1Drug AnalysisRyan Girgrah – CHEM 5801March 18, 2015

2 Outline

Pharmacokinetics Matrices Review of Technologies Drug Discussion

Marijuana Cocaine Amphetamine Type Stimulants

Therapeutic Drug Monitoring Drugs in Sports

3 Pharmacokinetics (ADME)

Absorption/Administration Distribution Metabolism Excretion

4 Detectable Periods (in Hours)

Drug Saliva Urine Blood HairAmphetamine 20-50 - 46

months-years*

Methamphetamine

24 87±51 48

MDMA 24 48 24Cannabis 34 34 or 87 5 or 36Cocaine 5-24 48-72 12 or 48Heroin 0.5-8 11-54 20GHB 5 12 5

*Wood et al., 2006

Verstraete, 2004

5 Urine and Blood

Most xenobiotics end up in blood and urine Highest concentration of analytes A study looked at 132 drugs in whole blood using LC-MS/MS* Cytochrome P450 found in the liver

Differentially expressed across individuals and races Allylic oxidation Major enzyme in the metabolism of 25% of all clinical drugs

de la Torre, 2012*Di Rago, 2014

6 Hair as a Matrix

Pros Slow clearance from hair Substances can take months to years to dissipate Useful to establish long-term/historical use

Cons Hard to determine initial dose despite a correlation. Detection varies with dosage, melanin affinity, and lipophilicity

Xiang, 2011

7 Hair analysis

Less intrusive, less embarrassing Does not require refrigeration (stored indefinitely) Grows at an average rate of 1cm/mo.

Cooper (2012)

8 Strategies for Sample Preparation

Purification Derivatization Extraction Pre-concentration Blanks Standards

9 Typical Strategies for Drug Analysis

Mobile Phase considerations Liquid chromatography (LC) or gas chromatography (GC) Tandem Mass Spectrometry (MS/MS)

Immunoassays (cross-reactivity)

10 Review of GC

Separate/Retain analytes based on their adsorption to the packing materials in the column

Mobile phase: helium, nitrogen, or argon gas Retention times depend on:

Polarity of molecule Temperature of column

11 Review of LC

Separate/Retain analytes based on hydrophobicity (Reverse Phase) Column is relatively short. Mobile phase comprised of a mix of solvents A and B

Solvent A: Ultrapure water or buffered solution Solvent B: Organic solvent miscible in water e.g. MeOH, ACN, IPA

12 Review of MS/MS

Harris, 2007

13 Review of MS/MS

Harris, 2007

14 Why MS?

149.12 Da

Monoisotopic mass135.10 Da

165.12 Da

169.07 Da??????

15 Actual Patient Analysis (HPLC)

Eichhorst, 2012

16 Substance Abuse

Definition: Using a substance (drug) outside of its intended use.

Why does it happen? Stress relief Social circle Competitive advantage

17 Drugs in the Media

Scarface (1983) Fear and Loathing in Las Vegas (1998) Super Troopers (2001) American Gangster (2007) Pineapple Express (2008) Limitless (2011)

The Wire (2002–2008) Breaking Bad (2008-2013)

imdb.com

18 Marijuana Overview

1,400 tons produced in 2012 worldwide

UNODC, 2014

19

Corn Potatoes Rice Wheat Marijuana0

50

100

150

200

250

300

350

North American Production of Common Commodities (2013)

Production in Tonnes Value/tonne

Prod

uctio

n in

Mill

ions

of T

onne

s

http://faostat.fao.org/

10, 000, 000 USD/tonne4, 000 USD/tonne

20 Metabolism of Cannabinoids

Watanabe et al., 2007

21 Active Compounds

Smoked: Δ9-tetrahydrocannabinol (THC) (A) Oral: 11-hydroxyl-Δ9-tetrahydrocannabinol (11-OH-THC) (B) Metabolite: 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) (C)

A B C

22 Routes of Entry

Smoking about 34mg of THC Ingesting about 15mg of THC

Grotenhermen, 2003

23 Chemical Properties

LogP = 6.97 Vapour pressure = 6.17 x 10-9 kPa Thermolabile and photolabile Half-life: 25-36 hours (orally) 11-OH-THC crosses the blood brain barrier more easily than THC

Grotenhermen, 2003

24 Analysis

Ruppel (Perkin Elmer), 2008

25

26 Cocaine Between 800 and 1,000 tonnes of cocaine produced per year worldwide Extracted from coca leaves

UNODC, 2008

27

Cocaine Cowboys

Griselda Blanco (aka La Madrina, Black Widow, Cocaine Godmother) Drug lord of the Medellin Cartel Pioneered cocaine trafficking into the USA Shipped over 3,400 pounds per month, about 40 murders (1979-1984) Deported from the US in 2004

http://bostinno.streetwise.co/2012/09/04/griselda-blanco-the-godmother-of-cocaine-killed-in-colombia-video/

28

18UNODC, 2014

29

Fries et al., 2008

30 Structure

c

31 Chemical Properties

LogP = varies greatly between salt and free base forms Free base is slightly volatile Solubility (salt): 200 g per 100 mL Solubility (free base): 0.17 g per 100 mL Pockets of hydrophilicity and lipophilicity Half-life: 1 hour Original function: Insecticide*

*Nathanson et al.,1993

Budavari, 1989

32 Biosynthesis of Cocaine

Leete et al., 1988

33 Biosynthesis of Cocaine

Leete et al., 1988

34 Metabolism

Snozek et al., 2012

75-90%**Jatlow, 1988

35 Analysis Snozek et al., 2012

36 Amphetamines

MDMA Methamphetamine

Amphetamine

37 Chemical Properties

LogP = 2.07 Solubility: 50 g per 100 mL (HCl salt) Very soluble in ethanol and ether Light sensitive

Neurotoxin

Hansch et al., 1995

38

Drug Enforcement Agency, 2013UNODC, 2014

c

39

Fries et al., 2008

40 The metabolism of MDMA

de la Torre, 2012

41 Therapeutic Drug Monitoring Tamoxifen is used to battle breast cancer

Tchu, 2012

42 Therapeutic Drug Monitoring

43 Therapeutic Drug Monitoring

44 Performance Enhancing Drugs

“There are gold medallists at this meet who are on drugs, that [100 metres] race will be looked at for many years, for more

reasons than one.” –Carl Lewis

(after losing in at the 1987 World Championships race to Johnson)

Sport Organisations require drug testing for two main reasons The health and safety of the athletes (short-term and long-term side

effects) Deterrent to athletes who face mounting pressure to take PEDs

Carroll, 2011

45 Olympics (Doping)

IOC Factsheet, OC Comm. (2014)

46 References1. Ahrens, B. D.; Starcevic, B.; Butch, A. W. In LC-MS in Drug Analysis;

Langman, L. J., Snozek, C. L. H., Eds.; Humana Press: New York, 2012; Vol. 902, pp 115–128.

2. Carroll, M. S. Drug Testing. Encyclopedia of Sports Management and Marketing, 2011, 412–414.

3. Cooper, G. A. A. Forensic Sci. Int. 2012, 218, 20–24.(4) 4. de la Torre, R.; Yubero-Lahoz, S.; Pardo-Lozano, R.; Farré, M. Front.

Genet. 2012, 3, 235.5. Di Rago, M.; Saar, E.; Rodda, L. N.; Turfus, S.; Kotsos, A.; Gerostamoulos,

D.; Drummer, O. H. Forensic Sci. Int. 2014, 243, 35–43.6. Eichhorst, J. C.; Etter, M. L.; Hall, P. L.; Lehotay, D. C. In LC-MS in Drug

Analysis; Langman, L. J., Snozek, C. L. H., Eds.; Humana Press: New York, 2012; Vol. 902, pp 29–41.

47 References (con’t)

7. EMCDDA. Cocaine: A European Union perspective in the global context; Luxembourg, 2010.

8. Fries, A.; Anthony, R. W.; Cseko, A.; Gaither, C. C.; Schulman, E. Price and Purity of Illicit Drugs: 1981 – 2007; Alexandria, 2008.

9. Grotenhermen, F. Clin. Pharmacokinet. 2003, 42, 327–360.10.Hammett-Stabler, C.; Cotten, S. W. In LC-MS in Drug Analysis; Langman,

L. J., Snozek, C. L. H., Eds.; Humana Press: New York, 2012; Vol. 902, pp 1–13.

11.Jatlow, P. Yale J. Biol. Med. 61, 105–113.12.Middleberg, R. A.; Homan, J. In LC-MS in Drug Analysis; Langman, L. J.,

Snozek, C. L. H., Eds.; Humana Press: New York, 2012; Vol. 902, pp 105–114.

48 References (con’t)

13.Montenarh, D.; Hopf, M.; Warth, S.; Maurer, H. H.; Schmidt, P.; Ewald, A. H. Drug Test. Anal. 2015, 7, 214–240.

14.Montenarh, D.; Wernet, M. P.; Hopf, M.; Maurer, H. H.; Schmidt, P. H.; Ewald, A. H. Anal. Bioanal. Chem. 2014, 406, 5939–5953.

15.Nathanson, J. A.; Hunnicutt, E. J.; Kantham, L.; Scavone, C. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 9645–9648.

16.Peloquin, C. A. Drugs 2002, 62, 2169–2183.17.Perrone, J.; De Roos, F.; Jayaraman, S.; Hollander, J. E. Am. J. Emerg.

Med. 2001, 19, 49–51.18.Ping, X. Hair analysis for drugs of abuse, University of Central

Lancashire, 2011.

49 References (con’t)

19.Pragst, F.; Balikova, M. A. Clin. Chim. Acta. 2006, 370, 17–49.20.Quintela, O.; Crouch, D. J. In LC-MS in Drug Analysis; Snozek, L. J. L. and

C. L. H., Ed.; Humana Press: New York, 2012; Vol. 902, pp 75–90.21.Snozek, C. L. H.; Bjergum, M. W.; Langman, L. J. In LC-MS in Drug

Analysis; Langman, L. J., Snozek, C. L. H., Eds.; Humana Press: New York, 2012; Vol. 902, pp 91–103.

22.UNODC. World Drug Report 2014; United Nations Publication: Vienna, 2014.

23.Verstraete, A. G. Ther. Drug Monit. 2004, 200–205.24.Watanabe, K.; Yamaori, S.; Funahashi, T.; Kimura, T.; Yamamoto, I. Life

Sci. 2007, 80, 1415–1419.

50 Questions?I’M THE ONE WHO ASKS!!!