Authors: Matthew Quinn, B.S.; Michael Cain Jr., B.S. and Monica Joshi*, Ph.D.

*Corresponding author:
Monica Joshi, Ph.D.
Department of Chemistry
West Chester University of Pennsylvania
mjoshi@wcupa.edu

Date first published: June 30, 2017

This project was supported by Award No. 2014-R2-CX-K008, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the Department of Justice.

Introduction

A microcrystalline test is a precipitation reaction between a drug and a reagent, forming an insoluble drug-reagent complex that is unique to that specific test. These tests are quick, requiring minimal sample preparation and can be non-destructive. Therefore, they can be used as preliminary and confirmatory tests with expertise. Microcrystalline tests are one of the oldest analytical chemistry practices and their use for classic drugs such as cocaine, heroin and amphetamines is well-documented. However, there is very limited research on microcrystalline tests for the novel compounds encountered by law enforcement today. This research is an effort to increase understanding and promote use of microcrystalline tests for novel psychoactive substances.

Infrared spectroscopy is a well-established technique for the identification of drugs and adulterants and is categorized as a SWGDRUG category A technique, providing selectivity through structural information. Microcrystalline tests are a category B technique that provides selectivity by leveraging differences in physical and chemical characteristics of substances. An analytical scheme that combines these two techniques yields both visual data and structural information.

This library compiles the images of microcrystals and an infrared spectrum for the most characteristic microcrystal of that substance. The 30 substances included in the library represent the ever-evolving structural classes of cathinones, phenethylamines, piperazines, aminoindanes and opioids. The section for each substance includes a brief introduction of the substance, descriptions and photomicrographs of microcrystals observed with different test reagents, and infrared spectra of one or more microcrystal tests. The infrared spectra demonstrate that the microcrystal observed with a specific reagent is indeed representative of the structure of the substance being studied. The microcrystal characteristics combined with the infrared spectrum give both a SWGDRUG category A and category B test for that substance.

The sensitivity of the tests varies widely both between classes of substances and within a class. Characteristic crystals were observed with test amounts ranging between 1-30 µg of the substance. The microcrystalline tests that performed reliably with a practical crystal formation time are reported in the library.

The purpose of the library is to serve as a reference to analysts performing microcrystalline tests. We invite analysts working with typical forensic samples to provide feedback on the performance of the tests and any deviations from crystals observed with reference chemicals.

Procedures and Instrumentation

Chemical reference standards for all substances were purchased from Cayman Chemical (Ann Arbor, MI) as 1 mg/mL methanolic solutions or as 1 mg solids. The solids were dissolved in 1 mL methanol for the study. Reagents used in this study were made as per the recipes listed below. A drop of test solution was placed on a glass slide to achieve a dried residue amount between 1- 30 µg depending on the analyte. The residue was dissolved in 5-10 µL of water, 10% hydrochloric acid or 10% acetic acid solutions. To this drop, 10 µL of reagent was added and the drop mixed to stimulate crystal growth. Microcrystal formation time, shape, habit, and features under crossed polars were observed and documented.

A Leica DM 750P polarized light microscope and an Olympus BX43F polarized light microscope were used to observe the resulting microcrystals. Characteristic microcrystals were documented as photomicrographs in the Olympus cellSense Entry imaging software at 100-200x magnification. A Thermo Scientific™ Nicolet™iN™10 Infrared Microscope with a liquid nitrogen cooled mercury, Cadmium, Tellurium (MCT) detector was used for analysis. All data was processed and analyzed in the Thermo Scientific™ OMNIC™ Picta™ software. The spectra were collected in transmission mode on AMTIR windows. Crystals grown on glass slides were carefully moved to the AMTIR windows with Microtools (MiTeGen LLC, Ithaca, NY). Excess reagent was wicked away with fine paper wicks and the crystals were washed with chloroform. This procedure removed interferences in the spectra and improved the quality of the infrared spectrum collected for a microcrystal.

Reagents Evaluated

     1. Gold chloride: Two formulations were used.
          o 5% Aqueous HAuCl4
          o 5% HAuCl4 in 1:2 concentrated H2SO4: H20

     2. Gold bromide (HAuBr4): 1 g HAuCl4 + 0.76g NaBr in 5 mL glacial CH3COOH + 15 mL 2:3 concentrated H2SO4: H20

     3. Platinic chloride (H2PtCl6): 5% Aqueous H2PtCl6

     4. Platinic bromide (H2PtBr6): 1 g H2PtCl6in 1.7 mL 40% HBr + 20 mL 2:3 concentrated H2SO4: H20

     5. Mercuric chloride (HgCl2): Two formulations were used depending on the length of time for crystal growth. The          reagent crystallizes very quickly in the aqueous formulation and prevents growth of drug-reagent crystals.
          o 1 g HgCl2 in 100 mL water
          o 1 g HgCl2 2.5 mL 1:1 Glycerol: Water 14.2 mL water, 500 μL 3M HCl

     6. Mercuric iodide (HgI2): 1 g HgI2 in 20ml 27:73 HCl: H20

Novel Psychoactive Substances Studied

    I. Cathinones

    For more information about this class of substances:
    Synthetic cathinones drug profile (EMCDDA)
    Synthetic Cathinones ("Bath Salts") DrugFacts

         1. 3,4-Methylenedioxypyrovalerone (3,4-MDPV)
         2. Methcathinones
         3. Ethcathinone
         4. Pentedrone
         5. Mephedrone
         6. 2-Ethylmethcathinone (2-EMC)
         7. 3-Ethylmethcathinone (3-EMC)
         8. 3-Methylbuphedrone
         9. 4-Methylbuphedrone
         10. Butylone
         11. Methylone
         12. NRG-3
         13. 4-Fluoromethcathinone
         14. α-Pyrrolidinopentiophenone
         15. 4'-methyl-α-Pyrrolidinohexanophenone (MPHP)
         16. 4'-methyl-α-Pyrrolidinopropiophenone (4-MePPP)

    II. Phenethylamines

    For more information about this class of substances:
    Phenethylamines (UNODC)
    NFLIS Special Report: Emerging 2C-Phenethylamines, Piperazines, and Tryptamines in NFLIS, 2006-2011
    NFLIS Special Report: 2C-Phenethylamines, Piperazines, and Tryptamines Reported in NFLIS, 2011–2015

         1. 25B-NBOMe
         2. 2C-B
         3. 2C-B-BZP
         4. 2C-B-FLY
         5. Bromo-DragonFLY

    III. Piperazines

    For more information about this class of substances:
    Piperazines (UNODC)
    NFLIS Special Report: Emerging 2C-Phenethylamines, Piperazines, and Tryptamines in NFLIS, 2006-2011
    NFLIS Special Report: 2C-Phenethylamines, Piperazines, and Tryptamines Reported in NFLIS, 2011–2015

         1. BZP
         2. 1-(4-Methoxyphenyl) piperazine (4-MeOPP)
         3. 4-FluoroBZP

    IV. Aminoindanes

    For more information about this class of substances:
    Aminoindanes (UNODC)

         1. 5-IAI
         2. MDAI

    V. Opioids

    For more information about this class of substances:
    DEA Taxonomy: Narcotics (Opioids)
    DEA 2017 National Drug Threat Assessment
    UNODC World Drug Report 2017: Opioid market in a constant state of change

         1. Fentanyl
         2. FIBF
         3. Furanyl Fentanyl
         4. W-18

Overall Observations and Conclusions

Microcrystalline tests are quick tests to determine the presence of target substances in a given liquid or solid. Some substances give easily recognizable crystals and others give crystals that are amenable for infrared microspectroscopy. In some cases, the crystals are unique to that substance when studied as a pure substance but in mixtures or in unknown samples, the crystal form may be similar to other crystals of other substances with the same reagent. Therefore, the crystals need to be studied carefully for characteristic features and must be combined with instrumental techniques to improve selectivity.

Overall, the gold-based reagents gave multiple reactions and formed distinct crystals with several of the substances studied.

A study of positional isomers such as 2- EMC and 3-EMC, 3-Methylbuphedrone and 4-Methylbuphedrone demonstrates the strength of the microcrystalline tests. Crystals of both isomers are observed in a mix of the isomers. Each isomer gives distinct crystals with the appropriate test reagent. When combined with IR microspectroscopy, the isomers can be structurally identified.

Additional Resources

    1. The challenge of novel psychoactive substances, Global SMART Programme, United Nations Office on Drugs and Crime (UNODC), 2013

    2. McCrone Research Institute. A Modern Compendium of Microcrystal Tests for Illicit-Drugs and Diverted Pharmaceuticals, 2015.

    3. Leonie Elie, Mark Baron, Ruth Croxton, Mathieu Elie, Microcrystalline identification of selected designer drugs, Forensic Science International, Volume 214, Issue 1, 2012, Pages 182-188

    4. C.C. Fulton, Modern Microcrystal Tests for Drugs, first ed., John Wiley & Sons, Inc., New York, 1969

Follow

Browse the Library of Microcrystalline Tests for Novel Psychoactive Substances Collections:

1-(4-Methoxyphenyl) piperazine (4-MeOPP)

25B-NBOMe

2C-B

2C-B-BZP

2C-B-FLY

2-Ethylmethcathinone (2-EMC)

3,4-Methylenedioxypyrovalerone (3,4-MDPV)

3-Ethylmethcathinone (3-EMC)

3-Methylbuphedrone (3-methyl BP)

4-FluoroBZP

4-Fluoromethcathinone (4-FMC)

4-Methylbuphedrone (4-methyl BP)

4'-methyl-α-Pyrrolidinohexanophenone (MPHP)

4'-methyl-α-Pyrrolidinopropiophenone (4-MePPP)

5-IAI

Bromo-DragonFLY

Butylone

BZP

Ethcathinone

Fentanyl

FIBF

Furanyl Fentanyl

MDAI

Mephedrone

Methcathinone

Methylone

NRG-3

Pentedrone

W-18

α-Pyrrolidinopentiophenone (α-PVP)