Degradation and racemization of zopiclone enantiomers in plasma and partially aqueous solutions

Abstract

We investigated the degradation and racemization of zopiclone (ZOP) enantiomers in plasma and partially aqueous solutions (ethano1:phosphate buffer). Degradation and racemization increased with increasing pH and temperature. Degradation products were identified by means of mass spectrometry, which revealed hydrolysis of the carbamate function and opening of the pyrrolidone ring. In plasma, neither degradation nor racemization occurred after 6 months of storage at -20°C and subsequent extraction. KEY WORDS: chirality, cyclopyrrolone, isomers, racemization, stability, zopiclone Zopiclone (ZOP) is a c h a l cyclopyrrolone ( Fig. 1) with hypnotic properties. It is administered therapeutically as a racemic mixture. ' The racemate has been extensively studied, and several analytical methods based on liquid chromatography or gas chromatography have been proposed for its determination in plasma. 2-7 Two chiral methods have been reported for the study of ZOP enantiomers in plasma and urine, using liquid chromatography with spectrofluorimetric detection. ' ,' It has recently been demonstrated that ZOP pharmacokinetics are stereoselective: higher plasma concentrations of the (+) enantiomer were observed following oral administration of the racemic mixture to humans.Enantiomers of certain chiral compounds, such as oxazepam, 11*12 thalidomide, l3 hyoscyamine, l 4 scopolamine, l4 and thymidine glycol, l5 undergo racemization in vitro in aqueous or partially aqueous solutions. It has been shown that compounds such as thalidomide and nonsteroidal antiinflammatory agents can also racemize in vivo. 13*16 In vitro or in vivo racemization may complicate stereoselectivity studies. When studying the stereoselective kinetics of a chiral compound, it is essential to prove that racemization does not occur during storage or treatment of biological samples.Given the presence of a carbamate function on the asymmetric carbon, we investigated the stability and racemization of ZOP enantiomers and the racemic mixture in conditions usually employed for the study of ZOP pharmacokinetic stereoselectivity . EXPERIMENTAL Materials and MethodsSemipreparative chromatography. The HPLC system for semipreparative enantiomer separation consisted of an LC-6A pump (Shimadzu, Touzart et Matignon, Vitry, France), a Rheodyne 7125 injection valve equipped with a 1 ml loop, a Lambda-Max Model 480 UV detector (Waters, Mdford, USA), and a C-R6A integrator (Shimadzu). The column was a semipreparative Chiralcel OD (10 x 250 mm) (Daicel, J.T. Baker France, Paris, France). A 300 ~1 aliquot of freshly prepared ruc-ZOP solution in ethanol (2 mg/ml) was injected into the semipreparative column and eluted with ethanokhexane (6040, v/v) at a flow rate of 2 mumin.Mobile phase fractions containing (-)-ZOP or (+)-ZOP were separately evaporated to dryness under nitrogen. After reconstitution with ethanol, the purity of the separated enan-

Zopiclone degradation in biological samples : Characteristics and consequences in forensic toxicology

“…However, the concentration of ZOP in spiked samples decreased more after 12 months of storage at -20°C compared to authentic post-mortem femoral blood [43]. This is an interesting finding that might be explained by pH shift in the matrices where a low pH is favorable for ZOP stability [117]. The pH of post-mortem blood tends to be acidic, whereas an alkaline shift takes place in fresh heparinized blood after storage at 4°C and at 20°C but not at -20°C [33].…”

“…ethanol, acetonitrile, dichloromethane) [92,102]. Analysis of ZOP in biological specimens is complicated by its instability in certain solvents such as methanol, and under acid or basic pH conditions [101,117,118]. Standard solution should be prepared in acetonitrile and extraction must be done at neutral pH to improve stability [101,119].…”

“…temperature have an effect on ZOP stability and it has been shown that ZOP undergoes degradation by chemical hydrolysis at basic pH by ring opening and conversion to ACP [63,117,149,150].…”

External quality assessment of multi-analyte chromatographic methods in oral fluid

“…It is known that cyclopyrrolones can hydrolyse under different conditions to 2-amino-5-chloropyridine, which could explain the analytical problems. [9,10] Moreover, when the participating laboratories developed their confirmation methods, deuterated zopiclone was not yet available to be used as internal standard.…”

Stereoselective binding of zopiclone to human plasma proteins

“…All reagents were of analytical grade and purchased from local suppliers. ZOP enantiomers were prepared by semipreparative liquid chromatography according to the method described previously by Fernandez et al 13 …”