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Distribution of zopiclone and main metabolites in hair following a single dose
Forensic Science International 306 (2020) 110074
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Distribution of zopiclone and main metabolites in hair following a single dose Stine Lund Hansena , Sys Stybe Johansena,* , Marie Katrine Klose Nielsena , Gunnel Nilssonb , Robert Kronstrandb,c a b c
Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden Division of Drug Research, Department of Medical and Health Sciences, Linköping University, SE 581 85 Linköping, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 August 2019 Received in revised form 18 November 2019 Accepted 20 November 2019 Available online 22 November 2019
In forensic investigations, such as drug-facilitated crimes, reference values are useful for interpretation of hair results. The aim of this study was to establish levels of zopiclone and two main metabolites, N-desmethylzopiclone and zopiclone N-oxide, in hair after the administration of a single dose of zopiclone, as very limited data are published. A controlled study was performed, where 16 volunteers consumed either 5 or 10 mg zopiclone. Hair was sampled prior to consumption and 14, 30, 60, and 120 days after intake. The deposition of drug in hair segments of all sampling time points was followed in small hair segments of 5-mm, using a validated ultra-high performance liquid chromatography–tandem mass spectrometry method. In all participants, hair segments corresponding to the time of intake were positive for zopiclone, but also with lower concentrations in the neighbouring segments. The highest zopiclone concentrations were detected in samples collected 30 or 60 days after intake. For all sampling time points maximum values for the 5-mg dose ranged from 5.0–370 pg/mg for zopiclone and 5.4 to 300 ce:hsp sp="0.25"/>pg/mg for N-desmethylzopiclone, where the maximum values for the 10-mg dose ranged from 17 to 590 pg/mg for zopiclone and 25–410 pg/mg for N-desmethylzopiclone for all sampling time points. No significant difference in concentrations was found between the two dosing groups for either zopiclone or N-desmethylzopiclone. Almost half of the participants showed lower levels 14 days after intake than in the later sampling time points. The metabolite to parent drug ratio of N-desmethylzopiclone to zopiclone varied from 0.6 to 3.4 (median = 1.2) for the maximum levels of all sampling time points. N-desmethylzopiclone are suggested to serve as an additional marker to confirm the intake of zopiclone. Traces of zopiclone N-oxide were detected in hair from only eight participants. This study showed, that it was possible to follow zopiclone and N-desmethylzopiclone in hair for 4 months even though the drugs was divided into several segments in the latest collected hair samples, and no obvious wash-out effect between the sampling time points by e.g. personal hygiene could be discerned because the cumulated amount at each sampling time point was similar. We conclude that the analysis of short segments e.g. segments of 5-mm can help determine the time of a single intake of zopiclone and that obtaining a sample 1–2 months after a drug exposure provide the best conditions to detect and interpret the results. © 2019 Elsevier B.V. All rights reserved.
Keywords: Sampling time point Segmented hair analysis Z-drugs Zopiclone N-desmethylzopiclone UHPLC–MS/MS
1. Introduction Use of drugs to incapacitate a victim for criminal actions is a wellknown phenomenon. Hypnotics and sedatives are often used in drug-facilitated crimes (DFC) [1,2]. Zopiclone, a short-acting hypnotic used clinically to treat insomnia, is typical used in DFC [2,3]. The maximum plasma concentration after oral intake is
* Corresponding author. E-mail address: [email protected] (S.S. Johansen). http://dx.doi.org/10.1016/j.forsciint.2019.110074 0379-0738/© 2019 Elsevier B.V. All rights reserved.
reached within 0.5–2.0 h, and the elimination half-life is 3.5–6.5 h [4,5]. The metabolism of zopiclone involves oxidation and demethylation [6]. After biotransformation in the liver, zopiclone is transformed into two main metabolites, zopiclone N-oxide, which is less active than zopiclone, and N-desmethylzopiclone, which is inactive [5]. The administration of zopiclone can cause memory impairment for up to 2 h after administration [7]. In combination with alcohol, it has an additive effect on impairment of psychomotor function, which is observed approximately 1.5 h after intake [8]. Furthermore, alcohol enhances the sedative effects of zopiclone. All of these effects make zopiclone a candidate for DFC.
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In forensic toxicology, blood and urine are common matrices used to investigate previous drug intake. However, most drugs used in DFC can be rapidly metabolized and excreted by the body. This makes drug detection in blood and urine difficult, especially if there is a delay in collection of specimens from victims [9,10]. Hair is an alternative matrix with a detection window of weeks to years depending on hair length. After ingestion, the drug can be incorporated into the growing hair. Hair is segmented on the basis of the average growth rate of approximately 1 cm per month [11]. Segmental analysis of hair can make it possible to determine if a victim was exposed to drugs in an expected period and not in other periods, e.g. before and after an incident. It is though not possible to identify the exact day of intake [12]. Over the years, the sensitivity of analytical instruments has improved, making hair analysis useful for the detection of a single exposure to drug [10,13–15]. Reference values for a single dose of zopiclone in hair are valuable for future DFC cases, but only a few controlled studies (n = 8 subjects) and DFC cases (n = 14 subjects) have been published [2,3,14,16–18]. In these studies, zopiclone was reported in concentrations of 1.7–340 pg/mg in hair. Three of the studies considered hair concentrations of zopiclone following a single controlled dose [2,14,16]. Combined, these three studies reported zopiclone concentrations of 1.7–36 pg/mg in the hair of eight participants who received a single dose of 7.5 mg zopiclone. Chèze et al. [16] and Villain et al. [14] analysed hair segments of 2 cm, whereas Deveaux et al. [2] analysed a segment of 0.5 cm. No metabolites were included in any of the single-dose studies. Irving et al. [18] analysed hair cut in 1-cm segments from one DFC victim 17 months after assault and found zopiclone concentrations of 2–6 pg/mg (n = 8 segments), whereas the metabolite N-desmethylzopiclone was detected in three segments, where a median ratio of metabolite to parent drug was determined to be approximately 0.5. The aim of this study was to establish reference values for zopiclone and its main metabolites, N-desmethylzopiclone and zopiclone N-oxide, in hair following a single administration of zopiclone (5 or 10 mg). Furthermore, the temporal patterns of zopiclone and the metabolites concentrations in hair were investigated using segmental analysis of hair sampled at four different times after intake. 2. Material and methods 2.1. Chemicals and reagents Zopiclone was obtained from Lipomed GmbH (Bad Säckingen, Germany). Zopiclone N-oxide, N-desmethylzopiclone, and a deuterated internal standard zopiclone-D8 were obtained from Toronto Research Chemicals (Toronto, Canada). The purity of the reference standards was generally 98 % beside N-desmethylzopiclone, where the purity was 95 %. Water, methanol, acetonitrile, and 2-propanol of LC–MS grade were purchased from Fisher Scientific (Loughborough, United Kingdom). Formic acid (>98 %) was obtained from Merck (Darmstadt, Germany) and ammonium formate (>97 %) from Sigma-Aldrich (Fluka Analytical, Seelze, Germany). 2.2. Study design The study was designed to answer the following research questions: First, if it was possible to find a correlation between doses and the amount of drug found in hair; therefore, two doses were administered. Second, if it was possible to estimate the time of intake more precisely by the results of shorter segments and compare these with the results of traditionally recommended 1–2-
cm segments [19]. Finally, if it was possible to follow the drug in the growing hair over long time (months) and if there was a washout effect that was influenced over time. Therefore, samples at four different times after administration were obtained, where it could be investigated if a second sample was likely to yield the same results. Sixteen healthy adult volunteers (9 females and 7 males, 19–27 years old) with Swedish origin and no history of zopiclone consumption participated in this study and were randomized to receive either 5 or 10 mg zopiclone. Eight participants (subjects #6, #7, #9, #10, #11, #12, #13, and #15) ingested 5 mg zopiclone as a single dose, and eight participants (subjects #1, #2, #3, #4, #5, #8, #14, and #16) ingested 10 mg. Three participants had blonde hair (#1 (though after 60 days (T3), a change in hair colour was observed, so the hair was probably coloured), #4, and #10), whereas the remaining participants had brown hair. Hair samples were obtained as close to the scalp as possible from the posterior vertex and stored at room temperature. The samples were collected prior to (T0) and approximately 14 (T1), 30 (T2), 60 (T3), and 120 days (T4) after the drug intake. All participants gave their informed consent prior to their participation in this study. The study protocol was approved by the Regional Ethics Committee in Linköping, Sweden (Dnr: 2010/41-31). All available hair samples were segmented according to the plan given in Table 1A. The relevant segment(s) and neighbouring segments of each sampling time point were analysed. 2.3. Hair extraction The extraction method previously published by Montesano et al. [20] was used with minor modifications. After alignment of the hair tuft, the hair was cut in 2–12 segments of 0.5 cm, depending on sampling time point and hair length. A scheme of the cut segments and the expected drug-positive segments is provided in Table 1A. The hair from two subjects #4 and #8 was also cut into longer segments (1–2 cm) as given in Table 1B, following the same procedure as described by Wang et al. [21]. Approximately 10 mg hair was weighed into a Precellys tube (Bertin Technologies, Montigny-le-Bretonneux, France) and decontaminated by washing once with 1 mL isopropanol for 1 min and twice with 0.5 mL water for 1 min; the last aqueous wash was analysed to check for external contamination. After drying overnight, six steel beads were added, followed by 470 mL extraction media and 30 mL internal standard solution (0.016 mg/L). The hair extraction medium was a 25:29:46 (v/v/v) mixture of methanol/acetonitrile/2 mM ammonium formate (pH 5.3). The hair was pulverized (4 30 s, 6500 rpm) using a Precellys 24 ball mill (Bertin Technologies), centrifuged (10 min, 3600 rpm), and incubated for 18 h at 37 C. The extracts were centrifuged (3 min, 6500 rpm) and transferred to Mini UniPrep (Whatman Inc., Piscataway, NJ, USA) vials containing a polytetrafluoroethylene filter. The filtered extracts were transferred to glass tubes and evaporated to dryness under a stream of nitrogen at 40 C for 40 min, and 75 mL extraction media was used to reconstitute the extracts. The samples were mixed for 1 min and transferred to amber vials with insert and analysed immediately, where 5 mL was injected into the ultra-high performance liquid chromatography– tandem mass spectrometry (UHPLC–MS/MS) system. In each series, a calibration curve with spiked blank hair at six concentrations and three quality controls in duplicates were included. 2.4. Analytical method Chromatographic separation was performed using an ACQUITY UPLC HSS C18 UHPLC system (Waters Corporation, Milford, MA, USA), 150.0 mm 2.1 mm, 1.8 mm column at 50 C, and constant flow rate of 0.4 mL/min. Mobile phases consisted of 5 mM
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Table 1 Segmentation plans for hair samples from volunteers collected at five different sampling time points. A: 0.5 cm segments, B: 1–2 cm segments, s: segment, black: expected drug positive segments assuming a growth rate at 1.0 cm/month, grey: possible trace, X: not analysed.
ammonium formate (pH 3.0), and 0.1 % formic acid in water (solvent A) and 0.1 % formic acid in acetonitrile (solvent B). The gradient elution was 0.1 % B (0–0.2 min), 0.1 %–25 % B (0.2–7.0 min), and 25.0 %–99.9 % B (7.0–7.5 min); the column was then washed with 99.9 % B (7.5–8.5 min). A total runtime of 10 min was used. The autosampler was maintained at 12 C. Detection was performed on a triple quadrupole mass spectrometer (Xevo TQ-S; Waters Corporation), with electrospray ionization in the positive mode. Source conditions were set as follows: source temperature 150 C, desolvation temperature 600 C, capillary voltage 3.0 kV, cone gas flow 150 L/h, desolvation gas flow 1000 L/h, and collision gas flow 0.15 mL/min, corresponding to a collision cell pressure of 400 Pa. Nitrogen was used as the cone and desolvation gas, whereas argon was used as the collision gas. Analytes were detected by multiple-reaction monitoring, with two transitions for each analyte. The following transitions were used. For zopiclone: m/z 389 > 245, with collision energy (CE) 17 eV (quantifier, Q), and m/z 389 > 217, with CE 33 eV. For N-desmethylzopiclone: m/z 375 > 245, with CE 34 eV (Q), and m/z 375 > 217, with CE 22 eV. For zopiclone N-oxide: m/z 405 > 143, with CE 12 eV (Q), and m/z 405 > 112, with CE 59 eV. The internal standard, zopiclone-D8, was measured at m/z 397 > 245, with CE 20 eV. The cone voltage was 20 V for all analytes and the internal standard. MassLynx 4.1 SCN 714 software with TargetLynx (Waters Corporation) was used for data handling.
The limit of quantification (LLOQ) was 0.5 pg/mg (n = 9) for zopiclone and zopiclone N-oxide, with precision (CV%) less than 11 % and mean accuracies 100 % (n = 45). For N-desmethylzopiclone, LLOQ was 2 pg/mg. A linear range was established from LLOQ to 1000 pg/mg for the three analytes. For levels above LLOQ, the imprecision was less than 3 % and the mean accuracies were within 99 %–100 % for zopiclone and zopiclone N-oxide. The process efficiencies were 39 %–68 % for zopiclone and 32 %–48 % for zopiclone N-oxide. CVs of three quality control samples in soaked blank hair analysed in 20–27 separate analytical series within a 6–8 month period were less than 12 % for zopiclone, 17 % for zopiclone N-oxide, and 12 % for N-desmethylzopiclone. The test of the lower amount of hair (2 or 5 mg) demonstrated the similar acceptable ranges (<20 %) for all three analytes. Therefore, when applying a sample amount down to one-fifth of 10 mg, similar precision and accuracy were achieved, and because the concentrations in the hair samples were greater than LLOQ, this was acceptable for authentic samples with less amount of hair available. 2.6. Statistical data treatment Statistical analysis was performed using the statistical software Rstudio (version 1.1.456). The Friedmantest was used to test for significant differences between two groups e.g. the two dosing groups. Statistical significance was defined as P < 0.05.
2.5. Method validation 3. Results and discussion The method was validated according to Peters et al. [22] and the Society of Hair Testing (SoHT) guidelines [19], with slight modifications. In-house quality control samples were prepared in three different concentration levels by soaking washed, drugfree hair cut in 2- to 3- mm pieces with standard solutions containing zopiclone (0.005; 0.050 and 0.50 mg/L), zopiclone N-oxide (0.010; 0.10 and 1.0 mg/L), and N-desmethylzopiclone (0.016; 0.16 and 1.6 mg/L) in acetonitrile and water (1:1) in an ultrasonic bath wrapped in foil for 2 h. The control samples were washed four times with acetonitrile and then dried overnight. The corresponding levels in hair were in the range 1–700 pg/mg for the three analytes. In some authentic cases, only 3–7 mg hair was available instead of the specified 10 mg for analysis. Therefore, the accuracy and precision were tested using 2 and 5 mg of the soaked quality controls, with the lowest concentration in sextet and triplicate, respectively.
All 16 participants provided hair samples over the 4-month study period. In total, 79 hair samples were collected and 452 segments were analysed. For one participant (#7), no sample was collected at T2, 30 days after intake. Also, five hair samples were inadequate for segmental analysis, T4 for subject #15 and T3 and T4 for subjects #3 and #10 because of short hair length. Zopiclone and the metabolites were not detected in any of the segments collected from the participants before administration (T0), indicating that no zopiclone was ingested the last month before this study. Overall, the results showed that after a single 5- or 10-mg dose, both zopiclone and N-desmethylzopiclone were detected in segments corresponding to the time of intake, but also at lower concentrations in the neighbouring segments. Zopiclone N-oxide was detected in hair samples of eight participants in
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concentrations near LLOQ and not in all sampling time points. Four of these received a 5-mg dose and the other four received a 10-mg dose. The analysed aqueous wash of all of the segments showed no detectable levels of either zopiclone or the metabolites, suggesting no external contamination as expected. For subject #1, no analytes were detected in the last two sampling time points, but a change in hair colour was noticed, and a hair treatment of bleaching/dying could explain the negative results. 3.1. Evaluation of temporal patterns On a group level, the distribution of zopiclone and Ndesmethylzopiclone concentrations in the segments are shown in Fig. 1, which is separated into two dosing groups of each sampling time point. The maximum ranges for each sampling time point are given in Table 2. In the hair samples collected 14 days after administration (T1), the proximal segment (0–0.5 cm) contained the highest levels of
zopiclone and N-desmethylzopiclone in all 16 participants, as seen in Fig. 1A1 and A2. In the remaining segments, low concentrations were quantified in 12 participants for zopiclone and seven participants for N-desmethylzopiclone. Assuming that hair growth was 1 cm per month, the 0.5-cm segment represents a 14-day period and segment S1 was, therefore, expected to contain the highest amount, which was in accordance with the findings. After 30 days (T2), the highest levels of zopiclone and Ndesmethylzopiclone were observed in S2 (0.5–1.0 cm); thus, lower concentrations were observed in the neighbouring segments (see Fig. 1B1 and B2). S2 was expected to contain the highest concentrations. In the later segments (S4–S6), minor amounts were quantified in 10 participants for zopiclone and four participants for N-desmethylzopiclone. After 60 days (T3), the highest levels of zopiclone and Ndesmethylzopiclone were observed in S4 (1.5–2.0 cm), as seen in Fig. 1C1 and C2. Minor amounts were quantified in S1 and S2 in most participants and likewise in S7 and S8 in some. S4 was
Fig. 1. Box and whiskers plot showing means (X), medians (–), quantiles (25–50 %, 50–75 %), whiskers (0–25 %, 75–100 %), and ranges for zopiclone and N-desmethylzopiclone concentrations in hair (pg/mg) of each segment along with the number of participants. Values < LLOQ are not included. S: segment. (A1): Dose: 5 mg, collected 14 days after intake, (A2): Dose: 10 mg, collected 14 days after intake, (B1): Dose: 5 mg, collected 30 days after intake, (B2): Dose: 10 mg, collected 30 days after intake, (C1): Dose: 5 mg, collected 60 days after intake, (C2): Dose: 10 mg, collected 60 days after intake, (D1): Dose: 5 mg, collected 120 days after intake, (D2): Dose: 10 mg, collected 120 days after intake.
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Table 2 Ranges of maximum values including median values for zopiclone and N-desmethylzopiclone concentrations (pg/mg) in the subjects who ingested a single dose of 5 mg zopiclone, and the subjects who ingested a single dose of 10 mg zopiclone in hair collected at four sampling time points after intake, where n describes the number of subjects. T1 = 14 days after intake, T2 = 30 days after intake, T3 = 60 days after intake, T4 = 120 days after intake. Sampling time point
T1
Analyte
Maximum values
Median
T2 Maximum values
Median
Maximum values
Median
Maximum values
Median
5 mg dose Zopiclone N-desmethyl-zopiclone n
6.7‒150 5.4–110 8
26 26
5.0‒350 5.6–300 7
68 130
40‒370 18–280 7
93 110
8.2‒200 12–180 6
50 99
10 mg dose Zopiclone N-desmethyl-zopiclone n
17‒280 25–270 8
72 80
27‒530 55–400 8
160 180
55‒590 110–410 6
140 220
23‒490 43–280 6
64 100
expected to contain the highest concentrations. Zopiclone N-oxide was quantified in five participants in S4 and S5 (1.3–25 pg/mg) and for two of the participants in the neighbouring segments as well. Three of the participants received a 5-mg dose where the remaining two recived a 10-mg dose. In the hair samples collected 120 days after administration (T4), the segments with the highest concentration differed between the two dosing groups (Fig. 1D1 and D2). In the 5 mg group, the highest concentrations were noted in S10 (4.5–5.0 cm), and in the 10 mg group, the highest concentrations of zopiclone and N-desmethylzopiclone were observed in S8 (3.5–4.0 cm). S8 was expected to contain the highest amount. Zopiclone N-oxide was quantified in six participants in S8 and S9 (0.8–23 pg/mg) as well as in minor concentrations in the neighbouring segments. Two of these received a 5-mg dose and four received a 10- mg dose. The findings outside the expected segments can be explained by slower or faster growth rate than 1 cm per month and show the limitation of using a general growth rate in an individual case. On a group level, as shown by our results, it seems like a good approximation. Also, an uneven cut of the hair sample or uneven alignment can give an irregular distribution between segments [12,23]. Even though this study was carefully designed according to the alignment and cutting of hair samples by experienced personnel, these factors cannot be excluded. In the sampling time points after 14, 30, and 60 days, the highest concentrations that were found correspond very well with the expected, whereas the samples after 120 days showed more variation. One conclusion from these results is that the farther away from the exposure the hair sample is collected, the more difficult it is to estimate the time of the drug intake. The SoHT states that head hair cut into 1- to 3-cm segments can provide a detailed historical profile of an individual’s drug exposure but that, in cases of alleged DFC, where a single administration of a central nervous system depressant drug is suspected, the analysis of even smaller segments may improve the temporal interpretation [19]. Choosing 0.5 cm as the segment length makes it possible to follow the distribution of the drug concentration in more detail. However, a less complicated presentation of the temporal pattern can be achieve using longer segments. On the contrary, it is more difficult to estimate the time of the drug intake, because each 2-cm segment would represent a period of about 2 months. Increasing the segment length would also give a lower concentration in the hair of single-dose cases compared to the concentration obtained in shorter length. We used the hair samples from two participants to illustrate this. In Fig. 2, results from 1- to 2-cm segments (depending on sampling time point) are compared to those from 0.5-cm segments. As expected, less spread between segments are
T3
T4
observed in the longer segments, but low concentrations are detected in the neighbouring segments 60 and 120 days as well after intake. Kintz [24] suggested that as long as the measured concentration in the expected segment in a DFC is at least three times higher than that of the neighbouring segments when using 2-cm segments, it qualifies for a single exposure and our data support this. 3.2. Evaluation of quantitative results 3.2.1. Comparison between 5 and 10 mg dosing groups The maximum concentration ranges and medians of zopiclone and N-desmethylzopiclone are provided in Table 2 and Fig. 3. In general, the means and medians of zopiclone and N-desmethylzopiclone in the participants who ingested 10 mg zopiclone were higher than those in the participants who received 5 mg zopiclone. Although by comparing the maximum concentrations of zopiclone and/or N-desmethylzopiclone between the two dosing groups during each sampling time point, no significant differences (P > 0.05) were observed. The individual maximum concentrations for the eight participants for each dose were widely distributed, and therefore, it was not possible to estimate the dose on the basis of the concentration in hair. Because of the short segments and distribution between the segments, a cumulated amount of each sampling time point was calculated and compared the same way as the maximum concentrations of each sampling time point. The statistical tests showed no significant difference between the two dosing groups. By comparing the maximum values in each sampling time point for males and females, no significant difference in the hair concentrations was observed. 3.2.2. Influence of hair colour Some factors have a greater influence on the incorporation than the dose, e.g. the hair colour. Six participants (three from the 5 mg dosing group and three from the 10 mg dosing group) with brown hair showed higher concentrations than the remaining ten participants where seven also had brown hair and only three of the participants had blonde hair. Two of those ingested 10 mg zopiclone, which showed the two lowest zopiclone concentrations found in the 10 mg group. The last one of those ingested 5 mg zopiclone and showed the second lowest zopiclone concentration found in the 5 mg group. The same tendency was observed for N-desmethylzopiclone. The data indicate that zopiclone is better incorporated into dark hair, as zopiclone is a basic drug that binds better to dark hair because of the higher melanin content [13]. Thus, more studies are needed to obtain further knowledge of the incorporation of zopiclone and N-desmethylzopiclone into hair of different colours.
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Fig. 2. Zopiclone and N-desmethylzopiclone concentrations in hair (pg/mg) segments of 0.5 cm and 1 or 2 cm, respectively from subject #4 (A1 & A2) and #8 (B1 & B2) at the five different sampling time points after administration. T1 = 14 days after intake, T2 = 30 days after intake, T3 = 60 days after intake, T4 = 120 days after intake. S; segment. : indicates expected positive segments. Both subjects consumed 10 mg zopiclone.
Fig. 3. Box and whiskers plot showing means (X), medians (–), quantiles (25–50 %,50–75 %), whiskers (0–25 %, 75–100 %), numbers of participants, and ranges for the maximum values of zopiclone and N-desmethylzopiclone concentrations in hair (pg/mg) at four different sampling time points after single intake. Notice that the number of participants drops during the sampling time points because some subjects had too short hair. (A) Concentrations in hair from subjects who ingested a single dose of 5 mg zopiclone. (B) Concentrations in hair from subjects who ingested a single dose of 10 mg zopiclone. T1 = 14 days after intake, T2 = 30 days after intake, T3 = 60 days after intake, T4 = 120 days after intake.
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3.2.3. Comparison of concentration between the different sampling time points within and between participants The maximum concentrations for both zopiclone and N-desmethylzopiclone are described for each subject at each sampling time point. The results provided in Table 2 and Fig. 3 show that the mean maximum concentrations differ between 14 (T1) and 120 days (T4). The lowest concentrations were found 14 days after intake, and the highest concentrations were detected after 30 (T2) or 60 (T3) days. 12 of the 16 participants showed lower levels 14 days after intake than in the following sampling time points. It takes 7–10 days for the newly formed hair in the follicle to reach the scalp [16], and therefore, some positive hair may still be beneath the skin. At decrease in medians of zopiclone and metabolite concentrations from T2 and T3 to T4 was observed. As seen in Fig. 2A1 and B1, a spread within the segments was observed at the latest sampling time point. These findings support the recommendation that hair collection should be delayed 4–6 weeks after a suspected ingestion [19]. As shown in Fig. 3, the variations in the measured maximum concentrations of each sampling time points are quite wide between the participants, as demonstrated by the vertical lines in the figure, where a factor of 11–70 (median = 22) for zopiclone and 4–54 (median = 13) for N-desmethylzopiclone was observed. A smaller variation was observed within each subject, where a factor of 1–30 (median = 2) for zopiclone and 1–29 (median = 3) for N-desmethylzopiclone was obtained. 3.2.4. Comparison of drug distribution and wash-out effects during different sampling time points The distribution of analytes between segments was more spread out in the last sampling time points probably because of irregular hair growth and long-time effects of variations in hair alignment. We could measure zopiclone 4 months after a single intake of zopiclone, but a spread within the segments was observed as seriously blurred and the concentrations were also clearly reduced. Because of this, the time of intake was more difficult to determine. A clearer presentation regarding the time of intake was achieved using larger segments, as seen in Fig. 2A2 and
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B2. Here, all sampling time points show the intake in the expected segments. Positive neighbouring segments were observed in the latest sampling time point, indicating that longer sampling time points causes more drug spread out between the segments. Furthermore, the general recommendation on performing hair analysis within 6 months after an exposure is also supported by these results. As depicted in Fig. 4, no obvious wash-out effect by e.g. personal hygiene was observed from 1 (T2) and 2 (T3) month to 4 (T4) months after intake because the cumulated concentration for all segments after 1, 2 and 4 months, respectively, showed no significant differences (P > 0.05). Only one subject (#16) showed some signs of wash-out effect. Therefore, in terms of statistics a wash-out effect between the sampling time points was not significant; however, it cannot be excluded. 3.2.5. Comparison to published literature Villain et al. [14] have reported that after a single intake of 7.5 mg zopiclone (n = 2 subjects), zopiclone concentrations of 5.4 and 9.0 pg/mg were obtained in hair. Both participants had brown hair, and the hair collected after 1 month was cut into three segments of 2 cm each, where the proximal segment of both showed the highest concentration. In our study, the observed zopiclone concentrations in the hair were in general higher compared to those reported by Villain et al. [14]. Deveaux et al. [2] reported zopiclone concentrations at 1.7 and 5.3 pg/mg after the intake of 7.5 mg zopiclone (n = 2 subjects), where results only from the 5-mm proximal segment were described. The hair samples were collected 3–5 weeks after ingestion. As with Villain et al. [14], the observed zopiclone concentrations in the hair were in general higher in our study. A possible reason for the differences in the results might be due to the segment size. Deveaux et al. [2] analysed 0.5-cm segments similar to our study, and Villain et al. [14] analysed 2-cm segments. In theory, one of the 0.5-cm segments could contain a concentration of up to four times higher than that of the 2-cm segments, giving that only one of the 0.5-cm segments had the whole drug content and not the remaining three
Fig. 4. Cumulated amount of zopiclone (pg/mg) in all segments at each sampling time point for all subjects. T1 = 14 days after intake, T2 = 30 days after intake, T3 = 60 days after intake, T4 = 120 days after intake.
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Table 3 N-desmethylzopiclone/zopiclone ratios including range, median, relative standard division (%RSD) and number of measurements (n) for the maximum concentration of zopiclone in the four different time periods for all subjects individually and in total. *Reduced data as not all sampling time points were collected. Subject
#1
#2
#3
#4
#5
#6
#7
#8
Range Median %RSD n
1.5–2.9 2.2 – 2*
1.1–1.7 1.3 20 4
0.67–0.79 0.73 – 2*
1.9–2.4 2.0 12 4
0.57–0.73 0.63 12 4
0.90–4.6 2.1 65 4
2.1–3.2 2.8 20 3*
1.4–2.2 1.8 20 4
Subject
#9
#10
#11
#12
#13
#14
#15
#16
All
Range Median %RSD n
1.1–2.1 1.6 29 4
1.5–1.5 1.5 – 2*
0.66–1.5 1.2 31 4
0.68–0.81 0.76 7 4
0.91–1.2 1.0 11 4
0.74–0.90 0.85 8 4
0.72–0.87 0.76 9 3*
1.1–1.5 1.4 12 4
0.57–4.6 1.3 54 56
segments that would have been a part of the 2-cm segments. This relation was confirmed by our analysis of two participants segmented by 0.5 and 2.0 cm (see Fig. 2). Overall, the results that Deveaux et al. [2] and Villain et al. [14] found were around the lower end of those found in this study. An explanation could be differences in the hair preparation. Villain et al. [14] extracted cut hair, where in this study the hair was pulverized. The method in Deveaux et al. [2] were though not discribed. However, many other factors are known to contribute to the deposition of drugs in hair. As mentioned previous, hair colour, irregular hair growth, variations in alignment, and sweat-contamination can all affect the concentration in hair [12,13,16,23,24]. 3.2.6. Metabolite to parent drug ratio in hair The ratio of N-desmethylzopiclone to zopiclone varied from 0.57 to 4.6 with a median of 1.3, calculated for the maximum concentrations of zopiclone for all sampling time points in all participants (n = 56 segments). In Table 3, the ratios for each subject are provided, depicting a higher variation between all participants than within a subject, although only three participants showed a variation higher than 20 %, including subjects #4 and #8; the distributions are shown in Fig. 2. For four participants, the ratio medians were 2.0, where two had blonde hair and the other two brown. The median of the remaining 12 participants was in the range 0.57–1.8. Compared with the median ratio of 0.5 found by Irving et al. [18], higher ratio levels were observed here. However, the ratio found by Irving et al. [18] represents three segments from only one subject. Zopiclone N-oxide was quantified only in eight of the participants and only in one or two of the sampling time points. Here the ratio of zopiclone N-oxide to zopiclone ranged from 0.0012 to 0.38, with a median of 0.053 for all segments containing zopiclone N-oxide for all sampling time points (n = 31). A metabolite can be included to confirm drug intake. In this case, the metabolite N-desmethylzopiclone was positive in all relevant segments. At the same time, a small variation was observed in the metabolite to parent drug ratio in general. These qualities suggest N-desmethylzopiclone to serve as an additional marker to confirm the intake of zopiclone. Zopiclone N-oxide on the other hand is not a relevant marker for zopiclone consumption, because it was only found in half of the participants and in lower concentrations than N-desmethylzopiclone. 4. Conclusion The reference values for single-dose consumption of zopiclone in hair were established for 16 participants consuming either 5 or 10 mg zopiclone. The validated method demonstrated a high enough sensitivity to detect a single dose of zopiclone in hair. Concentrations in hair varied greatly between the participants, and no significant difference was found between the two doses for
either zopiclone or N-desmethylzopiclone. The distribution of drug concentrations in hair could be followed in detail by the small 5mm segments of the different sampling time points, and the drug findings along the hair shaft were in accordance with the expected positive segments. Sampling hair after 14 days could be too early in some cases as the concentrations were lower compared to the other sampling time points. One or two months after intake is recommended as the concentrations are the highest and the distribution is not spread out too much between the segments. No wash-out effect could be detected within the 4 months after the intake of zopiclone as the cumulated amount in segments after 30 days was similar to the amount in later sampling time points. The metabolite N-desmethylzopiclone was a good marker for the intake of zopiclone where the median metabolite to drug ratio in hair was 1.3, whereas the other metabolite zopiclone N-oxide was present only in trace levels in some participants’ hair. Author Contributions Study conception and sample collection: RK, GN. Study design: All. Acquisition of data: SLH, SSJ, MKN. Analysis and interpretation of data: All. Drafting of manuscript: All. Critical revision: All. References [1] P. Xiang, M. Shen, O.H. Drummer, Review: drug concentrations in hair and their relevance in drug facilitated crimes, J. Forensic Leg. Med. 36 (2015) 126–135, doi:http://dx.doi.org/10.1016/j.jflm.2015.09.009. [2] M. Deveaux, M. Cheze, G. Pepin, The role of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to test blood and urine samples for the toxicological investigation of drug-facilitated crimes, Ther. Drug Monit. 30 (2) (2008) 225–228, doi:http://dx.doi.org/10.1097/FTD.0b013e3181676186. [3] X. Wang, S.S. Johansen, M.K.K. Nielsen, K. Linnet, Hair analysis in toxicological investigation of drug-facilitated crimes in Denmark over a 8-year period, Forensic Sci. Int. 285 (2018) e1–e12, doi:http://dx.doi.org/10.1016/j.forsciint.2018.01.021. [4] C. Fernandez, C. Martin, F. Gimenez, R. Farinotti, Clinical pharmacokinetics of zopiclone, Clin. Pharmacokinet. 29 (6) (1995) 431–441, doi:http://dx.doi.org/ 10.2165/00003088-199529060-00004. [5] S. Noble, H.D. Langtry, H.M. Lamb, Zopiclone. An update of its pharmacology, clinical efficacy and tolerability in the treatment of insomnia, Drugs 55 (2) (1998) 277–302, doi:http://dx.doi.org/10.2165/00003495-199855020-00015. [6] D.R. Drover, Comparative pharmacokinetics and pharmacodynamics of shortacting hypnosedatives: zaleplon, zolpidem and zopiclone, Clin. Pharmacokinet. 43 (4) (2004) 227–238, doi:http://dx.doi.org/10.2165/00003088200443040-00002. [7] J. Wagner, M.L. Wagner, W.A. Hening, Beyond benzodiazepines: alternative pharmacologic agents for the treatment of insomnia, Ann. Pharmacother. 32 (6) (1998) 680–691, doi:http://dx.doi.org/10.1345/aph.17111. [8] A.N. Wadworth, D. McTavish, Zopiclone: a review of its pharmacological properties and therapeutic efficacy as an hypnotic, Drugs Aging 3 (5) (1993) 441–459, doi:http://dx.doi.org/10.2165/00002512-199303050-00006. [9] L.K. Bechtel, C.P. Holstege, Criminal poisoning: drug-facilitated sexual assault, Emerg. Med. Clin. North Am. 25 (2) (2007) 499–525, doi:http://dx.doi.org/ 10.1016/j.emc.2007.02.008.
S.L. Hansen et al. / Forensic Science International 306 (2020) 110074 [10] P. Kintz, M. Villain, B. Ludes, Testing for the undetectable in drug-facilitated sexual assault using hair analyzed by tandem mass spectrometry as evidence, Ther. Drug Monit. 26 (2) (2004) 211–214. [11] Recommendations for hair testing in forensic cases, Forensic Sci. Int. 145 (2–3) (2004), doi:http://dx.doi.org/10.1016/j.forsciint.2004.04.022. [12] M.A. LeBeau, M.A. Montgomery, J.D. Brewer, The role of variations in growth rate and sample collection on interpreting results of segmental analyses of hair, Forensic Sci. Int. 210 (1) (2011) 110–116, doi:http://dx.doi.org/10.1016/j. forsciint.2011.02.015. [13] F. Pragst, M.A. Balikova, State of the art in hair analysis for detection of drug and alcohol abuse, Clin. Chim. Acta 370 (1–2) (2006) 17–49, doi:http://dx.doi. org/10.1016/j.cca.2006.02.019. [14] M. Villain, M. Cheze, A. Tracqui, B. Ludes, P. Kintz, Testing for zopiclone in hair application to drug-facilitated crimes, Forensic Sci. Int. 145 (2–3) (2004) 117– 121, doi:http://dx.doi.org/10.1016/j.forsciint.2004.04.026. [15] M. Chèze, G. Duffort, M. Deveaux, G. Pépin, Hair analysis by liquid chromatography–tandem mass spectrometry in toxicological investigation of drug-facilitated crimes: report of 128 cases over the period June 2003–May 2004 in metropolitan Paris, Forensic Sci. Int. 153 (1) (2005) 3–10, doi:http://dx. doi.org/10.1016/j.forsciint.2005.04.021. [16] M. Chèze, J.M. Gaulier, drugs involved in drug-facilitated crimes (DFC): analytical aspects: 2—Hair, in: P. Kintz (Ed.), Toxicological Aspects of DrugFacilitated Crimes, Academic Press, Oxford, 2014, pp. 181–222.
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[17] M. Chèze, A. Muckensturm, G. Hoizey, G. Pépin, M. Deveaux, A tendency for reoffending in drug-facilitated crime, Forensic Sci. Int. 196 (1) (2010) 14–17, doi: http://dx.doi.org/10.1016/j.forsciint.2009.12.037. [18] R.C. Irving, S.J. Dickson, The detection of sedatives in hair and nail samples using tandem LC-MS-MS, Forensic Sci. Int. 166 (1) (2007) 58–67, doi:http://dx. doi.org/10.1016/j.forsciint.2006.03.027. [19] G.A. Cooper, R. Kronstrand, P. Kintz, T. Society of Hair, Society of Hair Testing guidelines for drug testing in hair, Forensic Sci. Int. 218 (1–3) (2012) 20–24, doi:http://dx.doi.org/10.1016/j.forsciint.2011.10.024. [20] C. Montesano, S.S. Johansen, M.K.K. Nielsen, Validation of a method for the targeted analysis of 96 drugs in hair by UPLC–MS/MS, J. Pharm. Biomed. Anal. 88 (2014) 295–306, doi:http://dx.doi.org/10.1016/j.jpba.2013.08.050. [21] X. Wang, S.S. Johansen, Y. Zhang, J. Jia, Y. Rao, F. Jiang, K. Linnet, Deposition of diazepam and its metabolites in hair following a single dose of diazepam, Int. J. Legal Med. 131 (1) (2017) 131–141, doi:http://dx.doi.org/10.1007/s00414-016-1429-x. [22] F.T. Peters, O.H. Drummer, F. Musshoff, Validation of new methods, Forensic Sci. Int. 165 (2) (2007) 216–224, doi:http://dx.doi.org/10.1016/j. forsciint.2006.05.021. [23] P. Kintz, Analytical and Practical Aspects of Drug Testing in Hair, Taylor & Francis, Boca Raton, FL, 2007. [24] P. Kintz, Issues about axial diffusion during segmental hair analysis, Ther. Drug Monit. 35 (3) (2013) 408–410, doi:http://dx.doi.org/10.1097/ FTD.0b013e318285d5fa.
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Distribution of zopiclone and main metabolites in hair following a single dose Stine Lund Hansena , Sys Stybe Johansena,* , Marie Katrine Klose Nielsena , Gunnel Nilssonb , Robert Kronstrandb,c a b c
Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden Division of Drug Research, Department of Medical and Health Sciences, Linköping University, SE 581 85 Linköping, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 August 2019 Received in revised form 18 November 2019 Accepted 20 November 2019 Available online 22 November 2019
In forensic investigations, such as drug-facilitated crimes, reference values are useful for interpretation of hair results. The aim of this study was to establish levels of zopiclone and two main metabolites, N-desmethylzopiclone and zopiclone N-oxide, in hair after the administration of a single dose of zopiclone, as very limited data are published. A controlled study was performed, where 16 volunteers consumed either 5 or 10 mg zopiclone. Hair was sampled prior to consumption and 14, 30, 60, and 120 days after intake. The deposition of drug in hair segments of all sampling time points was followed in small hair segments of 5-mm, using a validated ultra-high performance liquid chromatography–tandem mass spectrometry method. In all participants, hair segments corresponding to the time of intake were positive for zopiclone, but also with lower concentrations in the neighbouring segments. The highest zopiclone concentrations were detected in samples collected 30 or 60 days after intake. For all sampling time points maximum values for the 5-mg dose ranged from 5.0–370 pg/mg for zopiclone and 5.4 to 300 ce:hsp sp="0.25"/>pg/mg for N-desmethylzopiclone, where the maximum values for the 10-mg dose ranged from 17 to 590 pg/mg for zopiclone and 25–410 pg/mg for N-desmethylzopiclone for all sampling time points. No significant difference in concentrations was found between the two dosing groups for either zopiclone or N-desmethylzopiclone. Almost half of the participants showed lower levels 14 days after intake than in the later sampling time points. The metabolite to parent drug ratio of N-desmethylzopiclone to zopiclone varied from 0.6 to 3.4 (median = 1.2) for the maximum levels of all sampling time points. N-desmethylzopiclone are suggested to serve as an additional marker to confirm the intake of zopiclone. Traces of zopiclone N-oxide were detected in hair from only eight participants. This study showed, that it was possible to follow zopiclone and N-desmethylzopiclone in hair for 4 months even though the drugs was divided into several segments in the latest collected hair samples, and no obvious wash-out effect between the sampling time points by e.g. personal hygiene could be discerned because the cumulated amount at each sampling time point was similar. We conclude that the analysis of short segments e.g. segments of 5-mm can help determine the time of a single intake of zopiclone and that obtaining a sample 1–2 months after a drug exposure provide the best conditions to detect and interpret the results. © 2019 Elsevier B.V. All rights reserved.
Keywords: Sampling time point Segmented hair analysis Z-drugs Zopiclone N-desmethylzopiclone UHPLC–MS/MS
1. Introduction Use of drugs to incapacitate a victim for criminal actions is a wellknown phenomenon. Hypnotics and sedatives are often used in drug-facilitated crimes (DFC) [1,2]. Zopiclone, a short-acting hypnotic used clinically to treat insomnia, is typical used in DFC [2,3]. The maximum plasma concentration after oral intake is
* Corresponding author. E-mail address: [email protected] (S.S. Johansen). http://dx.doi.org/10.1016/j.forsciint.2019.110074 0379-0738/© 2019 Elsevier B.V. All rights reserved.
reached within 0.5–2.0 h, and the elimination half-life is 3.5–6.5 h [4,5]. The metabolism of zopiclone involves oxidation and demethylation [6]. After biotransformation in the liver, zopiclone is transformed into two main metabolites, zopiclone N-oxide, which is less active than zopiclone, and N-desmethylzopiclone, which is inactive [5]. The administration of zopiclone can cause memory impairment for up to 2 h after administration [7]. In combination with alcohol, it has an additive effect on impairment of psychomotor function, which is observed approximately 1.5 h after intake [8]. Furthermore, alcohol enhances the sedative effects of zopiclone. All of these effects make zopiclone a candidate for DFC.
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In forensic toxicology, blood and urine are common matrices used to investigate previous drug intake. However, most drugs used in DFC can be rapidly metabolized and excreted by the body. This makes drug detection in blood and urine difficult, especially if there is a delay in collection of specimens from victims [9,10]. Hair is an alternative matrix with a detection window of weeks to years depending on hair length. After ingestion, the drug can be incorporated into the growing hair. Hair is segmented on the basis of the average growth rate of approximately 1 cm per month [11]. Segmental analysis of hair can make it possible to determine if a victim was exposed to drugs in an expected period and not in other periods, e.g. before and after an incident. It is though not possible to identify the exact day of intake [12]. Over the years, the sensitivity of analytical instruments has improved, making hair analysis useful for the detection of a single exposure to drug [10,13–15]. Reference values for a single dose of zopiclone in hair are valuable for future DFC cases, but only a few controlled studies (n = 8 subjects) and DFC cases (n = 14 subjects) have been published [2,3,14,16–18]. In these studies, zopiclone was reported in concentrations of 1.7–340 pg/mg in hair. Three of the studies considered hair concentrations of zopiclone following a single controlled dose [2,14,16]. Combined, these three studies reported zopiclone concentrations of 1.7–36 pg/mg in the hair of eight participants who received a single dose of 7.5 mg zopiclone. Chèze et al. [16] and Villain et al. [14] analysed hair segments of 2 cm, whereas Deveaux et al. [2] analysed a segment of 0.5 cm. No metabolites were included in any of the single-dose studies. Irving et al. [18] analysed hair cut in 1-cm segments from one DFC victim 17 months after assault and found zopiclone concentrations of 2–6 pg/mg (n = 8 segments), whereas the metabolite N-desmethylzopiclone was detected in three segments, where a median ratio of metabolite to parent drug was determined to be approximately 0.5. The aim of this study was to establish reference values for zopiclone and its main metabolites, N-desmethylzopiclone and zopiclone N-oxide, in hair following a single administration of zopiclone (5 or 10 mg). Furthermore, the temporal patterns of zopiclone and the metabolites concentrations in hair were investigated using segmental analysis of hair sampled at four different times after intake. 2. Material and methods 2.1. Chemicals and reagents Zopiclone was obtained from Lipomed GmbH (Bad Säckingen, Germany). Zopiclone N-oxide, N-desmethylzopiclone, and a deuterated internal standard zopiclone-D8 were obtained from Toronto Research Chemicals (Toronto, Canada). The purity of the reference standards was generally 98 % beside N-desmethylzopiclone, where the purity was 95 %. Water, methanol, acetonitrile, and 2-propanol of LC–MS grade were purchased from Fisher Scientific (Loughborough, United Kingdom). Formic acid (>98 %) was obtained from Merck (Darmstadt, Germany) and ammonium formate (>97 %) from Sigma-Aldrich (Fluka Analytical, Seelze, Germany). 2.2. Study design The study was designed to answer the following research questions: First, if it was possible to find a correlation between doses and the amount of drug found in hair; therefore, two doses were administered. Second, if it was possible to estimate the time of intake more precisely by the results of shorter segments and compare these with the results of traditionally recommended 1–2-
cm segments [19]. Finally, if it was possible to follow the drug in the growing hair over long time (months) and if there was a washout effect that was influenced over time. Therefore, samples at four different times after administration were obtained, where it could be investigated if a second sample was likely to yield the same results. Sixteen healthy adult volunteers (9 females and 7 males, 19–27 years old) with Swedish origin and no history of zopiclone consumption participated in this study and were randomized to receive either 5 or 10 mg zopiclone. Eight participants (subjects #6, #7, #9, #10, #11, #12, #13, and #15) ingested 5 mg zopiclone as a single dose, and eight participants (subjects #1, #2, #3, #4, #5, #8, #14, and #16) ingested 10 mg. Three participants had blonde hair (#1 (though after 60 days (T3), a change in hair colour was observed, so the hair was probably coloured), #4, and #10), whereas the remaining participants had brown hair. Hair samples were obtained as close to the scalp as possible from the posterior vertex and stored at room temperature. The samples were collected prior to (T0) and approximately 14 (T1), 30 (T2), 60 (T3), and 120 days (T4) after the drug intake. All participants gave their informed consent prior to their participation in this study. The study protocol was approved by the Regional Ethics Committee in Linköping, Sweden (Dnr: 2010/41-31). All available hair samples were segmented according to the plan given in Table 1A. The relevant segment(s) and neighbouring segments of each sampling time point were analysed. 2.3. Hair extraction The extraction method previously published by Montesano et al. [20] was used with minor modifications. After alignment of the hair tuft, the hair was cut in 2–12 segments of 0.5 cm, depending on sampling time point and hair length. A scheme of the cut segments and the expected drug-positive segments is provided in Table 1A. The hair from two subjects #4 and #8 was also cut into longer segments (1–2 cm) as given in Table 1B, following the same procedure as described by Wang et al. [21]. Approximately 10 mg hair was weighed into a Precellys tube (Bertin Technologies, Montigny-le-Bretonneux, France) and decontaminated by washing once with 1 mL isopropanol for 1 min and twice with 0.5 mL water for 1 min; the last aqueous wash was analysed to check for external contamination. After drying overnight, six steel beads were added, followed by 470 mL extraction media and 30 mL internal standard solution (0.016 mg/L). The hair extraction medium was a 25:29:46 (v/v/v) mixture of methanol/acetonitrile/2 mM ammonium formate (pH 5.3). The hair was pulverized (4 30 s, 6500 rpm) using a Precellys 24 ball mill (Bertin Technologies), centrifuged (10 min, 3600 rpm), and incubated for 18 h at 37 C. The extracts were centrifuged (3 min, 6500 rpm) and transferred to Mini UniPrep (Whatman Inc., Piscataway, NJ, USA) vials containing a polytetrafluoroethylene filter. The filtered extracts were transferred to glass tubes and evaporated to dryness under a stream of nitrogen at 40 C for 40 min, and 75 mL extraction media was used to reconstitute the extracts. The samples were mixed for 1 min and transferred to amber vials with insert and analysed immediately, where 5 mL was injected into the ultra-high performance liquid chromatography– tandem mass spectrometry (UHPLC–MS/MS) system. In each series, a calibration curve with spiked blank hair at six concentrations and three quality controls in duplicates were included. 2.4. Analytical method Chromatographic separation was performed using an ACQUITY UPLC HSS C18 UHPLC system (Waters Corporation, Milford, MA, USA), 150.0 mm 2.1 mm, 1.8 mm column at 50 C, and constant flow rate of 0.4 mL/min. Mobile phases consisted of 5 mM
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Table 1 Segmentation plans for hair samples from volunteers collected at five different sampling time points. A: 0.5 cm segments, B: 1–2 cm segments, s: segment, black: expected drug positive segments assuming a growth rate at 1.0 cm/month, grey: possible trace, X: not analysed.
ammonium formate (pH 3.0), and 0.1 % formic acid in water (solvent A) and 0.1 % formic acid in acetonitrile (solvent B). The gradient elution was 0.1 % B (0–0.2 min), 0.1 %–25 % B (0.2–7.0 min), and 25.0 %–99.9 % B (7.0–7.5 min); the column was then washed with 99.9 % B (7.5–8.5 min). A total runtime of 10 min was used. The autosampler was maintained at 12 C. Detection was performed on a triple quadrupole mass spectrometer (Xevo TQ-S; Waters Corporation), with electrospray ionization in the positive mode. Source conditions were set as follows: source temperature 150 C, desolvation temperature 600 C, capillary voltage 3.0 kV, cone gas flow 150 L/h, desolvation gas flow 1000 L/h, and collision gas flow 0.15 mL/min, corresponding to a collision cell pressure of 400 Pa. Nitrogen was used as the cone and desolvation gas, whereas argon was used as the collision gas. Analytes were detected by multiple-reaction monitoring, with two transitions for each analyte. The following transitions were used. For zopiclone: m/z 389 > 245, with collision energy (CE) 17 eV (quantifier, Q), and m/z 389 > 217, with CE 33 eV. For N-desmethylzopiclone: m/z 375 > 245, with CE 34 eV (Q), and m/z 375 > 217, with CE 22 eV. For zopiclone N-oxide: m/z 405 > 143, with CE 12 eV (Q), and m/z 405 > 112, with CE 59 eV. The internal standard, zopiclone-D8, was measured at m/z 397 > 245, with CE 20 eV. The cone voltage was 20 V for all analytes and the internal standard. MassLynx 4.1 SCN 714 software with TargetLynx (Waters Corporation) was used for data handling.
The limit of quantification (LLOQ) was 0.5 pg/mg (n = 9) for zopiclone and zopiclone N-oxide, with precision (CV%) less than 11 % and mean accuracies 100 % (n = 45). For N-desmethylzopiclone, LLOQ was 2 pg/mg. A linear range was established from LLOQ to 1000 pg/mg for the three analytes. For levels above LLOQ, the imprecision was less than 3 % and the mean accuracies were within 99 %–100 % for zopiclone and zopiclone N-oxide. The process efficiencies were 39 %–68 % for zopiclone and 32 %–48 % for zopiclone N-oxide. CVs of three quality control samples in soaked blank hair analysed in 20–27 separate analytical series within a 6–8 month period were less than 12 % for zopiclone, 17 % for zopiclone N-oxide, and 12 % for N-desmethylzopiclone. The test of the lower amount of hair (2 or 5 mg) demonstrated the similar acceptable ranges (<20 %) for all three analytes. Therefore, when applying a sample amount down to one-fifth of 10 mg, similar precision and accuracy were achieved, and because the concentrations in the hair samples were greater than LLOQ, this was acceptable for authentic samples with less amount of hair available. 2.6. Statistical data treatment Statistical analysis was performed using the statistical software Rstudio (version 1.1.456). The Friedmantest was used to test for significant differences between two groups e.g. the two dosing groups. Statistical significance was defined as P < 0.05.
2.5. Method validation 3. Results and discussion The method was validated according to Peters et al. [22] and the Society of Hair Testing (SoHT) guidelines [19], with slight modifications. In-house quality control samples were prepared in three different concentration levels by soaking washed, drugfree hair cut in 2- to 3- mm pieces with standard solutions containing zopiclone (0.005; 0.050 and 0.50 mg/L), zopiclone N-oxide (0.010; 0.10 and 1.0 mg/L), and N-desmethylzopiclone (0.016; 0.16 and 1.6 mg/L) in acetonitrile and water (1:1) in an ultrasonic bath wrapped in foil for 2 h. The control samples were washed four times with acetonitrile and then dried overnight. The corresponding levels in hair were in the range 1–700 pg/mg for the three analytes. In some authentic cases, only 3–7 mg hair was available instead of the specified 10 mg for analysis. Therefore, the accuracy and precision were tested using 2 and 5 mg of the soaked quality controls, with the lowest concentration in sextet and triplicate, respectively.
All 16 participants provided hair samples over the 4-month study period. In total, 79 hair samples were collected and 452 segments were analysed. For one participant (#7), no sample was collected at T2, 30 days after intake. Also, five hair samples were inadequate for segmental analysis, T4 for subject #15 and T3 and T4 for subjects #3 and #10 because of short hair length. Zopiclone and the metabolites were not detected in any of the segments collected from the participants before administration (T0), indicating that no zopiclone was ingested the last month before this study. Overall, the results showed that after a single 5- or 10-mg dose, both zopiclone and N-desmethylzopiclone were detected in segments corresponding to the time of intake, but also at lower concentrations in the neighbouring segments. Zopiclone N-oxide was detected in hair samples of eight participants in
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concentrations near LLOQ and not in all sampling time points. Four of these received a 5-mg dose and the other four received a 10-mg dose. The analysed aqueous wash of all of the segments showed no detectable levels of either zopiclone or the metabolites, suggesting no external contamination as expected. For subject #1, no analytes were detected in the last two sampling time points, but a change in hair colour was noticed, and a hair treatment of bleaching/dying could explain the negative results. 3.1. Evaluation of temporal patterns On a group level, the distribution of zopiclone and Ndesmethylzopiclone concentrations in the segments are shown in Fig. 1, which is separated into two dosing groups of each sampling time point. The maximum ranges for each sampling time point are given in Table 2. In the hair samples collected 14 days after administration (T1), the proximal segment (0–0.5 cm) contained the highest levels of
zopiclone and N-desmethylzopiclone in all 16 participants, as seen in Fig. 1A1 and A2. In the remaining segments, low concentrations were quantified in 12 participants for zopiclone and seven participants for N-desmethylzopiclone. Assuming that hair growth was 1 cm per month, the 0.5-cm segment represents a 14-day period and segment S1 was, therefore, expected to contain the highest amount, which was in accordance with the findings. After 30 days (T2), the highest levels of zopiclone and Ndesmethylzopiclone were observed in S2 (0.5–1.0 cm); thus, lower concentrations were observed in the neighbouring segments (see Fig. 1B1 and B2). S2 was expected to contain the highest concentrations. In the later segments (S4–S6), minor amounts were quantified in 10 participants for zopiclone and four participants for N-desmethylzopiclone. After 60 days (T3), the highest levels of zopiclone and Ndesmethylzopiclone were observed in S4 (1.5–2.0 cm), as seen in Fig. 1C1 and C2. Minor amounts were quantified in S1 and S2 in most participants and likewise in S7 and S8 in some. S4 was
Fig. 1. Box and whiskers plot showing means (X), medians (–), quantiles (25–50 %, 50–75 %), whiskers (0–25 %, 75–100 %), and ranges for zopiclone and N-desmethylzopiclone concentrations in hair (pg/mg) of each segment along with the number of participants. Values < LLOQ are not included. S: segment. (A1): Dose: 5 mg, collected 14 days after intake, (A2): Dose: 10 mg, collected 14 days after intake, (B1): Dose: 5 mg, collected 30 days after intake, (B2): Dose: 10 mg, collected 30 days after intake, (C1): Dose: 5 mg, collected 60 days after intake, (C2): Dose: 10 mg, collected 60 days after intake, (D1): Dose: 5 mg, collected 120 days after intake, (D2): Dose: 10 mg, collected 120 days after intake.
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Table 2 Ranges of maximum values including median values for zopiclone and N-desmethylzopiclone concentrations (pg/mg) in the subjects who ingested a single dose of 5 mg zopiclone, and the subjects who ingested a single dose of 10 mg zopiclone in hair collected at four sampling time points after intake, where n describes the number of subjects. T1 = 14 days after intake, T2 = 30 days after intake, T3 = 60 days after intake, T4 = 120 days after intake. Sampling time point
T1
Analyte
Maximum values
Median
T2 Maximum values
Median
Maximum values
Median
Maximum values
Median
5 mg dose Zopiclone N-desmethyl-zopiclone n
6.7‒150 5.4–110 8
26 26
5.0‒350 5.6–300 7
68 130
40‒370 18–280 7
93 110
8.2‒200 12–180 6
50 99
10 mg dose Zopiclone N-desmethyl-zopiclone n
17‒280 25–270 8
72 80
27‒530 55–400 8
160 180
55‒590 110–410 6
140 220
23‒490 43–280 6
64 100
expected to contain the highest concentrations. Zopiclone N-oxide was quantified in five participants in S4 and S5 (1.3–25 pg/mg) and for two of the participants in the neighbouring segments as well. Three of the participants received a 5-mg dose where the remaining two recived a 10-mg dose. In the hair samples collected 120 days after administration (T4), the segments with the highest concentration differed between the two dosing groups (Fig. 1D1 and D2). In the 5 mg group, the highest concentrations were noted in S10 (4.5–5.0 cm), and in the 10 mg group, the highest concentrations of zopiclone and N-desmethylzopiclone were observed in S8 (3.5–4.0 cm). S8 was expected to contain the highest amount. Zopiclone N-oxide was quantified in six participants in S8 and S9 (0.8–23 pg/mg) as well as in minor concentrations in the neighbouring segments. Two of these received a 5-mg dose and four received a 10- mg dose. The findings outside the expected segments can be explained by slower or faster growth rate than 1 cm per month and show the limitation of using a general growth rate in an individual case. On a group level, as shown by our results, it seems like a good approximation. Also, an uneven cut of the hair sample or uneven alignment can give an irregular distribution between segments [12,23]. Even though this study was carefully designed according to the alignment and cutting of hair samples by experienced personnel, these factors cannot be excluded. In the sampling time points after 14, 30, and 60 days, the highest concentrations that were found correspond very well with the expected, whereas the samples after 120 days showed more variation. One conclusion from these results is that the farther away from the exposure the hair sample is collected, the more difficult it is to estimate the time of the drug intake. The SoHT states that head hair cut into 1- to 3-cm segments can provide a detailed historical profile of an individual’s drug exposure but that, in cases of alleged DFC, where a single administration of a central nervous system depressant drug is suspected, the analysis of even smaller segments may improve the temporal interpretation [19]. Choosing 0.5 cm as the segment length makes it possible to follow the distribution of the drug concentration in more detail. However, a less complicated presentation of the temporal pattern can be achieve using longer segments. On the contrary, it is more difficult to estimate the time of the drug intake, because each 2-cm segment would represent a period of about 2 months. Increasing the segment length would also give a lower concentration in the hair of single-dose cases compared to the concentration obtained in shorter length. We used the hair samples from two participants to illustrate this. In Fig. 2, results from 1- to 2-cm segments (depending on sampling time point) are compared to those from 0.5-cm segments. As expected, less spread between segments are
T3
T4
observed in the longer segments, but low concentrations are detected in the neighbouring segments 60 and 120 days as well after intake. Kintz [24] suggested that as long as the measured concentration in the expected segment in a DFC is at least three times higher than that of the neighbouring segments when using 2-cm segments, it qualifies for a single exposure and our data support this. 3.2. Evaluation of quantitative results 3.2.1. Comparison between 5 and 10 mg dosing groups The maximum concentration ranges and medians of zopiclone and N-desmethylzopiclone are provided in Table 2 and Fig. 3. In general, the means and medians of zopiclone and N-desmethylzopiclone in the participants who ingested 10 mg zopiclone were higher than those in the participants who received 5 mg zopiclone. Although by comparing the maximum concentrations of zopiclone and/or N-desmethylzopiclone between the two dosing groups during each sampling time point, no significant differences (P > 0.05) were observed. The individual maximum concentrations for the eight participants for each dose were widely distributed, and therefore, it was not possible to estimate the dose on the basis of the concentration in hair. Because of the short segments and distribution between the segments, a cumulated amount of each sampling time point was calculated and compared the same way as the maximum concentrations of each sampling time point. The statistical tests showed no significant difference between the two dosing groups. By comparing the maximum values in each sampling time point for males and females, no significant difference in the hair concentrations was observed. 3.2.2. Influence of hair colour Some factors have a greater influence on the incorporation than the dose, e.g. the hair colour. Six participants (three from the 5 mg dosing group and three from the 10 mg dosing group) with brown hair showed higher concentrations than the remaining ten participants where seven also had brown hair and only three of the participants had blonde hair. Two of those ingested 10 mg zopiclone, which showed the two lowest zopiclone concentrations found in the 10 mg group. The last one of those ingested 5 mg zopiclone and showed the second lowest zopiclone concentration found in the 5 mg group. The same tendency was observed for N-desmethylzopiclone. The data indicate that zopiclone is better incorporated into dark hair, as zopiclone is a basic drug that binds better to dark hair because of the higher melanin content [13]. Thus, more studies are needed to obtain further knowledge of the incorporation of zopiclone and N-desmethylzopiclone into hair of different colours.
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Fig. 2. Zopiclone and N-desmethylzopiclone concentrations in hair (pg/mg) segments of 0.5 cm and 1 or 2 cm, respectively from subject #4 (A1 & A2) and #8 (B1 & B2) at the five different sampling time points after administration. T1 = 14 days after intake, T2 = 30 days after intake, T3 = 60 days after intake, T4 = 120 days after intake. S; segment. : indicates expected positive segments. Both subjects consumed 10 mg zopiclone.
Fig. 3. Box and whiskers plot showing means (X), medians (–), quantiles (25–50 %,50–75 %), whiskers (0–25 %, 75–100 %), numbers of participants, and ranges for the maximum values of zopiclone and N-desmethylzopiclone concentrations in hair (pg/mg) at four different sampling time points after single intake. Notice that the number of participants drops during the sampling time points because some subjects had too short hair. (A) Concentrations in hair from subjects who ingested a single dose of 5 mg zopiclone. (B) Concentrations in hair from subjects who ingested a single dose of 10 mg zopiclone. T1 = 14 days after intake, T2 = 30 days after intake, T3 = 60 days after intake, T4 = 120 days after intake.
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3.2.3. Comparison of concentration between the different sampling time points within and between participants The maximum concentrations for both zopiclone and N-desmethylzopiclone are described for each subject at each sampling time point. The results provided in Table 2 and Fig. 3 show that the mean maximum concentrations differ between 14 (T1) and 120 days (T4). The lowest concentrations were found 14 days after intake, and the highest concentrations were detected after 30 (T2) or 60 (T3) days. 12 of the 16 participants showed lower levels 14 days after intake than in the following sampling time points. It takes 7–10 days for the newly formed hair in the follicle to reach the scalp [16], and therefore, some positive hair may still be beneath the skin. At decrease in medians of zopiclone and metabolite concentrations from T2 and T3 to T4 was observed. As seen in Fig. 2A1 and B1, a spread within the segments was observed at the latest sampling time point. These findings support the recommendation that hair collection should be delayed 4–6 weeks after a suspected ingestion [19]. As shown in Fig. 3, the variations in the measured maximum concentrations of each sampling time points are quite wide between the participants, as demonstrated by the vertical lines in the figure, where a factor of 11–70 (median = 22) for zopiclone and 4–54 (median = 13) for N-desmethylzopiclone was observed. A smaller variation was observed within each subject, where a factor of 1–30 (median = 2) for zopiclone and 1–29 (median = 3) for N-desmethylzopiclone was obtained. 3.2.4. Comparison of drug distribution and wash-out effects during different sampling time points The distribution of analytes between segments was more spread out in the last sampling time points probably because of irregular hair growth and long-time effects of variations in hair alignment. We could measure zopiclone 4 months after a single intake of zopiclone, but a spread within the segments was observed as seriously blurred and the concentrations were also clearly reduced. Because of this, the time of intake was more difficult to determine. A clearer presentation regarding the time of intake was achieved using larger segments, as seen in Fig. 2A2 and
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B2. Here, all sampling time points show the intake in the expected segments. Positive neighbouring segments were observed in the latest sampling time point, indicating that longer sampling time points causes more drug spread out between the segments. Furthermore, the general recommendation on performing hair analysis within 6 months after an exposure is also supported by these results. As depicted in Fig. 4, no obvious wash-out effect by e.g. personal hygiene was observed from 1 (T2) and 2 (T3) month to 4 (T4) months after intake because the cumulated concentration for all segments after 1, 2 and 4 months, respectively, showed no significant differences (P > 0.05). Only one subject (#16) showed some signs of wash-out effect. Therefore, in terms of statistics a wash-out effect between the sampling time points was not significant; however, it cannot be excluded. 3.2.5. Comparison to published literature Villain et al. [14] have reported that after a single intake of 7.5 mg zopiclone (n = 2 subjects), zopiclone concentrations of 5.4 and 9.0 pg/mg were obtained in hair. Both participants had brown hair, and the hair collected after 1 month was cut into three segments of 2 cm each, where the proximal segment of both showed the highest concentration. In our study, the observed zopiclone concentrations in the hair were in general higher compared to those reported by Villain et al. [14]. Deveaux et al. [2] reported zopiclone concentrations at 1.7 and 5.3 pg/mg after the intake of 7.5 mg zopiclone (n = 2 subjects), where results only from the 5-mm proximal segment were described. The hair samples were collected 3–5 weeks after ingestion. As with Villain et al. [14], the observed zopiclone concentrations in the hair were in general higher in our study. A possible reason for the differences in the results might be due to the segment size. Deveaux et al. [2] analysed 0.5-cm segments similar to our study, and Villain et al. [14] analysed 2-cm segments. In theory, one of the 0.5-cm segments could contain a concentration of up to four times higher than that of the 2-cm segments, giving that only one of the 0.5-cm segments had the whole drug content and not the remaining three
Fig. 4. Cumulated amount of zopiclone (pg/mg) in all segments at each sampling time point for all subjects. T1 = 14 days after intake, T2 = 30 days after intake, T3 = 60 days after intake, T4 = 120 days after intake.
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S.L. Hansen et al. / Forensic Science International 306 (2020) 110074
Table 3 N-desmethylzopiclone/zopiclone ratios including range, median, relative standard division (%RSD) and number of measurements (n) for the maximum concentration of zopiclone in the four different time periods for all subjects individually and in total. *Reduced data as not all sampling time points were collected. Subject
#1
#2
#3
#4
#5
#6
#7
#8
Range Median %RSD n
1.5–2.9 2.2 – 2*
1.1–1.7 1.3 20 4
0.67–0.79 0.73 – 2*
1.9–2.4 2.0 12 4
0.57–0.73 0.63 12 4
0.90–4.6 2.1 65 4
2.1–3.2 2.8 20 3*
1.4–2.2 1.8 20 4
Subject
#9
#10
#11
#12
#13
#14
#15
#16
All
Range Median %RSD n
1.1–2.1 1.6 29 4
1.5–1.5 1.5 – 2*
0.66–1.5 1.2 31 4
0.68–0.81 0.76 7 4
0.91–1.2 1.0 11 4
0.74–0.90 0.85 8 4
0.72–0.87 0.76 9 3*
1.1–1.5 1.4 12 4
0.57–4.6 1.3 54 56
segments that would have been a part of the 2-cm segments. This relation was confirmed by our analysis of two participants segmented by 0.5 and 2.0 cm (see Fig. 2). Overall, the results that Deveaux et al. [2] and Villain et al. [14] found were around the lower end of those found in this study. An explanation could be differences in the hair preparation. Villain et al. [14] extracted cut hair, where in this study the hair was pulverized. The method in Deveaux et al. [2] were though not discribed. However, many other factors are known to contribute to the deposition of drugs in hair. As mentioned previous, hair colour, irregular hair growth, variations in alignment, and sweat-contamination can all affect the concentration in hair [12,13,16,23,24]. 3.2.6. Metabolite to parent drug ratio in hair The ratio of N-desmethylzopiclone to zopiclone varied from 0.57 to 4.6 with a median of 1.3, calculated for the maximum concentrations of zopiclone for all sampling time points in all participants (n = 56 segments). In Table 3, the ratios for each subject are provided, depicting a higher variation between all participants than within a subject, although only three participants showed a variation higher than 20 %, including subjects #4 and #8; the distributions are shown in Fig. 2. For four participants, the ratio medians were 2.0, where two had blonde hair and the other two brown. The median of the remaining 12 participants was in the range 0.57–1.8. Compared with the median ratio of 0.5 found by Irving et al. [18], higher ratio levels were observed here. However, the ratio found by Irving et al. [18] represents three segments from only one subject. Zopiclone N-oxide was quantified only in eight of the participants and only in one or two of the sampling time points. Here the ratio of zopiclone N-oxide to zopiclone ranged from 0.0012 to 0.38, with a median of 0.053 for all segments containing zopiclone N-oxide for all sampling time points (n = 31). A metabolite can be included to confirm drug intake. In this case, the metabolite N-desmethylzopiclone was positive in all relevant segments. At the same time, a small variation was observed in the metabolite to parent drug ratio in general. These qualities suggest N-desmethylzopiclone to serve as an additional marker to confirm the intake of zopiclone. Zopiclone N-oxide on the other hand is not a relevant marker for zopiclone consumption, because it was only found in half of the participants and in lower concentrations than N-desmethylzopiclone. 4. Conclusion The reference values for single-dose consumption of zopiclone in hair were established for 16 participants consuming either 5 or 10 mg zopiclone. The validated method demonstrated a high enough sensitivity to detect a single dose of zopiclone in hair. Concentrations in hair varied greatly between the participants, and no significant difference was found between the two doses for
either zopiclone or N-desmethylzopiclone. The distribution of drug concentrations in hair could be followed in detail by the small 5mm segments of the different sampling time points, and the drug findings along the hair shaft were in accordance with the expected positive segments. Sampling hair after 14 days could be too early in some cases as the concentrations were lower compared to the other sampling time points. One or two months after intake is recommended as the concentrations are the highest and the distribution is not spread out too much between the segments. No wash-out effect could be detected within the 4 months after the intake of zopiclone as the cumulated amount in segments after 30 days was similar to the amount in later sampling time points. The metabolite N-desmethylzopiclone was a good marker for the intake of zopiclone where the median metabolite to drug ratio in hair was 1.3, whereas the other metabolite zopiclone N-oxide was present only in trace levels in some participants’ hair. Author Contributions Study conception and sample collection: RK, GN. Study design: All. Acquisition of data: SLH, SSJ, MKN. Analysis and interpretation of data: All. Drafting of manuscript: All. Critical revision: All. References [1] P. Xiang, M. Shen, O.H. Drummer, Review: drug concentrations in hair and their relevance in drug facilitated crimes, J. Forensic Leg. Med. 36 (2015) 126–135, doi:http://dx.doi.org/10.1016/j.jflm.2015.09.009. [2] M. Deveaux, M. Cheze, G. Pepin, The role of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to test blood and urine samples for the toxicological investigation of drug-facilitated crimes, Ther. Drug Monit. 30 (2) (2008) 225–228, doi:http://dx.doi.org/10.1097/FTD.0b013e3181676186. [3] X. Wang, S.S. Johansen, M.K.K. Nielsen, K. Linnet, Hair analysis in toxicological investigation of drug-facilitated crimes in Denmark over a 8-year period, Forensic Sci. Int. 285 (2018) e1–e12, doi:http://dx.doi.org/10.1016/j.forsciint.2018.01.021. [4] C. Fernandez, C. Martin, F. Gimenez, R. Farinotti, Clinical pharmacokinetics of zopiclone, Clin. Pharmacokinet. 29 (6) (1995) 431–441, doi:http://dx.doi.org/ 10.2165/00003088-199529060-00004. [5] S. Noble, H.D. Langtry, H.M. Lamb, Zopiclone. An update of its pharmacology, clinical efficacy and tolerability in the treatment of insomnia, Drugs 55 (2) (1998) 277–302, doi:http://dx.doi.org/10.2165/00003495-199855020-00015. [6] D.R. Drover, Comparative pharmacokinetics and pharmacodynamics of shortacting hypnosedatives: zaleplon, zolpidem and zopiclone, Clin. Pharmacokinet. 43 (4) (2004) 227–238, doi:http://dx.doi.org/10.2165/00003088200443040-00002. [7] J. Wagner, M.L. Wagner, W.A. Hening, Beyond benzodiazepines: alternative pharmacologic agents for the treatment of insomnia, Ann. Pharmacother. 32 (6) (1998) 680–691, doi:http://dx.doi.org/10.1345/aph.17111. [8] A.N. Wadworth, D. McTavish, Zopiclone: a review of its pharmacological properties and therapeutic efficacy as an hypnotic, Drugs Aging 3 (5) (1993) 441–459, doi:http://dx.doi.org/10.2165/00002512-199303050-00006. [9] L.K. Bechtel, C.P. Holstege, Criminal poisoning: drug-facilitated sexual assault, Emerg. Med. Clin. North Am. 25 (2) (2007) 499–525, doi:http://dx.doi.org/ 10.1016/j.emc.2007.02.008.
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