Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Summary

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

Abstract

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Introduction

The illicit drug market operates similarly to a traditional business by continuing to evolve and adapt to a changing marketplace. Advances in modern technology, specifically, the global proliferation of powerful communication has seen increased online purchases via the Dark Net¹ and extensive knowledge sharing among users via online forums². Combined with advances in chemistry, the rapid emergence of new psychoactive substances (NPS) created a serious challenge for international and national drug control.

NPS are potentially dangerous substances of abuse that have similar effects to drugs under international control. Initially marketed as "legal" alternatives, 739 NPS were reported to the United Nations Office on Drugs and Crime (UNODC) between 2009 and 2016³. According to the most recent annual report, a record number of NPS were seized at the Australian border, with the majority of those analyzed, further identified as synthetic cathinones⁴. On a global scale, seizures of synthetic cathinones have been steadily increasing since first reported in 2010, and are one of the most commonly seized NPS⁵.

The challenges posed by NPS have been a largely published topic of discussion^6,7. Forensic laboratories and law enforcement personnel were left at a disadvantage without appropriate methods in place to detect and identify NPS during their rapid emergence. Extensive research into the detection of NPS, including synthetic cathinones, in seized material, has employed gas chromatography-mass spectrometry (GC-MS)⁸ and liquid chromatography-high resolution mass spectrometry (LC-HRMS)⁹ for confirmatory analysis. Increasing demand for minimal sample preparation has seen infrared and Raman spectroscopy¹⁰ studies as well as ambient ionisation mass spectrometric analyses, such as direct analysis in real time mass spectrometry (DART-MS)^11,12. The need for rapid, sensitive analysis in the field has also seen the incorporation of paper spray ionization-mass spectrometry (PSI-MS) into portable devices for use by law enforcement¹³. Many instrumental techniques offer confirmatory analysis with sensitive detection and quantitative results. However, for high-throughput analysis, they can be time-consuming due to sample preparation, run times, and instrument training and maintenance.

Presumptive color tests are designed to suggest the presence or absence of certain drug classes in a test sample¹⁴. The Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) classifies color testing as the lowest discriminating power technique, alongside ultraviolet spectroscopy and immunoassays¹⁵. However, they are still widely employed by law enforcement and other security personnel as a means to provide rapid results at a significantly lower cost compared to other techniques. The main advantage offered by color spot test methods is the ability to perform them in the field using portable test kits.

The selectivity of color tests relies on individual chemical reactions occurring between the test reagent and the drug class of interest to create a color change. Current presumptive testing protocols lack a particular test for detecting synthetic cathinones only; commonly used reagents that lack specificity and contain hazardous substances are often employed. Other recommended reagents have not been screened on a large number of possible synthetic cathinone substances¹⁶.

The aim of this work is to present a simple color test protocol that can be easily employed by interested parties for the preliminary screening of synthetic cathinones in illicit substances of unknown composition. Interested parties would include law enforcement, border protection agencies, forensic laboratories, and other relevant security personnel. The proposed methods employ a reduction-oxidation reaction occurring between the electron-accepting copper complex reagent and the electron rich synthetic cathinone drug molecules. Using these chemical methods developed, one can apply them in the form of a presumptive color test to suggest the presence of synthetic cathinones.

Protocol

1. Preparation of Color Test Reagent Solutions

NOTE: Weigh 0.12 g of copper nitrate trihydrate into a dry 100 mL beaker. Add 30 mL of deionized (DI) water and carefully swirl it at room temperature to dissolve all solids. Pour this solution into a 100 mL volumetric flask and fill up to the calibrated mark with DI water. This prepared solution is reagent 1.
NOTE: Reagent 1 can be prepared using other copper(II) salts, e.g. copper(II) chloride.

1. Weigh 0.11 g of 2,9-dimethyl-1,10-phenanthroline (neocuproine) hemihydrate into a dry 100 mL beaker. Add 50 mL of 0.10 mol/L hydrochloric acid (HCl) and use a glass stirring rod to promote dissolution of solids at room temperature. Pour this solution into a 100 mL volumetric flask and fill up to the calibrated mark with 0.10 mol/L HCl. This prepared solution is reagent 2.
   CAUTION: Neocuproine is acutely toxic can cause skin irritation and serious eye damage. Wear gloves and safety glasses while handling to minimize the risk of exposure.
   NOTE: Neocuproine is only slightly soluble in water, therefore, dilute acid is used to prepare this reagent and ensure all solids dissolve.
2. Weigh 16.4 g of sodium acetate into a dry 100 mL beaker. Add 50 mL of DI water and use a glass stirring rod to promote dissolution of solids at room temperature. Pour this solution into a 100 mL volumetric flask and fill up to the calibrated mark with DI water. This prepared solution is reagent 3.
   NOTE: The protocol can be paused here. The reagents are highly stable and can be stored for up to 12 months at room temperature.

2. Color Testing

1. Collect one clean porcelain spot plate, three disposable pipettes, three reagent solutions prepared in step 2.1, one clean spatula, an electric hotplate and the sample/seized material to be tested.
2. Using the spatula, place a small, pin-head sized amount (approximately 0.1-0.2 mg) of the unknown sample into three separate wells of a porcelain spot plate. Leave three adjacent wells empty (blank control) and another three wells with equal amounts of 4-methylmethcathinone HCl (4-MMC), a synthetic cathinone reference sample (positive control).
   NOTE: The preferred test surface is a porcelain spot plate. If these are not available, use plastic microwell plates or semi micro test tubes.
3. Using a disposable pipette, add 5 drops of the copper nitrate solution (Reagent 1) to each sample well, in addition to the blank and positive control wells.
4. Using a second disposable pipette, add 2 drops of the neocuproine solution (Reagent 2) to each sample well, in addition to the blank and positive control wells.
5. Using a third disposable pipette, add 2 drops of the sodium acetate solution (Reagent 3) to each sample well, in addition to the blank and positive control wells.
   NOTE: The solution turns light blue.
6. Place the porcelain spot plate directly onto an electric hotplate set at 80 °C.
   NOTE: Do not heat plastic microwell plates directly on the hotplate. Prepare a shallow boiling water bath to set the plastic plate. Heat semi-micro test tubes in a small boiling water bath. The precise time required to observe a color change will depend on the thickness and composition of the spot plate.
   CAUTION: Take care when handling spot plates to prevent burn injuries.
7. After heating for 10 min, observe by naked eye and note the final color change or take a photo of the final color change.
   NOTE: Use a white background to better visualize the color changes.

Results

The test protocol has been validated through several studies, the results of which are described in Philp et al.¹⁷. The color test method is able to presumptively detect synthetic cathinones in an unknown sample through a color change from light blue to yellow-orange (Figure 1). Yellow and orange color changes occuring after the heating period are considered positive test results and any other color change, including very weak...

Discussion

This color test protocol was adapted from experimental work published by Al-Obaid et al.¹⁸ in which the authors demonstrated a color change occurs in the presence of cathinone extracted from the khat plant. Modifications to the published protocol were necessary to foresee its application in presumptive illicit drug detection. The most important consideration was to reduce the scale of the reaction. The protocol described in the present paper is designed to be applied to street samples and...

Materials

Name	Company	Catalog Number	Comments
Chemicals
Reagents and solvents
neocuproine hemihydrate	Sigma-Aldrich	72090	≥99.0%. Acute toxicity
copper(II) nitrate trihydrate	Sigma Aldrich	61197	98.0%-103%
sodium acetate	Ajax Finechem	AJA680	anhydrous
hydrochloric acid	RCI Labscan	RP 1106	36%. Corrosive
Name	Company	Catalog Number	Comments
Powders
ascorbic acid	AJAX Finechem UNIVAR	104	L
benzocaine	Sigma-Aldrich	E1501
benzoic acid	Sigma-Aldrich	242381	≥99.5%
boric acid	Silform Chemicals	R27410
caffeine	Sigma-Aldrich	C0750
cellulose	Sigma-Aldrich	435236	microcrystalline
calcium chloride	AJAX Finechem UNILAB	960
citric acid	AJAX Finechem UNIVAR	160
codeine phosphate	Glaxo	-	Acute toxicity
cysteine	Sigma-Aldrich	168149	L
dimethylsulfone	Sigma-Aldrich	M81705	98%
ephedrine HCl	Sigma-Aldrich	285749	99%. Acute toxicity
glucose	AJAX Finechem UNIVAR	783	D, anhydrous
glutathione	AJAX Finechem UNILAB	234
glycine	AJAX Finechem UNIVAR	1083
lactose	Sigma	L254	D, monohydrate
levamisole HCl	Sigma-Aldrich	PHR1798	Acute toxicity
magnesium sulphate	Scharlau	MA0080	anhydrous, extra pure
maltose	AJAX Finechem LABCHEM	1126	Bacteriological
mannitol	AJAX Finechem UNIVAR	310
O-acetylsalicylic Acid	Sigma-Aldrich	A5376
phenethylamine	Sigma-Aldrich	241008
phenolphthalein	AJAX Finechem LABCHEM	368	Acute toxicity
potassium carbonate	Chem-Supply	PA021	AR, anhydrous
sodium carbonate	Chem-Supply	SA099	AR, anhydrous
sodium chloride	Rowe Scientific	CC10363
starch	AJAX Finechem UNILAB	1254	soluble
stearic acid	AJAX Finechem UNILAB	1255
sucrose	AJAX Finechem UNIVAR	530
tartaric acid	AJAX Finechem UNIVAR	537	(+)
Name	Company	Catalog Number	Comments
Household products
artificial sweetener	ALDI Be Light	n/a	Contains aspartame
brown sugar	CSR	n/a
icing sugar	CSR	n/a
caster sugar	CSR	n/a
paracetamol tablet	Panadol	n/a
protein powder	Aussie Bodies ProteinFX	n/a
self-raising	Woolworths Australia Homebrand	n/a
plain flour	Woolworths Australia Homebrand	n/a
Name	Company	Catalog Number	Comments
Reference compounds			controlled or illegal substances
Cathinone-type substances
1-(4-methoxyphenyl)-2-(1-pyrrolidinyl)-1-propanone HCl (MOPPP)	Australian Government National Measurement Institute (NMI)	D1024	Acute toxicity potential
1-phenyl-2-methylamino-pentan-1-one HCl	Lipomed	PTD-1507-HC	Acute toxicity potential
2,3-dimethylmethcathinone HCl (2,3-DMMC)	Chiron Chemicals	10970.12	Acute toxicity potential
2,4,5-trimethylmethcathinone HCl (2,4,5-TMMC)	Chiron Chemicals	10927.13	Acute toxicity potential
2,4-dimethylmethcathinone HCl (2,4-DMMC)	Chiron Chemicals	10971.12	Acute toxicity potential
2-benzylamino-1-(3,4-methylenedioxyphenyl)-1-butanone HCl (BMDB)	Chiron Chemicals	10925.18	Acute toxicity potential
2-fluoromethcathinone HCl (2-FMC)	LGC Standards	LGCFOR 1275.64	Acute toxicity potential
2-methylmethcathinone HCl (2-MMC)	LGC Standards	LGCFOR 1387.02	Acute toxicity potential
3,4-methylenedioxy-α-pyrrolidinobutiophenone (MDPBP) HCl	Australian Government National Measurement Institute (NMI)	D973	Acute toxicity potential
3,4-dimethylmethcathinone HCl (DMMC)	Australian Government National Measurement Institute (NMI)	D962	Acute toxicity potential
3,4-methylenedioxymethcathinone HCl (MDMC)	Australian Government National Measurement Institute (NMI)	D942	Acute toxicity potential
3,4-methylenedioxy-N,N-dimethylcathinone HCl	Australian Government National Measurement Institute (NMI)	D977	Acute toxicity potential
3,4-methylenedioxypyrovalerone HCl (MDPV)	Australian Government National Measurement Institute (NMI)	D951b	Acute toxicity potential
3-bromomethcathinone HCl (3-BMC)	Australian Government National Measurement Institute (NMI)	D1035	Acute toxicity potential
3-fluoromethcathinone HCl (3-FMC)	Australian Government National Measurement Institute (NMI)	D947b	Acute toxicity potential
3-methylmethcathinone HCl (3-MMC)	LGC Standards	LGCFOR 1387.03	Acute toxicity potential
4-bromomethcathinone HCl (4-BMC)	LGC Standards	LGCFOR 1387.11	Acute toxicity potential
4-fluoromethcathinone HCl	Australian Government National Measurement Institute (NMI)	D969	Acute toxicity potential
4-methoxymethcathinone HCl	Australian Government National Measurement Institute (NMI)	D952	Acute toxicity potential
4-methylethylcathinone HCl	Australian Government National Measurement Institute (NMI)	D968	Acute toxicity potential
4-methylmethcathinone HCl (4-MMC)	Australian Government National Measurement Institute (NMI)	D937b	Acute toxicity potential
4-methyl-N-benzylcathinone HCl (4-MBC)	Australian Government National Measurement Institute (NMI)	D1026	Acute toxicity potential
4-methyl-pyrrolidinopropiophenone HCl	Australian Government National Measurement Institute (NMI)	D964	Acute toxicity potential
4-methyl-α-pyrrolidinobutiophenone HCl	Australian Government National Measurement Institute (NMI)	D974	Acute toxicity potential
cathinone HCl (bk-amphetamine)	Australian Government National Measurement Institute (NMI)	D929	Acute toxicity potential
dibutylone HCl (bk-DMBDB)	Australian Government National Measurement Institute (NMI)	D1027	Acute toxicity potential
iso-ethcathinone HCl	Chiron Chemicals	10922.11	Acute toxicity potential
methcathinone HCl	Australian Government National Measurement Institute (NMI)	D724	Acute toxicity potential
methylenedioxy-α-pyrrolidinopropiophenone HCl	Australian Government National Measurement Institute (NMI)	D960	Acute toxicity potential
N,N-diethylcathinone HCl	Australian Government National Measurement Institute (NMI)	D957	Acute toxicity potential
N,N-dimethylcathinone HCl	Australian Government National Measurement Institute (NMI)	D958	Acute toxicity potential
naphthylpyrovalerone HCl (naphyrone)	Australian Government National Measurement Institute (NMI)	D981	Acute toxicity potential
N-ethyl-3,4-methylenedioxycathinone HCl	Australian Government National Measurement Institute (NMI)	D959	Acute toxicity potential
N-ethylbuphedrone HCl	Australian Government National Measurement Institute (NMI)	D1013	Acute toxicity potential
N-ethylcathinone HCl	Australian Government National Measurement Institute (NMI)	D938b	Acute toxicity potential
pentylone HCl	Australian Government National Measurement Institute (NMI)	D992	Acute toxicity potential
pyrovalerone HCl	Australian Government National Measurement Institute (NMI)	D985	Acute toxicity potential
α-dimethylaminobutyrophenone HCl	Australian Government National Measurement Institute (NMI)	D1011	Acute toxicity potential
α-dimethylaminopentiophenone HCl	Australian Government National Measurement Institute (NMI)	D1006	Acute toxicity potential
α-ethylaminopentiophenone HCl	Australian Government National Measurement Institute (NMI)	D1005	Acute toxicity potential
α-pyrrolidinobutiophenone HCl (α-PBP)	Australian Government National Measurement Institute (NMI)	D1012	Acute toxicity potential
α-pyrrolidinopentiophenone HCl	Australian Government National Measurement Institute (NMI)	D986b	Acute toxicity potential
α-pyrrolidinopropiophenone HCl	Australian Government National Measurement Institute (NMI)	D956	Acute toxicity potential
β-keto-N-methyl-3,4-benzodioxyolylbutanamine HCl (bk-MBDB)	Australian Government National Measurement Institute (NMI)	D948	Acute toxicity potential
Name	Company	Catalog Number	Comments
Other substances
(-)-ephedrine HCl	Australian Government National Measurement Institute (NMI)	M924	Acute toxicity potential
(-)-methylephedrine HCl	Australian Government National Measurement Institute (NMI)	M243	Acute toxicity potential
(+)-cathine HCl	Australian Government National Measurement Institute (NMI)	M297	Acute toxicity potential
(+/-)- 3,4-methylenedioxyamphetamine HCl (MDA)	Australian Government National Measurement Institute (NMI)	D842	Acute toxicity potential
(+/-)- N-methyl-3,4-methylenedioxyamphetamine HCl (MDMA)	Australian Government National Measurement Institute (NMI)	D792c	Acute toxicity potential
(+/-)-methamphetamine HCl	Australian Government National Measurement Institute (NMI)	D816e	Acute toxicity potential
(+/-)-N-ethyl-3,4-methylenedioxyamphetamine HCl (MDEA)	Australian Government National Measurement Institute (NMI)	D739c	Acute toxicity potential
(+/-)-N-methyl-1-(3,4-methylenedioxyphenyl)-2-butylamine HCl	Australian Government National Measurement Institute (NMI)	D450a	Acute toxicity potential
(+/-)-phenylpropanolamine HCl	Australian Government National Measurement Institute (NMI)	M296	Acute toxicity potential
(2S*,3R*)-2-methyl-3-[3,4-(methylenedioxy)phenyl]glycidic acid methyl ester	Australian Government National Measurement Institute (NMI)	D903	Acute toxicity potential
1-(3-chlorophenyl)piperazine HCl (mCPP)	Australian Government National Measurement Institute (NMI)	D907	Acute toxicity potential
1-[3-(trifluoromethyl)phenyl]piperazine HCl (TFMPP)	Australian Government National Measurement Institute (NMI)	D906	Acute toxicity potential
1-benzylpiperazine HCl (BZP)	Australian Government National Measurement Institute (NMI)	D905	Acute toxicity potential
2,5-dimethoxy-4-iodophenylethylamine HCl	Australian Government National Measurement Institute (NMI)	D922	Acute toxicity potential
2,5-dimethoxy-4-methylamphetamine HCl (DOM)	Australian Government National Measurement Institute (NMI)	D470b	Acute toxicity potential
2,5-dimethoxy-4-propylthio-phenylethylamine HCl	Australian Government National Measurement Institute (NMI)	D919	Acute toxicity potential
2,5-dimethoxyamphetamine HCl	Australian Government National Measurement Institute (NMI)	D749	Acute toxicity potential
2-bromo-4-methylpropiophenone	Synthesised in-house	n/a	Acute toxicity potential
2-fluoroamphetamine HCl	Australian Government National Measurement Institute (NMI)	D946	Acute toxicity potential
2-fluoromethamphetamine HCl	Australian Government National Measurement Institute (NMI)	D933	Acute toxicity potential
3,4-dimethoxyamphetamine HCl	Australian Government National Measurement Institute (NMI)	D453b	Acute toxicity potential
3,4-methylenedioxyphenyl-2-propanone (MDP2P)	Australian Government National Measurement Institute (NMI)	D810b	Acute toxicity potential
4-bromo-2,5-dimethoxyamphetamine HCl	Australian Government National Measurement Institute (NMI)	D396b	Acute toxicity potential
4-bromo-2,5-dimethoxyphenethylamine HCl	Australian Government National Measurement Institute (NMI)	D758b	Acute toxicity potential
4-fluoroamphetamine HCl	Australian Government National Measurement Institute (NMI)	D943b	Acute toxicity potential
4-fluorococaine HCl	Australian Government National Measurement Institute (NMI)	D854b	Acute toxicity potential
4-fluoromethamphetamine HCl	Australian Government National Measurement Institute (NMI)	D934	Acute toxicity potential
4-hydroxyamphetamine HCl	Australian Government National Measurement Institute (NMI)	D824b	Acute toxicity potential
4-methoxyamphetamine HCl (PMA)	Australian Government National Measurement Institute (NMI)	D756	Acute toxicity potential
4-methoxymethamphetamine HCl (PMMA)	Australian Government National Measurement Institute (NMI)	D908b	Acute toxicity potential
4-methylmethamphetamine HCl	Australian Government National Measurement Institute (NMI)	D963	Acute toxicity potential
4-methylpropiophenone	Sigma-Aldrich	517925	Acute toxicity potential
5-methoxy-N,N-diallyltryptamine	Australian Government National Measurement Institute (NMI)	D954	Acute toxicity potential
amphetamine sulphate	Australian Government National Measurement Institute (NMI)	D420d	Acute toxicity potential
cocaine HCl	Australian Government National Measurement Institute (NMI)	D747b	Acute toxicity potential
dimethamphetamine (DMA)	Australian Government National Measurement Institute (NMI)	D693d	Acute toxicity potential
gamma-hydroxy butyrate	Australian Government National Measurement Institute (NMI)	D812b	Acute toxicity potential
heroin HCl	LGC Standards	LGCFOR 0037.20	Acute toxicity potential
ketamine HCl	Australian Government National Measurement Institute (NMI)	D686b	Acute toxicity potential
methoxetamine HCl	Australian Government National Measurement Institute (NMI)	D989	Acute toxicity potential
methylamine HCl	Sigma-Aldrich	M0505	Acute toxicity potential
phencyclidine HCl	Australian Government National Measurement Institute (NMI)	D748	Acute toxicity potential
phentermine HCl	Australian Government National Measurement Institute (NMI)	D781	Acute toxicity potential
triethylamine	Sigma-Aldrich	T0886	Acute toxicity, corrosive, flammable
Name	Company	Catalog Number	Comments
Equipment
12-well porcelain spot plates	HomeScienceTools	CE-SPOTP12
96-well microplates	Greiner Bio-One	650201
Hot plate	Industrial Equipment and Control Pty Ltd.	CH1920 (Scientrific)
100 mL glass volumetric flasks	Duran	24 678 25 54
Soda lime glass Pasteur pipettes	Marienfeld-Superior	3233050	230 mm length