Chapter 1

Color Tests for the Preliminary Identification of New

Psychoactive Substances
Kaitlyn Toole, Morgan Philp, Nadine Krayem, Shanlin Fu,
Ronald Shimmon, and Sonia Taflaga

Abstract
Color tests are a key tool for the rapid and simple identification of seized illicit drugs. This chapter outlines
a series of color tests that can be used for the preliminary identification of new psychoactive substances
such as cathinones, piperazines, tryptamines, and amphetamine-type stimulants.

Key words New psychoactive substances, Novel psychoactive substances, NPS, Illicit drugs, Color test,
Preliminary test, Presumptive test, Cathinones, Piperazines, Tryptamines

1  Introduction

The rapid and simple identification of seized drugs is a key analytical

challenge for law enforcement and border control agencies. For
example, in the decade to June 2015 in Australia (a nation with a
relatively small population), the weight of illicit drugs seized increased
by 263.1% to 23.5 tonnes per annum [1]. While laboratory-­based
instrumental techniques remain critical for confirmatory analysis,
field-based presumptive testing is essential. A critical tool for such
presumptive testing is color testing, which utilizes a chemical reaction
between a drug and a reagent (or series of reagents) to produce a
readily observable colored product. Color tests have long been estab-
lished and applied to many classes of drugs; however, the emergence
of new psychoactive substances has posed a new challenge.

1.1  New “New psychoactive substances” (NPS) are defined by the European
Psychoactive Union as “narcotic or psychotropic drugs that are not scheduled
Substances under the United Nations 19611 or 19712 Conventions, but which

1
 Single Convention on Narcotic Drugs of 1961.
2
 Convention on Psychotropic Substances of 1971.

Rabi A. Musah (ed.), Analysis of Drugs of Abuse, Methods in Molecular Biology, vol. 1810,
https://doi.org/10.1007/978-1-4939-8579-1_1, © Springer Science+Business Media, LLC, part of Springer Nature 2018

1
2 Kaitlyn Toole et al.

may pose a threat to public health comparable to scheduled sub-

stances” [2, 3]. These drugs are often designed to mimic the psy-
chotropic effects of illicit drugs while circumventing legal controls.
The growth of NPS use has been driven in part by the flourishing
sale and distribution of these drugs via online marketplaces.
An enormous range of substances fall under the category of
NPS. For example, the United Nations Office on Drugs and Crime
(UNODC) reported that between 2008 and 2015, 102 countries
reported 644 NPS. The UNODC further describes the market in
the following terms [4];
UNODC monitoring of NPS since 2008 has so far shown a rather
dynamic supply situation with elements of persistence (a small num-
ber of substances emerge, spread and stay for several years) and change
(a considerable number of substances appear for a short time or only
locally).
Key classes of NPS which can be tested using the methodologies
described in this chapter include the following:

1.1.1  Cathinones Cathinones are the β-keto analogs of amphetamines, with the
generic structure shown in Fig. 1. The cathinones, which are stimu-
lants with empathogenic effects, are among the most common of
the NPS. In Europe they are the second most common class of NPS
(after synthetic cannabinoids) with over 8000 seizures totaling over
1 tonne in 2015 [5], while in Australia cathinones accounted for
71.1% of the mass of NPS seized in 2014–2015 [1]. New synthetic
cathinones continue to be reported, including over 20 in 2015 [4, 5].
Common cathinone analogs include those substituted at the amine
group (methcathinone and ethcathinone), ring substituted com-
pounds (2-, 3-, and 4-fluoromethcathinone (collectively known as
flephedrone) and 4-methoxymethcathinone (methedrone)), methy-
lenedioxy-substituted compounds (3,4-methylenedioxymethcathi-
none (methylone), 3,4-­ methylendioxypyrovalerone ­(3,4-MDPV)
and β-keto-N-­ methylbenzodioxolylbutanamine (butylone)), and
those with multiple substitutions (4-methlymethcathinone
(mephedrone)).

1.1.2  Piperazines Piperazines are synthetic stimulants that produce comparable effects
to amphetamine and 3,4-­methylenedioxymethamphetamine, and
may, in fact, be sold as the latter [6, 7]. There are two primary
subclasses of piperazines. Benzylpiperazines, for which the generic
structure is shown in Fig. 2, include N-benzylpiperazine (BZP) and

Fig. 1 Generic structure of cathinones

 Color Tests for the Preliminary Identification of New Psychoactive Substances 3

Fig. 2 Generic structure of benzylpiperazines

Fig. 3 Generic structure of phenylpiperazines

Fig. 4 Generic structure of tryptamines

1-(3,4-methylenedioxybenzyl)piperazine (MDBZP).
Phenylpiperazines, for which the generic structure is shown in
Fig.  3, include 1-(3-chlorophenyl)piperazine (mCPP),
1-(3-­
trifluoromethylphenyl)piperazine (TFMPP), and
1-(4-methoxyphenyl)piperazine (MeOPP).

1.1.3  Tryptamines Tryptamines are drugs with largely hallucinogenic effects. Naturally
occurring tryptamines include molecules endogenous to the
human body (e.g., serotonin and melatonin) as well as molecules
found in hallucinogenic mushrooms (e.g., psilocibin and psilocin)
[8]. The generic structure of tryptamines is shown in Fig. 4. Some
tryptamines considered new psychoactive substances have actually
long been part of religious rituals, such as N,N-dimethyltryptamine
which is found in ayahuasca; however, their recreational use is a
more recent phenomenon. Other tryptamines used recreationally
include diethyltryptamine (DET), 5-methoxy-N,N-­
diisopropyltryptamine (5-MeO-DiPT or Foxy methoxy), and
5-methoxy-N,N-diallyltryptamine (5-MeO-DALT).

1.1.4  Amphetamine-­ Amphetamine, methamphetamine and 3,4-­methylenedioxymethamp

Type Stimulants hetamine (MDMA) have long been mainstays of the illicit drug mar-
ket. However, in recent years novel amphetamine analogs featuring
substitution of the aromatic ring, as shown in Fig. 5, have emerged.
4 Kaitlyn Toole et al.

Fig. 5 Generic structure of ring-substitute amphetamines

These have included fluorination (2- and 4-­fluoroamphetamine and

fluoromethamphetamine), addition of methoxy groups (4-methoxy-
amphetamine, 4-­ methoxymethamphetamine, 2,5-dimethoxyam-
phetamine and 3,4-dimethoxyamphetamine) and more complex
substitutions (2,5-dimethoxy-4-propylthiophenylethylamine (2C-
T-7), 2,5-dimethoxy-4-iodophenylethylamine (2CI), 4-bromo-
2,5-­
dimethoxyamphetamine (DOB), and
2,5-dimethoxy-4-­methylamphetamine (DOM)).

1.2  Applying Color tests are simple, rapid, cost-effective and can be performed
Color Tests by personnel with limited training [9]; all critical features when
performing screening of suspected illicit materials. A further advan-
tage is that color tests are generally effective even in the case of
drug mixtures or in the presence of diluents or excipients.
The greatest disadvantage of color tests is that, as presumptive
tests, there is the potential for both false positive and false negative
results; where known, these are reported in the notes for this chap-
ter. As the mechanism of many color tests is not fully understood,
their applicability to new drugs and any potential interfering com-
pounds is best elucidated by empirical testing.
Generally, a sequence of color tests will be applied to provide
greater discrimination between drugs and confidence in results.
The methods given in this chapter could be readily integrated into
a color testing sequence utilizing other tests for common drug
classes. Color testing may also be combined with other presump-
tive testing methods such as attenuated total reflectance Fourier
transform infrared spectroscopy (ATR-FTIR) or thin layer chro-
matography (TLC).
The methods given are drawn from [10–15].

2  Materials

Prepare all solutions using deionized water. Prepare and store all
reagents at room temperature (unless otherwise indicated).

2.1  Marquis Reagent 1. Very slowly add 90 mL of concentrated sulfuric acid to 10 mL
Color Test for NPS of 37% formaldehyde aqueous solution in an ice bath, agitating
gently. Store at 6 °C, in a bottle wrapped with aluminum foil
(see Note 1).
 Color Tests for the Preliminary Identification of New Psychoactive Substances 5

2.2  Liebermann’s 1. Very slowly add 1 g of sodium nitrite (see Note 2) to 10 mL of
Reagent Color Test concentrated sulfuric acid (see Note 3) in an ice bath (see
for NPS Note 4), agitating gently (see Note 5). Store in a bottle
wrapped with aluminum foil (see Note 6).

2.3  Naphthoquinone- 1. NQS color test buffer: 0.1 M NaHCO3-NaOH, pH 10.8. Add
4-Sulfonate (NQS) approximately 20 mL water to a 100 mL graduated cylinder.
Color Test for Weigh 0.84 g of sodium hydrogen carbonate and transfer to
Piperazine Analogs the cylinder. Add water to a volume of 80 mL. Mix and adjust
pH with NaOH (see Note 7). Make up to 100 mL with water.
2. NQS reagent solution: 2.0 mM NQS. Dissolve 0.052 g
1,2-Naphthoquinone-4-sulfonic acid sodium salt in 100 mL
water (see Note 8). Store at 6 °C, in a bottle wrapped with
aluminum foil (see Note 9).

2.4  Copper-­ 1. Copper(II) nitrate reagent solution: 5.0 mM Cu(NO3)2.

Neocuproine Color Dissolve 0.094 g copper nitrate in 100 mL water (see Note 10).
Test for Synthetic 2. Neocuproine reagent solution: 5.1 mM neocuproine. Dissolve
Cathinones 0.11 g neocuproine hemihydrate (see Note 11) in 100 mL
0.1 M HCl (see Note 12).
3. Sodium acetate reagent solution: 2.0 M acetate. Dissolve
16.4 g sodium acetate in 100 mL water.

2.5  Color Testing 1. Porcelain spotting well plate (see Note 13).
2. Disposable pipettes.
3. Electric hot plate.

2.6  Drug Standards 1. Certified reference materials of high purity (see Note 14).
and Unknown Samples 2. Unknown samples received for presumptive testing.

3  Methods

Carry out all procedures at room temperature unless otherwise

specified.
For all tests;
1. Place a small amount of the unknown powder (see Note 15)
into a well of a porcelain well plate using a spatula (see Note 16).

3.1  Marquis Reagent 1. Add 2–3 drops of the Marquis reagent solution.
Color Test for NPS 2. Agitate gently and record the color change immediately.
●● Methylenedioxy-substituted cathinones give a bright yel-
low color change as show in Fig. 6 (see Note 17).
●● All other cathinones do not give a color change.
●● Piperazines do not give a color change.
6 Kaitlyn Toole et al.

Fig. 6 Product of reaction of Marquis reagent with 3,4-MDMC (bottom) compared

to negative control (top)

●● Tryptamines give a brown color change.

●● Amphetamine type stimulants give a range of color
changes;
–– Maroon—4-hydroxyamphetamine.
–– Red—4-fluoro-substituted amphetamines.
–– Orange—dimethylamphetamine, 2-fluoro substituted
amphetamines, 3,4-dimethoxyamphetamine,
4-­methylamphetamine (rapidly turns bright red) (see
Note 18).
–– Light yellow—DOM.
–– Green—2C-I, 2,5-dimethoxyamphetamine, DOB (see
Note 19).

3.2  Liebermann’s 1. Add 2–3 drops of the Liebermann’s reagent solution.

Reagent Color Test 2. Agitate gently and record the color change immediately.
for NPS ●● Cathinones which are not methylenedioxy-substituted
give a yellow color change as shown in Fig. 7 (see
Note 20). 3,4-­MDPV will initially give a yellow color
change but this turns to brown.
●● Butylone gives a green-brown color change.
●● 4-Methoxymethcathinone and 3,4-dimethylmethcathi-
none give an orange color change (see Note 21). Methylone
will initially give an orange color change but this turns to
brown.
 Color Tests for the Preliminary Identification of New Psychoactive Substances 7

Fig. 7 Product of reaction of Liebermann’s reagent with CAT (bottom row) com-

pared to negative controls (top two rows)

●● BZP gives a yellow color change.

●● mCPP gives a pink color change.
●● Tryptamines give a brown color change.
●● Amphetamine-type stimulants give a range of color
changes;
–– Violet—2C-T-7.
–– Orange—fluoro-substituted amphetamines, dimethyl-
amphetamine and 4-methylamphetamine (see
Note 22).
–– Maroon—4-methoxy-amphetamine and methamphet-
amine, 4-hydroxyamphetamine.
–– Brown—2C-I, 3,4-dimethoxyamphetamine (see
Note 23).
–– Green—2,5-dimethoxyamphetamine,DOB,2,5-dimethoxy-
4-methylamphetamine.

3.3  NQS Color Test 1. Add five drops of the NQS buffer solution and mix for a few
seconds.
2. Add four drops of the NQS reagent solution.
3. Record the color change after 2 min.
●● Piperazine analogs give an orange-red color change as
shown in Fig. 8 (see Note 24).

3.4  Copper– 1. Add five drops of the copper nitrate solution (see Note 25).
Neocuproine Color 2. Add two drops of the neocuproine solution.
Test
3. Add two drops of the sodium acetate solution (see Note 26).
8 Kaitlyn Toole et al.

Fig. 8 Product of reaction of NQS test with control (top) and BZP (bottom)

Fig. 9 Products of reaction of neocuproine test with (l-r) blank reagent, MMC, FMC, and methylone

4. Heat the plate on a hot plate set at 80 °C (see Note 27).
5. Record the color change before 10 min.
●● Synthetic cathinones give a yellow-orange color change as
shown in Fig. 9 (see Note 28).

4  Notes

1. The reagent can be stored for 1 month after preparation.

2. Higher (15%) or lower (10%) nitrite concentrations will lead in
some cases to reaction with the sulfuric acid solvent which may
produce different colored products.
3. Adding sodium nitrite to sulfuric acid, rather than sulfuric acid
to sodium nitrite, will reduce the production of fumes and
heat.
Color Tests for the Preliminary Identification of New Psychoactive Substances 9

4. This reaction is highly exothermic and must be performed

with cooling.
5. Agitate to facilitate absorption of the brown fumes which will
be generated by this reaction.
6. This reagent can be stored for up to 3 months; however,
monthly preparation is recommended.
7. It is best to use NaOH concentrations of approximately 5 M to
reach the required pH. Using solutions too concentrated can
lead to a sudden rise in pH.
8. Thoroughly mix the solution to make sure the NQS salt is
completely dissolved.
9. It is recommended that the NQS solution be prepared fresh on
the day of use, and only stored at 6 °C in-between testing done
that day. The activity of the reagent diminishes after storage for
more than 1 week in the refrigerator.
10. The calculated mass of copper nitrate is based on anhydrous
Cu(NO3)2. Adjust accordingly if using copper(II) nitrate trihy-
drate, i.e., use 0.12 g of Cu(NO3)2·3H2O.

11.
A synonym of neocuproine is 2,9-dimethyl-1,10-­
phenanthroline. This chemical has acute toxicity causing skin
irritation and serious eye damage if proper controls are not in
place.
12. The reagent solution is made up using dilute HCl due to its
poor solubility in water.
13. If porcelain well plates are not available, plastic micro well
plates may be used. It is recommended to place a white sheet
of paper underneath the clear plastic plate in order to visualize
the color change more easily.
14. In accordance with general operational presumptive color test-
ing procedures, negative and positive control standards are
tested alongside the seized material sample to be tested. The
certified reference material is the positive control sample.
Performing the test without the addition of a sample is the
negative control.
15. The sample may be a powder, crystal, or free base liquid. The
amount of sample required is not weighed out but is estimated
using a ‘small pin head sized amount’ which is approximately
0.1–0.2 mg.
16. Each test should be performed in triplicate alongside negative
(blank reagent) and positive control (depends on the color test
performed and target drug class) samples.
17. The operational limit of detection for methylone is 50 μg.
18. All known false positives for this test are mostly amphetamines,
1,3-dimethylpentlyamine, phentermine, and levamisole.
10 Kaitlyn Toole et al.

19. The known false positive for this test is 4-bromo-2,5-­
dimethoxyphenylethylamine (2C-B or Nexus).
20. The operational limit of detection for mephedrone is 400 μg.
All known false negatives for this test are 3-­fluoromethcathinone
and ethylcathinone. The known false positive for this test is
cocaine.
21. All known false positives for this test are amphetamine, meth-
amphetamine and pseudoephedrine.
22. All known false positives for this test are N-methylephedrine,
cathine, phentermine, and levamisole.
23. All known false positives for this test are 3,4-­methylenedioxyp
henylpropan-­2-one (MDMA precursor), heroin, and MDMA.
24. Benzylpiperazine (BZP) produces a very bright orange-red
color easily distinguishable from other piperazine analogs. The
operational limit of detection for BZP is 40 μg. Common cut-
ting agents, excipients, and diluents (e.g., caffeine, paracetamol,
levamisole, dimethylsulfone, and sugars) do not react with the
NQS reagent at room temperature. There are no known false
positives or false negatives.
25. Copper(II) chloride can also be used in place of copper(II)
nitrate. However, the nitrate solution gives a more stable col-
ored product and is considered ideal.
26. Following addition of the acetate solution, the test solution
will turn blue. All three reagents are crucial to forming the
colored complex.
27. The precise temperature setting on the hot plate will depend
on the thickness and composition of the porcelain well plates
used, as well as the individual hot plate capabilities. If a plastic
well plate was used, do not heat using a hot plate. Instead, heat
the plate using a simmering shallow water bath, or transfer test
solutions to semimicro test tubes and place in a beaker water
bath.
28. Synthetic cathinones will give yellow-orange and light yellow-­
orange color changes. Many cathinones will show a color
change after 5 min. The operational limit of detection of this
test for mephedrone is 40 μg. All known false negatives for this
test are 3,4-methylenedioxy-α-pyrrolidinobutiophenone,
3,4-methylenedioxypyrovalerone (MDPV),
­4-methyl-α-­ pyrrolidinobutiophenone, pyrovalerone, and
α-pyrrolidinopentiophenone. Paracetamol, codeine phosphate,
1-[3-(trifluoromethyl)phenyl]piperazine (TFMPP), and
5-methoxy-N,N-diallyltryptamine are known false positives.
No common cutting agents react with this copper–neocupro-
ine reagent.
 Color Tests for the Preliminary Identification of New Psychoactive Substances
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