ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT i907 2001 © The Japan Society for Analytical Chemistry

Chiral HPLC Determination of dl-Fenfluramine and dl- Norfenfluramine in Biological Fluids Using DIB-Cl as a Label

Amal KADDOUMI, Mihoko N. NAKASHIMA, Masakatsu TAKAHASHI, and Kenichiro NAKASHIMA†

Department of Analytical Research for Pharmacoinformatics, Graduate School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852, Japan (E-mail: [email protected])

An HPLC with fluorescence detection for the simultaneous determination of sympathomimetic amines including ephedrine (E), norephedrine (NE), 2-phenylethylamine (2-PEA), 4-bromo-2,5-dimethoxyphenylethylamine (2-CB), phentermine (Phen) and dl-fenfluramine (dl-Fen) in spiked human plasma has been developed using 4-(4,5-diphenyl-1H- imidazole-2-yl)benzoyl chloride (DIB-Cl) as a label (method I). The method was then modified to determine Phen and Fen, simultaneously, and applied to rat microdialysates (method II). Furthermore, a chiral HPLC method for the separation of d-Fen (dexfenfluramine) and l-Fen (levofenfluramine) in addition to their active metabolites d- and l- norfenfluramine (Norf) is developed (method III). The fluorescence intensity was monitored at 430 nm with excitation at 325 nm. The separation was achieved on C18 column or a chiral column (CHIRALCEL OD-R). The methods were fully validated, and shown to have excellent linearity, sensitivity and precision. The chiral method has been applied for the determination of Phen, d- and l-enantiomers of Fen and Norf in rat plasma after simultaneous administration of Phen and dl-Fen.

(Received on August 7, 2001; Accepted on September 13, 2001)

Fenfluramine (Fen) and phentermine (Phen) have been widely In this work, simple and sensitive HPLC method with prescribed in combination for short-term management of obesity. fluorescence detection using 4-(4,5-diphenyl-1H-imidazole-2- However, dl-Fen and the active enantiomer dexfenfluramine (d- yl)benzoyl chloride (DIB-Cl) as a label for the simultaneous Fen) were withdrawn from the US market in September 1997 determination of the DIB derivatives of Fen and Phen in human after many cases reported the development of serious side effects spiked plasma was developed. In addition, other 4 including valvular heart disease and primary pulmonary sympathomimetic amines, i.e., ephedrine (E), norephedrine (NE), hypertension1,2 either after the administration of Fen alone or in 2-phenylethylamine (2-PEA), and 4-bromo-2,5- combination with Phen. Yet, in spite of the withdrawal, Fen and dimethoxyphenylethylamine (2-CB), and fluoxetine (FLX) as an Phen are still being prescribed and widely abused, suggesting the internal standard were also examined (method I). development of a method for their simultaneous determination in Furthermore, the chromatographic conditions were modified biological fluids will be helpful in monitoring their levels. for the simultaneous determination of Fen, Norf and Phen, which The methods developed for the independent determination of was then applied for their determination in blood and brain Phen and Fen in biological fluids, used either GC3-5 or HPLC.6, 7 microdialysates following the administration of a single dose of In these methods, the minimum sample volume used for their 5 mg/kg of Phen and Fen to rats, intraperitonealy (i.p.) (method quantitation in plasma or urine was 1 ml with minimum II). Moreover, sensitive and reliable chiral-HPLC method for the quantitation limits of 10 ng/ml (67 nM) and 2.5 ng/ml (11 nM) simultaneous determination of Phen, dl-Fen and dl-Norf, in for Phen7 and Fen,4 respectively. plasma was developed. The application of the method to the The reports concerning the chiral separation of d-Fen, l-Fen, in monitoring of Phen and the enantiomers of Fen in rat plasma addition to their metabolites d-norfenfluramine (d-Norf) and l- samples after the administration of a single i.p. dose of 1 mg/kg norfenfluramine (l-Norf) in biological fluids are few. Most of the of Phen and racemic Fen is also described (method III). methods utilized GC coupled either with ECD8, 9 or FID.10 Among these, the most sensitive method reported by Srinivas et al 9 with detection limit of 2 ng/ml for Fen enantiomers. On the Experimental other hand, only one paper described a chiral-HPLC method with UV detection for the determination of dl-Fen and dl-Norf in Chemicals and materials 11 plasma and urine. The separation of 3,5-dinitrophenylurea dl-Fen.HCl, d-Fen.HCl and Phen.HCl were obtained from derivatives of Fen and Norf enantiomers was achieved on a Sigma Chemical Co. (St. Louis, MO, USA). dl-FLX.HCl was Pirkle-type chiral column. In this method 1 ml plasma was used purchased from Tocris Cookson Ltd. (Bristol, UK). E was with quantitation limit of 10 ng/ml (43 nM) per enantiomer and obtained from Dainippon Pharmacy (Osaka, Japan), 2-PEA from detection limit of 1 ng/ml (4.3 nM).

i908 ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT

Nacalai Tesque (Kyoto, Japan) and NE from Aldrich Results and discussion (Milwaukee WI, USA). DIB-Cl was synthesized in our laboratory12 and 2-CB was synthesized according to DeRuiter et al.13 dl-Norf and d-Norf were enzymatically prepared by Method I. incubating dl-Fen and d-Fen, respectively, with rat liver The derivatives were well separated within 50 min. The microsomal preparation.14 recoveries of the target compounds from plasma samples were Human blood samples were drawn from healthy volunteers in above 85%. The standard curves in spiked plasma were linear in our laboratory. Male Wistar rats (290-345 g) were used in the the studied ranges, i.e., for E, 2-CB and Fen (0.01 - 5 µM), Phen experiments. Blood samples were centrifuged at 2000 g for 10 (0.005 - 2.5 µM), NE (0.1 - 5 µM) and 2-PEA (0.05 - 5 µM.) min and obtained plasma samples were kept frozen at -20oC with the detection limits from 6 to 476 fmol/inj at a signal-to- prior to use. noise ratio of 3 (S/N=3). The intra- and inter-assay precisions were less than 13.3 and 12.2%, respectively, for the compounds. Extraction of plasma samples15 To 100 µl of plasma, 10 µl of an aqueous solution of 5 µM Method II. FLX (IS) were added, followed by the addition of 200 µl of 0.1 The above method was modified for the simultaneous M borate buffer (pH 10.6) and 750 µl of ethyl acetate. The determination of Phen, Fen and Norf. The separation was mixture was vortex mixed for 1 min and centrifuged. To 600 µl completed within 28 min, isocratically. As well, the of the organic layer, 10 µl of acetic acid were added, evaporated derivatization conditions with DIB-Cl were also modified where and then derivatized with DIB-Cl as described below. 2 mM of DIB-Cl was used compared to 1.5 mM for methods I and III in order to compensate for the reduction in the reaction Blood and brain microdialysates mixture volume and the direct derivatization of the dialysates. Dialysis probes used were CMA/PC10 (4 mm membrane The method was applied for the monitoring of Phen and Fen in length), and CMA/PC12 (2 mm membrane length) for blood and blood and brain microdialysates obtained from rats administered with single dose of 5 mg/kg of Phen and Fen, simultaneously. brain microdialysis, respectively. The blood probe was implanted within the jugular vein. Brain probe was implanted in The estimated average in vitro probes recoveries of Phen and the frontal cortex with the coordinates: AP 3.5 mm, LAT 1.3 mm, Fen were 22 and 24%, respectively, for brain probe and 33 and DV 5.6 mm relative to the bregma. Both probes were perfused 40%, respectively, for blood probe. Figures 1 and 2 show a with an artificial cerebrospinal fluid had the following chromatogram obtained from rat brain microdialysate of the fifth interval (80-100 min) from drugs administration and brain and composition: 145 mM NaCl, 1.5 mM KCl, 1.5 mM MgCl2, 1.25 mM CaCl , 10 mM glucose, 1.5 mM K HPO , adjusted to pH blood microdialysates levels of Fen after a single dose of Phen 2 2 4 and Fen (5 mg/kg each), respectively. 7.0 with flow rate of 1 µl/min.

Derivatization procedure

The residues of the evaporated plasma were derivatized as follows: 150 µl of 1.5 mM DIB-Cl suspension in acetonitrile and

50 µl of 0.01 M carbonate buffer (pH 9.0) were added to the residue, vortex mixed and then incubated at 60oC (at r.t. for the 15 enantiomeric separation) for 10 min. Microdialysate samples were derivatized directly where to the dialysates (20 µl), 5 µl of 0.1 M carbonate buffer (pH 9.0) were added followed by 75 µl of 2 mM DIB-Cl suspension in acetonitrile. Samples were vortex mixed and incubated at r.t. for 10 min. The reaction was stopped by adding 10 µl of 25% ammonia solution. Plasma samples were then diluted with acetonitrile (1:1, v/v) and 20 µl of the resultant solution were injected onto the achiral column. For microdialysate samples, 20 µl were injected onto the column and for the chiral system, samples were applied for SPE as described below.

SPE for DIB-derivative samples15 Cartridges were conditioned with 5 x 1 ml of acetonitrile and then 5 x 1 ml of deionized water. Samples were diluted with water (1:1, v/v) and loaded onto the cartridges and then were washed with 8 x 1 ml of acetonitrile-methanol-water (5:50:45, v/v/v) mixture. The DIB-derivatives were eluted with 2 x 75 µl acetonitrile, from which 20 µl were injected into the chiral HPLC system.

HPLC systems and chromatographic conditions Method I & III. A detailed description of the HPLC system can be found elsewhere for the simultaneous separation of the DIB- derivatives of sympathomimetic amines as well as the chiral separation.15 Both methods were performed using gradient HPLC systems. Method II. The same above HPLC system was used for the Fig. 1 Chromatogram of rat brain microdialysate sample (fifth microdialysates except being performed isocratically. Mobile fraction) after the simultaneous administration of single i.p. dose phase was 65% acetonitrile in water and the flow rate was set at of Phen and dl-Fen (5 mg/kg each). 1 ml/min.

ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT i909

2000 Brain dialysate 1750 References Blood dialysate M 1500 1250 1. L. Abenhaim, Y. Moride, F. Brenot, S. Rich, J. Benichou, X.

1000 Kurz, T. Higenbottam, C. Oakley, E. Wouters, M. Aubier, G. Simonneau, and B. Begaud, N. Eng. J. Med., 1996, 335, 750 609 500 2. M. Delcroix, X. Kurz, D. Walckiers, M. Demedts, and R. 250 Naeije, Eur. Respir. J., 1998, 12, 271 Concentration, n 3. B. Saletu, M. J. Barbanoj, P. Anderer, W. Sieghart, and J. 0 0 60 120 180 240 300 360 420 480 540 600 Grunberger. Methods Find. Exp. Clin. Pharmacol., 1993, 15, 291. Time, min 4. R. P. Richards, B. H. Gordon, J. Ings, D. B. Campbell, and

Fig. 2 Fen concentrations in brain and blood microdialysates L. J. King. Xenobiotica, 1989, 19, 547. after a single i.p. dose of of Phen and dl-Fen (5 mg/kg each). 5. A. K. Cho, B. J. Hodshon, B. Lindeke, and G. T. Miwa, J. Pharm. Sci., 1973, 62, 1491. Method III. 6. M. J. Bogusz, M. Kala, and R. Maier, J. Anal. Toxicol., The method was validated using human and rat plasma. The 1997, 21, 59. recoveries of Phen and the enantiomers following SPE were 69% 7. J. E. O'Brien, W. Zazulak, V. Abbey, and O. Hinsvark, J. and 76% for d- and l-Norf, respectively, 79% and 84% for d- and Chromatogr. Sci., 1972, 10, 336. l-Fen, respectively, and 59% for Phen. The detection limits 8. A. Jori, P. DePonte, and S. Caccia, Xenobiotica, 1978, 8, (S/N=3) were 27, 19 and 47 fmol/inj for d-Fen, Phen and l-Fen, 583. respectively. The intra- and inter-assay precisions were less than 9. N. R. Srinivas, J. W. Hubbard, J. K. Cooper, and K. K. 11% for Phen and the enantiomers of Fen in both human and rat Midha, J. Chromatogr., 1988, 433, 105. plasma. The method was applied for the monitoring of Phen and 10. D. B. Campbell, J. Chromatogr., 1970, 49, 442. the enantiomers of Fen and Norf in rat plasma after a single 11. J. Zeng, L. dou, M. Duda, and H. H. Stuting, J. Chromatogr. simultaneous administration of 1 mg/kg of Phen and racemic Fen B, 1994, 654, 231. to rats. It was possible to detect Phen, dl-Norf and dl-Fen to 10 h 12. K. Nakashima, H. Yamasaki, N. Kuroda, and S. Akiyama, with average concentrations (n=3) of 159, 420, 497, 23 and 20 Anal. Chim. Acta, 1995, 303,103. nM for Phen, d-Norf, l-Norf, d-Fen and l-Fen, respectively. 13. J. DeRuiter, C. R. Clark, and F. T. Noggle, J. Chromatogr. Sci., 1995, 33, 583. Both, the achiral (methods I and II) and chiral methods were 14. A. Kaddoumi, A. Kubota, M. N. Nakashima, M. Takahashi, simple and highly sensitive, and needed small sample size using K. Nakashima, Biomed. Chromatogr., 2001, 15, 379. DIB-Cl as the fluorescence reagent for the simultaneous 15. A. Kaddoumi, M. N. Nakashima, and K. Nakashima, J. determination of Phen and Fen in biological fluids. Chromatogr. B, in press.