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Институт теоретической и экспериментальной биофизики Российской академии наук.


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199406, Санкт-Петербург, ул.Гаванская, д. 49, корп.2

ISSN 1999-6314

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ТОМ 4, СТ. X (сc. X) // Апрель, 2003 г.


L. Khodasevitch*, J. Volgram* and J. Rotbergs**
* Latvian State Center for Forensic Medical Examinations, Riga, Latvia
** Latvian Medical Academy / P. Stradin University of Riga, Latvia


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Zopiclone (sin.Imovan) is cyclopyrrolone hypnosedative, widely used and abused in Latvia. The purpose of our study was to identify zopiclone and its alkaline hydrolysis products by TLC, GC-FID and GC-ECD in blood and urine samples. 2-amino-5-chloropyridine, the main product of alkaline hydrolysis of zopiclone, should be a target substance during toxicological analysis of cases involving zopiclone. TLC data and retention indices of zopiclone and its decomposition products are presented in this article.


Zopiclone is a non-benzodiazepine hypnotic drug that has been shown in insomniac patients to posess rapid onset of action and few associated side affects. Zopiclone is widely used and abused in Latvia. Several methods have been described to detect zopiclone in plasma and /or urine such as HPLC /1,2,3/, column liquid chromatography after solid-phase extraction/4/, FPIA /5/, gas chromatography/6/, gas-chromatography-mass-spectrometry /7,8/. Approximately 31% of initial dose was recovered by urinary excretion /9/. Only 5% was represented by unchanged drug.This meant that the major part of initial dose, which was converted to 2-amino-5-chloropyridine after alkaline hydrolysis, resulted from the metabolites N-desmethylzopiclone (15%), and zopiclone N-oxide (11%) /9/. The main purpose of our study was to detect native zopiclone, its decomposition products and 2-amino-5-chloropyridine in blood and urine by means thin-layer chromatography and gas chromatography (FID and ECD).

Materials and methods

The isolation of the anylate was performed by liquid/liquid extraction of urine and blood samples (5,0 ml) with dichloromethane-acetone (4:1,v/v) at pH 8,0 (two times with 10,0 ml, extraction time 5 min.). Organic phases were combined and after centrifugation extract was evaporated and residue was redissolved in 20µ L acetonitrile. A 2 µ L aliquot was then injected. 0,2 ml 10M sodium hydroxide solution was added to a 5 ml urine sample. The sample was hydrolyzed at 100 C in dry-block for 25 min, then cooled to room temperature and extracted with dichloromethane-acetone (4:1,v/v) as described above. 7,5 mg of zopiclone were dissolved in 10,0 ml of destiled water, than 0,5 ml 10 M sodium hydroxide was added and hydrolyzed in dry-block at 100 C 60 min. After cooling hydrolysis product was extracted by 25,0 ml of dichlo-romethane-acetone mixture (4:1). Products of hydrolysis consist of 2-amino-5-chloropyridine and negligible amount of "compound V". (Fig. 1) / 3 / A recovery of zopiclone from blood and urine was 86 and 92%. Aliquots of blood, urine samples and zopiclone hydrolysis products were first analyzed by thin-layer chromatography in solvent system benzene-1,4-dioxane-conc. ammonia (60:35:5). Silica gel plates 60 F 254 (Merck) were used. Zopiclone spots (Rf=0,34) could be easily identified as a white spots under UV light (366 nm, Desaga), its degradation product-as a violet spots (Rf=0,63) and 2-amino-5-chloropyridine-as blue-violet spots (Rf=0,95). Fast Black K /1 N NaOH and Dragendorff/Jodplatinate reagents can be used for zopiclone and its alkaline hydrolysis products detection. Fast Black K/1 N NaOH reagent gave red coloured spots with native zopiclone (Rf=0,34), violet-with alkaline hydrolysis product (Rf=0,63) and blue-violet spot (Rf=0,95) with 2-amino-5-chloropyridine.

Figure 1. Degradation scheme of zopiclone towards 2-amino-5-chloropyridine.

(LOD=0,5 µ g) Dragendorff/Jodplatinate reagent gave orange/brown spots. (LOD=0,3µ g). Zopiclone and its hydrolysis products were identified also by means GC. A gas chromatograph Hewlett-Packard 5890 ser. II equipped with FID was used. A 10 m fused silica capillary column HP-1 was used. Injector and detector temperatures were 270 C and 280 C. Oven progamme was initial 110 C (2 min.), rate 6 C/min untill 280 C (kept 15 min.). Carrier gas-nitrogen-1 ml/min. 2-amino-5 chloropyridine has a Rt=1,434 min (RI=1055,30), zopiclone degradation product-Rt=17,850 min. (RI=2400,68) and zopiclone Rt=27,786 min. (RI=3054,61); Zopiclone and its hydrolysis products were identified by means GC-ECD. A gas chromatograph Varian 3700 (USA) with capillary 10 m fused silica. HP-17 column was used. Injector and detector temperatures were 250 C and 280 C.LOD of zopiclone - 30 ng/ml, LOQ=50 ng/ml. Oven temperature programme was: 200 C (1 min.), rate 10 C/min untill 280 C (kept 5 min.). Carrier gas-nitrogen (1 ml/min). Zopiclone can be detected as a sharp peak with Rt= 13.5 min, 2-amino-5 chloropyridine- Rt=1,36 min, zopiclone degradation product- Rt=9,5 min. Linearity: 20-2000 ng/ml; LOD=10 ng/ml; LOQ=25 ng/ml.

Results and discussion

Zopiclone and its hydrolysis products can be easily detected on the TLC plates by means Fast Black K/1 N. NaOH reagent or Dragendorff/Jod-platinate reagents, but colours produced are not very specific, so another method of detection and confirmation will be necessary. For this purpose gas chromatography is a very suitable method (with flame ionization and electron capture detection systems).

Zopiclone, as a cyclopyrrolone derivative is unstable substance, especially in nucleophilic solvents such as methanol or ethanol. Acetonitrile was used for the preparation of standards and for redissolving purposes. Also ther-mal GC conditions are critical for degradation of zopiclone to "compound V" /3,7/. The structures of degradation products were confirmed by GC-MS /7,8/ In our experience, decomposition product peak was negligible on chromatogramm when acetonitrile was used for redissolving of extracts.

Another cyclopyrrolone-suriclone-is unknown in the market of pharma- ceuticals in Latvia. This substance during alkaline hydrolysis gives another products. So, 2-amino-5-chloropyridine could be a target substance during routine forensic toxicology analysis, especially when use of a low dose of zopiclone is suspected.


Zopiclone and its alkaline hydrolysis products (2-amino-5-chloropyridine etc) can be identified by means TLC and gas chromatography (FID and ECD) in blood and urine samples. 2-amino-5-chloropyridine could be a target substance during toxicological analysis of cases involving zopiclone.


/1/ A. Le Liboux, A. Frdman and J. Gaillot. Simultaneous determination of zopiclone and its two major metabolites in human biological fluids by reversed-phase HPLC. J. Chromatogr. 417:151-58 (1987)

/2/ P. J. Boniface, Martin I. C, Nolan S. L. Development of an HPLC method for analysing for zopiclone in biological samples.Proceeding of the 29th International TIAFT meeting, June 24th-27th 1991, Copenhagen, Denmark, p. 334-339.

/3/ Erik Mannaert, Jan Tytgat, and Paul Daenens. Detection of 2-amino-5-chlo- ropyridine in urine as a parameter of zopiclone(Imovane) intake using HPLC with diode array detection. J. Anal. Toxicology, v.21, p. 208-12(1997).

/4/ R. N. Gupta. Simultaneous determination of zopiclone and its two major metabolites (N-oxyde and N-desmethyl) in human biological fluids by column liquid chromatography after solid-phase extraction. J. Liq. Chrom. Rel. Technol. 19(5) 699-709 (1996).

/5/ E. Mannaert and P. Daenens.Development of fluorescence polarisation immunoassay for the routine detection of N-desmethyl-zopiclone in urine samples. Analyst 121, 857-61(1996)

/6/ Y. Gaillard, J. P. Gay-Montchamp and M. Ollagnica. Gas chromatographic determination of zopiclone in plasma after solid-phase extraction. J.Chrom. 619:310-14(1993).

/7/ A. Dona, S. Athanaselis, C. Maravelias, A. Koutselinis. Detection of "uncommon" tranquilizers-sedatives during screening toxicological analysis. Forensic Science International. 99 71-77, (1999).

/8/ C. Fernandes, F. Gimenez, J. Mayrague et al. Degradation and racemization of zopiclone enantiomers in plasma and partially aqueous solutions. Chirality 7, 267-71(1995).

/9/ J. Gaillot, D. Heusse, G. W. Hougton, J. M. Aurell and J. F. Dreyfus. Pharmacokinetics and metabolism of zopiclone.Pharmacology 27 (suppl. 2), 76-91 (1983).


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