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O22: Quantification of 22 synthetic cannabinoids and 10 metabolites in human hair
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Wednesday 11 June 2004 / Toxicologie Analytique & Clinique (2014) 26, S4-S14
O22
Quantification of 22 synthetic cannabinoids and 10 metabolites in human hair A. Salomone (1), E. Gerace (1), C. Luciano (1), D. DI Corcia (1), M. Vincenti (1, 2) (1) Centro regionale antidoping e di tossicologia “A. Bertinaria”, Orbassano (TO), Italy; (2) Università degli studi di Torino, dipartimento di chimica, Turin, Italy. Introduction. – Synthetic cannabinoids are the most common drugs among an expanding array of compounds that mimic the effects of traditional illicit psychoactive substances. A few methods have been developed for the determination of the parent synthetic cannabinoids in the keratin matrix, opening the scientific debate about how to interpret the quantitative results. In particular, the discrimination between (i) passive or active exposure, and (ii) chronic consumption and occasional use, appears to be essential within the forensic context. Just as occurred for the traditional drugs of abuse, the detection of drug metabolites is likely to represent the key factor to support the decision procedure. Therefore, we updated our existing method [A. Salomone et al., Drug Testing and Analysis, 2014, 6, 126-134] in order to complement the determination of 22 synthetic cannabinoids in human hair with 10 of their metabolites. Afterwards, we applied the new method to 15 real hair samples which previously tested positive for at least one synthetic cannabinoid [A. Salomone et al., cited], in order to verify the presence of metabolites. Methods. – The analytical method was validated in accordance with the criteria and recommendations of national and international guidelines. The following parameters were investigated: selectivity, specificity, linearity range, detection and quantification limits (LOD and LOQ), intra-assay and inter-assay precision and accuracy. Carry-over effect, recovery and matrix effects were also investigated. Two groups of subjects, namely driving re-licensing and drug abuse/withdrawal control subjects, provided hair samples that had previously resulted positive to at least one common drug of abuse and one synthetic cannabinoid. In this procedure, the samples were added with 1 mL NaOH 1N and subsequently incubated at 95 °C for 10 min. Immediately afterwards, the samples were extracted with 5 mL of n-hexane/ethylacetate 90:10 (v/v). The aqueous phase was newly added with glacial CH3COOH to reach a pH of 3.5-4.0 and again extracted with n-hexane/ethylacetate 90:10. The two organic phases were unified, dried under a nitrogen flow at 70 °C and reconstituted with 50 L of methanol. An aliquot of 1 μL methanol solution was directly injected into the UHPLC-MS/MS system. Results. – The optimized UHPLC – MS/MS method allowed the simultaneous determination of 22 synthetic cannabinoids, 10 metabolites and 6 internal standards. The whole chromatographic run, comprehensive of the time required for column re-equilibration before the following injection, was completed in 8.0 min. Retention times ranged between 2.2 min (WIN 48,098) and 5.5 min (JWH020). Two samples out of 15 resulted positive to one metabolite, namely the N-(5-hydroxypentyl) metabolite of JWH-122. The calculated concentrations were 2.5 pg/mg and 0.72 pg/mg. In the same samples, the drug parent concentrations were, respectively, 2800 pg/mg and 760 pg/mg. Conclusion. – The present preliminary study describes for the first time an extensive search of metabolites of synthetic cannabinoids in human hair. Undoubtedly, the detection of metabolites in hair may represent the decisive factor to support the interpretation of hair analyses, particularly with respect to potential sources of
external contamination. More samples will be analyzed in order to (i) verify to what extent the presence of metabolites can prove the active consumption, (ii) evaluate the concentration ratios between parent drugs and metabolites, and (iii) elaborate tentative cut-offs for parent drug and metabolites. O23
Hair analysis for synthetic cannabinoids: How does handling of herbal mixtures during forensic analysis affect the analyst’s hair concentrations? V. Auwärter, M. Hutter, M.A. Neukamm, B. Moosmann Institute of forensic medicine, forensic toxicology, university medical center Freiburg, Albertstr. 9, 79104 Freiburg, Germany. Introduction. – If narcotics police officers or other persons handling drug material at work are suspected of consuming drugs, hair analysis may be used to prove or refute such suspicion. However, it is known for many drugs that differentiation of actual drug use from external contamination can be challenging or sometimes impossible. The aim of this study was to evaluate the extent of external contamination caused by handling of synthetic cannabinoid containing drug material under realistic conditions in a forensic laboratory. Methods. – Hair of laboratory workers was systematically analyzed for synthetic cannabinoids with a validated LC-MS/MS method after a big seizure of legal high products had to be analyzed in our laboratory. Hair samples were taken two days after the last exposure and again one week later. In addition, hair samples of laboratory staff not directly in contact with the drug material and close relatives of exposed subjects were analyzed to check for cross contamination. Results. – All samples of persons who were in direct contact with drug material were tested positive for at least one of the synthetic cannabinoids (JWH-018, JWH-073, JWH-081, JWH-122, JWH-210, JWH-307, JWH-368, AM-2201, AM-2201 indazole derivative, AM2232, MAM-2201, RCS-4, XLR-11, 5F-PB-22, RCS-4 ortho isomer). Concentrations ranged from trace amounts up to a maximum of 170 pg/mg (JWH-210) and roughly reflected duration and intensity of exposition. There was no significant decline in concentrations from sample 1 to sample 2 (one week later). Unexpectedly, also subjects without direct contact to drug material showed measurable hair concentrations. In one case, a hair sample (21 cm) was taken 10 weeks after the last exposition with plant material. In this case, relevant concentrations of 5F-PB-22 were detected with an increase of concentrations from distal to proximal segments (7.9 – 20 pg/mg). Conclusion. – Depending on duration and intensity of exposition, relevant concentrations of synthetic cannabinoids may be found in hair samples of persons exposed to these drugs at work. Unexpectedly, even cross contamination from an exposed person to a close relative may occur and lead to (false) positive hair findings. Concentrations caused by contamination are in the typical range found in known users of these drugs and could lead to wrong conclusions. In contrast, detection of metabolites could strongly suggest an actual consumption. However, we did not detect such metabolites so far even in samples of known consumers of synthetic cannabinoids showing extremely high concentrations of the unchanged compounds. Therefore, body fluids have to be analyzed to unambiguously prove use of these drugs.
Wednesday 11 June 2004 / Toxicologie Analytique & Clinique (2014) 26, S4-S14
O22
Quantification of 22 synthetic cannabinoids and 10 metabolites in human hair A. Salomone (1), E. Gerace (1), C. Luciano (1), D. DI Corcia (1), M. Vincenti (1, 2) (1) Centro regionale antidoping e di tossicologia “A. Bertinaria”, Orbassano (TO), Italy; (2) Università degli studi di Torino, dipartimento di chimica, Turin, Italy. Introduction. – Synthetic cannabinoids are the most common drugs among an expanding array of compounds that mimic the effects of traditional illicit psychoactive substances. A few methods have been developed for the determination of the parent synthetic cannabinoids in the keratin matrix, opening the scientific debate about how to interpret the quantitative results. In particular, the discrimination between (i) passive or active exposure, and (ii) chronic consumption and occasional use, appears to be essential within the forensic context. Just as occurred for the traditional drugs of abuse, the detection of drug metabolites is likely to represent the key factor to support the decision procedure. Therefore, we updated our existing method [A. Salomone et al., Drug Testing and Analysis, 2014, 6, 126-134] in order to complement the determination of 22 synthetic cannabinoids in human hair with 10 of their metabolites. Afterwards, we applied the new method to 15 real hair samples which previously tested positive for at least one synthetic cannabinoid [A. Salomone et al., cited], in order to verify the presence of metabolites. Methods. – The analytical method was validated in accordance with the criteria and recommendations of national and international guidelines. The following parameters were investigated: selectivity, specificity, linearity range, detection and quantification limits (LOD and LOQ), intra-assay and inter-assay precision and accuracy. Carry-over effect, recovery and matrix effects were also investigated. Two groups of subjects, namely driving re-licensing and drug abuse/withdrawal control subjects, provided hair samples that had previously resulted positive to at least one common drug of abuse and one synthetic cannabinoid. In this procedure, the samples were added with 1 mL NaOH 1N and subsequently incubated at 95 °C for 10 min. Immediately afterwards, the samples were extracted with 5 mL of n-hexane/ethylacetate 90:10 (v/v). The aqueous phase was newly added with glacial CH3COOH to reach a pH of 3.5-4.0 and again extracted with n-hexane/ethylacetate 90:10. The two organic phases were unified, dried under a nitrogen flow at 70 °C and reconstituted with 50 L of methanol. An aliquot of 1 μL methanol solution was directly injected into the UHPLC-MS/MS system. Results. – The optimized UHPLC – MS/MS method allowed the simultaneous determination of 22 synthetic cannabinoids, 10 metabolites and 6 internal standards. The whole chromatographic run, comprehensive of the time required for column re-equilibration before the following injection, was completed in 8.0 min. Retention times ranged between 2.2 min (WIN 48,098) and 5.5 min (JWH020). Two samples out of 15 resulted positive to one metabolite, namely the N-(5-hydroxypentyl) metabolite of JWH-122. The calculated concentrations were 2.5 pg/mg and 0.72 pg/mg. In the same samples, the drug parent concentrations were, respectively, 2800 pg/mg and 760 pg/mg. Conclusion. – The present preliminary study describes for the first time an extensive search of metabolites of synthetic cannabinoids in human hair. Undoubtedly, the detection of metabolites in hair may represent the decisive factor to support the interpretation of hair analyses, particularly with respect to potential sources of
external contamination. More samples will be analyzed in order to (i) verify to what extent the presence of metabolites can prove the active consumption, (ii) evaluate the concentration ratios between parent drugs and metabolites, and (iii) elaborate tentative cut-offs for parent drug and metabolites. O23
Hair analysis for synthetic cannabinoids: How does handling of herbal mixtures during forensic analysis affect the analyst’s hair concentrations? V. Auwärter, M. Hutter, M.A. Neukamm, B. Moosmann Institute of forensic medicine, forensic toxicology, university medical center Freiburg, Albertstr. 9, 79104 Freiburg, Germany. Introduction. – If narcotics police officers or other persons handling drug material at work are suspected of consuming drugs, hair analysis may be used to prove or refute such suspicion. However, it is known for many drugs that differentiation of actual drug use from external contamination can be challenging or sometimes impossible. The aim of this study was to evaluate the extent of external contamination caused by handling of synthetic cannabinoid containing drug material under realistic conditions in a forensic laboratory. Methods. – Hair of laboratory workers was systematically analyzed for synthetic cannabinoids with a validated LC-MS/MS method after a big seizure of legal high products had to be analyzed in our laboratory. Hair samples were taken two days after the last exposure and again one week later. In addition, hair samples of laboratory staff not directly in contact with the drug material and close relatives of exposed subjects were analyzed to check for cross contamination. Results. – All samples of persons who were in direct contact with drug material were tested positive for at least one of the synthetic cannabinoids (JWH-018, JWH-073, JWH-081, JWH-122, JWH-210, JWH-307, JWH-368, AM-2201, AM-2201 indazole derivative, AM2232, MAM-2201, RCS-4, XLR-11, 5F-PB-22, RCS-4 ortho isomer). Concentrations ranged from trace amounts up to a maximum of 170 pg/mg (JWH-210) and roughly reflected duration and intensity of exposition. There was no significant decline in concentrations from sample 1 to sample 2 (one week later). Unexpectedly, also subjects without direct contact to drug material showed measurable hair concentrations. In one case, a hair sample (21 cm) was taken 10 weeks after the last exposition with plant material. In this case, relevant concentrations of 5F-PB-22 were detected with an increase of concentrations from distal to proximal segments (7.9 – 20 pg/mg). Conclusion. – Depending on duration and intensity of exposition, relevant concentrations of synthetic cannabinoids may be found in hair samples of persons exposed to these drugs at work. Unexpectedly, even cross contamination from an exposed person to a close relative may occur and lead to (false) positive hair findings. Concentrations caused by contamination are in the typical range found in known users of these drugs and could lead to wrong conclusions. In contrast, detection of metabolites could strongly suggest an actual consumption. However, we did not detect such metabolites so far even in samples of known consumers of synthetic cannabinoids showing extremely high concentrations of the unchanged compounds. Therefore, body fluids have to be analyzed to unambiguously prove use of these drugs.