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Detection of “uncommon” tranquillizers–sedatives during screening toxicological analysis
Forensic Science International 99 (1999) 71–77
Letter to the Editor
Detection of ‘‘uncommon’’ tranquillizers–sedatives during screening toxicological analysis A. Dona*, S. Athanaselis, C. Maravelias, A. Koutselinis Department of Forensic Medicine and Toxicology, University of Athens, Medical School, 75, M. Asias str., Goudi, Athens, Greece Received 13 March 1998; received in revised form 22 September 1998; accepted 1 October 1998
The determination of some ‘‘uncommon’’ tranquillizers-sedatives during a toxicological analysis is often difficult particularly when the analysis of biological samples concern cases of mild intoxications such as one tablet intake, driving under the influence of drugs, or when no case history exists. Therefore, we investigated the possibility of identifying the intake of subtoxic doses of four anxiolytic and / or sedative drugs, namely, zolpidem, zopiclone, chlormethiazole and buspirone, during the screening procedures for drug detection in biological fluids followed in our laboratory [1]. Urine from 20 healthy volunteers, who took one tablet of each drug (5 samples for each drug) were analyzed in this study. Urine samples were collected approximately 3 hours after the intake of a single tablet or capsule of each drug. Nine urine samples from known cases of individuals that had taken subtoxic doses of these drugs were also analyzed. The urine sampling took place 2 to 4 hours after the ingestion of the drugs. No cross reactivity was observed during urine screening by fluoroscence polarization immunoassayTDx (Abbott), by enzyme immunoassay ETS Plus system (Syva), or by Triage (Merck) for opiates, benzodiazepines, cocaine, amphetamines and cannabinoids. A general cross-reactivity assessment was not performed because this was not in the purposes of our study. 10 ml of the urine samples were extracted through Chem-Elut extraction columns (Varian, Harbor City, CA), as described by Lillsunde and Korte [2]. After the extraction, the eluent was evaporated to dryness under a stream of N 2 and was used for TLC and GC–MS analysis after reconstitution in 25 ml of MeOH. TLC screening was performed according to Lillsunde and Korte [2]. All four drugs or their metabolites could not be detected in urine. The urine extracts were further analyzed by GC–MS according to a standardized method that we use in our laboratory [1]. *Corresponding author. 0379-0738 / 99 / $ – see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 98 )00170-4
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A. Dona et al. / Forensic Science International 99 (1999) 71 – 77
Zolpidem was eluted at Rt 15.7 min and identified by comparing its retention time and mass spectrum to those of a standard. It should be noticed that its spectrum cannot be identified by the libraries NIST62 and PMWTOX2, because its mass spectrum is not included. Therefore, one should enrich the laboratory’s libraries with the mass spectrum of zolpidem. This spectrum (Fig. 1) was identical to the one reported in the literature [3]. It should be emphasized that the peak of zolpidem obtained in cases of mild intoxications is very weak and can be missed due to background interferences or noise (Fig. 1). Thus, the analyst should perform a SIM analysis looking for the characteristic ions of the drug (m / z 235, 307, 219). It should be noticed that during the screening analysis of urine samples in our lab we were not able to detect the two major metabolites of zolpidem 2-(4-carboxy-phenyl)-N,N,6-trimethylimidazo-[1,2-a]-pyridine-3-acetamide and 2-(4-methyl-phenyl)-(N,N,dimethyl-6-carboxy)-imidazo-(1,2-a]-pyridine-3-acetamide [4]. This is because zolpidem metabolites, due to their polar and amphoteric character, cannot be eluted through Chem-Elut columns. Although someone could use anion exchange columns or extractive alkylation with CH 3 J [5] and have much better results, these methods could be applied specifically for determination and quantitation of zolpidem and not for general screening. Moreover, gas chromatographic methods are not sensitive enough for the determination of these metabolites. That is why the analytical methods proposed in the literature for the identification and quantitation of zolpidem require complicated analytical tools, such as thermospray LC–MS / MS or on-column switching HPLC [3,6]. Zopiclone shows thermal decomposition during the GC–MS analysis to a decomposition product called ‘‘compound V’’ which is (6-(5-chloro-2-pyridyl)-6,7-dihydro[5H]-
Fig. 1. TIC of the urine extract after one tablet intake of zolpidem and its mass spectrum.
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pyrrolo[3,4-b]pyrazine-5-one [7]. Zopiclone’s ratio to this decomposition product depends on the injector port temperature, the condition of the liner and the injection mode (split or splitless). Furthermore, this ratio is also dependent on the injection solvent, as zopiclone appears to be unstable in nucleophilic solvents’, such as methanol [7–9]. In the screening method applied in our lab, the extraction solvent used is a mixture of methylene chloride and isopropanol (9:1) and the injection solvent is methanol. Thus, it was expected that zopiclone would decompose to ‘‘compound V’’. Indeed, during the analysis of urine samples with our screening method only compound V (m / z 246, 217, 191) was detected at Rt 12,4 while no zopiclone was found (Fig. 2). Due to these problems the methodology was changed and n-butyl chloride was chosen as the extraction solvent and acetonitrile as the injection solvent. With acetonitrile as a solvent, compound V was negligible [7,10]. According to the existing case history if zopiclone were one of the drugs found near the intoxicated patient or if it were one of the prescribed drugs by his or her physician the screening methodology followed should be the one described above. In this case zopiclone (m / z 245, 217, 143) elutes at Rt 20,1 (Fig. 3). If there is no history, the general screening methodology is followed and if compound V of zopiclone is detected the methodology with the above changes is applied. The mass spectrum of compound V that has been added in our libraries was extremely helpful for the confirmation of zopiclone intake. The intake of zopiclone could also be detected by determining a decomposition product of zopiclone, 2-amino-5chloropyridine, which is formed after alkaline hydrolysis [11], or when the urine samples are not properly conserved [11–13]. This product was not formed under the
Fig. 2. TIC of the urine extract after one tablet intake of zopiclone and compound’s V mass spectrum.
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Fig. 3. TIC of the urine extract after one tablet intake of zopiclone and its mass spectrum.
conditions of our screening procedures during the analysis of our urine samples which was performed the same day of sampling. During the GC–MS analysis of urine samples concerning cases of intoxication with chlormethiazole the only substance identified after screening analysis, by both libraries NIST62 and PMWTOX2 was chlormethiazole-M (2-OH-) or 2-hydroxy-4-methyl-5chloro-ethylthiazole, which is a metabolite of chlormethiazole satisfactorily extracted only from alkaline medium. It should be noticed that chlormethiazole itself and all the rest of its major metabolites are extracted only from acidic pH 1-3 [14,15], therefore, they cannot be detected according to the screening methodology applied in our lab. 2-OH-chlormethiazole (m / z 128, 177, 73) was eluted at Rt 5,8 (Fig. 4). Buspirone is excreted in urine primarily as its metabolites (29 to 63 percent of a single dose) and mainly as 1-(2-pyrimidinyl)piperazine (1-PP). Due to the extremely low concentrations of the drug and its metabolites in urine following the administration of a normal therapeutic dose of 5 mg their detection requires methods of extremely low detection limit [16]. Since we were not able to find any methods of analysis of buspirone in urine in the literature and since we failed to detect the parent drug or 1-PP during the screening procedures, we tried to confirm the presence of 1-PP by derivatization of the urine extract with pentafluoropropionic anhydride (PFPA) at 658C for 20 min. The PFP derivative of buspirone metabolite (1-PP) (m / z 108, 134, 122, 310) yields a sharp peak which elutes at Rt 8.15 (Fig. 5) and is easily observable. This observation was certified by the derivatization of 5 ng of 1-PP that were chromatographed under the same analytical conditions with our samples. It is obvious that when no case history exists buspirone intake can be missed if derivatization is not applied. Limits of detection of each drug or their metabolites were not established since we didn’t establish a new method for their determination. We checked the possibilities for their detection during the screening procedures followed in our laboratory and we
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Fig. 4. TIC of the urine extract after one tablet intake of chlormethiazole and mass spectrum of chlormethiazole-M-2-OH.
Fig. 5. TIC of the urine extract after one tablet intake of buspirone derivatized with PFPA and the mass spectrum of 1-PP derivative.
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focused on the therapeutic use or the one tablet intake of each drug (intentional or accidental), which can be detected – directly or indirectly – as it was described above. It has to be mentioned that the therapeutic dose (one tablet or capsule) of the above drugs is relatively low. It ranges from 5 to 10 mg (buspirone 10 mg, zopiclone 5 mg and zolpidem 10 mg), while for chlormethiazole is 192 mg. The knowledge of the complete analytical behavior of the above drugs can be of great importance in a routine analysis especially in cases where suspicions of CNS depressant drug intake exists, but the history is not quite clear. Each laboratory involved in emergency or forensic toxicology should check the behavior of the above four drugs and their metabolites during its own screening procedures, and check carefully their presence in cases where drug intake in subtoxic doses is a possibility. The enrichment of their GC / MS libraries with the mass spectra of the above uncommon drugs and / or their metabolites, if not available, would also be useful.
References [1] A.D. Athanaselis, C. Maravelias, A. Koutselinis, Analysis and identification of zipeprol and its metabolites during drug abuse screening, J. Anal. Toxic. 20 (1996) 564–567. [2] Lillsunde, T. Korte, Comprehensive drug screening in urine solid-phase extraction and combined TLC and GC–MS identification. J. Anal. Toxicol. 15 (1991) 71–81. [3] S. Vajta, J.P. Thenot, F. De Maack, G. Devant, M. Lesieur, Thermospray liquid chromatography tandem mass spectrometry: Application to the elucidation of zolpidem metabolism, Biomed. Mass Spectrom. 15 (1988) 223–228. [4] P. Thenot, A. Hermann, J. Durand, J. Burke, D. Allen, S. Garrirou, H. Vajta, J. Albin, G.O. Thebault, G. Olive, S. Warrington, in: J. Sauvent, S. Langer, P. Morselli (Eds.), Imidazopyridines in sleep disorders. Raven Press, New York, 1988, pp. 139–153. [5] M. Augsburger, C. Giroud, P. Lucchini, L. Rivier, Suicide involving zolpidem and hypothermia. Proceedings of the 31st International Meeting of TIAFT, Leipzig, 1993. [6] V. Ascalone, L. Flaminio, P. Guinebault, J.P. Thenot, L. Morselli, Determination of zolpidem a new sleep-inducing agent and its metabolites in biological fluids: pharmacokinetics, drug metabolism and overdosing investigations in humans, J. Chromatogr. 581 (1992) 237–250. [7] I. Boniface, M.S. Nolan, S. To Tan, Development of a method for the determination of zopiclone in whole blood, J. Chromatogr. 584 (1992) 199–206. [8] H. Ahrens, H.S. Schutz, G. Weiler, Screening, identification and determination of the two new hypnotics zolpidem and zopiclone. Arzneim. Forsch. / Drug Res. 44(II)7 (1994) 799–802. [9] J. Debruyne, B. Lacotte, H. de Ligny, M. Moulin, Determination of zolpidem and zopiclone in serum by capillary column gas chromatography, J. Pharm. Sciences 80(1) (1991) 71–74. [10] J. Van Boxclaer, E. Meyer, K. Clauwert, W. Lambert, M. Piette, A. De Leenheer, Analysis of zopiclone (Imovane ) in postmortem specimens by GC–MS and HPLC with diode-array detection, J. Anal. Toxicol. 20 (1996) 52–54. [11] J.T. Mannaert, J. Tytgat, P. Daenens, Detection of 2-amino-5-chloropyridine in urine as a parameter of zopiclone (Imovane ) intake using HPLC with diode-array detection, J. Anal. Toxicol. 21 (1997) 208–212. [12] J.T. Mannaert, Development of stereospecific and non-stereospecific immunochemical assays for the bio-analysis of zopiclone. Ph.D. thesis, Catholic University of Louvain, Leuven, Belgium, 1996. [13] F.G. Fernandez, J. Mayrargue, A. Thuillier, R. Farinotti, Degradation and racemization of zopiclone enantiomers in plasma and partially aqueous solutions, Chirality 7 (1995) 267–271.
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[14] G. Ende, G.R. Spiteller, R. Heipertz, Urinary metabolites of clomethiazole, Arzneim. Forsch. 29(11) (1979) 1655–1658. [15] A. Grupe, G. Spiteller, Unexpected metabolites produced from clomethiazole, J. Chromatogr. 230 (1982) 335–344. [16] R. Gammans, E. Kerns, W. Bullen, Capillary gas chromatographic-mass spectrometric determination of buspirone in plasma, J. Chromatogr. 345 (1985) 285–297.
Letter to the Editor
Detection of ‘‘uncommon’’ tranquillizers–sedatives during screening toxicological analysis A. Dona*, S. Athanaselis, C. Maravelias, A. Koutselinis Department of Forensic Medicine and Toxicology, University of Athens, Medical School, 75, M. Asias str., Goudi, Athens, Greece Received 13 March 1998; received in revised form 22 September 1998; accepted 1 October 1998
The determination of some ‘‘uncommon’’ tranquillizers-sedatives during a toxicological analysis is often difficult particularly when the analysis of biological samples concern cases of mild intoxications such as one tablet intake, driving under the influence of drugs, or when no case history exists. Therefore, we investigated the possibility of identifying the intake of subtoxic doses of four anxiolytic and / or sedative drugs, namely, zolpidem, zopiclone, chlormethiazole and buspirone, during the screening procedures for drug detection in biological fluids followed in our laboratory [1]. Urine from 20 healthy volunteers, who took one tablet of each drug (5 samples for each drug) were analyzed in this study. Urine samples were collected approximately 3 hours after the intake of a single tablet or capsule of each drug. Nine urine samples from known cases of individuals that had taken subtoxic doses of these drugs were also analyzed. The urine sampling took place 2 to 4 hours after the ingestion of the drugs. No cross reactivity was observed during urine screening by fluoroscence polarization immunoassayTDx (Abbott), by enzyme immunoassay ETS Plus system (Syva), or by Triage (Merck) for opiates, benzodiazepines, cocaine, amphetamines and cannabinoids. A general cross-reactivity assessment was not performed because this was not in the purposes of our study. 10 ml of the urine samples were extracted through Chem-Elut extraction columns (Varian, Harbor City, CA), as described by Lillsunde and Korte [2]. After the extraction, the eluent was evaporated to dryness under a stream of N 2 and was used for TLC and GC–MS analysis after reconstitution in 25 ml of MeOH. TLC screening was performed according to Lillsunde and Korte [2]. All four drugs or their metabolites could not be detected in urine. The urine extracts were further analyzed by GC–MS according to a standardized method that we use in our laboratory [1]. *Corresponding author. 0379-0738 / 99 / $ – see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 98 )00170-4
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A. Dona et al. / Forensic Science International 99 (1999) 71 – 77
Zolpidem was eluted at Rt 15.7 min and identified by comparing its retention time and mass spectrum to those of a standard. It should be noticed that its spectrum cannot be identified by the libraries NIST62 and PMWTOX2, because its mass spectrum is not included. Therefore, one should enrich the laboratory’s libraries with the mass spectrum of zolpidem. This spectrum (Fig. 1) was identical to the one reported in the literature [3]. It should be emphasized that the peak of zolpidem obtained in cases of mild intoxications is very weak and can be missed due to background interferences or noise (Fig. 1). Thus, the analyst should perform a SIM analysis looking for the characteristic ions of the drug (m / z 235, 307, 219). It should be noticed that during the screening analysis of urine samples in our lab we were not able to detect the two major metabolites of zolpidem 2-(4-carboxy-phenyl)-N,N,6-trimethylimidazo-[1,2-a]-pyridine-3-acetamide and 2-(4-methyl-phenyl)-(N,N,dimethyl-6-carboxy)-imidazo-(1,2-a]-pyridine-3-acetamide [4]. This is because zolpidem metabolites, due to their polar and amphoteric character, cannot be eluted through Chem-Elut columns. Although someone could use anion exchange columns or extractive alkylation with CH 3 J [5] and have much better results, these methods could be applied specifically for determination and quantitation of zolpidem and not for general screening. Moreover, gas chromatographic methods are not sensitive enough for the determination of these metabolites. That is why the analytical methods proposed in the literature for the identification and quantitation of zolpidem require complicated analytical tools, such as thermospray LC–MS / MS or on-column switching HPLC [3,6]. Zopiclone shows thermal decomposition during the GC–MS analysis to a decomposition product called ‘‘compound V’’ which is (6-(5-chloro-2-pyridyl)-6,7-dihydro[5H]-
Fig. 1. TIC of the urine extract after one tablet intake of zolpidem and its mass spectrum.
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pyrrolo[3,4-b]pyrazine-5-one [7]. Zopiclone’s ratio to this decomposition product depends on the injector port temperature, the condition of the liner and the injection mode (split or splitless). Furthermore, this ratio is also dependent on the injection solvent, as zopiclone appears to be unstable in nucleophilic solvents’, such as methanol [7–9]. In the screening method applied in our lab, the extraction solvent used is a mixture of methylene chloride and isopropanol (9:1) and the injection solvent is methanol. Thus, it was expected that zopiclone would decompose to ‘‘compound V’’. Indeed, during the analysis of urine samples with our screening method only compound V (m / z 246, 217, 191) was detected at Rt 12,4 while no zopiclone was found (Fig. 2). Due to these problems the methodology was changed and n-butyl chloride was chosen as the extraction solvent and acetonitrile as the injection solvent. With acetonitrile as a solvent, compound V was negligible [7,10]. According to the existing case history if zopiclone were one of the drugs found near the intoxicated patient or if it were one of the prescribed drugs by his or her physician the screening methodology followed should be the one described above. In this case zopiclone (m / z 245, 217, 143) elutes at Rt 20,1 (Fig. 3). If there is no history, the general screening methodology is followed and if compound V of zopiclone is detected the methodology with the above changes is applied. The mass spectrum of compound V that has been added in our libraries was extremely helpful for the confirmation of zopiclone intake. The intake of zopiclone could also be detected by determining a decomposition product of zopiclone, 2-amino-5chloropyridine, which is formed after alkaline hydrolysis [11], or when the urine samples are not properly conserved [11–13]. This product was not formed under the
Fig. 2. TIC of the urine extract after one tablet intake of zopiclone and compound’s V mass spectrum.
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Fig. 3. TIC of the urine extract after one tablet intake of zopiclone and its mass spectrum.
conditions of our screening procedures during the analysis of our urine samples which was performed the same day of sampling. During the GC–MS analysis of urine samples concerning cases of intoxication with chlormethiazole the only substance identified after screening analysis, by both libraries NIST62 and PMWTOX2 was chlormethiazole-M (2-OH-) or 2-hydroxy-4-methyl-5chloro-ethylthiazole, which is a metabolite of chlormethiazole satisfactorily extracted only from alkaline medium. It should be noticed that chlormethiazole itself and all the rest of its major metabolites are extracted only from acidic pH 1-3 [14,15], therefore, they cannot be detected according to the screening methodology applied in our lab. 2-OH-chlormethiazole (m / z 128, 177, 73) was eluted at Rt 5,8 (Fig. 4). Buspirone is excreted in urine primarily as its metabolites (29 to 63 percent of a single dose) and mainly as 1-(2-pyrimidinyl)piperazine (1-PP). Due to the extremely low concentrations of the drug and its metabolites in urine following the administration of a normal therapeutic dose of 5 mg their detection requires methods of extremely low detection limit [16]. Since we were not able to find any methods of analysis of buspirone in urine in the literature and since we failed to detect the parent drug or 1-PP during the screening procedures, we tried to confirm the presence of 1-PP by derivatization of the urine extract with pentafluoropropionic anhydride (PFPA) at 658C for 20 min. The PFP derivative of buspirone metabolite (1-PP) (m / z 108, 134, 122, 310) yields a sharp peak which elutes at Rt 8.15 (Fig. 5) and is easily observable. This observation was certified by the derivatization of 5 ng of 1-PP that were chromatographed under the same analytical conditions with our samples. It is obvious that when no case history exists buspirone intake can be missed if derivatization is not applied. Limits of detection of each drug or their metabolites were not established since we didn’t establish a new method for their determination. We checked the possibilities for their detection during the screening procedures followed in our laboratory and we
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Fig. 4. TIC of the urine extract after one tablet intake of chlormethiazole and mass spectrum of chlormethiazole-M-2-OH.
Fig. 5. TIC of the urine extract after one tablet intake of buspirone derivatized with PFPA and the mass spectrum of 1-PP derivative.
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focused on the therapeutic use or the one tablet intake of each drug (intentional or accidental), which can be detected – directly or indirectly – as it was described above. It has to be mentioned that the therapeutic dose (one tablet or capsule) of the above drugs is relatively low. It ranges from 5 to 10 mg (buspirone 10 mg, zopiclone 5 mg and zolpidem 10 mg), while for chlormethiazole is 192 mg. The knowledge of the complete analytical behavior of the above drugs can be of great importance in a routine analysis especially in cases where suspicions of CNS depressant drug intake exists, but the history is not quite clear. Each laboratory involved in emergency or forensic toxicology should check the behavior of the above four drugs and their metabolites during its own screening procedures, and check carefully their presence in cases where drug intake in subtoxic doses is a possibility. The enrichment of their GC / MS libraries with the mass spectra of the above uncommon drugs and / or their metabolites, if not available, would also be useful.
References [1] A.D. Athanaselis, C. Maravelias, A. Koutselinis, Analysis and identification of zipeprol and its metabolites during drug abuse screening, J. Anal. Toxic. 20 (1996) 564–567. [2] Lillsunde, T. Korte, Comprehensive drug screening in urine solid-phase extraction and combined TLC and GC–MS identification. J. Anal. Toxicol. 15 (1991) 71–81. [3] S. Vajta, J.P. Thenot, F. De Maack, G. Devant, M. Lesieur, Thermospray liquid chromatography tandem mass spectrometry: Application to the elucidation of zolpidem metabolism, Biomed. Mass Spectrom. 15 (1988) 223–228. [4] P. Thenot, A. Hermann, J. Durand, J. Burke, D. Allen, S. Garrirou, H. Vajta, J. Albin, G.O. Thebault, G. Olive, S. Warrington, in: J. Sauvent, S. Langer, P. Morselli (Eds.), Imidazopyridines in sleep disorders. Raven Press, New York, 1988, pp. 139–153. [5] M. Augsburger, C. Giroud, P. Lucchini, L. Rivier, Suicide involving zolpidem and hypothermia. Proceedings of the 31st International Meeting of TIAFT, Leipzig, 1993. [6] V. Ascalone, L. Flaminio, P. Guinebault, J.P. Thenot, L. Morselli, Determination of zolpidem a new sleep-inducing agent and its metabolites in biological fluids: pharmacokinetics, drug metabolism and overdosing investigations in humans, J. Chromatogr. 581 (1992) 237–250. [7] I. Boniface, M.S. Nolan, S. To Tan, Development of a method for the determination of zopiclone in whole blood, J. Chromatogr. 584 (1992) 199–206. [8] H. Ahrens, H.S. Schutz, G. Weiler, Screening, identification and determination of the two new hypnotics zolpidem and zopiclone. Arzneim. Forsch. / Drug Res. 44(II)7 (1994) 799–802. [9] J. Debruyne, B. Lacotte, H. de Ligny, M. Moulin, Determination of zolpidem and zopiclone in serum by capillary column gas chromatography, J. Pharm. Sciences 80(1) (1991) 71–74. [10] J. Van Boxclaer, E. Meyer, K. Clauwert, W. Lambert, M. Piette, A. De Leenheer, Analysis of zopiclone (Imovane ) in postmortem specimens by GC–MS and HPLC with diode-array detection, J. Anal. Toxicol. 20 (1996) 52–54. [11] J.T. Mannaert, J. Tytgat, P. Daenens, Detection of 2-amino-5-chloropyridine in urine as a parameter of zopiclone (Imovane ) intake using HPLC with diode-array detection, J. Anal. Toxicol. 21 (1997) 208–212. [12] J.T. Mannaert, Development of stereospecific and non-stereospecific immunochemical assays for the bio-analysis of zopiclone. Ph.D. thesis, Catholic University of Louvain, Leuven, Belgium, 1996. [13] F.G. Fernandez, J. Mayrargue, A. Thuillier, R. Farinotti, Degradation and racemization of zopiclone enantiomers in plasma and partially aqueous solutions, Chirality 7 (1995) 267–271.
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[14] G. Ende, G.R. Spiteller, R. Heipertz, Urinary metabolites of clomethiazole, Arzneim. Forsch. 29(11) (1979) 1655–1658. [15] A. Grupe, G. Spiteller, Unexpected metabolites produced from clomethiazole, J. Chromatogr. 230 (1982) 335–344. [16] R. Gammans, E. Kerns, W. Bullen, Capillary gas chromatographic-mass spectrometric determination of buspirone in plasma, J. Chromatogr. 345 (1985) 285–297.