präsentation salsa toxicological screening...präsentation_salsa_toxicological screening author...

Berlin, 8.12.2015

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Case presentation

At 1:00 a.m. a 23 year old male patient is admitted to the emergency department of the

University Hospital in Basel. He was very agitated, had a combative violent behaviour and

hallucination. He has been found in his home standing on his bed complaining of mice

everywhere. He was given the antidote for opiate intoxications without any effect.

The classical drugs of abuse screening tests in the laboratory were negative. The physicians

then suspected the intake of legal highs or research chemicals and sent a urine and a blood

sample to the laboratory.sample to the laboratory.

Opiate antidote

Classical Drugs of Abuse

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What is a „drug“?

„A chemical that changes the way your body works“

Definitions

What is „Drug misuse (abuse)“?

„Non-medical administration of a drug for its psychoactive effect,

intoxication or altered body image (despite knowing the risk involved)“

Toxicology

Pharmacology

„How a chemical (drug)

changes the way your

body works“

Pharmacodynamics

„How drugs effect the body“

Pharmacokinetics

„How the body effects the

drug“

Matthew Russel, University of Auckland, PGDip in Forensic Sci., Forensic 702 2015.

Toxicology

„The study of the adverse effects of the drugs, chemicals, biological toxins

on the body“

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Drug classification

Misused drugs

Psychoactive drugs Steroids

Stimulants

Uppers

Antidepressants

Hallucinogens

Somewhere else

Sedatives

DownersAntidepressants

• ATS

• cocaine

• nicotine

• caffeine

Somewhere else

Psychedelics

Psychotics• Tryptamines (LSD,

psilocybin)

• ketamine

• can also contain ATS

Downers

Depressants

Narcotics / Opiates• cannabis

• ethanol

• benzodiazepines

• heroin


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...getting the drug into the body

ORAL INJECTION

Drug administration and absorption

„movement of the drug into the

bloodstream and its transport to

the receptor sites“

subcutaneous, intramuscular,

intravenous

INHALATION NASAL

Matthew Russel, M. Bauer, University of Auckland, PGDip in Forensic Sci., Forensic 702 2015.

NASAL

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extraction?

chemical form of the drug?

unknown drugs?

mixture of compounds?

metabolism?

exact structure?

Analytical challenges

standards? comparison?

analysis time consuming?

customer?

cost/time benefit?


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Legal highs

Synthetic cannabinoids

Tryptamines PiperazinesPhenylethyl-

amines

Classification

- “Spice”

- Generates

- Imitation of

serotonin

- Influences the

concentration

- (Dopamin, Adrenalin,

Amphetamines)- Generates

Cannabis similar

euphoria

THC

serotonin

Tryptamine

Piperazine

concentration

of serotonin

and dopamine

Phenylethylamine

Amphetamines)

- Many amphetamine-like

derivatives

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Switzerland Germany

• Available as herbal mixtures, bath salts, air freshener or

cleaning agents

• Ingredients and their amount on the cover of the product

undeclared

highlifestyle.ch

Legal situation

Switzerland Germany

• 2013: European Court of Justice rules that legal highs belong not to the Pharmaceutical law

• List of legal highs that are forbidden within the Narcotics law (BtMG)

• 2011: 52 substances and 7 derivatives

prohibited

• 2012: 46 more substances prohibited

• 2015: Draft law that prohibits the

production, the import, the offer and the

trade of complete families of psychotropic

substances

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Growth of Legal Highs

Analyst, 2015, 140, 4932-4948

New psychoactive substances notified to the EWS between 2005–2014

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• Similarity to endogenous neurotransmitters

Mechanism of action

https://www.cnsforum.com/educationalresources/imagebank/substance_abuse/drug_amphet_high

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• Binding to transport systems

and thus inhibiting reuptake

• Mephedrone

Mechanism of action

https://www.cnsforum.com/educationalresources/imagebank/substance_abuse/drug_amphet_high

https://www.boundless.com/physiology/textbooks/boundless-anatomy-and-physiology-textbook/nervous-tissue-11/neurophysiology-113/types-of-

neurotransmitters-by-function-619-3349/images/major-elements-in-neuron-to-neuron-communication/

• Mephedrone

• Increases the dopamine

and serotonin

concentration in synaptic

space

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Mephedrone MDMA (Ecstasy)

Agitation + +

Violent behaviour + +

Tachycardia + +

Clinical Effects

Hallucinations + +

Paranoia + +

Hypertension + +

Routine analysis - +

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Drug screening methods

1. Preliminary screening

– Goal: Identify all presumptive POSITIVES (true + false

positives)

2. Confirmatory step

– Goal: among all positives, identify the TRUE positives– Goal: among all positives, identify the TRUE positives

– highly specific technology (eg. GC-MS, LC-MS)

– accurate recognition and quantification of the target

compounds

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Journal of Chromatography A, 1287 (2013) 84– 95

Sampling

Common samples

– Urine

non-invasive; drug metabolites can be detected for a longer time

– Blood serum– Blood serum

– Saliva

– Hair

larger surveillance window; can differentiate between chronic and single use

– Stool

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Am J Clin Pathol 2002;117:325-329

Preliminary screening techniques

1. Immunochemical/Immunoassay

2. Chemical

3. Atomic absorption spectroscopy (AAS)

4. Electrophoresis

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- Antibodies used for drug detection and quantification

- ELISA, EMIT, RIA

2. Chemical (spot tests)


Screening techniques


4. Electrophoresis

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T. Teerinen, et al., “A paper-based lateral flow assay for morphine,” Analytical and Bioanalytical Chemistry, 2014, 406: 24, pp. 5955-5965.

http://www.synchronium.net/2009/09/14/drug-testing-all-you-need-to-know/

Example of a lateral flow immunoassay (LFIA),

also called: dipstick assay, "pregancy test"-like assay

1. Immunochemical/Immunoassays

– Prone to cross-reactivity

– Example: chronic use of ibuprofen may result in false-positives for

cannabinoids or barbiturates

June 2006 Volume 37 Number 6 LABMEDICINE


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So can immunochemical tests for illegal drugs

detect “legal high” analogs?

• Some legal highs might be struc-tur-ally related to illegal drugs, but they’re still not

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June 2006 Volume 37 Number 6 LABMEDICINE

http://www.synchronium.net/2009/09/14/drug-testing-all-you-need-to-know/

• Some legal highs might be struc-tur-ally related to illegal drugs, but they’re still not

exactly the same; while some may cross-react, others do not

• Other legal highs share no closer sim-il-ar-ities with their illegal coun-ter-parts,

they just act the same receptors in your brain.

Immunoassays in drug screening

Substrate Substrate reacting

w/ Enzyme

1. EMIT (Enzyme multiplied immunoassay)

2. ELISA (Enzyme-linked immunosorbent assay)

3. FPIA (Fluorescence polarization immunoassay)

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Substrate

Enzyme w/ Drug

Attached

AntibodyDrug

Drug Blocking Drug

Attached to Enzyme

w/ Enzyme

http://www.boomer.org/c/p3/c03/c0308.html

Fluorescence polarization

Immunoassay

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2. Chemical spot tests

– „color tests“

– Based on simple chemical reactions which produce visible change

– Inexpensive and readily available reagents

– Easy to do w/o extensive training


– Easy to do w/o extensive training


4. Electrophoresis

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Analyst, 2015, 140, 4932-4948

Chemical spot tests

Example: Marquis reagent

• Presumptively identify alkaloids as well as other compounds

• ~2% (v/v) formaldehyde: concentrated sulfuric acid

• Found in ecstasy kits

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Color chart for some

of the compounds

that can react with

Marquis reagent

https://en.wikipedia.org/wiki/Marquis_reagent

Forensic Science International 109 (2000) 189–201




– Pharma: used to detect minute amounts of catalysts that remain in


– Pharma: used to detect minute amounts of catalysts that remain in

the drug product

– Toxic metals can be present in herbal drugs

4. Electrophoresis

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Pak J Pharm Sci. 2011 Jul;24(3):353-8; https://en.wikipedia.org/wiki/Atomic_absorption_spectroscopy




4. Electrophoresis


4. Electrophoresis

– Electrokinetic separation methods performed in submillimeter

diameter capillaries and in micro- and nanofluidic channels

– High separation efficiency and selectivity

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Pak J Pharm Sci. 2011 Jul;24(3):353-8; https://en.wikipedia.org/wiki/Atomic_absorption_spectroscopy

Capillary Electrophoresis

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Saliva from volunteer after

methamphetamine dosage

Standard mixture of drugs of abuse in

buffer and methanol

1: amphetamine; 2:methamphetamine;

3-13: other drugs Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2007, 151(1):31–36.

Other techniques

Surfaced enhanced Raman

spectroscopy

Electrochemistry based

sensing protocol

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Anal. Chem., 2014, 86 (19), pp 9985–9992; http://www.dropsens.com/en/screen_printed_electrodes_pag.html; Analyst, 2013, 138, 118-122

Anal. Chem., 2014, 86 (19), pp 9985–9992; http://www.dropsens.com/en/screen_printed_electrodes_pag.html; Analyst, 2013, 138, 118-122; http://www.mmu.ac.uk/news/news-items/2752/

Challenges

1. Need for faster, more sensitive screening

2. Absence of library or reference data for newly synthesized

compounds

3. Legal highs are sold as mixtures. How do these mixtures get

metabolized?

– Even if some have similar structure to a more understood

illegal drug; will they be metabolized in the same way if they

are mixed with others?

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Chromatographic Separation

Techniques

• TLC

• HPLC-DAD

• HPLC-MS

• HPLC-MS-MS

• GC-MS• GC-MS

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GC-MS

• Technique for the analysis and quantification of organic volatile and

semi-volatile compounds

• Gold standard in full screening of drugs

• Valid for court evidence

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• In general, liquid chromatography (LC) separates the components of a

sample based on differences in their affinity for the stationary phase or

mobile phase

• Detectors - UV, fluorescence, MS

Liquid chromatographic techniques

Mass

Chromatogram

IntensityChromatogram

(UV, TIC, MIC)

Mass Spectrum

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LC-MS-MS

Tandem mass spectrometry in combination with LC can be used for

confirmation and for quantification

– rapid analysis

– selectivity

– simple sample preparation

– attractive for high throughput

– not suitable for truly unknown drugs– not suitable for truly unknown drugs

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HPLC-MS GC-MS LC-MS/MS

Sample preparation simple may be difficult simple

Screening limited possible limited

Quantification good very good very good

Detection limits ng/mL ng/mL ng/mL

Comparison of methods

Detection limits ng/mL ng/mL ng/mL

Time 2 h (fully

automated)

2 h (fully

automated)

2 h (fully

automated)

Matrix blood, plasma urine, blood, serum blood, plasma

Requirements Soluble in mobile

phase

Volatile, thermaly

stable

Soluble in liquid

phase

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Case resolution

• After all this information, how would you do it?

• Immunochemically?

• With the use of chromatography? Which type?

• Will it change the way we treat the patients?

• Is the correct identification just for legal purposes? Can we actually

combat the growth of legal highs? Should we address the fact that is a

chemist vs chemist battle, therefore a moral issue?

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Thank you for your attention!

Questions?

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