Analytical Challenges in Identifying Structural Isomers of Drugs

JOHN GOODPASTER

DONNA ROSKOWSKI

ZACK ROBERSON

Forensic Chemistry ReduxIdentification

◦ Qualitative: Given an unknown, determine the presence or absence of a particular chemical species in the sample

◦ Quantitative: Given an unknown, determine the amount of a particular chemical species in the sample

◦ Keys to success are sensitivity, selectivity and specificity

Comparison◦ Compare the physical and chemical

characteristics of a questioned sample (Q) to the of a known sample (K)

◦ Provides circumstantial links between a suspect, victim and crime scene

◦ Keys to success are a large and diverse population of items, highly discerning analytical methods, and statistical analysis of the results

“cat and mouse synthetic drugs” (185,000 results on Google)

Analytical ChallengesThe analysis of synthetic drugs (cannabinoids, cathinones, etc.) is inherently more challenging because:

◦ The analysis is more complex and time consuming

◦ Complex mixtures are routine

◦ Closely related substances can appear very similar during analysis

◦ Reference materials may not be available

Drug OriginsNatural

◦ Marijuana

◦ Cocaine

Semi-synthetic◦ Heroin (from morphine)

◦ LSD (from lysergic acid)

Synthetic◦ Methamphetamine

◦ MDMA (“Ecstasy”)

Synthetic Drugs of Concern:◦ Cannabinoids (“Synthetic Marijuana”)

◦ Examples: “Spice”, “K2”

◦ A wide variety of compounds that target the cannabinoid recepters in the brain (CB! And CB2)

◦ Cathinones (“Bath Salts”)◦ Methcathinone

◦ 4-MET

◦ Opiates◦ fentanyl

◦ carfentanyl

◦ acetylfentanyl

◦ Psychedelic Hallucinogens◦ 25I-NOBMe

◦ 2C-I

◦ 25I-NBOH

IsomersChemically, one of two or more compounds having the same molecular formula (i.e., empirical formula), but different structures.

Structural Isomers (Constitutional Isomers)◦ Skeletal: Differ in 1o, 2o, or 3o configurations of the same functional group

◦ Positional (Regioisomers): Differ only in the location of a particular functional group

◦ Functional: Differ in functional groups (e.g., ethanol and dimethyl ether)

Stereoisomers (Spatial Isomers)◦ Diastereomers (non-superimposable, non-mirror images)

◦ Enantiomers (non-superimposable, mirror images)

Positional IsomersJWH-302

C22H25NO2

MW = 335.439 g/molNOT FEDERALLY SCHEDULED*

ANALGESIC (LESS POTENT)

JWH-250C22H25NO2

MW = 335.439 g/molDEA SCHEDULE I

ANALGESIC

* https://www.deadiversion.usdoj.gov/schedules/orangebook/c_cs_alpha.pdf

“meta”

“ortho”

Positional Isomers3,4-MDMAC11H15NO2

MW = 193.25 g/molDEA SCHEDULE I

PSYCHEDELIC / STIMULANT

2,3-MDMAC11H15NO2

MW = 193.25 g/molNOT SCHEDULED*

WEAKER EFFECTS ON SEROTONIN

* https://www.deadiversion.usdoj.gov/schedules/orangebook/c_cs_alpha.pdf

What Will It Take to “Catch the Mouse”?SCIENTIFIC INNOVATION

Increase the SPECIFICITY of analytical determinations

Develop instrumental methods that can separate and identify drugs, including as many isomers as possible.

STRUCTURE-BASED CONTROLS

Pass laws that control a “family” of compounds that is sufficiently specific to target the drugs of abuse yet avoid controlling everything

Requires direct participation of chemists in writing legislation

Scientific Innovation

Specificity / Discriminating PowerSpecificity is the ability of an analytical technique to unambiguously identify an analyte.

Highly specific methods can distinguish very subtle differences between analytes, including structural isomers.

Hierarchy of Results:1. Chemical Structure

(confirmatory)

2. Compound Class

3. Functional Groups (presumptive)

Confirmatory data must be reviewable:

◦ Printed spectra

◦ Printed chromatograms

SWGDRUG Classifications

Goodpaster Group ResearchAutomated Derivatization and Identification of Controlled Substances via Total Vaporization Solid Phase Microextraction (TV-SPME) and Gas Chromatography/Mass Spectrometry (GC/MS) (NIJ Award 2015-DN-BX-K058)

Goal: Developing SPME-based GC/MS methods for thermally unstable drugs “as is” or by forming derivatives on-fiber

Coupling Gas Chromatography (GC) and Vacuum Ultraviolet (VUV) Spectroscopy for Forensic Applications (NIJ Award 2017-R2-CX-0018)

Goals: Assessing the sensitivity, selectivity and specificity of a GC/VUV system with a focus on differentiating structural isomers of controlled substances

GC/VUV analysis

0.0

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0 2 4 6 8

AB

SOR

BA

NC

E

RETENTION TIME (min)

125 - 160 nm 140 - 160 nm 170 - 200 nm 125 - 240 nm

Methamphetamine (C-I)Phentermine (C-IV)

Methcathinone (C-I)

Pseudoephedrine (BTC)GC Column: Agilent HP-5 30m x 0.32mm x 0.25µmInjector Mode: Splitless - 0.5 min, 250°C, 1µLGC Oven: 50°C, hold 1 min, 15°C/min to 150°CTransfer Line and Flow Cell: 275°CColumn Flow Rate: 5 mL/min H2

Makeup Gas Pressure: 0.25 PSI N2

EI Mass Spectra (Category “A”)

58 58

9191

91

5858

91

(M-15)134

Formula: C10H15N

Molecular weight: 149.2328

(M-15)134

Formula: C10H15N

Molecular weight: 149.2328

These two compounds are structural (i.e., skeletal) isomers

VUV Spectra (130 – 240 nm)

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130 150 170 190 210 230NO

RM

ALI

ZED

AB

SOR

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WAVELENGTH (nm)

(±)-Methamphetamine Phentermine

These two compounds are structural (i.e., skeletal) isomers

EI Mass Spectra (Category “A”)

58

91

58

91

134

•Formula: C10H13NO

•Molecular weight: 163.2163

Formula: C10H15NO

Molecular weight: 165.2322

58 58

7777105 105, 106

587758

77

These two compounds are NOT isomers

105

VUV Spectra (130 – 240 nm)

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130 155 180 205 230

NO

RM

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WAVELENGTH (nm)

S(-)-Methcathinone HCl R,R(-)-Pseudoephedrine

These two compounds are NOT isomers

Measured Characteristics of the VUV Detector

1. Wavelength Accuracy: Meets expected specifications of ±2 nm

2. Wavelength Repeatability: Exceeds expected specifications of ±1 nm, observed ±0.5 nm

3. Baseline Noise Level (Root Mean Square Noise): Exceeds expectations of 0.001 A.U., observed 0.0005 A.U.

4. Baseline Stability: Meets expected specification of < 0.003 A.U. h-1

5. Assessed sensitivity, detection limit and linear range using cinnamaldehyde as a model compound

Chemical formula C9H8O

Molar mass 132.16 g/mol

Sensitivity, Linearity, Detection Limit

Linear range: 3 – 750 ppm

y = 0.0116x - 0.0014R² = 0.9998

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1 10 100 1000

SUM

MED

AB

SOR

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E AT

MA

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UM

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AV

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GTH

CONCENTRATION (ppm)

y = 0.8931x + 2.1793R² = 0.9047

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S/N

)

log(S) (peak area)

Phenylethylamine VUV Reference Spectra Acquired Thus Far (120 – 430 nm)

1. N-benzeneethanamine (C8H11N, 121.2 g/mol)

2. Amphetamine (C9H13N, 135.2 g/mol)

3. Methamphetamine (C10H15N, 149.2 g/mol)

4. Ephedrine (C10H15NO, 165.2 g/mol)

5. Pseudoephedrine (C10H15NO, 165.2 g/mol)

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120 220 320 420

NO

RM

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WAVELENGTH (nm)

Amphetamine Methamphetamine

N-benzeneethanamine Ephedrine

Pseudoephedrine

drug RN Rα Rβ R2 R3 R4 R5 R6

Benzeneethanamine H H H H H H H H

Amphetamine* CH3 H H H H H H H

Methamphetamine CH3 CH3 H H H H H H

Ephedrine CH3 CH3 OH H H H H H

pseudoephedrine CH3 CH3 OH H H H H H

The Role of Multi-variate Statistics

Sauzier, G.; Reichard, E.; Lewis, S.; van Bronswiijk, W., Goodpaster, J.V. Improving the confidence of “questioned versus

known” fiber comparisons using microspectrophotometry and chemometrics. Forensic Chemistry 2016; 2:15-21

Structure-Based Controls

Structure-Based Control of Synthetic DrugsControls the base molecule with certain allowable substitutions

Controls a large number of substances◦ Including their isomers and analogs

◦ Getting more difficult and expensive to make things that are not included.

◦ New drugs emerging to get around the law

What About Skeletal + Positional + Functional Isomers?

4-Methylethcathinone (4-MET)C12H17NO

MW = 191.27DEA SCHEDULE I

STIMULANT

N,N-Diethyl-meta-toluamide (DEET)C12H17NO

MW = 191.27NOT SCHEDULED

INSECT REPELLANT

meta-, 3o amide para-, ketone + 2o amine

Synthetic Cannabinoids (Included Structures)Any compound structurally derived from 3-(1-naphthoyl)indole or 1H-indol-3-yl-(1-naphthyl)methane by substitution at the nitrogen atom of the indole ring by alkyl, haloalkyl, alkenyl, cycloalkylmethyl, cycloalkylethyl, 1-(N-methyl-2-piperidinyl)methyl or 2-(4-morpholinyl)ethyl group, whether or not further substituted in the indole ring to any extent and whether or not substituted in the naphthyl ring to any extent.

Synthetic Cannabinoids Base StructuresNaphthoylindoles

◦ [3-(1-naphthoyl)indole]

Napthoylpyrroles◦ [3-(1-naphthoyl) pyrrole]

Naphthylmethylindenes◦ [1-(1-naphthylmethyl)indene]

Phenylacetylindoles◦ [3-phenylacetylindole ]

HydroxyCyclohexylphenols◦ [2-(3-hydroxycyclohexyl)phenol ]

Benzoylindoles◦ [3-(benzoyl)indole ]

As of June 2018, the following JWH compounds are controlled (DEA Schedule I): JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-250, JWH-398 (these compounds are either naphthoylindoles or phenylacetylindoles)

Naphthylindole (JWH-018)

napthyl ring

indole ring

alkyl substituent

CathinonesSubstituted 2-aminopropan-1-ones

Aka “Bath Salts”

Same type of wording in the law◦ Contains the base structure

◦ Included modifications, or additions to the base structure

CathinoneC9H11NO

MW = 149.1897DEA SCHEDULE I

STIMULANT

Substituted Cathinones

Methylenedioxypyrovalerone (MDPV)

SummaryThe “go to” analytical technique of GC/MS is sensitive, selective and specific, but not for ALL compounds

The issue of dominant fragmentation mechanisms means that several isomeric and/or closely related compounds cannot be distinguished by GC/MS

GC/VUV shows promise for discriminating drugs

Synthetic drug law controls a large number of substances, but not ALL of them.

Ultimately, the analysis and identification of a specific isomer may be difficult.