TY - JOUR
AU - Kristian, Linnet,
AB - Abstract Analysis of drugs in hair differs from their analysis in other tissues due to the extended detection window, as well as the opportunity that segmental hair analysis offers for the detection of changes in drug intake over time. The antipsychotic drug chlorprothixene is widely used, but few reports exist on chlorprothixene concentrations in hair. In this study, we analyzed hair segments from 20 deceased psychiatric patients who had undergone chronic chlorprothixene treatment, and we report hair concentrations of chlorprothixene and its metabolite desmethylchlorprothixene. Three to six 1-cm long segments were analyzed per individual, corresponding to ~3–6 months of hair growth before death, depending on the length of the hair. We used a previously published and fully validated liquid chromatography-tandem mass spectrometry method for the hair analysis. The 10th–90th percentiles of chlorprothixene and desmethylchlorprothixene concentrations in all hair segments were 0.05–0.84 ng/mg and 0.06–0.89 ng/mg, respectively, with medians of 0.21 and 0.24 ng/mg, and means of 0.38 and 0.43 ng/mg. The estimated daily dosages ranged from 28 mg/day to 417 mg/day. We found a significant positive correlation between the concentration in hair and the estimated daily doses for both chlorprothixene (P = 0.0016, slope = 0.0044 [ng/mg hair]/[mg/day]) and the metabolite desmethylchlorprothixene (P = 0.0074). Concentrations generally decreased throughout the hair shaft from proximal to distal segments, with an average reduction in concentration from segment 1 to segment 3 of 24% for all cases, indicating that most of the individuals had been compliant with their treatment. We have provided some guidance regarding reference levels for chlorprothixene and desmethylchlorprothixene concentrations in hair from patients undergoing long-term chlorprothixene treatment. Introduction Chlorprothixene, a first generation antipsychotic, was first used clinically in 1962 and is still widely prescribed, with recommended daily doses ranging from 20 to 600 mg (1). The therapeutic blood concentration of chlorprothixene ranges from 0.02 to 0.3 mg/L, and the toxic concentration is 0.4 mg/L or higher (2–4). Chlorprothixene can be lethal, typically when the blood concentration is 0.8 mg/L or above (1, 2). Several fatalities have been reported with chlorprothixene present as the only drug in blood (4). Chlorprothixene is metabolized via N-demethylation, N-oxidation, sulphoxidation and ring hydroxylation (1). The elimination half-life of chlorprothixene in blood is ~8–12 h, but this can be markedly increased in older individuals (1, 5). Chlorprothixene is not likely to undergo major postmortem redistribution in femoral blood (4). Patients in antipsychotic treatment have an increased risk of death when compared with the background population (6). Poor compliance behavior has long been associated with antipsychotic therapy due to its severe adverse effects. The adverse effects from chlorprothixene therapy are dry mouth, confusion, tachycardia, urinary retention and constipation (1, 5, 7). Compliance can be determined by measurement of the intake of a prescribed drug through blood or urine sampling, but the hair matrix is another source that provides a longer and more retrospective window on drug use (8, 9). Postmortem segmental hair analysis is particularly beneficial when looking back in time is desired, as it allows the study of drug exposure over an extended time period up to the time of death. Reference values for drugs in hair are valuable in forensic toxicology and for assessment of drug compliance. Several studies regarding antipsychotic drug concentrations in hair have been published (9–13), including three studies that among others reported chlorprothixene concentrations in hair. For example, Shen et al. reported a chlorprothixene concentration of 30 ng/mg in the hair of a psychiatric patient treated daily with 50 mg chlorprothixene (9). Two other publications reported chlorprothixene concentrations in hair but without dose information (14, 15). Nielsen et al. (14) found chlorprothixene in hair in two deceased drug addicts, one with a concentration of 1.1 ng/mg and one where the concentration was below the lower limit of quantification (LLOQ) of 0.05 ng/mg. Chlorprothixene has been reported by Wang et al. in a drug-facilitated sexual assault case, where 0.006 ng/mg chlorprothixene and 0.009 ng/mg desmethylchlorprothixene were detected in the victim’s hair (15). In the present study, concentrations of chlorprothixene were examined in hair segments from 20 postmortem cases. Our aim was to obtain knowledge on postmortem chlorprothixene concentrations in hair and to study the relation between the administered dose, postmortem blood concentrations and hair concentrations. To our knowledge, this is the first study to report segmental analysis of both chlorprothixene and desmethylchlorprothixene in hair. Experimental Chemicals and reagents Chlorprothixene was purchased from Sigma-Aldrich (St Louis, USA) (99% purity). Desmethylchlorprothixene was kindly donated by H. Lundbeck A/S (Copenhagen, DK). The purity of desmethylchlorprothixene was 96%. Amitriptyline-d6 was used as internal standard for both chlorprothixene and desmethylchlorprothixene and was purchased from C/D/N Isotopes Inc. (Quebec, CA) (99% purity). Methanol, acetonitrile and water were of LC–MS grade obtained from Fisher Scientific (Leicestershire, UK). Ammonium formate was from Fluka (Buchs, CH) (≥97% purity), and formic acid (98–100%) was obtained from Merck (Darmstadt, DE). Postmortem cases Cases were selected from the ‘SURVIVE’ project population: a Danish National forensic autopsy-based study of deceased individuals with diagnosed or suspected mental illness collected in the period from 2013 to 2015 (16). The ‘SURVIVE’ study was approved by the Danish National Committee on Research Ethics (reference number: 1305373) and the Danish Data Protection Agency (reference number: SUND-2016-16). Consent for this research was collected from the next of kin for each subject. Cases (n = 20) that showed chlorprothixene in postmortem blood and/or that had a medical report suggesting chlorprothixene treatment were selected for analysis if head hair was also available. Apparatus Analysis was performed by chromatographic separation using an ACQUITY ultra-high performance liquid chromatography (UHPLC) system (Waters Corporation, Milford, USA) with an ACQUITY UPLC® HSS C18 150 mm × 2.1 mm, 1.8-μm column and detection was performed on a Waters® TQ Detector tandem quadrupole spectrometer (Waters Corporation, Milford, USA) with electrospray ionization in the positive mode. Methods Hair extraction A previously published extraction method by Montesano et al. was used with minor modifications (17). The extraction medium volume was increased from 200 to 500 μL, as this study concerned chronic consumers. Briefly, hair samples were aligned and cut into 1-cm segments (double determinations). Three to six segments were analyzed per individual, depending on the hair length. Segment 1 (S1) was the segment closest to the scalp. Approximately 10 mg of hair was weighed into a Precellys tube (Bertin Technologies, Montigny-le-Bretonneux, FR) and decontaminated by washing once with 1 mL 2-propranol, twice with 500 μL purified water and then once more with 1 mL 2-propanol. Each washing step lasted 5 min including centrifugation. The last aqueous wash was analyzed to check for external contamination. After drying overnight, six steel beads and 500 μL extraction medium consisting of methanol, acetonitrile and 2 mM ammonium formate (25:29:46, v/v/v) were added. The hair was pulverized (4 × 30 s at 6,500 rpm) using a Precellys 24 ball mill (Bertin Technologies) and incubated overnight at 37°C. The extracts were filtered and diluted 1:1 with water before analysis. The injection volume was 10 μL. UHPLC–MS-MS The analytes were detected by multiple reaction monitoring with two transitions: a quantifier and a qualifier. The transitions were m/z 316 > 231, collision energy 33 eV, cone voltage 43 V (quantifier), m/z 316 > 271, collision energy 20 eV, cone voltage 43 V (qualifier) for chlorprothixene and m/z 302 > 271, collision energy 19 eV, cone voltage 37 V (quantifier), m/z 302 > 221, collision energy 35 eV, cone voltage 37 V (qualifier) for desmethylchlorprothixene. The amitriptyline-d6 internal standard was measured at one transition m/z 284 > 91 with collision energy 25 eV and cone voltage of 37 V. Analytical procedures and parameters were published previously by Montesano et al. (17). Validation The method was validated previously for single-dose studies (17). The extraction medium volume was increased by a factor of 2.5, as this study concerns chronic consumers. The limit of detection (LOD), LLOQ and upper limit of quantification (ULOQ) were therefore re-evaluated, and desmethylchlorprothixene was added to the method and validated according to Society of Hair Testing guidelines (18) and Peters et al. (19). Bias and precision was evaluated in eight replicates and general acceptance criteria were bias in the range 80–120% near LLOQ and 85–115% above LLOQ and precision with maximum values of 20% near LLOQ and 15% above LLOQ. The LOD was 0.01 ng/mg, and the linear range from LLOQ to ULOQ was 0.025–25 ng/mg for both analytes. The precision for desmethylchlorprothixene was 14% at LLOQ and maximum 13% above LOQ, and the bias was 78% at LLOQ and 80% above LLOQ and so bias was slightly outside the limit. This was accepted on the basis of the satisfactory results of the control hair samples (Table I). The intermediate precision of soaked control hair samples, analyzed in 14 series over 5 months, was below 20% for both analytes at low and high levels (see Table I). Table I. Intermediate precision of soaked control hair samples analyzed in 14 series over 5 months QC low (ng/mg) Intermediate precision (%) QC high (ng/mg) Intermediate precision (%) Chlorprothixene 0.22 11.7 7.0 10.7 Desmethylchlorprothixene 0.30 18.4 11 19.3 QC low (ng/mg) Intermediate precision (%) QC high (ng/mg) Intermediate precision (%) Chlorprothixene 0.22 11.7 7.0 10.7 Desmethylchlorprothixene 0.30 18.4 11 19.3 Table I. Intermediate precision of soaked control hair samples analyzed in 14 series over 5 months QC low (ng/mg) Intermediate precision (%) QC high (ng/mg) Intermediate precision (%) Chlorprothixene 0.22 11.7 7.0 10.7 Desmethylchlorprothixene 0.30 18.4 11 19.3 QC low (ng/mg) Intermediate precision (%) QC high (ng/mg) Intermediate precision (%) Chlorprothixene 0.22 11.7 7.0 10.7 Desmethylchlorprothixene 0.30 18.4 11 19.3 Analysis of postmortem femoral blood Femoral blood samples were taken at the beginning of the autopsy. The samples were preserved in 1% sodium fluoride and stored at −20°C until analysis. A previously published method was used for quantification (20). The linear ranges were 0.002–1.0 mg/kg for chlorprothixene and 0.005–0.50 mg/kg for desmethylchlorprothixene (not included in the previous publication). Sample preparation was performed by solid-phase extraction with a fully automated robotic system. Chromatographic separation was performed using an UHPLC system (Waters Corporation, Milford, USA) with an ACQUITY UPLC® BEH C18 100 mm × 2.1 mm, 1.7-μm column and detection was performed on a Waters® TQ Detector tandem quadrupole spectrometer (Waters Corporation, Milford, USA) with electrospray ionization in the positive mode equivalent to the detection of chlorprothixene in hair. Statistical analysis Statistical analysis was performed using the statistical software R version 3.4.0. Correlations were evaluated by using the squared Pearson correlation coefficient (R2). Estimated doses The project has permission from the Danish Data Protection Agency (reference number: SUND-2017–30) to use the Danish medicinal drug register, which contains data from all Danish pharmacies and hospital pharmacies, to extract data for some specified pharmaceutical drugs for up to 3 years before time of death. We extracted data on chlorprothixene prescriptions for each individual. The prescribed doses were not registered, therefore daily chlorprothixene doses were estimated by multiplying the number of tablets collected times the chlorprothixene amount in the tablet. This was averaged for the last 6 months prior to death. Results and discussion Subject characteristics We present results for 20 chlorprothixene-positive hair samples. The individuals were 12 men and 8 women, aged 23–68 years, with a median age of 46 years. All individuals suffered from psychiatric illnesses, such as schizophrenia, mental and behavioral disorder, anxiety and depression. Causes of death were drug poisoning in 12 cases, natural in 6 cases (disease of the circulatory system, disease of the digestive system, infection or malignant neoplasm), trauma in 1 case and a combination of natural causes and poisoning in 1 case. For 80% of the individuals, substances of abuse were found in hair and/or postmortem femoral blood, or they were registered as substance abusers: seven abused illicit drugs, three abused illicit drugs and medicine, three abused illicit drugs and alcohol, two abused alcohol and one abused alcohol and medicine. All the hair samples contained additional drugs other than chlorprothixene. The relative frequencies of drug classes among the investigated cases were: 85% opioids, 80% benzodiazepines, 70% antidepressants, 50% illicit drugs (amphetamine, ketamine, cocaine, benzoylecgonine or 6-monoacetylmorphine), 50% other antipsychotics, 50% z-hypnotics, 35% anti-epileptics and 35% sedating antihistamines. Three individuals were assessed as having dyed or bleached their hair, based on color changes along the hair shaft and discoloration of the wash fractions. We found no correlation between age and the concentration in hair. Furthermore, no difference in the concentration in hair for men and women or for pigmented and non-pigmented hair was found. We expected increased concentrations of chlorprothixene in pigmented hair compared with non-pigmented hair, as Rothe et al. found significant variation between white and pigmented hair with a white/pigmented hair ratio of 0.5 for the same individual (n = 1) (21). Sato et al. also found much lower concentrations of chlorpromazine in white hair, with <10% of the concentration in black hair for the same individual (n = 5) (22). Chlorprothixene and chlorpromazine are structurally similar, differing only by a substitution of a carbon atom carrying an exocyclic double bond for a nitrogen in the central ring. However, in our study, the cohort was small and the individuals’ hair color had little variation, therefore no trends could be discerned. Chlorprothixene concentrations in hair The chlorprothixene concentrations in all hair segments of each individual are illustrated in Figure 1. The 10th–90th percentiles of chlorprothixene concentrations in all hair segments were 0.05–0.84 ng/mg, with a median of 0.21 ng/mg and a mean of 0.38 ng/mg. Higher chlorprothixene concentrations were generally observed in the proximal segment (S1), with 10th–90th percentiles of 0.15–1.5 ng/mg, a median of 0.36 ng/mg and mean of 0.57 ng/mg. The mean concentrations of chlorprothixene for each hair segment for all 20 individuals, in the order from the proximal segment (S1) to the distal segment (S6), were: 0.57, 0.42, 0.34, 0.33, 0.25 and 0.24 ng/mg (median 0.36, 0.21, 0.20, 0.18, 0.14 and 0.13 ng/mg). The mean decreases in concentration were 24, 24, 30, 44 and 48% from the proximal to distal segments from S1 to S2–S6. The 10th–90th percentiles of desmethylchlorprothixene concentrations in all hair segments were 0.06–0.89 ng/mg, with a median of 0.24 ng/mg and mean of 0.43 ng/mg. The mean concentrations of desmethylchlorprothixene in hair for each segment for all 20 individuals, in the order from the proximal segment (S1) to the distal segment (S6), were 0.60, 0.43, 0.36, 0.34, 0.26 and 0.24 ng/mg (median 0.33, 0.21, 0.22, 0.23, 0.17 and 0.20 ng/mg), and so the decrease throughout the segments was analogous to that of chlorprothixene. Additionally, the mean ratios of chlorprothixene to desmethylchlorprothixene were 1.27, 1.43, 1.41, 1.56, 1.10 and 1.07 from S1 to S6, with ratios of the 10th–90th percentiles of 0.66–2.43 for all segments for a median ratio of 1.07; the ratio throughout the hair segments for each individual varied only slightly. The concentration of chlorprothixene and desmethylchlorprothixene in the last aqueous wash was negligible in all cases. Figure 1. View largeDownload slide Chlorprothixene concentrations in all hair segments for each individual, 3–6 hair segments per individual. S1–S3:2, 7, 8, 10, 16. S1–S4:1, 9, 11. S1–S5:4, 14. S1–S6:3, 5, 6, 12, 13, 15, 17, 18, 19, 20. Figure 1. View largeDownload slide Chlorprothixene concentrations in all hair segments for each individual, 3–6 hair segments per individual. S1–S3:2, 7, 8, 10, 16. S1–S4:1, 9, 11. S1–S5:4, 14. S1–S6:3, 5, 6, 12, 13, 15, 17, 18, 19, 20. These results are in line with the previously reported concentration of 1.1 ng/mg in the hair of a deceased drug addict (14). All concentrations were above the concentration of 0.006 ng/mg previously reported after a single-dose drug-facilitated sexual assault (15). This finding was expected, as all individuals in this study had been undergoing chronic chlorprothixene treatment. Both reference cases were previously reported from our lab following the same extraction method used in this study. Our results are much lower than those from one case reported by Shen et al., who found a chlorprothixene concentration of 30 ng/mg in the hair of a psychiatric patient treated with 50 mg chlorprothixene per day (9). This difference may have arisen due to variations in extraction and analysis methods, as well as in hair color and treatment for a Chinese population compared with a Danish population. Kidwell et al. describe how cultural bias can give rise to significantly different concentrations in hair in people taking the same drug dose due to differences in hair color as well as in personal habits like hair care and hygiene, which can vary among cultural groups (23). Daily dose of chlorprothixene The average daily dose over the last 6 months before death could be estimated for 13 individuals. Nine were men and four were women. Doses could not be estimated for the remaining seven individuals, as prescription data were not available or the individual had collected chlorprothixene only once. The estimated dosages were from 28 to 417 mg/day, with a median of 72 mg/day and a mean of 128 mg/day. Thus, the doses were within the recommended daily dose ranges of 20–600 mg for all individuals (1). We defined low-, medium- and high-dose groups based on the estimated doses. The ranges of chlorprothixene concentrations in hair S1 were 0.06–0.49 ng/mg at low doses (28–52 mg/day, n = 6); 0.23–0.65 ng/mg at medium doses (72–154 mg/day, n = 4) and 1.29–2.03 ng/mg at high doses (244–417 mg/day, n = 3). We investigated the correlation between the estimated daily dose and the concentration of chlorprothixene in hair S1 (Figure 2). A significant positive correlation between dose and concentration was observed for both chlorprothixene and desmethylchlorprothixene in the 13 individuals in which the dosage could be estimated (chlorprothixene: R2 = 0.74, P = 0.0016, slope = 0.0044 [ng/mg hair]/[mg/day]; desmethylchlorprothixene: R2 = 0.49, P = 0.0074, slope = 0.0057 [ng/mg hair]/[mg/day]). We used the concentration in S1 in the calculation because of the varied decreases in concentration from the proximal to the distal segments. Positive significant correlations were also observed between the estimated dose and the mean concentration of S1–S3 (chlorprothixene: R2 = 0.68, P = 0.0005, slope = 0.0032 [ng/mg hair]/[mg/day]; desmethylchlorprothixene: R2 = 0.45, P = 0.0169, slope = 0.0039 [ng/mg hair]/[mg/day]). Therefore, a trend towards an increased concentration in hair when the daily dose is increased seemed evident. Figure 2. View largeDownload slide Correlation between the estimated daily dose and the concentrations of chlorprothixene and desmethylchlorprothixene in hair segment 1 (S1) for each individual (n = 13). Chlorprothixene: R2 = 0.74, P = 0.0016, slope = 0.0044 (ng/mg hair)/(mg/day), intercept = 0.0997 ng/mg. Desmethylchlorprothixene: R2 = 0.49, P = 0.0074, slope = 0.0057 (ng/mg hair)/(mg/day), intercept = 0.0186 ng/mg. Figure 2. View largeDownload slide Correlation between the estimated daily dose and the concentrations of chlorprothixene and desmethylchlorprothixene in hair segment 1 (S1) for each individual (n = 13). Chlorprothixene: R2 = 0.74, P = 0.0016, slope = 0.0044 (ng/mg hair)/(mg/day), intercept = 0.0997 ng/mg. Desmethylchlorprothixene: R2 = 0.49, P = 0.0074, slope = 0.0057 (ng/mg hair)/(mg/day), intercept = 0.0186 ng/mg. From the slope in Figure 2, we estimated an average increase of 0.0044 ng/mg hair when the daily dose of chlorprothixene was increased by 1 mg. We observed large variations in the concentration in hair for individuals treated with equal doses, making determination of a daily dosage impossible based on the concentration measured in hair. This concurs with previous observations by others who have studied the relationship between drug dose and concentration in hair for other drugs (26, 29–31). Inter-individual variation may arise from variations in hair color, physical hair treatment, hair growth rate, hair structure, sex, age, ethnicity, drug metabolism, etc. and these variables should be considered when interpreting drug concentrations measured in hair (24, 29). Chlorprothixene distribution in hair segments suggesting constant intake In 17 cases, the concentrations in hair either generally decreased from the proximal to distal segments, or remained constant throughout the hair segments, suggesting no change in chlorprothixene intake within the last 3–6 months before death (24). The chlorprothixene concentrations in each segment for these 17 cases are illustrated in Figure 3. In the remaining three cases, discussed later, the segmental analysis indicated a change in chlorprothixene intake within the last 6 months before death. The mean decrease in chlorprothixene concentration for each hair segment for the 17 individuals with presumed constant intake were 21, 20, 31, 40 and 43%, in the order of proximal to distal segments from S1 to S2–S6, corresponding to an average linear decline rate of 0.087 (ng/mg)/segment. Figure 3. View largeDownload slide Chlorprothixene concentrations in all hair segments for individuals with constant or declining chlorprothixene concentrations from proximal to distal segments (S1–S6), suggesting unchanged chlorprothixene intake (n = 17). Figure 3. View largeDownload slide Chlorprothixene concentrations in all hair segments for individuals with constant or declining chlorprothixene concentrations from proximal to distal segments (S1–S6), suggesting unchanged chlorprothixene intake (n = 17). Decreases in concentration from proximal to distal segments from the washout effect and physical treatment of the hair are well known and are not necessarily a sign of non-compliance (18, 25, 26). For example, Drooger et al. observed a steep linear decrease of 54% from S1 to S3 for patients receiving the same constant dose of tamoxifen and attributed the decrease to effects of hair washing and UV exposure (27). Chlorprothixene is also sensitive to light, so UV exposure could contribute to the decrease we observed in this study (28). However, Kronstrand et al. defined a loss of more than 33% from S1 (0–1 cm) to S3 (2–3 cm) as indicating non-compliance when reporting concentrations of methamphetamine in hair from individuals undergoing long-term selegiline treatment (26). No general agreement exists regarding the loss of drug throughout segments. In our study, the mean decrease from S1 to S3 was 24% but in 9 of the 17 cases, we observed a decrease of 34–69%, although we believe these subjects had unchanged chlorprothixene intake. These nine cases are marked with dashed lines in Figure 2. Steep decreases were observed in all cases where the concentration in S1 exceeded 1 ng/mg, as well as in five cases where the concentration in S1 was low. The results from our study suggest the prevalence of a decrease in chlorprothixene concentration from the proximal to the distal segments in hair. Chlorprothixene distribution in hair segments indicating changes in intake Inconsistent chlorprothixene concentrations throughout the segments, which we believed to indicate changes in chlorprothixene intake, were observed in three cases (Figure 4). Two of these cases (case nos 8 and 12) showed a steep decrease from S1 to S2, followed by a stable plateau, indicating changes to a higher intake shortly before death. It was evident from the drug register that these two individuals started treatment <50 days before death. Thus, S3–S6 corresponded to a period before they started treatment. The chlorprothixene concentrations were more than nine times lower in S3–S6 (0.03–0.05 ng/mg) when compared with S1 (0.37–0.38 ng/mg) in both cases. The concentrations measured in each segment of these cases are illustrated in Figure 4. Figure 4. View largeDownload slide Chlorprothixene concentrations in all hair segments for individuals with concentrations indicating a change in chlorprothixene intake in the last 6 months before death. Figure 4. View largeDownload slide Chlorprothixene concentrations in all hair segments for individuals with concentrations indicating a change in chlorprothixene intake in the last 6 months before death. This internal contamination of the distal segments might be caused by sweat/sebum or physical treatment of the hair and represents an example of the complicated nature of data interpretation from hair analysis. In a previous study of quetiapine in hair, we saw the same degree of internal contamination of distal segments for two individuals who had started quetiapine treatment less than a month before death (32). In the present two cases, we did not detect the metabolite desmethylchlorprothixene in segments 3–6, whereas the metabolite was detected above the LOD in all segments in all other cases. Even in one case (case no. 18), where the chlorprothixene concentrations throughout segments were 0.04–0.06 ng/mg (i.e., the same low level as the distal segments in these two cases), we detected the metabolite in all segments, indicating a long-term regular intake of chlorprothixene, which was also confirmed by data from the drug register. Case no. 11 showed an increase in the concentration of chlorprothixene in hair from the proximal to distal segment, indicating non-regular intake or changes to a lower intake within the last 4 months before death (24). These examples illustrate that segmental hair analysis is useful because the patients serve as their own controls. Thus, the development of concentrations throughout the hair segments can be more informative than the actual concentration in the hair. Chlorprothixene and desmethylchlorprothixene in postmortem femoral blood The 10th–90th percentiles of chlorprothixene concentrations in postmortem femoral blood were 0.007–0.10 mg/L, with a median of 0.045 mg/L and a mean of 0.055 mg/L for chlorprothixene. In six cases, the concentrations in blood were below the lower limit commonly observed during therapy, at 0.02 mg/L (2, 4), and in one case (case no. 7) neither chlorprothixene nor desmethylchlorprothixene were detected in blood, but both substances were detected in hair. The concentrations were below the commonly regarded toxic level of 0.4 mg/L in all cases (3, 4). Desmethylchlorprothixene was detected below 0.005 (LLOQ) in two cases. The 10th–90th percentiles of desmethylchlorprothixene in blood were 0.01–0.099 mg/L, with a median of 0.021 mg/L and a mean of 0.046 mg/L. The ratios between the drug and metabolite in blood in the 10th–90th percentiles ranged from 0.56 to 2.9, with a median of 1.2 and a mean of 1.7, which is the same order of magnitude as reported by Skov et al. (4). The correlation between the concentrations of chlorprothixene and desmethylchlorprothixene in hair (S1) and their respective concentrations in postmortem femoral blood were not significant (P = 0.097 and P = 0.093 for chlorprothixene and desmethylchlorprothixene, respectively). This could be explained by the variation in the time between intake and blood sampling for the individuals, as the timing of the last drug dose before death was unknown. Conclusion We presented concentrations of chlorprothixene and desmethylchlorprothixene in 3–6 hair segments from 20 deceased psychiatric patients who had undergone chronic chlorprothixene treatment. A trend towards increased chlorprothixene and desmethylchlorprothixene concentrations in hair when the chlorprothixene dose is increased is evident from these results. Furthermore, our results indicate that chlorprothixene concentration decreases steadily in hair from proximal to distal segments for individuals who are chronically treated with unchanged chlorprothixene doses for several months. Lastly, our results show that distal segments can be internally contaminated with chlorprothixene but not with the metabolite desmethylchlorprothixene. These findings contribute new information to the field of hair analysis by reporting reference concentrations for chlorprothixene and desmethylchlorprothixene in hair, and they illustrate how segmental hair analysis can add information regarding chronic drug intake over time. Acknowledgments The authors would like to acknowledge the Forensic Technicians for sampling the hair during the autopsies. Funding This study received Danish Governmental seed-money by Satspuljen (7-604-10-11/5 KAD, 3-3019-5/2 KAD). References 1 Baselt , R.C. Disposition of Toxic Drugs and Chemicals in Man , 11th ed. Biomedical Publications : Seal Beach, CA , 2017 . 2 Schulz , M. , Schmoldt , A. ( 2003 ) Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics . Die Pharmazie , 58 , 447 – 474 . Google Scholar PubMed 3 Winek , C.L. , Wahba , W.W. , Winek , C.L. , Balzer , T.W. ( 2001 ) Drug and chemical blood-level data 2001 . Forensic Science International , 122 , 107 – 123 . Google Scholar Crossref Search ADS PubMed 4 Skov , L. , Johansen , S.S. , Linnet , K. ( 2015 ) Postmortem femoral blood reference concentrations of aripiprazole, chlorprothixene, and quetiapine . Journal of Analytical Toxicology , 39 , 41 – 44 . Google Scholar Crossref Search ADS PubMed 5 Käferstein , H. , Sticht , G. , Madea , B. ( 2013 ) Chlorprothixene in bodies after exhumation . Forensic Science International , 229 , 30 – 34 . Google Scholar Crossref Search ADS 6 Tiihonen , J. , Lönnqvist , J. , Wahlbeck , K. , Klaukka , T. , Niskanen , L. , Tanskanen , A. , et al. . ( 2009 ) 11-year follow-up of mortality in patients with schizophrenia: a population-based cohort study (FIN11 study) . The Lancet , 374 , 620 – 627 . Google Scholar Crossref Search ADS 7 Awad , A.G. , Voruganti , L.N.P. ( 2004 ) New antipsychotics, compliance, quality of life, and subjective tolerability—are patients better off? Canadian Journal of Psychiatry , 49 , 297 – 302 . Google Scholar Crossref Search ADS PubMed 8 Kintz , P. , Villain , M. , Cirimele , V. ( 2006 ) Hair analysis for drug detection . Therapeutic Drug Monitoring , 28 , 442 – 446 . Google Scholar Crossref Search ADS PubMed 9 Shen , M. , Xiang , P. , Wu , H. , Shen , B. , Huang , Z. ( 2002 ) Detection of antidepressant and antipsychotic drugs in human hair . Forensic Science International , 126 , 153 – 161 . Google Scholar Crossref Search ADS PubMed 10 Matsuno , H. , Uematsu , T. , Nakashima , M. ( 1990 ) The measurement of haloperidol and reduced haloperidol in hair as an index of dosage history . British Journal of Clinical Pharmacology , 29 , 187 – 194 . Google Scholar Crossref Search ADS PubMed 11 Cirimele , V. , Kintz , P. , Gosselin , O. , Ludes , B. ( 2000 ) Clozapine dose–concentration relationships in plasma, hair and sweat specimens of schizophrenic patients . Forensic Science International , 107 , 289 – 300 . Google Scholar Crossref Search ADS PubMed 12 Binz , T.M. , Yegles , M. , Schneider , S. , Neels , H. , Crunelle , C.L. ( 2014 ) Time resolved analysis of quetiapine and 7-OH-quetiapine in hair using LC/MS-MS . Forensic Science International , 242 , 200 – 203 . Google Scholar Crossref Search ADS PubMed 13 Schneider , S. , Sibille , E. , Yegles , M. , Neels , H. , Wennig , R. , Muhe , A. , et al. . ( 2009 ) Time resolved analysis of risperidone and 9-hydroxy-risperidone in hair using LC/MS-MS . Journal of Chromatography. B: Analytical Technologies in the Biomedical and Life Sciences , 877 , 2589 – 2592 . Google Scholar Crossref Search ADS 14 Nielsen , M.K. , Johansen , S.S. , Dalsgaard , P.W. , Linnet , K. ( 2010 ) Simultaneous screening and quantification of 52 common pharmaceuticals and drugs of abuse in hair using UPLC-TOF-MS . Forensic Science International , 196 , 85 – 92 . Google Scholar Crossref Search ADS PubMed 15 Wang , X. , Johansen , S.S. , Nielsen , M.K.K. , Linnet , K. ( 2018 ) Hair analysis in toxicological investigation of drug-facilitated crimes in Denmark over a 8-year period . Forensic Science International , 2018 . doi:10.1016/j.forsciint.2018.01.021 . 16 Banner , J. ( 2013 ) Survive: let the dead help the living . Scandinavian Journal of Forensic Science , 19 , 2 – 2 . 17 Montesano , C. , Johansen , S.S. , Nielsen , M.K. ( 2014 ) Validation of a method for the targeted analysis of 96 drugs in hair by UPLC-MS/MS . Journal of Pharmaceutical and Biomedical Analysis , 88 , 295 – 306 . Google Scholar Crossref Search ADS PubMed 18 Cooper , G.A. , Kronstrand , R. , Kintz , P. ( 2012 ) Society of Hair Testing guidelines for drug testing in hair . Forensic Science International , 218 , 20 – 24 . Google Scholar Crossref Search ADS PubMed 19 Peters , F.T. , Drummer , O.H. , Musshoff , F. ( 2007 ) Validation of new methods . Forensic Science International , 165 , 216 – 224 . Google Scholar Crossref Search ADS PubMed 20 Nielsen , M.K.K. , Nedahl , M. , Johansen , S.S. , Linnet , K. ( 2018 ) Validation of a fully automated solid-phase extraction and ultra-high-performance liquid chromatography-tandem mass spectrometry method for quantification of 30 pharmaceuticals and metabolites in post-mortem blood and brain samples . Drug Testing and Analysis , 2018 . doi:10.1002/dta.2359 . 21 Rothe , M. , Pragst , F. , Thor , S. , Hunger , J. ( 1997 ) Effect of pigmentation on the drug deposition in hair of grey-haired subjects . Forensic Science International , 84 , 53 – 60 . Google Scholar Crossref Search ADS PubMed 22 Sato , H. , Uematsu , T. , Yamada , K. , Nakashima , M. ( 1993 ) Chlorpromazine in human scalp hair as an index of dosage history: comparison with simultaneously measured haloperidol . European Journal of Clinical Pharmacology , 44 , 439 – 444 . Google Scholar Crossref Search ADS PubMed 23 Kidwell , D.A. , Lee , E.H. , DeLauder , S.F. ( 2000 ) Evidence for bias in hair testing and procedures to correct bias . Forensic Science International , 107 , 39 – 61 . Google Scholar Crossref Search ADS PubMed 24 Pragst , F. , Balikova , M.A. ( 2006 ) State of the art in hair analysis for detection of drug and alcohol abuse . Clinica Chimica Acta , 370 , 17 – 49 . Google Scholar Crossref Search ADS 25 Psillakis , T. , Tsatsakis , A.M. , Christodoulou , P. , Michalodimitrakis , M. , Paritsis , N. , Helidonis , E. ( 1999 ) Carbamazepine levels in head hair of patients under long-term treatment: a method to evaluate the history of drug use . Journal of Clinical Pharmacology , 39 , 55 – 67 . Google Scholar Crossref Search ADS PubMed 26 Kronstrand , R. , Ahlner , J. , Dizdar , N. , Larson , G. ( 2003 ) Quantitative analysis of desmethylselegiline, methamphetamine, and amphetamine in hair and plasma from Parkinson patients on long-te . Journal of Analytical Toxicology , 27 , 135 – 141 . Google Scholar Crossref Search ADS PubMed 27 Drooger , J.C. , Jager , A. , Lam , M.H. , den Boer , M.D. , Sleijfer , S. , Mathijssen , R.H.J. , et al. . ( 2015 ) Development and validation of an UPLC-MS/MS method for the quantification of tamoxifen and its main metabolites in human scalp hair . Journal of Pharmaceutical and Biomedical Analysis , 114 , 416 – 425 . Google Scholar Crossref Search ADS PubMed 28 Kopelent-Frank , H. , Mittlböck , M. ( 1996 ) Stability-indicating high-performance liquid chromatographic assay for the simultaneous determination of dixyrazine and chlorprothixene in intravenous admixtures . Journal of Chromatography. A , 729 , 201 – 206 . Google Scholar Crossref Search ADS 29 Tracqui , A. , Kintz , P. , Mangin , P. ( 1995 ) Hair analysis: a worthless tool for therapeutic compliance monitoring . Forensic Science International , 70 , 183 – 189 . Google Scholar Crossref Search ADS PubMed 30 Williams , J. , Patsalos , P.N. , Mei , Z. , Schapel , G. , Wilson , J.F. , Richens , A. ( 2001 ) Relation between dosage of carbamazepine and concentration in hair and plasma samples from a compliant inpatient epileptic population . Therapeutic Drug Monitoring , 23 , 15 – 20 . Google Scholar Crossref Search ADS PubMed 31 Pirro , V. , Fusari , I. , Di Corcia , D. , Gerace , E. , De Vivo , E. , Salomone , A. , et al. . ( 2014 ) Hair analysis for long-term monitoring of buprenorphine intake in opiate withdrawal . Therapeutic Drug Monitoring , 36 , 796 – 807 . Google Scholar Crossref Search ADS PubMed 32 Günther , K.N. , Johansen , S.S. , Nielsen , M.K.K. , Wicktor , P. , Banner , J. , Linnet , K. ( 2018 ) Post-mortem quetiapine concentrations in hair segments of psychiatric patients—correlation between hair concentration, dose and concentration in blood . Forensic Science International , 285 , 58 – 64 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Segmental Analysis of Chlorprothixene and Desmethylchlorprothixene in Postmortem Hair
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bky038
DA - 2018-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/segmental-analysis-of-chlorprothixene-and-desmethylchlorprothixene-in-l1cpRsEO30
SP - 642
VL - 42
IS - 9
DP - DeepDyve
ER -