detection of drugs-of-abuse by tandem mass spectrometry

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: 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 Flunitrazepam, Diazepam

Opiods Codeine, Heroine, methadone, morphine, pethidine etc

Marijuana Various forms of Cannabis sativa containing tetrahydrocannabinol (THC)

Barbiturates Phenobarbital, secobarbital

Alcohol Alcoholic Beverages

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

“Orange Juice HUBLY-BUBLY”

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

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

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

The Cell

Microsomes and Lysosomes

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

Metabolism-Opiates

NCH3

CH3COO OOCCH3O

NCH3

OH OOCCH3O

NCH3

OH OHO

NCH3

CH3O OHO

Heroin

6-Acetylmorphine Morphine Codeine

Conjugation

Metabolism Cocaine

CH3NO

O

O OMe

CH3NO

O

O OH

CH3N OH

O OMe

CH3N OH

O OH

Cocaine Ecgonine Methyl Ester

Benzoylecgonine Ecgonine

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

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 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

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

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

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

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

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

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

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.

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

Operation 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.