1. A rational toxicology-oriented approach to monitor NPS in the population
F. Tagliaro, University of Verona, Italy
in collaboration with
- Dr. S. Savchuk and Dr. S. Appolonova, “I.M. Sechenov” First Moscow State Medical University, Moscow, Russia
- Prof. Cristiano Bertolucci and Prof. Matteo Marti, University of Ferrara, Italy

2.  What are the sources of toxicological information ?
Patent drug abuse:
subjects admitted to hospital for acute intoxication
subjects driving under the influence of drugs or injured/deceased in drug-related traffic accidents
drug facilitated sexual assaults
deceased persons for drug overdosing
addicts/chronic users enrolled in treatment programs
Hidden drug abuse:
persons injured/deceased in traffic accidents (apparently non drug-related)
subjects holding safety sensitive jobs under toxicological monitoring

3. What clinical/pathological information is relevant?
Living persons:
anagraphic information
case-history (anamnesis)
clinical symptoms
response to pharmacological treatments (ex adiuvantibus)
outcome
Deceased subjects:
police information
circumstances of death (and paraphernalia)
pathological data (autopsy)

4. Suitability of the biological specimen
Blood/serum/(oral fluid)
investigation of a condition of “present” intoxication of an individual
Urine
identification of drug intake in the recent past
Keratinized tissues (hair and nails)
investigation of drug abuse histories

5. Points of weakness
Blood/saliva: low drug concentrations, narrow chronological window of detectability (related to the drug half-life)
Urine: prevalence of metabolites, poor detectability in case of extensive metabolism (e.g. JWH’s)
Hair/nails: medium-to-low concentrations, contamination from the environment (less relevant in epidemiological studies)

6. What sources of information from Forensic Toxicology are relevant for an Early Warning System?
- Evaluation of “harm”:
Analysis of blood
- Evaluation of “use” in the population:
Analysis of hair/nails and urine

7. Toxicological analysis of hair/nails, potential usefulness
Field of application
Monitoring treatments
Application of special law provisions (e.g. home detention, suspended sentence...) for promoting rehabilitation of drug addicts convicted for drug related crimes
Divorce/child custody
Certification of fitenss-to-work in the context of the “safety sensitive” jobs
Certification of fitness-to-drive
Epidemiological surveys
Analytical strategy: targeted/broad spectrum analysis
Analytes of interest
Parent drug (and metabolites)

8. Points of strength of hair/nail analysis vs. urine analysis
Hair/nail analysis explores a wide time-window, which is important to detect NPS use which is typically non regular
Hair/nails embed primarily the parent drug, even in the presence of extensive metabolism limiting the usefulness of urine analysis
Hair/nail matrix protect drugs from degradation
Hair/nail samples pose no problems of collection and shipping to reference labs.

9. Example: application of hair analysis in a survey on the use of synthetic cannabinoids in north-east Italy
# 600 hair samples from subjects undergoing toxicological screening to check their fitness for re-granting of the driving license having a “past” history of cannabis use
Area: Verona, Vicenza and Bolzano
Time:
All subjects had tested negative for THC and THC-COOH in urine and hair in seriate at the time of hair collection

10. confirmation and quantification by LC-ESI-QQQ MS or GC-QQQ MS
ANALYTICAL STRATEGY
HRMS screening by using LC-ESI-QTOF MS
based of accurate mass and isotopic ratio
confirmation and quantification
by LC-ESI-QQQ MS or GC-QQQ MS

11. Results Sample ID Age Year Results 1 21 2009
JWH-250: 3 pg/mg, JWH-073: 17 pg/mg, JWH-018: 6 pg/mg 2 40
2010
JWH-081: 16 pg/mg 3 22
JWH-122: 125 pg/mg 4 29
JWH-081: 1280 pg/mg 5 20
JWH-018: 10 pg/mg 6 28
2011
JWH-081: 24 pg/mg 7 27
JWH-018: 11 pg/mg 8 19
JWH-210: 1.2 pg/mg 9 45
JWH-081: 11 pg/mg 10
JWH-015: 10 pg/mg, JWH-073: 13 pg/mg 11
JWH-081: 37 pg/mg 12 30
2012
JWH-081: 14 pg/mg 13 32
JWH-210: 1 pg/mg 14 23
2013
JWH-073: 4pg/mg, JWH-081: 132 pg/mg, JWH-018: 4 pg/mg

12. Most frequent JWH compounds found in hair

13. Most frequent NPS found in seized materials in 2009-2013 (n=963 samples)

14. Toxicological data do not fit well with the information from seized material:
AM (present in the market, not in the hair samples)
AM-694 (present in the market, not in the hair samples)
JWH-018 (frequently detected in the hair samples, rare in the market)
Possible explanation:
toxicological data do not reflect only the availability of the compounds but also the preference of the «customers»

15. Why nail analysis could be useful ?
Easy availability when hair is lacking
Much smaller surface/volume ratio easier cleaning procedures
More compact structure less risk of penetration of drugs from the surface
For toenails: less risk of contamination from the environment

16. GC-MS determination of NPS in hair and nails of multi-drug user (case report, Ulan-Ude, Buryatia, Russia)
Purpose: checking the correspondence of the drug incorporation pattern between hair and nail
Approch: a sequential six stage procedure of analysis was tested aimed at distinguishing between loosely bound (external contamination) and firmly bound compounds (drugs embedded into the hair matrix)

17. Sample preparation: mg of finely cut samples (hair or nails) washed 5 times with 2 ml of methanol, and finally incubated with further 3 ml of methanol and sonicated on an ultrasonic bath for 6 h. All extracts were evaporated to dryness, added with 150 ml of ACN and analysed by GC-MS (and LC-MS/MS).
Analysis
GC-MS: Agilent 5975, 5977, full scan
Column: HP-5MS 30m, 0.25 mm, 0.25 mm. Const Pressure 85 kpa
Method 1 “SCREEN”: 1000С (1 min), 350С /min, 3000С (10 min)
Method 2“DOAS”: 1000С (1 min), 150С /min, 2800С (30 min)

18. Results (preliminary)
Hair
Toenails

19. Conclusions
Keratinized tissue analysis is a good strategy to study the “use” of a psychoactive drug in the population:
presence and stability of the parent drug (no metabolism)
wide window of detectability
only chronic or repeated use give positive results (occasional use may be of less concern)
the problem of “contamination” is less relevant in this context
Nail analysis is potentially a good alternative to hair testing

20. Zebrafish as a model to study NPS
Zebrafish (Danio rerio) is rapidly becoming a new popular model organism in biomedical research.
The utility of both adult and larval zebrafish in neuroscience has grown markedly in the past decades because this vertebrate species shows:
high physiological and genetic homology to humans
simple genetic manipulation
well characterized genome
possessing both rapid development and a relatively long lifespan
availability of multiple zebrafish strains
close parallels between mammalian and zebrafish behavioral
high cost-efficiency (particularly using larval zebrafish
easy to breed

21. Zebrafish as a model of drug-related disorders
Zebrafish is a valid translational model because it shows:
reward sensitivity
drug abuse
high sensitivity to various drugs
as well as tolerance
and clear preference for these agents
withdrawal symptoms

22. Material & methods Fish Treatment
96 wild type zebrafish larvae (aged 5 days) housed at Department of Life Sciences and Biotechnology (SVeB), University of Ferrara, were studied
Treatment
Fish were placed into a 96 multiwell plate in the DanioVision Observation Chamber and locomotory activity was recorded and analyzed by EthoVision XT software.
After a day of locomotory activity record, animals were treated with Methiopropamine (MPA), a thiophene ring-based structural analog of methamphetamine. Locomotory activity has been monitored for the 2 sequent days.
MPA has been reported as a new psychoactive substance by the European Monitoring Centre for Drugs and Drugs Addiction (EMCDDA) Early Warning System.
For all this set of experiments, less than 2 mg of the tested compound were used.
High-throughput machine analysis: DanioVision chamber

23. Material & methods
Individuals were divided into 4 growing concentration experimental groups:
saline, medium of larvae incubation and drug vehicle (n=12)
0,001 mM MPA (n=18)
0,01 mM MPA (n=18)
0,1 mM MPA (n=18)
1 mM MPA (n=18)
Methiopropamine (MPA)

24. Results Daily variations of total locomotor activity (mean ± SD)
Pre –Treatment (basal activity)
Treatment with MPA
Post–Treatment (after MPA)

25. Analysis during the diurnal activity
saline

26. Analysis of the overall diurnal activity (AUC analysis)**

27. Conclusions
Two-way ANOVA analysis show that the locomotor activity of MPA treated larvae differ significantly from controls.
The AUC analysis reveals an increase of total activity: 37%; 56%; 42% for the 0.001; and 0.1 mM concentrations, respectively. Conversely, the highest concentration (1 mM) is associated with a reduction of locomotor activity of about 25%.
The effect persists the day after treatment, with an increased activity of 17% (0.001 mM) and of 12% (0.01 mM) in the lower doses and a reduction of 24% (0.1 mM) and of 54% (1mM) in the upper ones.
The 1 mM MPA concentration caused the death of all the zebrafish larvae after 30 hours from drug administration, showing its potential TOXICITY
Finally, on the basis of the present data, the larval Zebrafish model looks very promising as a rapid, simple (reduced requirements for amount of drug to be used in the study and also in terms of ethical approval) and low cost screening test for NPS.