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Transcript
Novel Psychoactive
Substances: Challenges and
Choices in Forensic Toxicology
Julia M. Pearson, PhD, F-ABFT
Chief Forensic Toxicologist
Hillsborough County Medical Examiner
Tampa, FL
Forensic TOXICOLOGY
The Study of Poisons for Forensic Toxicology
The study of the effects of chemical substances on
living systems
Forensic test results are subject to challenge in our
adversarial justice system
For forensic toxicology use only.
All substances are poisons; there is none which is not a
poison. The right dose differentiates a poison and a
remedy
Paracelsus (1493-1541)
Poisons and medicines are often times the same
substance given with different intents.
Peter Mere Latham (1789-1875)
For forensic toxicology use only.
Types of Substances Analyzed in Forensic Toxicology
Volatiles
Gases
Acid/neutral/basic compounds
Hydrocarbons
Halogens
Polar and non-polar
Lipophilic and hydrophilic
Conjugates or glucuronides
Small and large molecules
Thermally stable and labile
Heavy metals
Extremely potent drugs (ng/mL or pg/mL range)
There is not a single analytical instrument that can analyze for all these
various analytes
For forensic toxicology use only.
Forensic Toxicology Analysis
 Screen
 An initial test to distinguish negatives from presumptive positives
 Immunoassay, color tests, GCMS, LC-MS/MS, LC-TOF
 Confirmation
 A second test used to definitively identify a drug or chemical
 Usually mass spectral based techniques
 Quantitation
 A test used to identify the amount of drug present in the specimen
 Immunoassay, chromatographic or mass spectral based techniques
 General Rule in Forensic Analysis
 The detection of drugs should be confirmed (whenever possible) by a second
analysis preferably based on a different analytical principle.
For forensic toxicology use only.
Analytical Method Validation Parameters
 Accuracy (bias)
 Precision
 Limit of detection
 Limit of quantitation
 Interference studies
 Calibration model
 Carryover
 Ionization suppression/enhancement (LC techniques)
 Dilution integrity
 Stability
Data from United Nations Office of Drugs and Crime Early Warning Advisory on New Psychoactive Substances (Jan 2015)
Analytical Challenges Associated with
Novel Psychoactive Substances
 Is a standard available?
 Cost of the analytical standard?
 Mass spectral library?
 Published analytical methods?
 Published case studies to help interpret results?
 Staffing resources available to analytical forensic method
development and validation (estimated 3-6 months)?
 Is it cost effective to develop forensic method or send to a private
reference laboratory?
For forensic toxicology use only.
Immunoassays
 Inexpensive, rapid tests to screen for specific drugs or drug classes
 Can be very sensitive (especially kits developed for blood and oral fluid)
 Requires a known standard and antibody to develop an immunoassay
(make take several months to years to develop a new immunoassay to a
new psychoactive substance)
 Manufactured commercially
 Some new designer drugs cross-react with immunoassays for other similar
classes of drugs
 Cathinones may cross-react with methamphetamine immunoassay
 Fentanyl may cross-react with fentanyl immunoasssay
For forensic toxicology use only.
Some Designer Stimulants
Cathinones
Tryptamines
Benzofurans
Methylone
5-MeO-DALT
5 APDB
Mephedrone
5-MeO-DIPT
6 APDB
Methedrone
5-MeO-DMT
5 APB
MDPV
DMT
6 APB
Butylone
Ethylone
Pentylone
Fentanyl, Acetyl Fentanyl
and Butyryl Fentanyl
Fentanyl
Butyryl Fentanyl
For forensic toxicology use only.
Full Scan Electron Impact (EI)
Gas Chromatography
Mass Spectrometry (GCMS)
EI full scan GCMS
 Used extensively for decades in analytical forensic toxicology laboratories, ‘gold
standard’
 All analytes eluting from column are hit with a beam of electrons at 70 eV so
fragmentation pattern is identical on all instruments
 Several hundred thousands of chemicals are published in searchable full scan
GCMS libraries (NIST, SWGDRUG, AAFS, Maurer Wiley and websites)
 Libraries must be periodically upgraded to include new compounds
 Analysis performed is gas phase so analytes amenable to GCMS analysis are
typically small, volatile, thermally stable and non-polar
 To maximize sensitivity, targeted analysis can be conducted using selected ion
monitoring (SIM) mode
 Straightforward, simple operation (or more so than LC-MS/MS or TOF)
 Sensitivity µg/mL to ng/mL range
 Cost ~ $50,000 to $150,000
For forensic toxicology use only.
CASE STUDIES IN WHICH GCMS
ANALYSIS IDENTIFIED THE NPS
LSD
Methylone and 5-APDB
25I-NBOMe
Fentanyl analogs
For forensic toxicology use only.
LSD Poisoning Case
• A family of four (including a mother in her ninth month of pregnancy)
became ill after consuming a home cooked meal of steak fajitas.
• Initial symptoms included tingling in arms and legs, nausea, dizziness,
tachycardia, dilated pupils and hallucinations.
• The family drove to a nearby hospital where they were admitted.
• All victims (including the newborn infant) recovered spontaneously with 24
hours after admission and were released within a few days.
• Hospital urine drug test results were negative.
For forensic toxicology use only.
LSD Poisoning Case
 All four victims’ hospital blood and urine samples were analyzed for 12
classes of drugs of abuse by immunoassay
 Acetaminophen, barbiturates, benzodiazepines, carisoprodol/meprobamate,
cocaine metabolite (benzoylecgonine), cannabinoids, fentanyl, methadone,
methamphetamine, opiates, oxycodone and salicylates.
 All results were negative.
 All four victims’ hospital blood and urine samples were analyzed by full
scan electron impact GCMS.
 All results were negative.
For forensic toxicology use only.
The suspected ‘tainted’ meat package
For forensic toxicology use only.
LSD Poisoning Case
• LSD was identified on the ingested steak fajita meat by GCMS
analysis.
• Targeted analysis for LSD in urine by LC-MS/MS confirmed the
presence of LSD in three of the victims’ samples (the fourth victim’s
urine sample had insufficient volume for LSD analysis).
• Once the LSD was identified on the steak fajita meat, the case was
taken over by the FDA for specialized testing.
• FDA has confirmed the presence of LSD on the steak fajita meat and
the case is the source of the LSD was never determined.
For forensic toxicology use only.
Methylone Case 1
 23 year old male
 Walking in and out of traffic at major intersection,
banging on cars with fists, screaming profanities,
yelling rap songs
 Refuse to cooperate with police, combative,
resisted arrest, police used taser (missed) and
tackled him to ground
 Transported to ER
 Body temperature 105.9° F
 Seizures
 Cardiac arrest
 Rhabdomyolysis
 Multiple organ failure
 Died 24 hrs after admission
 Post Mortem Samples were tested
Methylone Case 2
 19 yr old female at club in Kansas City
 Witnessed to take a pill called “Molly”
 Collapsed with seizures
 Taken to ER
Body temperature 103.9° F
Asystole
Despite resuscitation efforts pronounced dead 8
minutes after admission
 Post Mortem Samples were tested
For forensic toxicology use only.
Methylone Case 3
 23 yr old male after after-hours club
 Witnessed to take “LSD”
 Acting irrational, sweating, bad trip, freaking out
 Management facilitated his removal by “saran
wrapping” him to chair and place him in chair into the
back of his friend’s van
 4 hours later another friend found him in the van
 Transported to ER
Body temperature 107° F
Heart rate 132
Acidotic
Pronounced dead 45 minutes after admission
 Post Mortem Samples were tested
For forensic toxicology use only.
Methylone Analysis
 In all three cases, immunoassay was positive for methamphetamines
yet no amphetamines or methamphetamines were detected.
 GCMS identified an early eluting unidentified peak that was
determined to be methylone
For forensic toxicology use only.
Designer Drug Cases
Designer Drug Cases
12
10
10
8
6
6
4
4
2
0
2
2
1
1
0
0
0
2011
2012
2013
Methylone
Ethylone
0
2014
Alpha-PVP
2015
Synthetic LSD
For forensic toxicology use only.
Case Report
25I-NBOMe










20 yr old male
Usually the “trip sitter” for friends while they did “LSD”
On date of incident, deceased decided to use “LSD”
Went crazy, freaking out, running through woods
Several hours later someone reported hearing “Oh shit” and
thud
Deceased found lying on pavement
surrounding swimming
th
pool. It is believed he fell from 7 floor balcony.
Drug screen (including LSD) were negative.
25I-NBOMe not detected using standard toxicology
procedures
25I-NBOME was identified on “LSD” blotter paper by GCMS
Targeted analysis by LC-MS/MS confirmed 25I-NBOMe in
biological specimens
For forensic toxicology use only.
Fentanyl GCMS Full Scan EI
14_07590_URINE #4764
RT: 15.26
T: {0,0} + c EI Full ms [43.00-550.00]
AV: 1
SB: 2 15.17 , 15.32
NL: 2.28E7
146.1
100
95
90
85
245.2
80
75
70
Relative Abundance
65
60
55
50
189.2
45
40
35
30
25
105.1
44.1
57.0
20
77.1
132.1
70.1
10
91.1
158.1
246.2
202.1
118.1
5
159.1
203.1
0
50
Fentanyl
96.1
15
100
150
244.1
200
m/z
For forensic toxicology use only.
247.2
250
282.0
298.0
300
316.1
331.0
340.1
350
Acetyl Fentanyl GCMS Full Scan EI
14_07590_URINE #4588 RT: 14.96
T: {0,0} + c EI Full ms [43.00-550.00]
AV: 1
SB: 243 14.62-14.89 , 15.02-15.16
NL: 2.03E7
231.2
100
95
146.1
90
85
80
75
70
Relative Abundance
65
60
55
50
188.1
45
40
35
44.1
30
105.1
158.1
25
77.1
20
189.2
96.1
118.1
91.1
79.1
15
232.2
132.1
70.1
10
69.1
5
159.1
172.2
0
50
100
150
207.1
208.1
233.2
200
m/z
For forensic toxicology use only.
250
266.1
282.1
295.1
300
315.0
343.0
350
Butyryl Fentanyl GCMS Full Scan EI
15-06440_D #4691-4720 RT: 14.63-14.69
T: {0,0} + c EI Full ms [43.00-550.00]
AV: 30
SB: 1227 13.47-14.49 , 14.87-15.98
NL: 7.44E4
100
95
90
85
80
75
70
146.2
44.1
Relative Abundance
65
60
105.1
55
50
45
77.1
40
55.1
35
189.2
30
71.1
25
259.3
91.1
20
Butyryl Fentanyl
98.2
15
118.1
10
132.2
82.1
153.4
5
144.3
158.2
172.2
186.3
190.1
199.2
216.3
260.3
236.3 246.4
270.6
0
60
80
100
120
140
160
180
200
m/z
220
240
For forensic toxicology use only.
260
309.0
287.1
280
300
323.1 333.2
320
340
354.2
Liquid Chromatography Tandem Mass
Spectrometry (LC-MS/MS)
LC-MS/MS
 Becoming more popular since they are easier to use and more affordable
than before
 Extremely sensitive ng/ml to pg/mL range
 Can analyze larger, non-volatile, thermally labile and polar molecules
(unlike GCMS)
 Targeted analysis scanning for precursor ions and then fragmenting to
product ions (selected reaction monitoring (SRM) or multiple reaction
monitoring (MRM)
 Subject to ion suppress/enhancement from matrix components that can
interfere with ionization during electrospray
 Since the voltages on electrospray and collision energies vary depending
on analytes, LC-MS/MS libraries are not transferable between instruments
and must be established on each instrument
 LC-MS/MS better suited for targeted analysis and not unknown screening
 Price range approximately <$200,000 to $300,000
For forensic toxicology use only.
LC-MS/MS Forensic Applications
 Cocaine, Cocaethylene, benzoylecgonine quantitation
 Opiate quantitation
 Benzodiazepine quantitation
 Cannabinoid quantitation
 Targeted 25I-NBOMe analysis of biological specimens (pg/mL)
 Targeted LSD analysis in biological specimens (pg/mL)
 Targeted fentanyl analogue analysis in biological specimens
(pg/mL)
For forensic toxicology use only.
LC-MS/MS Forensic Analysis of blank
blood versus 0.5 ng/mL fentanyl control
For forensic toxicology use only.
Liquid Chromatography High Resolution Mass
Spectrometry (LC-HRMS)
Liquid Chromatography Time of Flight (LC-TOF)
or Quadrupole Time of Flight (Q-TOF)
Liquid Chromatography Orbitrap MS or
Quadrupole Exactive (Q Exactive) MS
For forensic toxicology use only.
Liquid Chromatography High Resolution
Mass Spectrometry (LC-HRMS)
 GCMS and LC-MS/MS typically have mass resolution of approximately 1
amu (285 ± 1 amu)
 HRMS has mass resolution of 0.0001 to 0.001 amu (1-10 ppm) or better
 Can be used for general unknown screening to identify the structure of an
unknown analyte
 HRMS can distinguish between chemicals with similar masses
 Morphine (285.1365) and 7-aminoclonazepam (285.0669)
 Usually used in tandem with a quadrupole and collision cell to get some
fragmentation
 Newer instruments, laboratories inexperienced with the technology, pretty
complicated to use
 Approximate cost >$200,000 - $300,000
For forensic toxicology use only.
Schematic of LC Q Exactive (Focus)MS
For forensic toxicology use only.
Schematic of LC-QTOF
Liquid Chromatography High Resolution
Mass Spectrometry (LC-HRMS)
 HRMS cannot distinguish between isobaric compounds
 Morphine (285.1365) and hydromorphone (285.1365)
 Cocaine (303.1470) and scopolamine (303.1470)
 Hydrocodone (299.1521) and codeine (299.1521)
 Phentermine (149.1204) and methamphetamine (149.1204)
 JWH-019 (355.1939) and JWH-122 (355.1939)
 JWH-016 (341.1779) and JWH-018 (341.1779)
 Butylone (221.1051) and Ethylone (221.1051)
 Amitriptyline (277.1830) and EDDP (277.1830)
 For isobaric compounds must use retention time and fragmentation or other
mass spectral technique for definitive identification
For forensic toxicology use only.
Comparison of GCMS, LC-MS/MS and LC-HRMS
GCMS
LC-MS/MS
LC-HRMS
Screening
Full scan
Targeted
Full scan
Analytes
Small, volatile,
thermally stable,
non-polar
Small or large,
non-volatile,
thermally unstable
and polar
Small or large,
non-volatile,
thermally unstable
and polar
Resolution
1 amu
1 amu
0.001-0.0001 amu
Libraries
Large,
transferable
Smaller, nontransferable
Smaller, nontransferable
Analyte
identification
EI full scan spectra MS/MS
fragmentation
and retention
time
and retention
time
Exact mass,
isotope pattern,
fragmentation
and retention
time
Cost
$
$$-$$$
$$
For forensic toxicology use only.
Designer Drugs
They are the wave of the future
They are here to stay
They will continue to be a legal and forensic
laboratory nightmare
Synthetic drug chemists will always have an
advantage over legislation and labs
Economics 101: Supply and Demand