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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
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