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Home , Clomifene, Enclomifene, Zuclomifene

In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (16). Sport und Buch Strauß - Köln 2008

F. Oueslati, M. Maatki, Z. Osman, H. Loueslati

Identification by LC-ESI-MS/MS of new metabolites in

National Laboratory of Drug and Doping control, Tunis, Tunisia

1. Introduction Clomifene is a synthetic anti-, structurally related to , and is a first choice therapy to treat women with absent or irregular [1]. It is also used by male abusers to bind the receptors for blocking unwanted effects of estrogens and to restores the natural production of . Recently X. de la Torre [2] has reported the presence of four glucoconjugate metabolites of and six sulfoconjugate metabolites of in urine by mean of liquid chromatography-mass spectrometry using electrospray ionization. However, the investigation of a single dose of 50mg of clomifene (Serpafar®) in man and women using a triple quadripole LC-ESI- MS/MS shows the presence of new glucoconjugates metabolites in addition to those recently identified. Excretion studies were collected over one week and the identification of metabolites was based on the retention time, ES-MS and MS-MS mass spectra. 2. Materials and Methods 2.1. Sample preparation To 2 mL of urine, 20µL of 17α- (20µg/mL) as internal standard were added, 200 µL of phosphate buffer pH=7 and 25 µL of beta-glucuronidase from E. coli were added and hydrolysis was performed for 1 h at 55°C. The buffered solution was then alkalinized with 300 µL of carbonate buffer. The sample was treated with 5 ml of a tert- butylmethyl ether and thoroughly homogenized by shaking on a mechanical shaker for 10 min, followed by centrifugation at 2500 rpm for 5 min to allow the separation of the organic phase. The aqueous phase was discarded and the organic phase was then evaporated to dryness under a Nitrogen stream. The dry residue was dissolved in mobile phase. 2.2. Urine Samples Excretion study was performed on one healthy female volunteer. One single dose of Serpafar® (50 mg of clomifene) was administrated. For 6 times a day all urine samples were collected.

333 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (16). Sport und Buch Strauß - Köln 2008

2.3. LC-MS/MS parameters Instrument: Agilent 1100 HPLC with Quattro micro (Micromass, UK) Ionization mode: ES(+) Data acquisition mode: MRM Methyltestosterone 303/107 Met1, Met2 and Met3 Met4, Met5 and Met6 394>72, 422>100 and 486>100 440>100, 452>100 and 424>72 respectively. High voltage electrodes: 3500V Source temperature: 120ºC Desolvation temperature: 350 ºC Desolvation gaz flow: 500L/h Neubilisation gaz flow: 90 psi. Collision gaz maintained at 2 10 -3 mbar Column: Zorbax RX C8 (2.1×150 mm, 5 µm) at 60 ºC. Mobile phase: Acetonitrile (A)/Ammonium Formate (B) (5 mM pH = 3.5) at 0.4 mL/min flow rate. 0 to 4 min 60% (A), 4 to 10 min 90% (A), 10 to 15 min 90% (A).

3. Results and Discussion The full scan mass spectrum of extracted urine samples after ingesting of 50 mg of clomifene was compared with that of blank urine sample to find out the probable metabolites. Then, these compounds were analyzed by LC-ESI (+)-MS/MS. Their retention-times, changes in observed mass (M) and MS/MS spectra were compared with the substructural ‘template’ of Clomifene standard to identify metabolites and elucidate their structures. Based on the method mentioned above, the parent drug and its main metabolites were found in human urine after ingesting of 50 mg of Clomifene. The LC-ES (+)–MS chromatograms showed the presence of

six peaks appeared at tR = 4.28 (m/z=486), 5.6 (m/z=440), 8.8 (m/z=394), 10.91 (m/z=422), 11.5 (m/z=424) and 12.01 min (m/z=452). In agreement with previous studies, the main metabolic reactions of clomifene were oxidation in both phenyl rings, hydroxylation in para position and methoxylation in 3-meta position [2-3]. The 4-hydroxyclomifene and 3- methoxy-4-hydroxyclomifene were identified in urine as glucoconjugate (Fig. 1A and B). However, the presence of 4-hydroxy-desethylclomifene and 3-methoxy-4-hydroxy- desethylclomifene with a characteristic product ion at m/z=72 (Fig. 1C and D) in glucoconjugate fraction, indicate that clomifene undergo N-desalkylation and oxidation reactions. Whereas, the dissociation of pseudo-molecular ion at m/z= 486 and m/z= 440 gave four product ions at m/z=486/422, 450/404, 100 and 72. The presence of the m/z =100 and 72 ions, which were produced by the partial losses of the side chain (Fig. 1E and F), indicate the similarity of these compounds to clomifene. In addition, the presence of fragment ions at m/z= 468 (respectively 422) and m/z= 450 (respectively 404) suggest a spontaneous losses of two hydroxyl groups. This finding indicates that these metabolites could be the N-oxide-α-

334 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (16). Sport und Buch Strauß - Köln 2008

60C 60C

clomifene urineMSMS1clomifene urineMSMS2 24 (12.683) Sm (SG, 2x0.50) CH3 blanc urin clom if 2: MRM of 4 Channels ES+ 6: Daughters of 452ES+ N 422 > 100 452.254.85e5 100 3CH 100 10.57 11.11 10.35 11.48 11.73 2.04e3 O 9.78 9.95 A % 100.05 Cl 95 sujet 1ech 2 2: MRM of 4 Channels ES+ 10.91 100 422 > 100 1.13e6 % OH %

99.23 0 Time 10.00 11.00 12.00 86.03 343.26 72.08 351.07 378.94 449.35 0 clomifene urineMSMS1clomifene urineMSMS2 15 (11.499) Sm (SG, 2x0.50) CH3 blanc urin clomif 2: MRM of 4 Channels ES+ 5: Daughters of 422ES+ N 452 > 100 422.57 2.49e5 100 3CH 100 2.20e3 422.07 O 13.99 11.87 14.95 100.11 11.11 B % 9.53 13.31 421.56

88 Cl sujet 1ech 2 2: MRM of 4 Channels ES+ 12.01 100 452 > 100 6.11e5

O % % CH 3 13.14 13.42 OH 0 Time

313.18 420.43 72.08 99.10 101.25 229.36 257.62 274.77 312.55 320.68 419.67 151.11 241.28 349.12 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

60C 60C blanc urin clomif F2 clomifene urineMSMS1clomifene urineMSMS3 28 (10.923) Sm (SG, 2x0.50) NH CH3 394 > 72 5: Daughters of 424ES+ 72.08 100 100 2.16e3 3.75e5 10.52 11.14 11.4811.53 % O

C 89 sujet 2 ech 6 2: MRM of 4 Channels ES+ Cl 100 424 > 72 7.22e4

% %

O 3 Time CH3 10.50 11.00 11.50 423.96 OH 317.34 316.96 342.44 423.33 316.02 70.69 258.94 283.10 254.84 351.20 366.46 422.57 0 clomifene urineMSMS1clomifene urineMSMS3 52 (10.179) Sm (SG, 2x0.50) blanc urin clomif F2 4: Daughters of 394ES+ NH CH 72.11 3 10.43 2.91e5 100 100 394 > 72 10.23 2.16e3 10.52 12.18 9.11 9.98 11.81 % D O

89 Cl sujet 2 ech 6 2: MRM of 4 Channels ES+ 100 394 > 72 8.64e4 %

%

OH 2 Time 394.03 10.00 12.00 71.22 287.39 72.80 286.32 69.58 219.37 322.21 392.46 207.02 313.45 347.58 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420

60C 60C clomifene urineMSMS1clomifene urineMSMS3 19 (6.278) Sm (SG, 2x0.50) blanc urin clomif F1 3: Daughters of 440ES+ 100.11 100 486 > 100 2.66e5 100 2.26e3

4.11 4.76 % 3.52 4.17 5.38 F Cl 5.47 88 sujet 2 ech 6 1: MRM of 4 Channels ES+ 100 486 > 100 O 7.89e4 3.74 % 440.29 3CH N CH3 % O OH 3 Time 404.54 3.00 4.00 5.00 99.10 404.17 71.95 98.22 165.26 57.56 268.09 313.25 422.32 0 clomifene urineMSMS1clomifene urineMSMS3 7 (4.157) Sm (SG, 2x0.50); Cm (4:7) blanc urin clomif F1 1: Daughters of 486ES+ 100.11 100 6.23 440 > 100 8.68e4 100 6.14 6.28 2.35e3 6.03 7.02 5.52 % 4.65 E Cl 486.30 OH 86 sujet 2 ech 6 1: MRM of 4 Channels ES+ O 100 440 > 100 1.06e5 % 6.51 3CH N CH3 O % O CH3 OH 468.14 2 Time 449.99 99.29 5.00 6.00 7.00 449.42 98.85 466.95 448.16 72.02 85.59 358.96 393.39 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 Figure 1: Mass Spectra (ES+, MS2 [M+H]+) of (A) 4-hydroxyclomifene, (B) 3-methoxy-4-hydroxyclomifene, (C) 4-hydroxy-desethylclomifene, (D) 3-methoxy-4-hydroxy-desethylclomifene, (E) N-oxide-α-hydroxy-3- methoxy-4-hydroxyclomifene and (F) the N-oxide-4-hydroxyclomifene.

335 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (16). Sport und Buch Strauß - Köln 2008

hydroxy-3-methoxy-4-hydroxyclomifene and the N-oxide-4-hydroxyclomifene respectively in agreement with a previous study which showed the presence of α–hydroxy-tamoxifene, N- oxide and α-hydroxy-N-desmethyltamoxifen metabolite in the urine of patients on therapy [4]. Figure 2 illustrated the excretion study results. The maximum concentration of the two metabolites (Met E) N-oxide-α-hydroxy-3-methoxy-4-hydroxyclomifene and (Met F) the N-oxide-4-hydroxyclomifene has been detected in the urine sample collected 9 hours after administration; and they still exist all over the week.

met.3 met.7

o 7,00 Met F 6,00 5,00 4,00 3,00 2,00 (SG Corrected) 1,00 Met E

Analyte/IS Peak Area Rati Area Peak Analyte/IS 0,00 0 20 40 60 80 100 120 140 160 180 Hours (after administration)

Figure 2: Excretion profiles of clomifene metabolites: (Met.E) and (Met.F)

4. Conclusion Oral administration of clomifene resulted in detectable urinary elimination of six drug’s metabolites; 4-hydroxyclomifene was identified as an important metabolite of clomifene. Other metabolites: 3-methoxy-4-hydroxy-clomifene, 4-hydroxy-desethylclomifene and 3- methoxy-4-hydroxy-desethylclomifene and the N-oxide-α-hydroxy-3-methoxy-4-hydroxy- clomifene, N-oxide-4-hydroxyclomifene are also detectable in human urine after oral administration of a single 50 mg dose of Serpafar®. The detection of an abuse even of a single therapeutical application of clomifene is possible over a long time period.

5. References [1] Crewe H. K, Ghobadi C, Gregory A, Lennard M S. (2007) Determination by liquid chromatography–mass spectrometry of clomiphene in the plasma of patients undergoing treatment for the induction of ovulation. J. Chromatogra B 847, 296–299. [2] Vitoriano B, De la Torre X. (2007) Study of Clomiphene metabolism by LC/MS/MS. In: Schanzer W, Geyer H, Gotzmann A, Mareck U. (eds.) Recent Advences in Doping Analysis (15), Köln, pp 113-122. [3] Lim C. K, Yuan Z-X, Jones R. M, Smith L. L. (1997) Identification and mechanism of formation of potentially genotoxic metabolites of tamoxifen: study by LC-MS/MS. J. Pharm. Biom. Anal 15, 1335 1342. [4] Jordan V. C. (2007) New insights into the metabolism of tamoxifen and its role in the treatment and prevention of cancer. 72, 829–842.

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