Review: Development and Validation of a Presumptive Color Spot Test Method for the Detection of Synthetic Cathinones in Seized Illicit Materials

Emily C. Lennert

Category: Chemistry

Keywords: bath salts, plant food, DANPS, cathinone, synthetic cathinones, mephedrone, presumptive test, selectivity

Article to be reviewed:

1. Philp, M.; Shimmon, R.; Tahtouh, M.; Fu, S. “Development and validation of a presumptive color spot test method for the detection of synthetic cathinones in seized illicit materials.” Forensic Chemistry. 2016, 1, 39–50.

Additional references:
2. Al-Obaid, A.; Al-Tamrah, S.; Aly, F.; Alwarthan, A. “Determination of (S)(−)-cathinone by spectrophotometric detection.” Journal of Pharmaceutical and Biomedical Analysis. 1998, 17 (2), 321–326.

Disclaimer: The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.


This study sought to address a gap in current presumptive tests. Drug Analogues and New Psychoactive Substances (DANPS) are substances that share similar structures to prohibited or scheduled compounds, or are substances that have a similar psychoactive and pharmacological effects as a prohibited or scheduled drug.1 DANPS have become more prevalent in the drug market, and are often referred to by street names such as “bath salts” or “plant food” and marked “not for human consumption” in attempts to avoid legal conflict. These substances are more properly known as synthetic cathinones, and are among the most common DANPS. Synthetic cathinones share a common general structure, with variations in peripheral groups on the general structure, which are referred to as R groups, such as in figure 1 within the study.

Positive identification and quantification of synthetic cathinones is performed in a laboratory, either by Gas Chromatography – Mass Spectrometry (GC-MS) or High Performance Liquid Chromatography – Mass Spectrometry (HPLC-MS).1 GC-MS and HPLC-MS are highly specific and reliable techniques that work well for drug identification and quantification. However, due to a number of factors (i.e. cost of the instrument, supplies, and maintenance; lengthy analysis time; and the need for a trained analyst to run the instrument and analyze data), GC-MS and HPLC-MS are not ideal for high throughput analysis, or analysis of a large number, of unknown substances. Presumptive testing allows for rapid screening of an unknown sample to determine if the sample should be sent for instrumental analysis, which can save both time and money. Color tests are a common method of presumptive testing that are simple and rapid. A number of commercially available presumptive tests are available for synthetic cathinones, but the tests either use hazardous chemicals, lack sensitivity, cannot identify synthetic cathinones, or have not been tested on a large number of available synthetic cathinones.

This study tested the suitability of a reagent from a method developed by researchers at Istanbul University, called the Cupric Reducing Antioxidant Capacity method (CUPRAC), for application as a presumptive test for synthetic cathinones, or “bath salts”.1 CUPRAC was originally developed to measure antioxidant compounds, due to the reduction-oxidation reaction that occurs through the method. CUPRAC uses a copper(II)- neocuprine reagent, which is reduced in the presence of antioxidants to copper(I)-neocuprine chelate complex, which results in a color change. The authors of this study investigated the use of a copper(II)-neocuprine reagent as color test reagent for the detection of synthetic cathinones, modelling its method after a method previously used by Al-Obaid et al.2 for the detection of cathinone from khat plants.

Method development was performed using 4-methylmethcathinone hydrochloride (4-MMC HCl), also known as mephedrone, which was synthesized in laboratory. The identity and purity of the synthesized 4-MMC HCl was confirmed by GC-MS, nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FTIR), and UV-visible spectrophotometry. Starch, glucose, caffeine, sucralose, codeine phosphate, and ephedrine were also tested during method development to determine if a color response occurred with common cutting agents, drugs from other classes, and other substances that were not the target compound. In the presence of 4-MMC HCl, a yellow-orange color developed. This was easily distinguished from the original color of the reagent, which was a light clear with a slight blue tint. For most other substances testing during method development, a light green-blue color formed, except for codeine phosphate, which produced a yellow color that was still distinguishable from the yellow-orange of the synthetic cathinone. In mixtures, the yellow-orange color dominated.

An optimized presumptive test method was determined through a series of trials, testing varying concentrations of reagent solutions, varying temperatures for test conditions, and varying amounts of buffer. A general recommended procedure was given for use on a solid sample.1

  1. Add 0.1-0.2 mg, a “pinhead sized amount,” of solid sample to a well of a test plate.
  2. Test a negative and positive control standard alongside the unknown sample.
  3. Add five drops of 5.00 x 10-3 M copper(II) nitrate solution.
  4. Add two drops of 5.12 x 10-3 M neocuprine solution.
  5. Add two drops of 2.00 M sodium acetate buffer.
  6. Heat the well plate. For a plastic well plate, use a boiling water bath, and for a porcelain well plate, use a hot plate set to 100 ̊C.
  7. Wait 10 minutes, and record the color change.

The method is simple enough for a non-scientist to perform, and the authors state that the test may be developed for field use in the future, using a chemical in place of the heating step.

The method was validated in this study as well, with over 120 test samples being examined following the general recommended procedure. Forty-four (44) cathinone analogues were tested, with 39 yielding definitively positive color responses. The five that did not respond were found to share a unique structural characteristic among the cathinone analogues that prevented the color change. Eighty-two (82) non-cathinone substances were also examined to determine the test’s specificity to cathinone analogues. Eight substances produced yellow color that could be interpreted as a positive result, as well as one substance that produced the yellow-orange color expected of cathinone analogues. The remaining substances produced either shades of yellow that were distinguishable from a positive result, orange that was also distinguishable from the positive result, or a blue-green color similar to non-cathinone samples. The presumptive test was determined to show good selectivity toward cathinone analogues by readily identifying them and not identifying non-cathinones. Even in the presence of a mixture, adulterant, or diluent, a positive color change was still observed for cathinone analogues.

Scientific Highlights:

  • Synthetic cathinones share a general structure, which allows a test such as this to work on many cathinone analogues.
  • The test procedure is relatively simple and does not require skilled training to perform. Implementation of this presumptive test may prevent unnecessary instrumental testing, such as by GC-MS or HPLC-MS, saving time and money.
  • The test displays good selectivity for cathinone analogues.
  • The copper(II)-neocuprine test reagent was found to be highly stable over time and at various storage temperatures.
  • The presumptive test’s color reaction requires heat. Any difference in heating/temperature will affect the result.
  • The test is not yet developed for field use.

Relevance: Synthetic cathinones and cathinone analogues have increased in prevalence due to their ability to be acquired relatively easily. These drugs produce similar psychoactive and pharmacological affects to prohibited and/or scheduled drugs, and many are fairly new with few existing presumptive tests. This presumptive test addresses that gap in presumptive drug testing.

Potential conclusions:

  • The copper(II)-neocuprine test reagent is a viable option for presumptive testing of unknown samples for cathinone analogues (i.e. “bath salts”).
  • The test utilizes a highly stable reagent that provides good selectivity for cathinone analogues, making it a good choice for a presumptive test.

Legal Brief: Development and validation of a presumptive color spot test method for the detection of synthetic cathinones in seized illicit materials

Steve Krejci

Field test kits are subject to challenges for admissibility. While the law is not clear regarding whether admissibility of these tests are subject to Daubert, the factual argument as to the reliability of the test would still apply. See Bell v. State, 179 So. 3d 349 (Fla. 5th DCA 2015); Turner v. State, 388 So. 2d 254 (Fla. 1st DCA 1980); 75 A.L.R.3d 717 (Originally published in 1977). The Daubert factors consider whether the technique has been tested, if it is capable of peer review, if the error rate is known, and whether the technique is generally accepted. Booker v. Sumter Cnty. Sheriff’s Office, 166 So. 3d 189 (Fla. 1st DCA 2015). If Daubert does apply, then the court must also consider whether the technique was reliably applied in the case at hand; in other words, whether the police officer used the field test kit properly. See id. However, even if Daubert is not required for admissibility, the foundational questions are excellent for cross examination regarding the reliability of the test itself. In Orlando, the infamous Donut/meth case gives a concrete example of exposing the limitations of these tests. Man Suing City After Doughnut Icing Test Postive for Meth During Orlando Traffic Stop, Oct. 21, 2016 5:43 PM, available at traffic-stop/459506995.