Detection of Drugs-of-Abuse by Tandem Mass Spectrometry. Dr Tim Laurens MSc.Chem(Pretoria), Ph.D. Chem (Pretoria), MSc.Toxicology (Surrey,UK) FRSChem, MFSSoc Email: laurensj@lancet.co.za / tim.laurens@up.ac.za Mobile: 082 891 4886
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
CNS Depressants Benzodiazepines piods Flunitrazepam, Diazepam Codeine, Heroine, methadone, morphine, pethidine etc Marijuana Various forms of Cannabis sativa containing tetrahydrocannabinol (THC) Barbiturates Alcohol Phenobarbital, secobarbital Alcoholic Beverages
Amphetamines Cocaine ther stimulants Inhalants Halocinogens PCP Anabolic steroids CNS Stimulants Speed methamphetamine Ecstacy (methylenedioxymethamphetamine) Free base crack and hydrochloride Ephidrine, Pseudoephidrine, ther substances Petrol, solvents, propane (LPG), LSD, Ecstacy, mescaline, psilocybin, scopolamine Phencyclidine Testosterone, stanozol
range Juice HUBLY-BUBLY
Routes of Administration ral intake - Amphetamines, EXTACY, Barbitirates, tricyclic antidepressants Inhalation - Cannabis, Methaqualone, Metcathinone, Methamphetamine, Cocaine Intravenous -Heroin, piates ral intake First pass metabolism Liver Inhalation / Intravenous directly in the bloodstream
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
Drug Half Life Half-Life Concentration (mg/l) 18 16 14 12 10 8 6 4 2 0 0 2 4 6 8 10 12 Time (days)
The Cell
Microsomes and Lysosomes
Metabolism Cannabis CH 3 H C H 3 C H 3 CH 3 Delta-9-Tetrahydrocannabinol (THC) CH 2 H CH H H C H 3 C H 3 CH 3 C H 3 C H 3 CH 3 11-Hydroxy-delta-9-THC 11-nor-9-Carboxy-delta-9-THC Conjugation
Metabolism- piates NCH 3 CH 3 C CCH 3 Heroin NCH 3 NCH 3 NCH 3 H CCH 3 H H CH 3 H 6-Acetylmorphine Morphine Codeine Conjugation
Metabolism Cocaine Me Me CH 3 N CH 3 N H Cocaine Ecgonine Methyl Ester CH 3 N H CH 3 N H H Benzoylecgonine Ecgonine
Metabolism Amphetamines H N H H N H N Methcathinone Ephedrine Methamphetamine H NH 2 NH 2 NH 2 Cathinone Norephedrine Amphetamine H p-hydrolation and Conjugation Benzoic acid Glucuronide and Glycine conjugation H N H N H N MDMA MDEA MBDB CH 3 H N NH 2 NH 2 H HMMA MDA BDB Conjugation
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
Relative Abundance in Biological Matrices Relative abundance of Cannabinoids in biological matrices [Staub et al J Chromatog B 733(1999)119] 100 90 80 70 % 60 50 40 THC THC-CH THC-H CBD CBN 30 20 10 0 Blood Saliva Sweat Hair
Drugs of Abuse in Saliva Concentrations of Drugs of Abuse in Saliva Schramm 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 piods Morphine 0.6 3h 24 h Codeine 0.6-120 3h - 36 h Barbiturates Amobarbital Hexobarbital 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
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 piates: Codeine, Heroin, Morphine, pium Poppy, xycodone, xymorphone, Barbiturates: Amobarbital, Butabarbital, Butalbital, Pentobarbital, Phenobarbital, Secobarbital Short acting Long acting 2 days 1 day 2-3 weeks Benzodiazepines Therapeutic dose 2 weeks Amphetamines, Metamphetamines, Methylenedioxyamphetamines(MDA, MDMA, MDEA), Pseudoephidrine and Ephidrine 2-4 days
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
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
Example of a Donor s Statement of Informed Consent 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 and I 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)
Transport to Laboratory 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.
Flaws in the Chain of Custody 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. nly 1 sample received 7. Insufficient sample for complete analysis 8. Leaking sample
Laboratory Analysis Test for adulteration To check that the sample submitted for testing is in fact urine. ph (Normal 4-9) Creatinine (5-20 mg/dl) utside this range Specific Gravity must be determined. Sample not suitable for analysis due to an unidentified interferant or poor sample quality such as turbidity.
Testing Philosophy Screening test first (Toxicology Lab/ Industrial Lab) then confirmation at specialized lab competent to give evidence in a court of law
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
peration of a tandem MS
Compounds included in LC MS/MS drug screen
Examples Ritalin Pseudo and nor-pseudo ephedrine
Conclusions Tandem Mass Spectroscopy (MS/MS) provides a fast cost effective selective and sensitive method to reliably analyse a large number of drugs simultaneously.