysis time and sensitivity. LC coupled with mass detector (LC-ESI-MS) was used to detect most of the metabolites of COX-2 inhibitors in human plasma and urine. The intrinsic fluorescence of some COX-2 inhibitors makes them good candidates for fluorescence detection. Fluorescence detection was highly sensitive and specific, and also gave good precision and a wide range of linear detector response, it is obligatory if only small sample volumes are available and for clinical specimens where interferences are likely to be present. In this review, I have discussed the present state-of-the art of the analytical methods for the determination of not only preferential COX-2 inhibitors viz., nimesulide and meloxicam, but also selective agents such as celecoxib, rofecoxib, and valdecoxib. In conclusion, HPLC has been proven in many laboratories to be an effective tool for monitoring of COX-2 inhibitors. Due to the inherent power of HPLC and the impact of better methods and equipment as detailed above, the role of HPLC in the therapeutic monitoring of COX-2 inhibitors may be expected to increase during the coming years. ABBREVIATION LIST NSAIDs COX 6-MNA HPLC 6-HNA RP APCI DAD TEA MRM LOQ ADS MRM SRM ESI = Non steroidal antinflammatory drugs = Cyclooxygenase = 6-Methoxy-2-naphtylacetic acid = High-performance liquid chromatography = 6-Hydroxy-2-naphtylacetic acid = Reversed - phase = Atmospheric pressure chemical ionization = Diode array detector = Triethylamine = Multiresolution mode = Limit of quantitation = Alkyl diol silica = Multiple reaction monitoring = Selected reaction monitoring = Electrospray ionization

Zorbax SB = Zorbax stable bond

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Received: June 24, 2008

Revised: July 28, 2008

Accepted: August 05, 2008