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-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.</li>
	<li>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.</li>
</ol>
2. Color Testing
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the porcelain spot plate directly onto an electric hotplate set at 80 &#176;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.</li>
	<li>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.</li>
</ol>

<ol>
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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.

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

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 ...

color-spot-test-as-presumptive-tool-for-rapid-detection-synthetic

Research

JoVE Journal

Chemistry

24.6K Views.  University of Technology Sydney (UTS). 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.

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

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate the content of kerosene in diesel/kerosene blends, a wide-spread activity to adulterate fuel. In solvents with low viscosity, the dyes rapidly deactivate via a so-called twisted intramolecular charge transfer state, efficiently quenching the fluorescence. Measurements of diesel/kerosene blends revealed a good linear correlation between the decrease in fluorescence and the increase of the fraction of the less viscous kerosene in diesel/kerosene blends. Immobilization of the hydroxy derivative 4-DNS-OH in cellulose paper yielded test strips that preserve the fluorescent indicator&#39;s behavior. Combination of the strips with a reader based on a smartphone and a controlling app allowed to create a simple field test. The method can reliably detect the presence of kerosene in diesel from 7 to 100%, outperforming present standard methods for diesel adulteration.

Here, we present a protocol to detect the adulteration of diesel with kerosene using test strips coated with a fluorescent viscosity probe together with a smartphone-based analysis system.

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate ...

Environment

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

Biochemistry

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Neuroscience

Harvesting the Orthogonality of LC and TIMS&#45;TOF MS&#47;MS to Screen Fentanyl Analogs

Fentanyl Analog Screening using LC-TIMS-TOF MS/MS

The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its analogs are the major contributors to fatal and nonfatal overdoses in the United States. Most recent cases of fentanyl-related overdose are linked to illicitly manufactured fentanyl and its associated extreme potency. In the present work, we describe a high-throughput analytical protocol for the screening of fentanyl analogs. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and tandem mass spectrometry allow for the separation and assignment of hundreds of fentanyl analogs from a single sample in a single scan. The described approach takes advantage of the recent development of data-dependent acquisition and data-independent acquisition using parallel accumulation in the mobility trap followed by sequential fragmentation using collision-induced dissociation. The fentanyl analogs are confidently assigned based on their retention time, mobility, and MS fragmentation pattern.

Author Spotlight: An Efficient Methodology to Confidently Differentiate and Characterize Fentanyl Analogs 

A method for the screening of fentanyl analogs based on their retention time, mobility, and mass spectrometry fragmentation pattern.

The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its ...

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

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

Summary

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Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Fentanyl Analog Screening using LC-TIMS-TOF MS/MS

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics