The authors would like to acknowledge the support provided to Morgan Philp through an Australian Government Research Training Program Scholarship.

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.
<ol>
	<li>Weigh 0.11 g of 2,9-dime...

<ol>
	<li>Martin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drugs on the Dark Net: How Cryptomarkets are Transforming the Global Trade in Illicit Drugs. , (2014).</li><li>Beharry, S., Gibbons, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+emerging+and+new+psychoactive+substances+in.">An overview of emerging and new psychoactive substances in.</a> the United Kingdom. Forensic Sci. Int. 267, 25-34 (2016).</li><li>United Nations Office on Drugs and Crime (UNODC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Drug Report 2017. , (2017).</li><li>Australian Criminal Intelligence Commission (ACIC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Illicit Drug Data Report 2014-2015. , (2016).</li><li>United Nations Office on Drugs and Crime (UNODC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Drug Report 2016. , (2016).</li><li>Chatwin, C., Measham, F., O'Brien, K., Sumnall, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+drugs,+new+directions?+Research+priorities+for+new+psychoactive+substances+and+human+enhancement+drugs.">New drugs, new directions? Research priorities for new psychoactive substances and human enhancement drugs.</a> Int. J. Drug Policy. 40, 1-5 (2017).</li><li>Reuter, P., Pardo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+psychoactive+substances:+Are+there+any+good+options+for+regulating+new+psychoactive+substances?.">New psychoactive substances: Are there any good options for regulating new psychoactive substances?.</a> Int. J. Drug Policy. 40, 117-122 (2017).</li><li>Elie, M. P., Elie, L. E., Baron, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+pace+with+NPS+releases:+fast+GC-MS+screening+of+legal+high+products.">Keeping pace with NPS releases: fast GC-MS screening of legal high products.</a> Drug Test. Anal. 5 (5), 281-290 (2013).</li><li>Strano Rossi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analytical+approach+to+the+forensic+identification+of+different+classes+of+new+psychoactive+substances+(NPSs)+in+seized+materials.">An analytical approach to the forensic identification of different classes of new psychoactive substances (NPSs) in seized materials.</a> Rapid Commun Mass Sp. 28 (17), 1904-1916 (2014).</li><li>Jones, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+and+Raman+screening+of+seized+novel+psychoactive+substances:+a+large+scale+study+of+&gt;200+samples.">Infrared and Raman screening of seized novel psychoactive substances: a large scale study of &gt;200 samples.</a> Analyst. 141 (3), 902-909 (2016).</li><li>Lesiak, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+in+real+time+mass+spectrometry+(DART-MS)+of+&amp;#34;bath+salt&amp;#34;+cathinone+drug+mixtures.">Direct analysis in real time mass spectrometry (DART-MS) of &amp;#34;bath salt&amp;#34; cathinone drug mixtures.</a> Analyst. 138 (12), 3424-3432 (2013).</li><li>Brown, H., Oktem, B., Windom, A., Doroshenko, V., Evans-Nguyen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Analysis+in+Real+Time+(DART)+and+a+portable+mass+spectrometer+for+rapid+identification+of+common+and+designer+drugs+on-site.">Direct Analysis in Real Time (DART) and a portable mass spectrometer for rapid identification of common and designer drugs on-site.</a> Forensic Chem. (Supplement C), 66-73 (2016).</li><li>Bruno, A. M., Cleary, S. R., O'Leary, A. E., Gizzi, M. C., Mulligan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+the+utility+and+legality+of+implementing+portable+mass+spectrometers+coupled+with+ambient+ionization+in+routine+law+enforcement+activities.">Balancing the utility and legality of implementing portable mass spectrometers coupled with ambient ionization in routine law enforcement activities.</a> Anal Methods-UK. 9 (34), 5015-5022 (2017).</li><li>United Nations Office on Drugs and Crime (UNODC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Recommended methods for the identification and analysis of amphetamine, methamphetamine and their ring-substituted analogues in seized materials. , (2006).</li><li>Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vol. 7.1. , (2016).</li><li>United Nations Office on Drugs and Crime (UNODC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Recommended methods for the identification and analysis of synthetic cathinones in seized materials. , (2015).</li><li>Philp, M., Shimmon, R., Tahtouh, M., Fu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+presumptive+color+spot+test+method+for+the+detection+of+synthetic+cathinones+in+seized+illicit+materials.">Development and validation of a presumptive color spot test method for the detection of synthetic cathinones in seized illicit materials.</a> Forensic Chem. 1, 39-50 (2016).</li><li>Al-Obaid, A. M., Al-Tamrah, S. A., Aly, F. A., Alwarthan, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+(S)(&#8722;)-cathinone+by+spectrophotometric+detection.">Determination of (S)(&#8722;)-cathinone by spectrophotometric detection.</a> J Pharmaceut Biomed. 17 (2), 321-326 (1998).</li><li>Namera, A., Kawamura, M., Nakamoto, A., Saito, T., Nagao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+review+of+the+detection+methods+for+synthetic+cannabinoids+and+cathinones.">Comprehensive review of the detection methods for synthetic cannabinoids and cathinones.</a> Forensic Toxicol. 33 (2), 175-194 (2015).</li><li>Isaacs, R. C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structure-reactivity+relationship+driven+approach+to+the+identification+of+a+color+test+protocol+for+the+presumptive+indication+of+synthetic+cannabimimetic+drugs+of+abuse.">A structure-reactivity relationship driven approach to the identification of a color test protocol for the presumptive indication of synthetic cannabimimetic drugs of abuse.</a> Forensic Sci. Int. 242, 135-141 (2014).</li></ol>

The authors have nothing to disclose.

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...

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 &#34;legal&#34; 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 discussion6,7. 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.

color spot test as a presumptive tool for the rapid detection of synthetic cathinones

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.

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...

Watch this Scientific Journal Video about Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones at JoVE.com

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

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.

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

The overall goal of this procedure is to presumptively determine the presence or absence of a synthetic cathinone within an unknown seized material using color testory agents and associated color changes. This method can help provide law enforcement agencies with rapid, cost-effective and simple suggestions as to whether or not a synthetic cathinone may be present in a seized material. The main advantage of this technique over other color tests available is its high selectivity to other synthetic cathinone class of elicit drugs.To begin, weigh 0.12 grams of copper nitrate trihydrate into a dry, 100mL beaker. Add 30mL of deionized, or DI water, and carefully swirl it at room temperature to dissolve all solids. Pour this solution into a 100mL volumetric flask.Fill the solution up to the calibrate mark with DI water. This prepared solution is Reagent One. Then, weigh 0.11 grams of necuproine hemihydrate into a dry, 100mL beaker.Add 50mL of 0.1 molar hydrochloric acid and use a glass stirring rod to promote dissolution of solids at room temperature. Pour this solution into a 100mL volumetric flask. Fill up the flask to the calibrated mark with 0.1 molar hydrochloric acid.This prepared solution is Reagent Two. Next, weigh 16.4 grams of sodium acetate into a dry, 100mL beaker. Add 50mL of DI water and use a glass stirring rod to promote dissolution of solids at room temperature.Pour this solution into a 100mL volumetric flask. And fill up to the calibrated mark with DI water. This prepared solution is Reagent Three.Using a spatula, place a small, pinhead-sized amount of the unknown sample into three separate wells of a porcelain spot plate. Leave three adjacent wells empty for the blank control And add equal amounts of 4-MMC HCL to another three wells. These wells serve as a positive control, representing a synthetic cathinone reference sample.Using a disposable pipette, add five drops of the copper nitrate solution to each sample well in addition to the blank and positive control wells. Then, using a second disposable pipette, add two drops of the neocuproine solution to each sample well in addition to the blank and positive control wells. Finally, using a third disposable pipette, add two drops of the sodium acetate solution to each sample well in addition to the blank and positive control wells.At this point, the solution turns light blue. Place the porcelain spot plate directly onto an electric hotplate set at 80 degrees Celsius. After heating for 10 minutes, observe the color change by the naked eye using a white background to better visualize the color changes.Make a note or take a photo of the final color change. Representative results of a blank control and 4-MCC HCL as a synthetic cathinone positive control are shown here. In general, a yellow-orange color change after heating is considered a positive result.However, this color can vary in intensity as seen with N, N-dimethylcathinone HCL which is dark colored as well as with 3 4-dimethylmethcathinone HCL which is light colored. Other positive results include 2 4 5-trimethylmethcathinone HCL in a piperazenic analog, a false positive. Conversely, representative compounds producing negative results are shown here.The first is a false negative result produced by MDPBP HCL, while the other three are true negative results of an amino acid, drug precursor, and amphetamine. Although the drug precursor is yellow-orange color, this occurred before the heating step. Finally, reactions with cathinone mixtures are not affected and show positive results.After watching this video, you should have a good understanding of how to perform a simple color test to detect the presence of synthetic cathinones in seized material. While attempting this procedure, it is important to remember that this protocol is intended to be used for presumptive identification. In a forensic context, sophisticated instrumentation, such as mass spectrometry, must be performed in order to confirm any identifications.

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24.0K 조회수.  University of Technology Sydney (UTS). The overall goal of this procedure is to presumptively determine the presence or absence of a synthetic cathinone within an unknown seized material using color testory agents and associated color changes. This method can help provide law enforcement agencies with rapid, cost-effective and simple suggestions as to whether or not a synthetic cathinone may be present in a seized material. The main advantage of this technique over other color tests available is its high selectivity to other synth...