The Effective Characterisation of Novel Pure Substance Reference

Materials while Meeting Users Needs in a Timely Fashion

Lindsey Mackay and Steve DaviesNational Measurement Institute, Australia

NMIA specialises in pure substance, organic reference materials in the areas of• Illicit drugs • Sports drug • Agricultural and veterinary chemicals

Our biggest challenge is the continued need for new materials in the areas of• New designer drugs • New prohibited substances

N

HN H

N OMe

OS

O

O

NH

OH

NN

HH

The Age of Designer Drugs

• Recent explosion in ‘Designer’ drugs

• Both Forensic and Drugs in Sport areas

• Specifically synthesised to avoid detection or to go beyond the boundaries of current legislation

How do we Detect Designer Drugs?

• NMIA has a two significant programs aimed at:

• Forensics• Within our forensic laboratory we endeavour to identify

unknown compounds found in seizures– Mass spectral and NMR investigation to elucidate structure

• Drugs in Sport• Research programs sourcing materials internationally

that are available for abuse by athletes

What is a Designer Drug?

• Substance with modification in molecular structure designed to circumvent detection or legislation

CH3

NHCH3

CH3

NHCH3

O

O

New Forensic ‘Designers’– what are they?

• Tryptamines

• Phenethylamines

• Piperazine derivatives

• Cathinone derivatives

• Synthetic cannabinoids

• ‘Research chemicals’

N

O

Tryptamines

N

HO

NH2

H N

N

H

OH

H3CCH3

SerotoninPsilocin

5'-Methoxy-N,N-diallyltryptamine

/4

/4

/4

/4

/4 /4

4.11

13

1.97

19

3.77

95

3.00

07

3.93

75

3.90

39

Inte

gral

3.86

74

3.25

593.

2398

(ppm)1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5

N

N

H

H3CO

Phenethylamines

CHCH2NH2

HO

OH

HOCH2CH2NH2

H3CO

OCH3

OCH3

Noradrenalin

Mescaline

The ‘Fly’ Compounds

O

O

NH2

BrO

O

NH2

Br

CH3

O

OH3C

NH2

Br

Br

NH2

O

O

2C-B-FLY Bromo-Dragon FLY

2CB-Butterfly

The Cathinone Family

O

O

CH3

NHCH3

O

CH3

NHCH3H3C

O

O

O NHCH3

O

CH3

CH3

NHCH2CH3

O

O

O N

O

CH3

MDPV ‘Plant Food’!

O

O N

O

CH3

MDPV as Soothing Bath Salts

FluoromethcathinoneGroup

CH3

NHCH3

OF

CH3

NHCH3

O

F

CH3

NHCH3

O

F

The “Spice” Products –Herbal Highs

• In the mid-2000s, a new range of herbal smoking products appeared on the market - The ‘Spice’range :

Spice DiamondSpice GoldSpice Silver, etc

• Distributed by ‘Psyche Deli’• Contents declared as a mixture

of the usual herbal high materials• “JWH” series

N

O

CH3

Drugs in Sport

• Our program has sourced about 200 “supplements” over the last few years– Trawling body building sites on the web– Purchasing materials– Identifying the contents– Carrying out metabolism studies– Developing methods for the metabolites in urine/blood– Preparing reference materials where needed

Sites

• Bodybuilding sites (bodybuilding.com)• Amazon.com

– supplements -184,114 products– sports supplements - 2,043 products– Many have no information on ingredients

Prostanozol

[3,2-c]pyrazole-5α-androstane-17α-methyl-17β-ol

HN

N

OH

H HN

N

OH

H

stanozolol

Superdrol

2α,17α-dimethyl-5α-androstane-3-oneOH

CH3

H3C

OH

OH

OH

Dihydrotestosterone (DHT)

2,3-Epithio-17α-methyl-5α-androst-17β-ol

OH

CH3

S

H

HH

Australian DrugAnalogue Legislation

• Section 314.4 (2) Border Controlled Drug– a stereoisomer; or– a structural isomer having the same constituent groups; or– an alkaloid;– a structural modification ……..– otherwise a homologue, analogue, chemical derivative or

substance substantially similar in chemical structure.

World Anti-Doping Agency Prohibited List

• Similarly open with respect to analogues• Bans anabolic agents

– Lists 47 specific compounds and then adds “and other substances with a similar chemical structure or similar biological effect”

• Bans diuretics and stimulants ...”and other substances with similar chemical structure or biological effect”

How do we Prepare Pure Calibrant Reference Materials

to Meet these Needs?

• Need fit-for-purpose standards available in a timely manner– Rapid certification – Identity confirmation is essential– Purity values and measurement uncertainty that meet

the requirements for prosecution

Our Approach• Purify seizures or synthesise in-house • Identity confirmation• Certification of purity by an array of techniques

to ensure cross checking of as much data as possible

Establishing Identity

Identity - Regiochemistry?

- Stereochemistry?

J = 8 Hz i.e β configuration

If available - comparison with reference spectra (1H and 13C NMR, IR, MS) and m.p.New compounds need thorough in-house assessment

HNHO

OO

Ha CO2H

H

H

OH

OH

H

HO

Hb

O2N

CHCl2

O

H

H

H

– Organic: Assay by GC-FID or HPLC-PDA/ELSD

– Volatile: Assay by TGA, Karl-Fischer

– Non-volatile: Assay by TGA (full combustion)

Cross check with 1H NMR, head space GC-MS and elemental analysis

Ultimate cross check QNMR

Comprehensive characterisation not solely relying on the mass balance approach

Assessment of Purity

mi4 5 6 7 8 9 10 11 12 13

counts

0

200

400

600

800

1000

1200

1400

1600

1800

FID1 A, (A:\COKE-H01.D)

9.9

33

11.

526

12.

682

Response Factor

Organic Purity = 99.2%

??

Identified by GC-MS

Assessment of Organic Impurities by GC-FID

Ensuring Minimal Bias in GC-FID

ret. time Area Area %7.560 4.704 0.167.677 2.940 0.108.376 16.533 0.568.581 2922.456 99.12

O

OH

OD620H

3 different types of column with different retention times

Three different GC-FID instruments

used for each certification17-Epioxandrolone

HPLC for Organic Assessment – Greater Care Required

Photo Diode Array Peak area – f([analyte] and extinction coefficient)

Cholesta-3,5-dieneλmax 235 nmεmax 19,000

Cholesta-2,4-dieneλmax 275 nmεmax 10,000

N,N -Dimethylanilineλmax 251 nmεmax 15,500

N

N

2-Methy l-N,N -dimethylanilineλmax 248 nmεmax 6,360

HPLC-PDA

H

OD853bOH

OH

HO

Estradiol OH

~ 1.4 – 1.5% ~ 97.9%

4-Hydroxynandrolone

/4

0.00

63

0.00

55

0.00

54

1.00

00

Inte

gral

(ppm)5.96.06.16.26.36.46.56.66.76.86.97.07.17.27.37.47.5

Cross Check HPLC Result with 1H NMR

H

OD853bOH

OH

~ 0.5-0.6% mass fraction

CHCl3

1H NMR also detected 0.5% ethyl acetate

HO

Estradiol OH

ThermoGravimetric Analysis - heat to 120ºC and observe weight loss.

Results compared with elemental analysis,

Karl Fischer analysis & 1H NMR (occluded solvents etc).

Volatile assessment of lactose5.07% H2O40 ºC

120 ºC

Assessment of Volatiles

TGA: < 0.2% volatiles at 120 ºC, 140 ºC and 160 ºC.

But.....

Karl Fischer moisture analysis indicates ~ 0.5% H2O mass fraction.

Underestimation of volatiles by TGAis commonly observed

ON

HO

AcO

CH3

But that’s not all.....

6-Monoacetylmorphine

Volatile Content Determination Problems

7.26

00

2.44

74

2.13

41

(ppm)1.01.52.02.53.03.54.04.55.05.56.06.57.0

1H NMR to the Rescue

Diethyl ether; 0.7% m/mDCM; 0.9% m/m

Epitestosterone: Another Case Study

OH

O

D547

5.71

55

4.83

63

3.67

243.

6575

3.01

13

2.19

522.

1904

2.18

122.

1742

2.16

782.

1559

2.14

642.

1376

2.13

242.

1251

2.12

022.

1123

2.09

952.

0915

2.08

662.

0787

1.24

37

0.73

30

(ppm)0.81.21.62.02.42.83.23.64.04.44.85.25.6

H3C CH3

O

TGA: ~ 0% volatiles at 120 ºC Water analysis by KF shows 0.4% H2O mass fraction

Acetone not detected by other

techniques

Polymerisation?In

tegr

al

(ppm)0.81.21.62.02.42.83.23.64.04.44.85.25.66.06.4

2H 1H34H

27Hs in 2005QNMR vs TPPO => Purity decrease over time not observed

by HPLC, TGA and KF

C21H28O2

1H QNMR Analysis• Every compound is subject to QNMR• Certified 5 very high purity materials to use as internal

standards• Compare multiple signals• Completely independent cross check of absolute purity

0.98

75

0.00

55

1.00

02

0.00

50

2.01

99

0.00

42

0.98

15

1.03

59

2.01

66

1.01

10

1.03

91

3.07

41

1.03

01

7.15

22

3.02

60

Inte

gral

8.98

59

7.04

097.

0201

6.50

956.

5031

6.48

876.

4820

6.42

52

6.02

38

3.53

023.

5104

3.49

00

3.35

13

2.71

822.

6913

2.68

032.

5091

2.50

472.

5000

2.49

562.

4909

1.84

00

1.25

631.

2408

0.65

44

(ppm)0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0

Certification• We no longer rely on a simple mass balance purity

approach– We use a range of techniques to cross check for bias in each

component

• Techniques like NMR provide enormous structural and purity information in a rapid way

• Each material is subject to full homogeneity assessment

• Use 20 years of experience in producing forensic and drugs in sport reference materials to estimate a stability uncertainty in the first year– Have over 150 drugs in sport pure materials and over 150 forensic

pure materials in our catalogue

Designer forensic drugs seen at NMIA in 2009/2010

• 2-fluoroamphetamine

• 4-fluoroamphetamine

• 2-fluoromethylamphetamine

• 4-fluoromethylamphetamine

• 1-(3-trifluoromethylphenyl)-piperazine

• 1-(3-chlorophenyl)-piperazine

• 1-(4-fluorophenyl)-piperazine

• Naphyrone

• JWH-018

• N-ethylcathinone

• 4'-methylmethcathinone

• 3'-fluoromethcathinone

• 4'-methoxymethcathinone

• 3',4'-methylenedioxymeth-cathinone

• 3',4'-methylenedioxypyrovalerone

• Butylone

• 5-methoxy-N,N-diallyltryptamine

Designer forensicdrugs seen at NMIA in 2009/2010

• 2-fluoroamphetamine

• 4-fluoroamphetamine

• 2-fluoromethylamphetamine

• 4-fluoromethylamphetamine

• 1-(3-trifluoromethylphenyl)-piperazine

• 1-(3-chlorophenyl)-piperazine

• 1-(4-fluorophenyl)-piperazine

• Naphyrone

• JWH-018

• N-ethylcathinone

• 4'-methylmethcathinone

• 3'-fluoromethcathinone

• 4'-methoxymethcathinone

• 3',4'-methylenedioxymeth-cathinone

• 3',4'-methylenedioxypyrovalerone

• Butylone

• 5-methoxy-N,N-diallyltryptamine

Conclusions

• Huge challenges exist to meet customer needs

• Produce materials of appropriate quality for legal proceedings in a timely manner

– Certificates must withstand legal scrutiny by defence experts– No point having the perfect reference material long after its actually

needed