- Email: [email protected]
Testing for zopiclone in hair application to drug-facilitated crimes
Forensic Science International 145 (2004) 117–121
Testing for zopiclone in hair application to drug-facilitated crimes M. Villaina,*, M. Che`zeb, A. Tracquia, B. Ludesa, P. Kintza a
Institut de Me´decine Le´gale, 11 Rue Humann, 67000 Strasbourg, France b Toxlab, 7 Rue Jacques Cartier, 75018 Paris, France Available online 15 June 2004
Abstract The use of a drug to modify a person’s behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused alarm in the general public. The drugs involved can be difficult to detect due to low dosages or chemical instability. They possess amnesic properties and can be quickly cleared from the body fluids. In these situations, blood or even urine can be of poor interest. This is the reason why this laboratory developed an original approach based on hair testing by LC–MS/MS. Zopiclone (Imovane), due to its short half-life associated with rapid hypnotic activity, is considered as a compound of choice to sedate victims. To document the detection of zopiclone in hair, we first tested specimens obtained from two volunteers who had ingested a single 7.5 mg Imovane tablet, and from repetitive consumers of zopiclone. After pH 8.4 buffer incubation and extraction with methylene chloride/diethyl ether (80/20 (v/v)), hair extracts were separated on a Xterra MS C18 column using a gradient of acetonitrile and formate buffer. Zopiclone and diazepam-d5, used as internal standard, were detected by tandem mass spectrometry. A single exposure to zopiclone was detectable in the first hair segment of two volunteers at concentration of 5.4 and 9.0 pg/mg, respectively. Hair from repetitive consumers tested positive for zopiclone at concentrations of 37 and 66 pg/mg. Hair analysis was applied to two authentic criminal cases. In the first one, zopiclone tested positive in the corresponding hair segment at 4.2 pg/mg, in accordance with a single exposure to the drug. In the other expertise, zopiclone was detected in the two segments analyzed, at 21.3 and 21.5 pg/mg, making unlikely the hypothetical single exposure to zopiclone. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Drug facilitated sexual assault; Zopiclone; Hair; LC–MS/MS
1. Introduction The use of a drug to modify a person’s behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault or DFSA, robbery) has caused alarm in the general public. Drugs used can be difficult to detect in blood and urine due to low dosages used. They possess amnesic properties and can be quickly cleared from the body fluids.
* Corresponding author. Tel.: þ33 3 90243356; fax: þ33 3 90243362. E-mail address: [email protected] (M. Villain).
Apart from ethanol and cannabis, benzodiazepines and hypnotics are the most frequently observed compounds in cases of alleged sexual assault [1,2]. Zopiclone is a cyclopyrrolone hypnotic agent, sold in France since 1987, that was believed not to have any abuse potential. Nevertheless, during the past few years there have been an increasing number of reports on the abuse and misuse of zopiclone [3]. As the drug is short acting (Tmax ¼ 1.75 h), it can impair an individual rapidly. Moreover, due to its amnesic properties [4], the victims are less able to accurately recall the circumstances under which the offence occurred. Blood and urine are the conventional specimens to document drug exposure [5]. However, in cases of DFSA, due to a frequent long delay between facts and police declaration
0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2004.04.026
118
M. Villain et al. / Forensic Science International 145 (2004) 117–121
(for example, Marc et al. reported a mean delay of about 17 h, [6]), blood and urine can be of poor interest. In order to enhance the window of detection, hair analysis has proved to be a solution. In the literature, DFSA cases have been documented using hair [7–9].
2. Materials and methods 2.1. Specimen Hair from two volunteers (male and female) were collected one month after they were administered a single oral dose of Imovane (a white 7.5 mg tablet). Strands of about 100 hairs were oriented (tied up at the root), cut in vertex posterior as close as possible to the scalp, and stored at room temperature. Subjects participated in the experimental part of the study through oral informed consent. Zopiclone free hair samples were obtained from laboratory personnel.
Table 1 HPLC gradient profiles for the analysis of zopiclone Time (min)
Acetonitrile (%)
Formiate buffer (%)
0 3 7 10 10.5 20
5 60 80 80 5 5
95 40 20 20 95 95
Table 2 MRM transitions and conditions for the measurement of zopiclone and the internal standard Compound
Parent ion (m/z)
Product ion (m/z)
Cone Collision voltage (V) energy (eV)
Zopiclone
389.0
245.1 345.1
35 35
15 9
Diazepam-d5
290.1
154.1 198.2
60 60
30 30
2.2. Chemicals and reagents Acetonitrile, diethyl ether and methylene chloride were HPLC grade (Merck, Darmstadt, Germany). Chemicals for the saturated phosphate buffer–(NH4)2HPO4, adjusted at pH 8.4 with orthophosphoric acid—were purchased from Fluka (Saint-Quentin Fallavier, France). Diazepam-d5 was purchased from Promochem (Molsheim, France). 2.3. Zopiclone extraction Hair strands were twice decontaminated using methylene chloride (5 mL, 2 min) and then segmented (three segments of 2 cm). Each segment was cut into small pieces (<1 mm). About 20 mg were incubated overnight in 1 mL of phosphate buffer at pH 8.4, in the presence of 1 ng of diazepam-d5 used as internal standard (IS). After a liquidliquid extraction with 5 mL of a mixture of methylene chloride/diethyl ether (80/20 (v/v)) and evaporation to dryness, the residue was reconstituted in 50 ml of acetonitrile/water (50/50 (v/v)). 2.4. LC–MS/MS procedure LC was performed using a Waters Alliance 2695 system. Chromatography was achieved using a XTerra MS C18 column (100 mm 2.1 mm, 3.5 mm) eluted with a gradient delivered at a flow rate of 0.2 mL/min (Table 1). An injection volume of 10 mL was used in all cases. A Quattro Micro triple-quadrupole mass spectrometer (Micromass-Waters) fitted with a Z-Spray ion interface was used for analyses. Ionization was achieved using electrospray in the positive ionization mode (ESþ).
The following conditions were found to be optimal for the analysis of zopiclone and the IS: capillary voltage, 1.0 kV; source block temperature, 120 8C; and desolvatation gas (nitrogen) heated to 350 8C and delivered at a flow rate of 550 L/h. In order to establish appropriate multiple reaction monitoring (MRM) conditions, the cone voltage was adjusted to maximize the intensity of the protonated molecular ion and collision induced dissociation (CID) of both species was performed. Collision gas (argon) pressure was maintained at 3.0 103 mbar and the collision energy (eV) was adjusted to optimize the signal for the two most abundant product ions. (Table 2). 2.5. Method validation A standard calibration curve was obtained by preparing spiked standards containing 1, 2, 5, 10, 20 and 50 pg/mg of zopiclone. Within-batch precision was determined using blank hair spiked with zopiclone at 10 pg/mg. The limit of detection (LOD) was evaluated by decreasing concentrations of zopiclone until a response equivalent to three times the background noise was observed. Recovery was established at 10 pg/mg.
3. Results and discussion 3.1. Validation Linearity was observed for zopiclone concentrations ranging from 1 to 50 pg/mg with a correlation coefficient of
M. Villain et al. / Forensic Science International 145 (2004) 117–121
119
case involving zopiclone exposure should be addressed through hair analysis. To better interpret these concentrations of zopiclone, we tested the hair of two chronic consumers. The first one (black hair) was taking one 7.5 mg tablet of zopiclone per day for 1 year and the concentration in the last 8 cm was 66 pg/mg. The other one (blond hair) was using zopiclone every day for 3 months. The proximal segment, (0–4 cm) corresponding to the period of drug intake, was positive for zopiclone at a concentration of 37 pg/mg, while the next segment was zopiclone free. The corresponding pubic hair was positive for zopiclone at 48 pg/mg. Head and pubic hair of an occasional consumer (no dosage available, but assuming four to six doses over 3 months) revealed zopiclone at 8.5 and 16.7 pg/mg, respectively. These concentrations are not very different from those observed after a single exposure. This can be due to a poor incorporation of the drug into the hair shaft. However, discrimination can be achieved using segmental analysis. Hair analysis was applied to two authentic criminal cases.
0.994. Within-batch precision at 10 pg/mg was 13.7 %, and the extraction recovery of 91.9 %. The limit of detection was 0.3 pg/mg. Under the chromatographic conditions used, there was no interference with the analytes by any extractable endogenous materials present in hair. 3.2. Zopiclone detection in hair It is accepted by the scientific community that hair growth of about 1 cm/month (range 0.7–1.4 cm per month). Assuming normal hair growth rate, it was the opinion of the authors, that in order to document any DFSA case, to collect the hair 3–5 weeks after the alleged offence and to cut the strand into three segments of 2 cm, that the administered drug present in the proximal segment (root) while not detected in the other segments, would support a single dose hypothesis. The hair of the two volunteers that had ingested a single zopiclone dose was segmented, analyzed and quantified. In the two cases (brown hair), zopiclone tested positive in each root segment, at concentrations of 5.4 and 9.0 pg/mg, respectively (Fig. 1). That demonstrates that a single 7.5 mg dose of zopiclone is detectable in hair. This is the lower amount that could be surreptitiously administrated to potential victim of sexual assault, therefore, each
3.3. Case 1 A 16-year-old girl claimed to have been raped during an afternoon, while sedated.
souchi_MD_zc, 0-2cm, 13mg 080104-8 Sm (SG, 2x3)
MRM of 22 Channels ES+ 389 > 345.1 867 Area
6.64;107
100
%
17
080104-8 Sm (SG, 2x3) 100
MRM of 22 Channels ES+ 389 > 245.1 1.87e3 Area
6.64;266
%
3 080104-8 Sm (SG, 2x3) 100
MRM of 22 Channels ES+ 290.1 > 154.1 3.78e4 Area
9.17;7284
%
-4
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Time
12.00
Fig. 1. Chromatogram of a hair extract from a subject exposed to a single zopiclone tablet. Zopiclone concentration is 5.4 pg/mg. On the top, the two daughter ions of zopiclone, on the bottom, the daughter ion of diazepam-d5.
120
M. Villain et al. / Forensic Science International 145 (2004) 117–121
03/1170, 0-3cm, 45.8mg B+H-051203-8 Sm (SG, 2x2) 100
MRM of 18 Channels ES+ 389 > 345.1 1.15e3 Area
6.57;106
%
9 B+H-051203-8 Sm (SG, 2x2)
MRM of 18 Channels ES+ 389 > 245.1 2.74e3 Area
6.60;299
100
%
1 B+H-051203-8 Sm (SG, 2x2)
MRM of 18 Channels ES+ 290.1 > 154.1 1.97e4 Area
9.12;2499
100
%
-0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Time
12.00
Fig. 2. Chromatogram of a hair extract from a victim of a alleged assault. Zopiclone concentration is 4.2 pg/mg. On the top, the two daughter ions of zopiclone, on the bottom, the daughter ion of diazepam-d5.
Due to the long delay (6 days) between the alleged event and her deposition to the police, there was no interest to sample blood or urine. We were requested to analyze the victim’s hair, collected 9 weeks after the offense. Benzodiazepines and hypnotics were tested by LC–MS/ MS and the first 3 cm segment was positive for zopiclone at the concentration of 4.2 pg/mg (Fig. 2), the second (3–5 cm) at 1.0 pg/mg, whereas the last segment (5–7 cm) was zopiclone free. This appears to be consistent with a single exposure to the drug. 3.4. Case 2 A 50-year-old University Professor went to a student party. During the night, he was admitted to the emergency unit of a local hospital, where he woke up the following day with no recall of the previous night. At the admission, blood alcohol concentration was about 3 g/l. He was accused of inappropriate behavior with female students so he privately requested analysis of hair, collected 4 weeks after the party, in order to demonstrate poisoning. Benzodiazepines and hypnotics were tested by LC–MS/ MS and the two segments, from the root to 2, and 2–4 cm, were positive for zopiclone. The concentrations were 21.3 and 21.5 pg/mg, respectively. This makes unlikely the
hypothetical single exposure to zopiclone, and therefore, inconclusive hair findings.
4. Conclusion Hair testing should be used to complement conventional blood and urine analysis as it increases the window of detection and can allow differentiation, by segmentation, of long-term therapeutic use from a single exposure. Selectivity and sensitivity of MS/MS are a pre-requisite. This method is availalable to other compounds such as zolpidem, flunitrazepam, clonazepam, bromazepam and alprazolam. Moreover, positive hair findings are of importance for the victim, particularly to start psychological follow-up. References [1] I. Hindmarch, M. ElSohly, J. Gambles, S. Salamone, Forensic urinalysis of drug use in cases of alleged sexual assault, J. Clin. Forensic Med. 8 (2001) 197–205. [2] F. Questel, G. Lagier, D. Fompeydie et al., Criminal use of psycho-active drugs: analysis of a cohort in Paris, Ann. Toxicol. Anal. 14 (2002) 371–380.
M. Villain et al. / Forensic Science International 145 (2004) 117–121 [3] G. Hajak, W.E. Mu¨ ller, H.U. Wittchen et al., Abuse and dependence potential for the non-benzodiazepine hypnotics zolpidem and zopiclone: a review of case reports and epidemiological data, Addiction 98 (2003) 1371–1378. [4] J.P. Goulle´ , J.P. Anger, Sedative-hypnotic drugs and amnesia. Review and cases, Ann. Toxicol. Anal. 14 (2002) 381–389. [5] M. LeBeau, W. Andollo, W.L. Hearn, Recommendations for toxicological investigations of drug-facilitated sexual assaults, J. Forensic Sci. 44 (1999) 227–230. [6] B. Marc, F. Baudry, P. Vaquero, Sexual assault under benzodiazepine submission in a Paris suburb, Arch. Gynecol. Obstet. 263 (2000) 193–197.
121
[7] J.P. Goulle´ , M. Che`ze, G. Pe´ pin, Determination of endogenous levels of GHB in human hair. Are there possibilities for the identification of GHB administration through hair analysis in cases of drug-facilitated sexual assault? J. Anal. Toxicol. 27 (2003) 574–580. [8] G. Frison, D. Favretto, L. Tedeschi, S.D. Ferrara, Detection of thiopental and pentobarbital in head and pubic hair in a case of drug-facilitated sexual assault, Forensic Sci. Int. 133 (2003) 171–174. [9] G. Pe´ pin, M. Che`ze, G. Duffort, F. Vayssette, Interest of hair and tandem mass spectrometry for chemical submission: about nine cases, Ann. Toxicol. Anal. 14 (2002) 395–406.
Testing for zopiclone in hair application to drug-facilitated crimes M. Villaina,*, M. Che`zeb, A. Tracquia, B. Ludesa, P. Kintza a
Institut de Me´decine Le´gale, 11 Rue Humann, 67000 Strasbourg, France b Toxlab, 7 Rue Jacques Cartier, 75018 Paris, France Available online 15 June 2004
Abstract The use of a drug to modify a person’s behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused alarm in the general public. The drugs involved can be difficult to detect due to low dosages or chemical instability. They possess amnesic properties and can be quickly cleared from the body fluids. In these situations, blood or even urine can be of poor interest. This is the reason why this laboratory developed an original approach based on hair testing by LC–MS/MS. Zopiclone (Imovane), due to its short half-life associated with rapid hypnotic activity, is considered as a compound of choice to sedate victims. To document the detection of zopiclone in hair, we first tested specimens obtained from two volunteers who had ingested a single 7.5 mg Imovane tablet, and from repetitive consumers of zopiclone. After pH 8.4 buffer incubation and extraction with methylene chloride/diethyl ether (80/20 (v/v)), hair extracts were separated on a Xterra MS C18 column using a gradient of acetonitrile and formate buffer. Zopiclone and diazepam-d5, used as internal standard, were detected by tandem mass spectrometry. A single exposure to zopiclone was detectable in the first hair segment of two volunteers at concentration of 5.4 and 9.0 pg/mg, respectively. Hair from repetitive consumers tested positive for zopiclone at concentrations of 37 and 66 pg/mg. Hair analysis was applied to two authentic criminal cases. In the first one, zopiclone tested positive in the corresponding hair segment at 4.2 pg/mg, in accordance with a single exposure to the drug. In the other expertise, zopiclone was detected in the two segments analyzed, at 21.3 and 21.5 pg/mg, making unlikely the hypothetical single exposure to zopiclone. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Drug facilitated sexual assault; Zopiclone; Hair; LC–MS/MS
1. Introduction The use of a drug to modify a person’s behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault or DFSA, robbery) has caused alarm in the general public. Drugs used can be difficult to detect in blood and urine due to low dosages used. They possess amnesic properties and can be quickly cleared from the body fluids.
* Corresponding author. Tel.: þ33 3 90243356; fax: þ33 3 90243362. E-mail address: [email protected] (M. Villain).
Apart from ethanol and cannabis, benzodiazepines and hypnotics are the most frequently observed compounds in cases of alleged sexual assault [1,2]. Zopiclone is a cyclopyrrolone hypnotic agent, sold in France since 1987, that was believed not to have any abuse potential. Nevertheless, during the past few years there have been an increasing number of reports on the abuse and misuse of zopiclone [3]. As the drug is short acting (Tmax ¼ 1.75 h), it can impair an individual rapidly. Moreover, due to its amnesic properties [4], the victims are less able to accurately recall the circumstances under which the offence occurred. Blood and urine are the conventional specimens to document drug exposure [5]. However, in cases of DFSA, due to a frequent long delay between facts and police declaration
0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2004.04.026
118
M. Villain et al. / Forensic Science International 145 (2004) 117–121
(for example, Marc et al. reported a mean delay of about 17 h, [6]), blood and urine can be of poor interest. In order to enhance the window of detection, hair analysis has proved to be a solution. In the literature, DFSA cases have been documented using hair [7–9].
2. Materials and methods 2.1. Specimen Hair from two volunteers (male and female) were collected one month after they were administered a single oral dose of Imovane (a white 7.5 mg tablet). Strands of about 100 hairs were oriented (tied up at the root), cut in vertex posterior as close as possible to the scalp, and stored at room temperature. Subjects participated in the experimental part of the study through oral informed consent. Zopiclone free hair samples were obtained from laboratory personnel.
Table 1 HPLC gradient profiles for the analysis of zopiclone Time (min)
Acetonitrile (%)
Formiate buffer (%)
0 3 7 10 10.5 20
5 60 80 80 5 5
95 40 20 20 95 95
Table 2 MRM transitions and conditions for the measurement of zopiclone and the internal standard Compound
Parent ion (m/z)
Product ion (m/z)
Cone Collision voltage (V) energy (eV)
Zopiclone
389.0
245.1 345.1
35 35
15 9
Diazepam-d5
290.1
154.1 198.2
60 60
30 30
2.2. Chemicals and reagents Acetonitrile, diethyl ether and methylene chloride were HPLC grade (Merck, Darmstadt, Germany). Chemicals for the saturated phosphate buffer–(NH4)2HPO4, adjusted at pH 8.4 with orthophosphoric acid—were purchased from Fluka (Saint-Quentin Fallavier, France). Diazepam-d5 was purchased from Promochem (Molsheim, France). 2.3. Zopiclone extraction Hair strands were twice decontaminated using methylene chloride (5 mL, 2 min) and then segmented (three segments of 2 cm). Each segment was cut into small pieces (<1 mm). About 20 mg were incubated overnight in 1 mL of phosphate buffer at pH 8.4, in the presence of 1 ng of diazepam-d5 used as internal standard (IS). After a liquidliquid extraction with 5 mL of a mixture of methylene chloride/diethyl ether (80/20 (v/v)) and evaporation to dryness, the residue was reconstituted in 50 ml of acetonitrile/water (50/50 (v/v)). 2.4. LC–MS/MS procedure LC was performed using a Waters Alliance 2695 system. Chromatography was achieved using a XTerra MS C18 column (100 mm 2.1 mm, 3.5 mm) eluted with a gradient delivered at a flow rate of 0.2 mL/min (Table 1). An injection volume of 10 mL was used in all cases. A Quattro Micro triple-quadrupole mass spectrometer (Micromass-Waters) fitted with a Z-Spray ion interface was used for analyses. Ionization was achieved using electrospray in the positive ionization mode (ESþ).
The following conditions were found to be optimal for the analysis of zopiclone and the IS: capillary voltage, 1.0 kV; source block temperature, 120 8C; and desolvatation gas (nitrogen) heated to 350 8C and delivered at a flow rate of 550 L/h. In order to establish appropriate multiple reaction monitoring (MRM) conditions, the cone voltage was adjusted to maximize the intensity of the protonated molecular ion and collision induced dissociation (CID) of both species was performed. Collision gas (argon) pressure was maintained at 3.0 103 mbar and the collision energy (eV) was adjusted to optimize the signal for the two most abundant product ions. (Table 2). 2.5. Method validation A standard calibration curve was obtained by preparing spiked standards containing 1, 2, 5, 10, 20 and 50 pg/mg of zopiclone. Within-batch precision was determined using blank hair spiked with zopiclone at 10 pg/mg. The limit of detection (LOD) was evaluated by decreasing concentrations of zopiclone until a response equivalent to three times the background noise was observed. Recovery was established at 10 pg/mg.
3. Results and discussion 3.1. Validation Linearity was observed for zopiclone concentrations ranging from 1 to 50 pg/mg with a correlation coefficient of
M. Villain et al. / Forensic Science International 145 (2004) 117–121
119
case involving zopiclone exposure should be addressed through hair analysis. To better interpret these concentrations of zopiclone, we tested the hair of two chronic consumers. The first one (black hair) was taking one 7.5 mg tablet of zopiclone per day for 1 year and the concentration in the last 8 cm was 66 pg/mg. The other one (blond hair) was using zopiclone every day for 3 months. The proximal segment, (0–4 cm) corresponding to the period of drug intake, was positive for zopiclone at a concentration of 37 pg/mg, while the next segment was zopiclone free. The corresponding pubic hair was positive for zopiclone at 48 pg/mg. Head and pubic hair of an occasional consumer (no dosage available, but assuming four to six doses over 3 months) revealed zopiclone at 8.5 and 16.7 pg/mg, respectively. These concentrations are not very different from those observed after a single exposure. This can be due to a poor incorporation of the drug into the hair shaft. However, discrimination can be achieved using segmental analysis. Hair analysis was applied to two authentic criminal cases.
0.994. Within-batch precision at 10 pg/mg was 13.7 %, and the extraction recovery of 91.9 %. The limit of detection was 0.3 pg/mg. Under the chromatographic conditions used, there was no interference with the analytes by any extractable endogenous materials present in hair. 3.2. Zopiclone detection in hair It is accepted by the scientific community that hair growth of about 1 cm/month (range 0.7–1.4 cm per month). Assuming normal hair growth rate, it was the opinion of the authors, that in order to document any DFSA case, to collect the hair 3–5 weeks after the alleged offence and to cut the strand into three segments of 2 cm, that the administered drug present in the proximal segment (root) while not detected in the other segments, would support a single dose hypothesis. The hair of the two volunteers that had ingested a single zopiclone dose was segmented, analyzed and quantified. In the two cases (brown hair), zopiclone tested positive in each root segment, at concentrations of 5.4 and 9.0 pg/mg, respectively (Fig. 1). That demonstrates that a single 7.5 mg dose of zopiclone is detectable in hair. This is the lower amount that could be surreptitiously administrated to potential victim of sexual assault, therefore, each
3.3. Case 1 A 16-year-old girl claimed to have been raped during an afternoon, while sedated.
souchi_MD_zc, 0-2cm, 13mg 080104-8 Sm (SG, 2x3)
MRM of 22 Channels ES+ 389 > 345.1 867 Area
6.64;107
100
%
17
080104-8 Sm (SG, 2x3) 100
MRM of 22 Channels ES+ 389 > 245.1 1.87e3 Area
6.64;266
%
3 080104-8 Sm (SG, 2x3) 100
MRM of 22 Channels ES+ 290.1 > 154.1 3.78e4 Area
9.17;7284
%
-4
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Time
12.00
Fig. 1. Chromatogram of a hair extract from a subject exposed to a single zopiclone tablet. Zopiclone concentration is 5.4 pg/mg. On the top, the two daughter ions of zopiclone, on the bottom, the daughter ion of diazepam-d5.
120
M. Villain et al. / Forensic Science International 145 (2004) 117–121
03/1170, 0-3cm, 45.8mg B+H-051203-8 Sm (SG, 2x2) 100
MRM of 18 Channels ES+ 389 > 345.1 1.15e3 Area
6.57;106
%
9 B+H-051203-8 Sm (SG, 2x2)
MRM of 18 Channels ES+ 389 > 245.1 2.74e3 Area
6.60;299
100
%
1 B+H-051203-8 Sm (SG, 2x2)
MRM of 18 Channels ES+ 290.1 > 154.1 1.97e4 Area
9.12;2499
100
%
-0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Time
12.00
Fig. 2. Chromatogram of a hair extract from a victim of a alleged assault. Zopiclone concentration is 4.2 pg/mg. On the top, the two daughter ions of zopiclone, on the bottom, the daughter ion of diazepam-d5.
Due to the long delay (6 days) between the alleged event and her deposition to the police, there was no interest to sample blood or urine. We were requested to analyze the victim’s hair, collected 9 weeks after the offense. Benzodiazepines and hypnotics were tested by LC–MS/ MS and the first 3 cm segment was positive for zopiclone at the concentration of 4.2 pg/mg (Fig. 2), the second (3–5 cm) at 1.0 pg/mg, whereas the last segment (5–7 cm) was zopiclone free. This appears to be consistent with a single exposure to the drug. 3.4. Case 2 A 50-year-old University Professor went to a student party. During the night, he was admitted to the emergency unit of a local hospital, where he woke up the following day with no recall of the previous night. At the admission, blood alcohol concentration was about 3 g/l. He was accused of inappropriate behavior with female students so he privately requested analysis of hair, collected 4 weeks after the party, in order to demonstrate poisoning. Benzodiazepines and hypnotics were tested by LC–MS/ MS and the two segments, from the root to 2, and 2–4 cm, were positive for zopiclone. The concentrations were 21.3 and 21.5 pg/mg, respectively. This makes unlikely the
hypothetical single exposure to zopiclone, and therefore, inconclusive hair findings.
4. Conclusion Hair testing should be used to complement conventional blood and urine analysis as it increases the window of detection and can allow differentiation, by segmentation, of long-term therapeutic use from a single exposure. Selectivity and sensitivity of MS/MS are a pre-requisite. This method is availalable to other compounds such as zolpidem, flunitrazepam, clonazepam, bromazepam and alprazolam. Moreover, positive hair findings are of importance for the victim, particularly to start psychological follow-up. References [1] I. Hindmarch, M. ElSohly, J. Gambles, S. Salamone, Forensic urinalysis of drug use in cases of alleged sexual assault, J. Clin. Forensic Med. 8 (2001) 197–205. [2] F. Questel, G. Lagier, D. Fompeydie et al., Criminal use of psycho-active drugs: analysis of a cohort in Paris, Ann. Toxicol. Anal. 14 (2002) 371–380.
M. Villain et al. / Forensic Science International 145 (2004) 117–121 [3] G. Hajak, W.E. Mu¨ ller, H.U. Wittchen et al., Abuse and dependence potential for the non-benzodiazepine hypnotics zolpidem and zopiclone: a review of case reports and epidemiological data, Addiction 98 (2003) 1371–1378. [4] J.P. Goulle´ , J.P. Anger, Sedative-hypnotic drugs and amnesia. Review and cases, Ann. Toxicol. Anal. 14 (2002) 381–389. [5] M. LeBeau, W. Andollo, W.L. Hearn, Recommendations for toxicological investigations of drug-facilitated sexual assaults, J. Forensic Sci. 44 (1999) 227–230. [6] B. Marc, F. Baudry, P. Vaquero, Sexual assault under benzodiazepine submission in a Paris suburb, Arch. Gynecol. Obstet. 263 (2000) 193–197.
121
[7] J.P. Goulle´ , M. Che`ze, G. Pe´ pin, Determination of endogenous levels of GHB in human hair. Are there possibilities for the identification of GHB administration through hair analysis in cases of drug-facilitated sexual assault? J. Anal. Toxicol. 27 (2003) 574–580. [8] G. Frison, D. Favretto, L. Tedeschi, S.D. Ferrara, Detection of thiopental and pentobarbital in head and pubic hair in a case of drug-facilitated sexual assault, Forensic Sci. Int. 133 (2003) 171–174. [9] G. Pe´ pin, M. Che`ze, G. Duffort, F. Vayssette, Interest of hair and tandem mass spectrometry for chemical submission: about nine cases, Ann. Toxicol. Anal. 14 (2002) 395–406.