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Elimination profiles of flurbiprofen and its metabolites in equine urine for doping analysis
Talanta 55 (2001) 1173– 1180 www.elsevier.com/locate/talanta
Elimination profiles of flurbiprofen and its metabolites in equine urine for doping analysis C. Tsitsimpikou a, M-H.E. Spyridaki a, I. Georgoulakis b, D. Kouretas b, M. Konstantinidou c, C.G. Georgakopoulos a,* a
Doping Control Laboratory of Athens, Olympic Athletic Center of Athens, Kifissias 37, 15123 Maroussi, Greece b School of Agriculture, Uni6ersity of Thessaly, Volos, Greece c School of Agriculture, Aristotelian Uni6ersity of Thessaloniki, Thessaloniki, Greece Received 11 June 2001; received in revised form 31 July 2001; accepted 8 August 2001
Abstract Flurbiprofen and its main acidic metabolites were detected in equine urine after a single-dose administration of 500 mg flurbiprofen to two 2.5–3.5-years-old mares, in order to be used in equine doping control routine analysis. The urine levels of the parent drug were determined using GC/MS. Five acidic metabolites were found in the urine. The structure of the proposed metabolites was confirmed by HRMS accurate mass measurements. The highest flurbiprofen concentration was 204 mg ml − 1 at 1–3 h post administration. Flurbiprofen could be detected for 24 – 37 h in urine using the standard screening procedure. All metabolites were present 25 h post administration, while 4%-hydroxyflurbiprofen could be traced for more than 48 h and it is regarded as the long-term metabolite of flurbiprofen in horse. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Flurbiprofen; Flurbiprofen metabolites; Equine urine
1. Introduction Flurbiprofen, racemic 2-(2-fluoro-4-biphenyl) propionic acid Fig. 1, is a non-steroidal anti-inflammatory drug with analgesic and antipyretic properties indicated for conditions associated with mild-to-moderate pain [1]. Structurally it contains a phenylalkanoic acid functionality and is classified in the profen-drugs class. According to * Corresponding author. Tel./fax: +30-1-6868549. E-mail address: [email protected] (C.G. Georgakopoulos).
literature, flurbiprofen is a potent inhibitor of prostaglandin synthesis [2,3]. The drug, when administered orally, has been shown to absorb rapidly and almost completely in humans [2] and other species studied, such as mouse, rat, dog and baboon [4,5]. Biotransformation of flurbiprofen via hydroxylation and methylation leads to three major metabolites in the urine Fig. 1: 4%-hydroxyflurbiprofen (I), 3%,4%-dihydroxyflurbiprofen (II) and 3%-hydroxy, 4%-methoxyflurbiprofen (III) [1–3]. Phase II metabolism of flurbiprofen is also important leading mainly to acylglucuronides and aminoacid esters [6,7].
0039-9140/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 1 ) 0 0 5 4 5 - 8
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A number of analytical methods are available for qualitative and quantitative analysis of flurbiprofen in biological specimens, including high performance liquid chromatography (HPLC) [8,9] and gas chromatography or capillary electrophoresis – mass spectrometric and tandem mass spectrometric analyses [7,10,11]. The pharmacokinetics of flurbiprofen in equine has not been extensively studied [3,12]. In the framework of the Doping Control Laboratory of Athens preparation project for the doping control analysis during the 2004 Olympic Games, the present work aimed at the detection of flurbiprofen and its metabolites in equine urine using a multi-residue screening procedure used for doping control analysis [10,12]. The excretion profile of the parent drug and metabolites was studied after oral administration of a 500 mg single dose of
flurbiprofen. GC/High-Resolution Mass Spectrometry (GC/HRMS) was also applied to confirm the structure of the metabolites.
2. Experimental
2.1. Materials Flurofen® was a BASF Pharma Knoll, Germany, product and flurbiprofen was purchased from Sigma-Aldich Chemie, Steinheim, Germany. All reagents and organic solvents used in the extraction procedures were of analytical grade. b-Glucuronidase from Escherichia coli [EC 3.2.1.31, 2130000 U mg − 1 protein] (Boehringer, Mannheim, Germany) and b-glucuronidase/arylsulphatase from Helix pomatia [EC 3.2.1.31,
Fig. 1. Structures of flurbiprofen (I) and flurbiprofen metabolites (II – VI).
C. Tsitsimpikou et al. / Talanta 55 (2001) 1173–1180
Fig. 2. Secreted Flurbiprofen concentration –time profile after oral administration of a single dose of 500 mg flurbiprofen to two scyrian mares. Analysis of the excretion study urine samples was performed according to the screening procedure for NSAIDs as described in Section 2.4 and detection by GC/MS. Acidic hydrolysis of the urine led to 30% less recovery of the conjugated metabolites, compared with the alkaline hydrolysis used in the screening procedure. The detection limit of the screening analytical method was 50 ng ml − 1 (Signal to Noise Ratio \ \ 3) and no interference was found from endogenous compounds. Quantification of flurbiprofen was performed by one level calibration using samples at 0.25 and 50 mg ml − 1, depending on the concentration of the sample.
127000 U ml − 1] (Sigma-Aldich Chemie, Steinheim, Germany) were used for enzymatic hydrolysis. Derivatization reagents, N-methyl-N-trimethylsilyltrifluoro-acetamide (MSTFA), were purchased from Macherey to Nagel, Du¨ ren, Germany.
2.2. Instrumentation A Hewlett –Packard 5890 gas chromatograph coupled with a 5970 quadropole mass spectrometric detector (MSD) with a crosslinked 5% phenyl–methylsilicone gum capillary column (12 m ×0.200 mm, I.D. 0.33 mm, HP Ultra2) was used. Helium, at a flow-rate of 1.0 ml min − 1, was used as carrier gas. 1 ml of sample was injected in the split mode (1:15). Temperatures of the injector and the transfer line were set at 250 and 310 °C, respectively. Initial oven temperature was 130 °C, then ramped at 15 °C min − 1 – 300 °C and and held for 5.0 min. The MSD was aquiring data in the full scan mode (mass range 73– 800).
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HRMS analysis was performed with a threesector (electrostatic–magnetic–electrostatic) reverse geometry double focusing mass spectrometer (Autospec Micromass) coupled to an HP 6890 gas chromatograph. A crosslinked methylsilicone gum capillary column (12 m× 0.200 mm, I.D. 0.33 mm, HP Ultra1) was employed with helium carrier gas (flow 1 ml min − 1). A 2 ml sample volume were injected (split 1:10) in the accurate mass measurement analysis. The GC temperature was ramped as follows: 150 °C for 0.5 min, 12.5 °C min − 1 to 310 °C, 2.5 min final time. The injection port was heated at 250 °C and the transfer line at 280 °C. In the accurate mass measurement analysis, the HRMS was acquiring data in the mass range m/z 100–500 using magnet scan in 7000 resolution.
2.3. Drug administration For experimental purposes two mares from the Greek island of Skyros aged 2.5–3.5-years-old were used. The mares had good performance, excellent nourishing conditions and were not in a pregnant condition. Prior to use of any medication, urine was collected with an elastic catheter within glass tubes. The two mares weighing 173 and 176 kg b.w. were each given 500 mg flurbiprofen per-os (Flurofen®, five tablets of 100 mg in levigation and mixed with a spoonful of honey). Following administration, urine samples were collected from the mares within similar tubes and always with the use of catheters, in a regular time span: 1, 3, 5, 9, 13, 24, 36, 48, 60, 72 and 84 h. The pH of urine was 4.5–5.5 and no preservatives were added. After each collection, the tubes with the urine were placed in a freezer − 20 °C, where they were kept until the end of the experiment. On the same day the excretion study was completed, they were transfered to the laboratory for analysis within an isothermic vessel for temperature preservation.
2.4. Sample preparation For the screening procedure of non-steroidal anti-inflammatory drugs (NSAID) excreted as free compounds and conjugates [10,12,13], 2.5 ml of
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urine were extracted with a mixture of hexane/ ethyl acetate/dichloromethane 4/3/3.5 (solvent A) at pH 3.0 using orthophosphoric buffer (nalidixic acid was used as internal standard), after chemical hydrolysis with NaOH 1M (20 min, room temper-
ature). The dry extract was derivatised using 100 ml of MSTFA and incubating at 80 °C for 45 min. After heating, the O-and N-trimethylsilyl (-OTMS, -NTMS) derivatives of the compounds of interest were analysed by GC/MS.
Fig. 3. Typical mass chromatograms of flurbiprofen: (1) ion chromatogram of m/z 316 (molecular ion) of flurbiprofen in the flurbiprofen standard sample, (2) ion chromatogram of m/z 316 (molecular ion) of flurbiprofen in an excretion study sample (9 h post-administration).
C. Tsitsimpikou et al. / Talanta 55 (2001) 1173–1180
Acidic hydrolysis of the urine was also tested using 6.0 M HCl in the presence of cysteine at 100 °C for 30 min. The organic extract was dried and derivatised as described above. In order to throw some light into the conjugation of flurbiprofen and its metabolites in horse, detected after urine excretion, 5.0 ml of urine were processed as follows: 1. extraction with solvent A prior to hydrolysis at the pH of urine (pH 4.5– 5.5) to obtain the free fraction; 2. enzymatic hydrolysis of the remaining urine with E. coli b-glucuronidase (pH 7.0, phos-
1177
phate buffer) followed by routine acidic extraction to obtain the glucuronide ethers fraction; 3. enzymatic hydrolysis of the remaining urine b-glucuronidase/aryl sulfatase from H. pomatia (pH 5.0, acetate buffer), followed by routine acidic extraction to obtain the sulfate esters fraction; 4. routine chemical hydrolysis and acidic extraction of the remaining urine, as previously summarised, to obtain the glucuronide esters and the aminoacid conjugates fraction. All organic extracts were separately dried, derivatised and analysed as described above.
Fig. 4. Mass spectrum and fragmentation pattern of TMS derivatives of flurbiprofen (I) and flurbiprofen metabolites (II– VI) in urine samples taken from a skyrian mare 5 h after oral administration of 500 mg flurbiprofen. The sample had been prepared according to the screening procedure for NSAIDs and derivatisation was carried out as described in Section 2.3.
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Fig. 4. (Continued)
3. Results and discussion Following oral administration of 500 mg of
flurbiprofen, five metabolites together with the parent compound were detected in equine urine. Flurbiprofen (I) itself was excreted for more than
Table 1 Calculated data of the exact mass measurements of the flurbiprofen and its metabolites (I–V) Compound
Calculated mass
Real mass
Dm/m (ppm)a
Flurbiprofen (I)
316.1271 301.1039 404.1633 389.1426 492.2090 477.1755 434.1769 419.1459 404.1635 389.1382
316.1296 301.1061 404.1640 389.1405 492.1985 477.1750 434.1746 419.1511 404.1640 389.1405
7.9 7.3 1.7 −5.4 −21.3 −1.0 −5.3 12.4 1.2 5.9
4%-OH-Flurbiprofen (II) 3%,4%-diOH-Flurbiprofen (III) 3%OH-4%-MeO-Flurbiprofen (IV) x%-OH-Flurbiprofen (V)
a
Dm/m =(Real mass−calculated mass/real mass)106.
C. Tsitsimpikou et al. / Talanta 55 (2001) 1173–1180
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Fig. 5. Percentage secreted 4%-OH-flurbiprofen (II)-time profile after oral administration of a single dose of 500 mg flurbiprofen to two scyrian mares. Analysis of the excretion study urine samples was performed according to the screening procedure for NSAIDs as described in Section 2.4 and detection by GC/MS. The % secreted flurbiprofen metabolite (II) was determined based on the maximum secreted amount of (II), as calculated by chromatographic data.
24 h post administration for both horses accounting for an average of 26% of the total dose Figs. 2 and 3. The drug level peaked at 1– 3 h, which is in accordance with previously published data [2,3,12]. All known metabolites of flurbiprofen have been identified. The major one appears to be 4%hydroxyflurbiprofen (II) Fig. 4. 3%,4%-Dihydroxyflurbiprofen (III) and 3%-hydroxy-4%-methoxyflurbiprofen (IV) that are regarded as abundant metabolites in several species including man [2,4], were also present and could be detected and identified by their mass spectra for 24 h post-administration Fig. 4. A second hydroxy-, probably 3%-hydroxyflurbiprofen (V), and a second hydroxy–methoxy–metabolite (VI) that were not mentioned in previous reports on horse metabolism of flurbiprofen [12,14], were detected in minor amounts and identified based on their mass spectral data, which were identical with those of (II) and (IV). All flurbiprofen metabolites’ structures proposed in the present study have been confirmed by accurate mass measurements of their molecular and other at least two diagnostic ions Table 1. The comparison of exper-
imental with proposed mass spectrum demonstrates differences below 10 ppm (ppm= Dm/m) in nearly all cases. Flurbiprofen metabolites follow the same excretion pattern with the parent compound, exhibiting a peak at 1–5 h post administration and remaining detectable for more than a day. 4%-hydroxyflurbiprofen (II) is detected for more than 50 h post-administration and could be used as a longterm metabolite for screening of the drug abuse in horses. It is interesting to note that horse B exhibited a biphasic excretion pattern both for flurbiprofen and its 4%-hydroxy metabolite Fig. 5. Finally, conjugation of flurbiprofen and its metabolites was also studied, since very limited data is available in the literature. In the present study all metabolites and the parent compound were detectable in the free fraction in considerable amounts (\ 30%). Less than 30% of the metabolites were excreted as glucuronide and sulfate conjugates. Glucuronide ethers represented only an average 5% of the conjugation for all metabolites. Glucuronide esters are generally not hydrolysed enzymatically and are, therefore, cleaved in the chemically hydrolysed fraction [15]. Sulfates rep-
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resented 15–25% of the total excreted compound for metabolites (I), (IV) and (VI), while for the rest never exceeded 5%. In our study, the glycine conjugation of flurbiprofen and its metabolites has proven to be quite important, as found from the GC/MS analysis of the hydrolysates. Such conjugation represents the major metabolic route for the carboxylic acid of the 2-arylpropionic acids in general [16,17].
4. Conclusions In this paper, the detection of flurbiprofen and its metabolites in equine for doping control analysis is discussed. Excretion profile of flurbiprofen after oral administration of 500 mg is also presented. The main metabolites were detected along with unchanged flurbiprofen for more than 24 h. 4%-Hydroxyflurbiprofen, which is detected for more than 50 h post-administration, could be used as a long-term metabolite for screening of the drug abuse in horses. Two new hydroxy- and hydroxy-methoxy metabolites are proposed. The structure of the main metabolites were confirmed by an accurate mass measurement HRMS experiment of diagnostic ions.
References [1] E.A. Cefali, W.J. Poynor, D. Sica, S. Cox, J. Clin. Pharmacol. 31 (1991) 808. [2] N.M. Davies, Clin. Pharmacokinet. 28 (1995) 100.
[3] R.K. Leavitt, R. Nespolo, S.D Timmings, P.M. Beaumier The determination of flurbiprofen in urine and plasma of the standardbred race horse using High Performance Liquid Chromatrography, in: T. Tobin, J. Blake, M. Potter T. Wood (Eds.), Proceedings of the 7th International Conference of Racing Analysts and Veterinarians, Kentucky, 1988, pp. 297 – 309. [4] P.C. Risdall, S.S Adams, E.L. Crampton, B. Marchant, Xenobiotica 11 (1978) 691. [5] D.G. Kaiser, C.D. Brooks, P.L. Lomen, Am. J. Med. 80 (1986) 10. [6] M.P. Knadler, D.C. Brater, S.D. Hall, Br. J. Clin. Pharmacol. 33 (1992) 369. [7] A.E. Ashcroft, H.J. Major, I.D. Wilson, Anal. Proc. 32 (1995) 459. [8] J.M. Hutzler, R.F. Frye, T.S. Tracy, J. Chromatogr. B Biomed. Sci. Appl. 749 (2000) 119. [9] T. Hirai, S. Matsumoto, I. Kishi, J. Chromatogr. B Biomed. Sci. Appl. 692 (1997) 375. [10] U.M. Laakkonen, A. Leinonen, L. Savonen, Analyst 119 (1994) 2695. [11] M. Fillet, I. Bechet, V. Piette, J. Crommen, Electrophoresis 20 (1999) 1907. [12] R. Nespolo, P. Beaumier, S. Timmings, R. Leavitt, Flurbiprofen, in: A.J. Stevenson, M.P. Weber, S. Kacew (Eds.), ‘‘Analytical Methodology for Detection and Confirmation of Drugs in Equine Body Fluids’’, Volume 3, 1996, pp. 106 – 111. [13] F. Jamali, B.W. Berry, M.R. Tehrani, A.S. Russell, J. Pharm. Sci. 77 (1988) 666. [14] S.A. Babhair, J. Liq. Chromatogr. 11 (1988) 463. [15] M.A. Seymour, P. Teale, M. Hoeijmakers, A. Coert, E. Houghton, The metabolism of vedaprofen in the horse, in: B. Laviolette and M.R. Koupai-Abyazani (Eds.), Proceedings of the 12th International Conference of Racing Analysts and Veterinarians, Vancouver, 1998, pp. 171 – 175. [16] A.J. Hutt, J. Caldwell, J. Pharm. Pharmacol. 35 (1983) 693. [17] H. Tsunematsu, S. Yoshida, K. Horie, M. Yamamoto, J. Drug Target 2 (1995) 517.
Elimination profiles of flurbiprofen and its metabolites in equine urine for doping analysis C. Tsitsimpikou a, M-H.E. Spyridaki a, I. Georgoulakis b, D. Kouretas b, M. Konstantinidou c, C.G. Georgakopoulos a,* a
Doping Control Laboratory of Athens, Olympic Athletic Center of Athens, Kifissias 37, 15123 Maroussi, Greece b School of Agriculture, Uni6ersity of Thessaly, Volos, Greece c School of Agriculture, Aristotelian Uni6ersity of Thessaloniki, Thessaloniki, Greece Received 11 June 2001; received in revised form 31 July 2001; accepted 8 August 2001
Abstract Flurbiprofen and its main acidic metabolites were detected in equine urine after a single-dose administration of 500 mg flurbiprofen to two 2.5–3.5-years-old mares, in order to be used in equine doping control routine analysis. The urine levels of the parent drug were determined using GC/MS. Five acidic metabolites were found in the urine. The structure of the proposed metabolites was confirmed by HRMS accurate mass measurements. The highest flurbiprofen concentration was 204 mg ml − 1 at 1–3 h post administration. Flurbiprofen could be detected for 24 – 37 h in urine using the standard screening procedure. All metabolites were present 25 h post administration, while 4%-hydroxyflurbiprofen could be traced for more than 48 h and it is regarded as the long-term metabolite of flurbiprofen in horse. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Flurbiprofen; Flurbiprofen metabolites; Equine urine
1. Introduction Flurbiprofen, racemic 2-(2-fluoro-4-biphenyl) propionic acid Fig. 1, is a non-steroidal anti-inflammatory drug with analgesic and antipyretic properties indicated for conditions associated with mild-to-moderate pain [1]. Structurally it contains a phenylalkanoic acid functionality and is classified in the profen-drugs class. According to * Corresponding author. Tel./fax: +30-1-6868549. E-mail address: [email protected] (C.G. Georgakopoulos).
literature, flurbiprofen is a potent inhibitor of prostaglandin synthesis [2,3]. The drug, when administered orally, has been shown to absorb rapidly and almost completely in humans [2] and other species studied, such as mouse, rat, dog and baboon [4,5]. Biotransformation of flurbiprofen via hydroxylation and methylation leads to three major metabolites in the urine Fig. 1: 4%-hydroxyflurbiprofen (I), 3%,4%-dihydroxyflurbiprofen (II) and 3%-hydroxy, 4%-methoxyflurbiprofen (III) [1–3]. Phase II metabolism of flurbiprofen is also important leading mainly to acylglucuronides and aminoacid esters [6,7].
0039-9140/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 1 ) 0 0 5 4 5 - 8
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C. Tsitsimpikou et al. / Talanta 55 (2001) 1173–1180
A number of analytical methods are available for qualitative and quantitative analysis of flurbiprofen in biological specimens, including high performance liquid chromatography (HPLC) [8,9] and gas chromatography or capillary electrophoresis – mass spectrometric and tandem mass spectrometric analyses [7,10,11]. The pharmacokinetics of flurbiprofen in equine has not been extensively studied [3,12]. In the framework of the Doping Control Laboratory of Athens preparation project for the doping control analysis during the 2004 Olympic Games, the present work aimed at the detection of flurbiprofen and its metabolites in equine urine using a multi-residue screening procedure used for doping control analysis [10,12]. The excretion profile of the parent drug and metabolites was studied after oral administration of a 500 mg single dose of
flurbiprofen. GC/High-Resolution Mass Spectrometry (GC/HRMS) was also applied to confirm the structure of the metabolites.
2. Experimental
2.1. Materials Flurofen® was a BASF Pharma Knoll, Germany, product and flurbiprofen was purchased from Sigma-Aldich Chemie, Steinheim, Germany. All reagents and organic solvents used in the extraction procedures were of analytical grade. b-Glucuronidase from Escherichia coli [EC 3.2.1.31, 2130000 U mg − 1 protein] (Boehringer, Mannheim, Germany) and b-glucuronidase/arylsulphatase from Helix pomatia [EC 3.2.1.31,
Fig. 1. Structures of flurbiprofen (I) and flurbiprofen metabolites (II – VI).
C. Tsitsimpikou et al. / Talanta 55 (2001) 1173–1180
Fig. 2. Secreted Flurbiprofen concentration –time profile after oral administration of a single dose of 500 mg flurbiprofen to two scyrian mares. Analysis of the excretion study urine samples was performed according to the screening procedure for NSAIDs as described in Section 2.4 and detection by GC/MS. Acidic hydrolysis of the urine led to 30% less recovery of the conjugated metabolites, compared with the alkaline hydrolysis used in the screening procedure. The detection limit of the screening analytical method was 50 ng ml − 1 (Signal to Noise Ratio \ \ 3) and no interference was found from endogenous compounds. Quantification of flurbiprofen was performed by one level calibration using samples at 0.25 and 50 mg ml − 1, depending on the concentration of the sample.
127000 U ml − 1] (Sigma-Aldich Chemie, Steinheim, Germany) were used for enzymatic hydrolysis. Derivatization reagents, N-methyl-N-trimethylsilyltrifluoro-acetamide (MSTFA), were purchased from Macherey to Nagel, Du¨ ren, Germany.
2.2. Instrumentation A Hewlett –Packard 5890 gas chromatograph coupled with a 5970 quadropole mass spectrometric detector (MSD) with a crosslinked 5% phenyl–methylsilicone gum capillary column (12 m ×0.200 mm, I.D. 0.33 mm, HP Ultra2) was used. Helium, at a flow-rate of 1.0 ml min − 1, was used as carrier gas. 1 ml of sample was injected in the split mode (1:15). Temperatures of the injector and the transfer line were set at 250 and 310 °C, respectively. Initial oven temperature was 130 °C, then ramped at 15 °C min − 1 – 300 °C and and held for 5.0 min. The MSD was aquiring data in the full scan mode (mass range 73– 800).
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HRMS analysis was performed with a threesector (electrostatic–magnetic–electrostatic) reverse geometry double focusing mass spectrometer (Autospec Micromass) coupled to an HP 6890 gas chromatograph. A crosslinked methylsilicone gum capillary column (12 m× 0.200 mm, I.D. 0.33 mm, HP Ultra1) was employed with helium carrier gas (flow 1 ml min − 1). A 2 ml sample volume were injected (split 1:10) in the accurate mass measurement analysis. The GC temperature was ramped as follows: 150 °C for 0.5 min, 12.5 °C min − 1 to 310 °C, 2.5 min final time. The injection port was heated at 250 °C and the transfer line at 280 °C. In the accurate mass measurement analysis, the HRMS was acquiring data in the mass range m/z 100–500 using magnet scan in 7000 resolution.
2.3. Drug administration For experimental purposes two mares from the Greek island of Skyros aged 2.5–3.5-years-old were used. The mares had good performance, excellent nourishing conditions and were not in a pregnant condition. Prior to use of any medication, urine was collected with an elastic catheter within glass tubes. The two mares weighing 173 and 176 kg b.w. were each given 500 mg flurbiprofen per-os (Flurofen®, five tablets of 100 mg in levigation and mixed with a spoonful of honey). Following administration, urine samples were collected from the mares within similar tubes and always with the use of catheters, in a regular time span: 1, 3, 5, 9, 13, 24, 36, 48, 60, 72 and 84 h. The pH of urine was 4.5–5.5 and no preservatives were added. After each collection, the tubes with the urine were placed in a freezer − 20 °C, where they were kept until the end of the experiment. On the same day the excretion study was completed, they were transfered to the laboratory for analysis within an isothermic vessel for temperature preservation.
2.4. Sample preparation For the screening procedure of non-steroidal anti-inflammatory drugs (NSAID) excreted as free compounds and conjugates [10,12,13], 2.5 ml of
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urine were extracted with a mixture of hexane/ ethyl acetate/dichloromethane 4/3/3.5 (solvent A) at pH 3.0 using orthophosphoric buffer (nalidixic acid was used as internal standard), after chemical hydrolysis with NaOH 1M (20 min, room temper-
ature). The dry extract was derivatised using 100 ml of MSTFA and incubating at 80 °C for 45 min. After heating, the O-and N-trimethylsilyl (-OTMS, -NTMS) derivatives of the compounds of interest were analysed by GC/MS.
Fig. 3. Typical mass chromatograms of flurbiprofen: (1) ion chromatogram of m/z 316 (molecular ion) of flurbiprofen in the flurbiprofen standard sample, (2) ion chromatogram of m/z 316 (molecular ion) of flurbiprofen in an excretion study sample (9 h post-administration).
C. Tsitsimpikou et al. / Talanta 55 (2001) 1173–1180
Acidic hydrolysis of the urine was also tested using 6.0 M HCl in the presence of cysteine at 100 °C for 30 min. The organic extract was dried and derivatised as described above. In order to throw some light into the conjugation of flurbiprofen and its metabolites in horse, detected after urine excretion, 5.0 ml of urine were processed as follows: 1. extraction with solvent A prior to hydrolysis at the pH of urine (pH 4.5– 5.5) to obtain the free fraction; 2. enzymatic hydrolysis of the remaining urine with E. coli b-glucuronidase (pH 7.0, phos-
1177
phate buffer) followed by routine acidic extraction to obtain the glucuronide ethers fraction; 3. enzymatic hydrolysis of the remaining urine b-glucuronidase/aryl sulfatase from H. pomatia (pH 5.0, acetate buffer), followed by routine acidic extraction to obtain the sulfate esters fraction; 4. routine chemical hydrolysis and acidic extraction of the remaining urine, as previously summarised, to obtain the glucuronide esters and the aminoacid conjugates fraction. All organic extracts were separately dried, derivatised and analysed as described above.
Fig. 4. Mass spectrum and fragmentation pattern of TMS derivatives of flurbiprofen (I) and flurbiprofen metabolites (II– VI) in urine samples taken from a skyrian mare 5 h after oral administration of 500 mg flurbiprofen. The sample had been prepared according to the screening procedure for NSAIDs and derivatisation was carried out as described in Section 2.3.
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Fig. 4. (Continued)
3. Results and discussion Following oral administration of 500 mg of
flurbiprofen, five metabolites together with the parent compound were detected in equine urine. Flurbiprofen (I) itself was excreted for more than
Table 1 Calculated data of the exact mass measurements of the flurbiprofen and its metabolites (I–V) Compound
Calculated mass
Real mass
Dm/m (ppm)a
Flurbiprofen (I)
316.1271 301.1039 404.1633 389.1426 492.2090 477.1755 434.1769 419.1459 404.1635 389.1382
316.1296 301.1061 404.1640 389.1405 492.1985 477.1750 434.1746 419.1511 404.1640 389.1405
7.9 7.3 1.7 −5.4 −21.3 −1.0 −5.3 12.4 1.2 5.9
4%-OH-Flurbiprofen (II) 3%,4%-diOH-Flurbiprofen (III) 3%OH-4%-MeO-Flurbiprofen (IV) x%-OH-Flurbiprofen (V)
a
Dm/m =(Real mass−calculated mass/real mass)106.
C. Tsitsimpikou et al. / Talanta 55 (2001) 1173–1180
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Fig. 5. Percentage secreted 4%-OH-flurbiprofen (II)-time profile after oral administration of a single dose of 500 mg flurbiprofen to two scyrian mares. Analysis of the excretion study urine samples was performed according to the screening procedure for NSAIDs as described in Section 2.4 and detection by GC/MS. The % secreted flurbiprofen metabolite (II) was determined based on the maximum secreted amount of (II), as calculated by chromatographic data.
24 h post administration for both horses accounting for an average of 26% of the total dose Figs. 2 and 3. The drug level peaked at 1– 3 h, which is in accordance with previously published data [2,3,12]. All known metabolites of flurbiprofen have been identified. The major one appears to be 4%hydroxyflurbiprofen (II) Fig. 4. 3%,4%-Dihydroxyflurbiprofen (III) and 3%-hydroxy-4%-methoxyflurbiprofen (IV) that are regarded as abundant metabolites in several species including man [2,4], were also present and could be detected and identified by their mass spectra for 24 h post-administration Fig. 4. A second hydroxy-, probably 3%-hydroxyflurbiprofen (V), and a second hydroxy–methoxy–metabolite (VI) that were not mentioned in previous reports on horse metabolism of flurbiprofen [12,14], were detected in minor amounts and identified based on their mass spectral data, which were identical with those of (II) and (IV). All flurbiprofen metabolites’ structures proposed in the present study have been confirmed by accurate mass measurements of their molecular and other at least two diagnostic ions Table 1. The comparison of exper-
imental with proposed mass spectrum demonstrates differences below 10 ppm (ppm= Dm/m) in nearly all cases. Flurbiprofen metabolites follow the same excretion pattern with the parent compound, exhibiting a peak at 1–5 h post administration and remaining detectable for more than a day. 4%-hydroxyflurbiprofen (II) is detected for more than 50 h post-administration and could be used as a longterm metabolite for screening of the drug abuse in horses. It is interesting to note that horse B exhibited a biphasic excretion pattern both for flurbiprofen and its 4%-hydroxy metabolite Fig. 5. Finally, conjugation of flurbiprofen and its metabolites was also studied, since very limited data is available in the literature. In the present study all metabolites and the parent compound were detectable in the free fraction in considerable amounts (\ 30%). Less than 30% of the metabolites were excreted as glucuronide and sulfate conjugates. Glucuronide ethers represented only an average 5% of the conjugation for all metabolites. Glucuronide esters are generally not hydrolysed enzymatically and are, therefore, cleaved in the chemically hydrolysed fraction [15]. Sulfates rep-
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resented 15–25% of the total excreted compound for metabolites (I), (IV) and (VI), while for the rest never exceeded 5%. In our study, the glycine conjugation of flurbiprofen and its metabolites has proven to be quite important, as found from the GC/MS analysis of the hydrolysates. Such conjugation represents the major metabolic route for the carboxylic acid of the 2-arylpropionic acids in general [16,17].
4. Conclusions In this paper, the detection of flurbiprofen and its metabolites in equine for doping control analysis is discussed. Excretion profile of flurbiprofen after oral administration of 500 mg is also presented. The main metabolites were detected along with unchanged flurbiprofen for more than 24 h. 4%-Hydroxyflurbiprofen, which is detected for more than 50 h post-administration, could be used as a long-term metabolite for screening of the drug abuse in horses. Two new hydroxy- and hydroxy-methoxy metabolites are proposed. The structure of the main metabolites were confirmed by an accurate mass measurement HRMS experiment of diagnostic ions.
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