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Detection of Drugs-of-Abuse byTandem Mass Spectrometry.
Dr Tim Laurens
MSc.Chem(Pretoria), Ph.D. Chem (Pretoria), MSc.Toxicology (Surrey,UK)FRSChem, MFSSoc
Email: [email protected] / [email protected]
Mobile: 082 891 4886
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Introduction
What are drugs of Abuse?.• Any substance that due to desirable effect is misused
for purposes other than intended• Effect on CNS
Any substance of which the possession or supply of which is restricted by law due to potential harmful effects on the user– Schedules of the Medicines and Related
Substances Control Act, Act 101 of 1965– Drugs and Drug Trafficking Act, Act 140 of 1992
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CNS Depressants
Benzodiazepines Flunitrazepam, Diazepam
Opiods Codeine, Heroine, methadone, morphine, pethidine etc
Marijuana Various forms of Cannabis sativa containing tetrahydrocannabinol (THC)
Barbiturates Phenobarbital, secobarbital
Alcohol Alcoholic Beverages
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CNS StimulantsAmphetamines “Speed” – methamphetamine
“Ecstacy”–(methylenedioxymethamphetamine)Cocaine Free base “crack” and hydrochlorideOther stimulants
Ephidrine, Pseudoephidrine,
Other substancesInhalants Petrol, solvents, propane (LPG), Halocinogens LSD, Ecstacy, mescaline, psilocybin, scopolaminePCP PhencyclidineAnabolic steroids Testosterone, stanozol
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“Orange Juice HUBLY-BUBLY”
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Routes of Administration
• Oral intake - Amphetamines, EXTACY, Barbitirates, tricyclicantidepressants
• Inhalation - Cannabis, Methaqualone, Metcathinone, Methamphetamine, Cocaine
• Intravenous -Heroin, Opiates
• Oral intake – First pass metabolism – Liver
• Inhalation / Intravenous – directly in the bloodstream
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Pharmacology
• Absorbtion• Distrubution• Metabolism (Liver, Lungs)• Excretion
“A.D.M.E.”• Metabolism takes place to increase water solubility of
a compound in the body to enhance urinary excretion
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Half-Life
0
2
4
6
8
10
12
14
16
18
0 2 4 6 8 10 12
Time (days)
Con
cent
ratio
n (m
g/L)
Drug Half Life
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The Cell
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Microsomes and Lysosomes
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Metabolism Cannabis
OCH 3
CH 3
O H
C H 3
C H 3
OCH 3
CH 3
O H
C H 2 O H
C H 3O
CH 3
CH 3
O H
C H 3
C O O H
D e lta -9 -T e tra h y d ro c a n n a b in o l (T H C )
1 1 -H y d ro x y -d e lta -9 -T H C 1 1 -n o r-9 -C a rb o x y -d e lta -9 -T H C
C o n ju g a t io n
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Metabolism-Opiates
NCH3
CH3COO OOCCH3O
NCH3
OH OOCCH3O
NCH3
OH OHO
NCH3
CH3O OHO
Heroin
6-Acetylmorphine Morphine Codeine
Conjugation
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Metabolism Cocaine
CH3NO
O
O OMe
CH3NO
O
O OH
CH3N OH
O OMe
CH3N OH
O OH
Cocaine Ecgonine Methyl Ester
Benzoylecgonine Ecgonine
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Metabolism Amphetamines
NH
OH
C H3 O
NH
O HNH
NH
O
NH
O
O
N H2O
O
NH
O
O
NH
O
O
N H2
O HN H2
N H2
O
O H
O
N H 2O
O
M ethca th inone Ephedrine M etham phetam ine
Cath inone Norephedrine A m phetam ine
Benzoic acid
p-Hydro la tion and C on juga tion
G lucuron ide and G lyc ine con juga tion
M D M A M D EA M B D B
H M M A M D A B D B
C on juga tion
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Choice of Bio sample Matrix
• Blood (Plasma, Serum)– Mandatory in cases of driving under the influence.
• Hair• Sweat• Saliva• Nails• Urine: Still the sample of choice since the
concentration levels are relatively high and samples can be taken non-invasively
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Relative abundance of Cannabinoids in biological matrices[Staub et al J Chromatog B 733(1999)119]
0
10
20
30
40
50
60
70
80
90
100
Blood Saliva Sweat Hair
%
THCTHC-COOHTHC-OHCBDCBN
Relative Abundance in Biological Matrices
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Concentrations of Drugs of Abuse in SalivaSchramm et al, Journal of Analytical Toxicology, 16(1992)1
Drug Class Analyte ng/ml Time of detection
Cannabinoids Delta – 9 - THC 5-330 4 – 14 hours
Cocaine Cocaine 1-10 12h – 10 days
Opiods Morphine 0.6 3h – 24 h
Codeine 0.6 - 120 3h - 36 h
Barbiturates AmobarbitalHexobarbital
100 - 8000 12 – 50 h
Methaqualone Methaqualone 20 - 300 24 h – 3 days
Diazepines Diazepam 2 - 700 5 h – 50 h
Amphetamine Amphetamine 2 -40 50h
Drugs of Abuse in Saliva
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Reported time during which drugs of abuse can be measured in URINE
Drug Comment Approximate time of detection
Cannabinoids Moderate smoker 10 days
Chronic use 30 days
Cocaine Benzoyecgonine metabolite 1 – 4 days
Phencyclidine Moderate use 8 days
Chronic use 8 – 30 days
Opiates: Codeine, Heroin, Morphine, Opium Poppy, Oxycodone, Oxymorphone,
2 days
Barbiturates: Amobarbital, Butabarbital, Butalbital, Pentobarbital, Phenobarbital, Secobarbital
Short acting 1 day
Long acting 2-3 weeks
Benzodiazepines Therapeutic dose 2 weeks
Amphetamines, Metamphetamines, Methylenedioxyamphetamines(MDA, MDMA, MDEA), Pseudoephidrine and Ephidrine
2-4 days
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Hair
• Drug incorporation in hair depends on:– Lipophilicity of drug– Melanine affinity– Membrane permeability
• Subjected to environmental contamination• Establish use weeks to months before
collection– Pre employment investigations
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Sampling procedure
– Steps to ensure that the specimen is freshly voided.
– Steps to protect against tampering and adulteration.
– Identification of the individual giving the specimen.
– Evidence of the written informed consent of the individual to the analysis of the specimen
– Disclosure of recent medication, or evidence that the individual was advised of the significance of recent medication
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I confirm that I have provided a freshly voided urine specimen to the specimen collector. “I have observed the specimen being placed and sealed in the specimen bottles and confirm that the information on this form and on the specimen labels is correct. I hereby give permission for a minimum of two sealed specimen containers to be sent to the laboratory andI consent that they be tested for evidence of drug use and for tests to be carried out to confirm the validity of the sample. I understand that the results will be communicated confidentially to the employer or a designated representative.I consent to the above.Donor’s Name (Block”Capitals) _____________________Donor’s Signature:________________________Date:________________________Donor’s identifier on the specimen labels (if different from above)_________
Example of a Donor’s Statement of Informed Consent
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1. Chain of custody
2. The minimum information required on the Chain of Custody Form is:
a) Information identifying the donor.
b) Date and time of collection.
c) Name of testing laboratory.
d) Names and signatures of all individuals who had custody of the sample during the collection process.
3. Document proceedings as close as possible, try to have a witness with you all the time.
Transport to Laboratory
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1. Barcode mismatch or absent
2. No documentation received with the sample
3. No written consent to test from the donor
4. Seals broken or tampered with on any bottle
5. No seals
6. Only 1 sample received
7. Insufficient sample for complete analysis
8. Leaking sample
Flaws in the Chain of Custody
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Laboratory AnalysisTest for adulteration
• To check that the sample submitted for testing is in fact urine.
• pH (Normal 4-9)
• Creatinine (5-20 mg/dl) – Outside this range – Specific Gravity must be determined.
• “Sample not suitable for analysis” due to an unidentified interferant or poor sample quality such as turbidity.
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Testing Philosophy
• Screening test first (Toxicology Lab/ Industrial Lab)
• then confirmation at specialized lab competent to give evidence in a court of law
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Analytical Techniques
• Screening
– Immuno assays, RIA, Enzyme-based assays, LC-DAD, GC –Selective detection
– Tandem Mass spectrometry does show some potential 200 drugs simultaneously NB!!! Quantitatively
– With direct infusion and “no chromatographic separation”
• Confirmation by GC-MS
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Operation of a tandem MS
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Compounds included in LC MS/MS drug screen
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Examples
• Ritalin• Pseudo and nor-pseudo ephedrine
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Conclusions
Tandem Mass Spectroscopy (MS/MS) provides a – fast – cost effective – selective and – sensitive
method to reliably analyse a large number of drugs simultaneously.