THCCOOH and ETG in hair: analytical challenges and interpretation of results in Forensic Toxicology. Luca Morini

Transcription

1 THCCOOH and ETG in hair: analytical challenges and interpretation of results in Forensic Toxicology. Luca Morini Department of Public Health, Experimental and Forensic Medicine University of Pavia Via Forlanini 12, Pavia, Italy

2 Metabolites in hair: why? Simple sample collection Wide window of detection Easy storage Advantages Segmental analysis Simple sample preparation Evaluation of a past use

3 Regional protocol New regional protocol for the determination of drugs of abuse and alcohol for the evaluation of drive license eligibility It has been developed and edited in 2014 It regards all the procedures and the tests that should be performed on drivers in Lombardy

4 Italy Lombardy - Pavia

5 Laboratories currently accredited for hair analysis in Lombardy Como (1): Hospital Como (1): Health Local Agency Bergamo (1): Sondrio (1): Health Local Agency Health Local Agency Varese (2): Health Local A gency Private Lab Milan (4): Forensic Toxicology lab 2 Hospitals Private Lab Brescia (3): Forensic Toxicology lab Health Local Agency Private lab Pavia (1): Forensic Toxicology lab Monza (2): Hospital Private lab Cremona (1): Health Local Agency

6 Regional Protocol: procedure Hair analysis: Opiates Cocaine Cannabinoids Amfetamine MDMA Methadone Buprenorphine Ketamine Ethyl glucuronide (**) Urine analysis Opiates Cocaine Cannabinoids Amfetamine MDMA Benzodiazepines Methadone Buprenorphine Ketamine Hair lenght for sample collection a) Hair length 6 cm - analyis on the 6 cm proximal hair segment - analysis on two segments (0-3 cm; 3-6 cm) b) Hair lenght 3cm and <6 cm - analysis of the total amount of samples c) Hair lenght < 3 cm - Postpone the sample collection - Collection of hair from other body area (*) d) Baldness - collection of hair from other body area (*) * Hair from other body area should be interprete with caution ** Pubic hair should not be used for EtG analysis. Hair from chest area should be preferred

7 Regional protocol: cutoff Class Compound Cut-off (ng/mg) Opiates Morphine acetylmorphine 0.2 Codeine 0.2 Cocaine Cocaine 0.2 Benzoylecgonine 0.05 Cocaethylene 0.05 Ecgonine methyl ester 0.05 Amfetamine Amfetamine 0.2 Metamfetamine 0.2 MDMA MDMA 0.2 MDA 0.2 MDEA 0.2 MBDB 0.2 Cannabinoids THC 0.05 THCCOOH Methadone Methadone 0.2 EDDP 0.2 Buprenorphine Buprenorphine 0.05 Norbuprenorphine 0.05 Ketamine Ketamine 0.5 Ethyl glucuronide Ethyl glucuronide 0.03

8 THC analysis: sample preparation Hair sample extracted in NaOH 5 M. Liquid/liquid extraction with hexane-ethyl acetate mixture (9:1) and 1 ml acetate buffer at ph 5 Solvent soaked under nitrogen stream. Addition of 50µl MSTFA, maintained at 75 C for 15 minutes: Chromatographic settings Gascromatography: HP 6890 Plus with HP 7683 Autosampler Column: HP 5 Ultra 2, (5%-phenyl)-methylpolysiloxane (12 m x 0.2 mm i.d. x 0.33 μm) Mass detector: HP 5973 Carrier gas: Helium (10.3 psi, 1 ml/min, constant flow) Injection volume: 1 μl Ramp: Injector: pulsed splitless, 1 min, 25 psi, 250 C Column: 100 C (2.5 min) 40 C /min 180 C 10 C/min 290 C (10 min) Interface: 280 C Source: 230 C

9 THCCOOH: sample preparation (L/L) The remaining solution from THC analysis is acidified with glacial acetic acid. Liquid/liquid extraction with hexane-ethyl acetate mixture (9:1) and 1 ml acetate buffer at ph 5 Solvent soaked under nitrogen stream. Addition of 50µl PFPA and 25µl HFIP and maintained at 75 C for 1 h: Solvent soaking followed by 50µ hexane addition Chromatographic settings Capillary column HP-5MS (5%-phenyl)-methylpolysiloxane) (30 m x 0,25mm i.d., 0,25 μm). Carrier: He at 1,2 ml/min NCI source at 150 C Source Gas: Methane Collision Gas: nitrogen CE: 10 ev -5 ev dwell time= 100 milliseconds

10 THCCOOH: sample preparation (SPE) The remaining solution from THC analysis is acidified with glacial acetic acid Samples extractred thorugh SPE procedure using Bond Elut Certify II cartridges Extraction procedure: 2ml methanol 2 ml sodium acetate buffer with 5% methanol Sample loading 2 ml methanol-water solution (1:1) Elution with 2ml of hexane-ethyl acetate solution (75:25) with 1% of glacial acetic acid.

11 THCCOOH: sample preparation (SPE) The remaining solution from THC analysis is acidified with glacial acetic acid Samples extractred thorugh SPE procedure using UCT cartridges Extraction procedure: 1 x 3 ml CH3OH 1 x 3 ml H2O 1 x 1 ml 100 mm HCl Load sample at 1-2 ml / minute 1 x 3 ml DI H2O 1 x 3 ml CH3CN /HCl (30:70) Dry column (5 minutes at > 10 inches Hg) 1 x 3 ml Hexanes/Ethyl acetate/acetic Acid (49:49: 2)

12 Instrumentation Capillary coumn HP-5MS 5% phenil metil silox (30 m x 0,25mm i.d., 0,25 μm). Carrier gas: He at 1,2 ml/min Soruce: NCI at 150 C Source Gas: Methane Collision Gas: Nitrogen CE: 10 ev -5 ev dwell time= 100 ms

13 THCCOOH derivatization THCCOOH DERIVATIZATION PFPA HFIP 1h 60 C THCCOOH-d3 (IS) THCCOOH-d3: m/z 623 m/z 495; (10 ev) THCCOOH-d3: m/z 623 m/z 386; (5 ev) THCCOOH: m/z 620 m/z 492; (10 ev) THCCOOH: m/z 620 m/z 383; (5eV)

14 THCCOOH: chromatograms Real positive sample (0.6 pg/mg THCCOOH)

15 Calibration curve LOD= 0,02 pg/mg LOQ= 0,04 pg/mg

16 THCCOOH: Real Samples Sample THC THCCOOH THC THCCOOH Sample (ng/mg) (pg/mg) (ng/mg) (pg/mg) Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

17 THCCOOH: Real Samples Sample THC THCCOOH THC THCCOOH Sample (ng/mg) (pg/mg) (ng/mg) (pg/mg) Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

18 THCCOOH: Real samples Only 8 out 40 (20%) tested positive for either THC and THCCOOH by using the proposed cut-off Decreasing the cut off to 0.2 pg/mg (the one suggested by the SoHT) we would assess a total of 21 cannabis consumers among the tested group The number of positive samples could be even more increased (75%) by using as cut-off the LOQ (0.04 pg/mg)

19 Literature

20 Literature

21 Questions still open for discussion The use of THCCOOH in hair is a reliable tool for discriminating cannabis users? What is the most reliable cutoff? Is it acceptable the use of a less sensitive method for THCCOOH detection in hair?

22 Ethyl glucuronide UDP-glucuronosyltransferase Ethanol β-d-ethylglucuronide (EtG) Direct ethanol metabolite Highly sensitive and specific markers for alcohol misuse diagnosis in the short, mid and long term, depending on the matrix evaluated.

23 LC-MS/MS: EtG ed EtS Direct Marker: high diagnostic sensitivity and specificty; Indicator of alcohol use/misuse in the short (blood/serum/plasma) mid (urine) and long (hair, nails) term; Determination of EtG in meconium is useful to evaluate intrauterine exposure to alcohol.

24 Regional Protocol: procedure Hair analysis: Opiates Cocaine Benzoylecgonine Cannabinoids Amfetamine MDMA Methadone Buprenorphine Ketamine Ethyl glucuronide (**) Urine analysis Opiates Cocaine Cannabinoids Amfetamine MDMA Benzodiazepines Methadone Buprenorphine Ketamine Hair lenght for sample collection a) Hair length 6 cm - analyis on the 6 cm proximal hair segment - analysis on two segments (0-3 cm; 3-6 cm) b) Hair lenght 3cm and <6 cm - analysis of the total amount of samples c) Hair lenght < 3 cm - Postpone the sample collection - Collection of hair from other body area (*) d) Baldness - collection of hair from other body area (*) * Hair from other body area should be interprete with caution ** Pubic hair should not be used for EtG analysis. Hair from chest area should be preferred

25 Regional protocol Biomarker Teetotaller or occasional user Grey zone Harmful consumption ( 60 g/day) EtG 7.0 pg/mg pg/mg 30 pg/mg CDT* * Depending on the analytical procedure the cut-off could be different. The procedure should be stated In case of a discordance between the two biomarker the EtG result should be preferred

26 Etg in hair: sample preparation Hair (30 mg) washed with dichlormethane and methanol Addition of 1 ml water with EtG-D 5 Ultrasonication for two hours: SPE extraction on Strata-X-A Phenomenex: 2 ml methanol; 2 ml water Sample loading 2 ml water (2% of NH 3 ) 2 ml methanol Elution with 2 ml methanol (5% formic acid)

27 EtG in hair: instrumental parameters Solvent soaking under nitrogen stream. Addition of 75µl PFPA, maintained at 50 C for 1 h: Solvent Soaked and remaining eluted in 50 µl hexane Chromatographic parameters Capillary column HP-5MS 5% phenyl methyl silox (30 m x 0,25mm i.d., 0,25 μm). Carrier gas: He at 1,2 ml/min Source NCI at 150 C Source Gas: Methane Collision Gas: Nitrogen CE: 10 ev -5 ev dwell time= 100 ms EtG-d5: m/z 352 m/z 163; (5 ev) EtG: m/z 347 m/z 163; (5 ev) EtG: m/z 347 m/z 119; (5eV)

28 EtG: chromatograms Real positive sample (7 pg/mg)

29 Calibration curve LOD= 2 pg/mg LOQ= 3 pg/mg

30 HEtG: literature The concentration of ethyl glucuronide in hair appears to correlate with EDI Ethyl glucuronide in hair: Is it a reliable marker of chronic high levels of alcohol consumption? Politi L, Morini L, Leone F, Polettini A. Addiction Oct;101(10): Variations in the EtG concentrations with respect to hair segments may provide an overview of the drinking history of patients. Moreover, EtG concentration in hair may help to estimate the daily alcohol intake Segmental determination of ethyl glucuronide in hair: a pilot study.appenzeller BM, Agirman R, Neuberg P, Yegles M, Wennig R. Forensic Sci Int Dec 20;173(2-3): Epub 2007 Mar 6 These findings suggest that measurements of EtG in hair may provide improved diagnostic information on alcohol abuse, due to a long retrospective time-window for detection and stability of EtG in hair in the decaying cadaver Comparison of ethyl glucuronide in hair with phosphatidylethanol in whole blood as post-mortem markers of alcohol abuse. Bendroth P, Kronstrand R, Helander A, Greby J, Stephanson N, Krantz P. Forensic Sci Int Mar 21;176(1): Epub 2007 Nov 19.

31 HEtG: literature Our results confirm further that HEtG is a sensitive and specific marker of chronic heavy drinking Ethyl glucuronide in hair. A sensitive and specific marker of chronic heavy drinking. Morini L, Politi L, Polettini A. Addiction Jun;104(6): Epub 2009 Apr 9 Our results indicate that HEtG, as compared to CDT measured using different methods, is a selective marker of ethanol heavy chronic use providing considerably higher sensitivity Comparison of ethyl glucuronide in hair with carbohydrate-deficient transferrin in serum as markers of chronic high levels of alcohol consumption. Morini L, Politi L, Acito S, Groppi A, Polettini A. Forensic Sci Int Jul 1;188(1-3): Epub 2009 May 1 The present assay appears convenient to carry out owing to the very small quantity of hair samples required to determine an effective heavy alcohol consumption Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction. Lamoureux F, Gaulier JM, Sauvage FL, Mercerolle M, Vallejo C, Lachâtre G. Anal Bioanal Chem Aug;394(7): Epub 2009 Jun 12.

32 HEtG : literature EtG analysis in hair is a useful tool for assessing fitness to drive in suspected drinking drivers; compared to CDT values it provides a direct and unequivocal marker for reliable abstinence monitoring over a period of several months, depending on the length of the hair Abstinence monitoring of suspected drinking drivers: ethyl glucuronide in hair versus CDT. Liniger B, Nguyen A, Friedrich-Koch A, Yegles M. Traffic Inj Prev Apr;11(2): It is recommended, that in context of driving ability diagnostics the EtG result is determinant Comparison of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) concentrations in hair for testing abstinence. Albermann ME, Musshoff F, Madea B. Anal Bioanal Chem Apr;400(1): Epub 2010 Dec 3. EtG can be used qualitatively to indicate alcohol consumption with a positive result providing strong evidence for an individual drinking within the past 3 months Comparison of ethyl glucuronide in hair with self-reported alcohol consumption. Lees R, Kingston R, Williams TM, Henderson G, Lingford-Hughes A, Hickman M. Alcohol Alcohol May-Jun;47(3): Epub 2012 Feb 14.

33 HEtG: cut-off e consensus Highly sensitive and specific marker for excessive chronic alcohol consumption Cutoff: 30 pg/mg (since 2012 also the cutoff at 7.0 pg/mg) Consensus on EtG and FAEEs approved during the SoHT meeting in Rome (2009) and revised every two years Consensus of the Society of Hair Testing on hair testing for chronic excessive alcohol consumption Kintz P. Forensic Sci Int Mar 20;196(1-3):2. Epub 2010 Jan 8.

34 HEtG: limitations Hair treatments could hydrolized/eliminate EtG from the matrix Effect of bleaching on ethyl glucuronide in hair: an in vitro experiment. Morini L, Zucchella A, Polettini A, Politi L, Groppi A. Forensic Sci Int May 20;198(1-3):23-7. Epub 2009 Dec 3. EtG in pubic hair tends to overestimate EDI Ethyl glucuronide determination: head hair versus non-head hair. Kerekes I, Yegles M, Grimm U, Wennig R. Alcohol Alcohol Jan-Feb;44(1):62-6. Epub 2008 Nov 28 EtG in axillary hair underestimates the concentration of EtG in head hair A study of distribution of ethyl glucuronide in different keratin matrices. Pirro V, Di Corcia D, Pellegrino S, Vincenti M, Sciutteri B, Salomone A. Forensic Sci Int Jul 15;210(1-3): Epub 2011 Apr 20

35 HEtG: limitations In a recent study EtG in patients with severe decreased kidney function is accumulated i hair in an higher ratio Higher Levels of Hair Ethyl Glucuronide in Patients with Decreased Kidney Function. Høiseth G, Morini L, Ganss R, Nordal K, Mørland J. Alcohol Clin Exp Res Jun 14. doi: /j x. [Epub ahead of print]

36 Grazie per l attenzione Thanks for the attention