Chapter 34

Quantitative Colorimetric Assays

for Methamphetamine
Aree Choodum1, Niamh NicDaeid2
1Faculty of Technology and Environment, Prince of Songkla University, Phuket, Thailand; 2Centre for Anatomy and Human Identification,
School of Science and Engineering, University of Dundee, Dundee, UK

Abbreviations in a powder form of its hydrochloride salt. This stimulant affects

the central nervous system (United Nations International Drug
AM  Amphetamine Control Programme, 1994) and has been linked to serious health
app  Application problems. MA has been classified as a Class A Schedule 2 drug
CCD  Charge-coupled device under The Misuse of Drugs Act 1971 in the United Kingdom
cm  Centimeter and a Schedule II substance under the United Nations Conven-
CMOS  Complementary metal oxide semiconductor tion on Psychotropic Substances in the United States of America.
DIC  Digital image colorimetry Although this drug has been strictly controlled, it has been increas-
dm  Decimeter ingly abused worldwide. Methods for the detection and quanti-
DSLR  Digital single-lens reflex fication of MA are necessary to combat criminal activity and in
FID  Flame ionization detector addition, the methods used to quantify the compound have the
GC  Gas chromatography potential for deployment as tools for the clinical study of MA.
GPS  Global positioning system The quantification of MA in various sample types is commonly
HPLC  High-performance liquid chromatography achieved by using gas chromatography (GC) (Choe et al., 2013;
L  Liter Khajeamiri, Faizi, Sohani, Baheri, & Kobarfard, 2012; Kuwayama
nm  Nanometer et al., 2008; Lee et al., 2008; Tsujikawa et al., 2013) combined
MA  Methamphetamine with a flame ionization detector (FID) or mass spectrometry (MS).
MDMA  3,4-Methylenedioxymethamphetamine Other analytical techniques such as high-performance liquid chro-
mg  Milligram matography (HPLC)/MS (del Mar Ramírez Fernández, Wille, di
mL  Milliliter Fazio, Gosselin, & Samyn, 2010; Nieddu, Boatto, Pirisi, & Baralla,
mmol  Millimole 2009) and electrochemiluminescence method (Cai et al., 2010;
MS  Mass spectrometry Dai, Wang, Wu, Zhang, & Chen, 2009) have also been reported.
RGB  Red green blue Although these techniques provided accurate results, they are
RSD  Relative standard deviation laboratory-based and require an expert to operate expensive instru-
TBPE  Tetrabromophenolphthalein ethyl ester ments, especially for MS. The complicated sample preparation
TBPE·H  Tetrabromophenolphthalein ethyl ester acid dichloroethane may also be required prior to analysis.
solution Presumptive tests are also commonly utilized to screen for
UK  United Kingdom the presence or absence of the drug quickly eliminating negative
UN  United Nations samples. Typical presumptive tests for MA involve the use of a
USA  United State of America colorimetric test (color test or spot test) which is the simplest and
v/v  Volume by volume quickest method to screen the presence of the drug in the sample
w/v  Weight by volume and such tests are recommended by the United Nations International
μg  Microgram Drug Control Programme (1994). Although colorimetric tests
μmol  Micromole have been widely used in forensic science laboratories, they are
not considered sufficient for the confirmation of the presence of
MA and, until recently, have been considered only as a qualitative
INTRODUCTION analysis where quantification still requires other complementary
Methamphetamine (MA) is a synthetic stimulant which has been methods such as spectrophotometry and chromatography. In this
used as the main component in many illegal drugs. It is called chapter, a new means of using color presumptive tests to develop
“Ice” in a clear crystal form, “Yaba” in a tablet form, and “Crank” quantitative colorimetric methods for MA will be described.

Neuropathology of Drug Addictions and Substance Misuse, Volume 2. http://dx.doi.org/10.1016/B978-0-12-800212-4.00034-0

Copyright © 2016 Elsevier Inc. All rights reserved. 349
350  PART | I Stimulants
COLORIMETRIC ASSAYS FOR MA
A colorimetric assay or test relies on a simple chemical reac-
tion between the analyte of interest and specific reagents to pro-
duce a visible product that can be observed by eye. The color
of the product is used to indicate the presence of the analyte in
the sample. Such colorimetric tests are usually the first chemi-
cal method that an analyst applies to a sample because it is the
simplest and quickest. The reagents and necessary materials
(such as spot plate, dropper) are also inexpensive and readily
available and many commercial test kits exist in the marketplace.
For MA, Marquis and Simon’s tests are recommended by the
United Nations International Drug Control Programme (1994)
and the United Nations Office on Drugs and Crime (Laboratory
and Scientific Section, 2006). These tests have also been used
for MA detection by many researchers (Choodum & Nic Daeid,
2011a; Choodum et al., 2014; Nakahara & Sekine, 1984; O’Neal,
Crouch, & Fatah, 2000; Pin, Chin, Hin, & Lim, 2010; Suzuki,
Inoue, & Niwaguchi, 1983) and also widely used in forensic sci-
ence laboratories.
The Marquis test is the most versatile and widely used colo-
rimetric assay for drug analysis. It has been used as an indica-
tive test by forensic and law enforcement bodies worldwide and
is currently used as presumptive test for amphetamine (AM),
morphine, and heroin (Pin et al., 2010). This test requires two
reagents, 2.5% v/v formaldehyde in glacial acetic acid (reagent
M1) and concentrated sulfuric acid (reagent M2) (Choodum
& Nic Daeid, 2011a; Laboratory and Scientific Section, 2006;
United Nations International Drug Control Programme, 1994).
Occasionally, the reagents can be prepared without glacial
acetic acid by mixing concentrated sulfuric acid in formalde-
hyde solution (40% v/v) with a ratio of 100:5 (reagent M3)
(Anderson, 2005; James, Saferstein, & Meloan, 2001; O’Neal
et al., 2000). When conducting the test, one drop of reagent
M1 is added to the suspected material, followed by either
one (­Laboratory and Scientific Section, 2006) or two (United
Nations International Drug Control Programme, 1994) drops of
reagent M2. When reagents are prepared without glacial acetic
acid, several drops of reagent M3 are added to the suspected
material (Anderson, 2005). An orange product which slowly
turns to brown (Figure 1(A)) indicates the presence of MA and/
or AM in the suspected material (Laboratory and Scientific
Section, 2006; United Nations International Drug Control Pro-
gramme, 1994).
The orange–brown product is the carbenium ion which is
formed due to the reaction of formaldehyde and aromatic com-
pound in acidic medium (Kovar & Laudszun, 1989). The mecha-
nism of the reaction (Figure 2) is complex and not completely
understood (Bell, 2006; Kovar & Laudszun, 1989). The carbo-
nium ion (A) formed from formaldehyde reacts with aromatic
ring of MA to produce the alcohol intermediate (B). Under
strong acidic conditions, the carbenium ion (C) is produced, and
it is further reacted with a second molecule of MA to produce a
dimer (D). Oxidation of the dimer is facilitated by trace metal
impurities in the sulfuric acid producing the product (E) as a col-
ored carbenium ion (F).
Although the Marquis test produces a differentiation for
MA from other drug types, e.g., opiates (morphine, codeine,
and diamorphine—which produce a violet product (Figure 3)),
as well as from other phenethylamine compounds such as
FIGURE 1  Color products from colorimetric assays of MA and AM.
Color products of MA and AM from (A) Marquis test and (B) Simon’s test.
3,4-methylenedioxymethamphetamine (MDMA; which produces
black product), an additional colorimetric assay is required to
differentiate MA from AM. The Simon’s test is usually used for
this purpose.
The Simon’s test is a test commonly used for secondary
amines including MA. It has been used for MA detection by
many researchers (Choodum & Nic Daeid, 2011a; Choodum
et al., 2014; Nakahara & Sekine, 1984; Suzuki et al., 1983). The
test commonly consists of two reagents, i.e., 10% v/v acetalde-
hyde in aqueous sodium nitroprusside solution (1% w/v) (reagent
S1) and 2% w/v sodium carbonate in water (reagent S2) (Bell,
2006; Choodum & Nic Daeid, 2011a; Choodum et al., 2014;
Laboratory and Scientific Section, 2009; O’Neal et al., 2000;
United Nations International Drug Control Programme, 1994).
Occasionally, acetaldehyde solution (50% in ethanol) (reagent
S3) has been prepared separately from an aqueous sodium nitro-
prusside solution (1% w/v) (reagent S1/1) (Laboratory and Sci-
entific Section, 2006). During testing, one drop of reagent S1 is
added to the suspected material followed by two drops of reagent
S2 (O’Neal et al., 2000; United Nations International Drug Con-
trol Programme, 1994). When the acetaldehyde solution was
prepared separately, one drop of reagent S2 was added to the
suspected materials with stirring followed by the addition of one
drop of reagent S1/1 and reagent S3 (Laboratory and Scientific
Section, 2006). A blue product was obtained if MA was present
within the sample (Figure 1(B)). Although, the Simon’s reagents
do not react with AM (which is a primary amine), other second-
ary amines, such as MDMA do produce similar color products to
MA. The combination of the Simon’s test, deployed in tandem
with a second colorimetric test such as the Marquis test is there-
fore advantageous.
The blue product obtained from the Simon’s test is com-
monly referred to as a Simon–Awe complex (Bell, 2006; Kovar
& Laudszun, 1989) (Figure 4). MA reacts with acetaldehyde
(through an addition–condensation reaction) and produces an
enamine (G) which further reacts with sodium nitroprusside to
produce an immonium salt intermediate (H). The intermediate
subsequently reacts with water to form the blue Simon–Awe
complex (I).
It has been reported that MA immediately produces a dark
brown product by reacting with 5% ammonium dichromate and
 Quantitative Color Test for Methamphetamine Chapter | 34  351

FIGURE 2  Mechanism of Marquis test for MA. (A) Carbonium ion, (B) alcohol intermediate, (C) carbenium ion, (D) dimer, (E) oxidation product,
and (F) carbenium ion. Adapted from Kovar and Laudszun (1989) with courtesy of publisher.

concentrated sulfuric acid (Laboratory and Scientific Section,

FIGURE 3  Color products from Marquis test of MA and other drugs.
Orange–brown products of MA and AM, and violet product of morphine
and codeine from Marquis test (A) after adding reagent M2 and (B) after
mixing. Unpublished data.
2009), while a dark green product is produced using the Mande-
lin test (1% ammonium vanadate in concentrated sulfuric acid)
(O’Neal et al., 2000; Pin et al., 2010). However, AM also pro-
duces the same color products using these assays. A red product
is also immediately produced from the reaction of MA and tet-
rabromophenolphthalein ethyl ester (TBPE) acid dichloroethane
solution (TBPE⋅H) which can be prepared by extracting aqueous
TBPE solution in acidic media using 1,2-dichloroethane (Sakai
& Ohno, 1986). However, this yellow reagent also reacts with
ephedrine and methylephedrine to produce a product which is
similar in color to the MA product. A bisazo dye with an attached
trifluoroacetyl group has also been found to reversibly form a
hemiaminal with AM, changing the color from blue to red, and
responds more significantly to the MA molecule (by a factor of
1.2) (Mohr, 2003; Mohr, Wenzel, Lehmann, & Czerney, 2002).
The dye, (4-[4-(4-trifluoroacetylphenylazo)-1-naphthylazo]-
N,N-dioctylaniline), can be prepared using a 5 h diazotization
procedure.
A number of colorimetric assays have been reported to identify
a presence or absence of MA in an illicit drug sample. Within these
tests, the use of the Marquis test in tandem with the Simon’s test
can differentiate MA from other drugs; however, these assays are
commonly used only for qualitative analysis.
352  PART | I Stimulants
FIGURE 4  Mechanism of Simon’s test for MA. (G) an enamine, (H) an immonium salt intermediate, and (I) a Simon–Awe complex. Adapted from
Kovar and Laudszun (1989) with courtesy of publisher.
QUANTIFICATION OF MA BY
COLORIMETRIC ASSAY
Quantification of MA is typically achieved using a range of instru-
mental analytical modalities including, for example, GC–FID,
GC–MS, HPLC. The use of spectroscopic methods (other than
ultraviolet–visible; UV–Vis) is more uncommon and there are
only a few publications that report the use of colorimetric methods
for quantitative analysis of MA which make use of spectrophoto-
metric methods even though this is a well-known methodology in
the field outside of forensic drug analysis.
Sakai and Ohno (1986) reported the use of a spectrophotomet-
ric method for quantification of MA, ephedrine, and methylephed-
rine where the absorption of the red product generated from MA
and TBPE⋅H was calibrated at 570 nm, revealing linearity in the
range of 2–12 μmol/dm3. This method revealed detection limits
of 0.15 μg/cm3 for MA. Similarly, the absorption of a red prod-
uct obtained from the chemical reaction of a bisazo dye (4-[4-
(4-trifluoroacetylphenylazo)-1-naphthylazo]-N,N-dioctylaniline)
and reported by Mohr et al. (2002); and Mohr (2003) was detected
at 630 nm with a detection limit of 0.1 mmol/l. Although the dye
was initially revealed in tests with AM the response obtained using
MA were more intense due presumably, in part, to the higher lipo-
philicity of the reactant.
Much more recently, attention has been focused on the inte-
gration of digital image analysis to functionalize the colorimetric
reaction products (so-called “digital image colorimetry” or DIC) in
order to deliver a rapid quantitative analysis of MA (Choodum &
Nic Daeid, 2011a; Choodum et al., 2014). This method is based on
the measurement of the intensity of the basic red green blue (RGB)
color values derived from a digital image of the colorimetric prod-
uct. During the imaging process, the photon from the reflected
light of the colored product is separated into three channels
(400–500 nm (blue), 500–580 nm (green), and 580–700 nm (red),
respectively) (Cantrell, Erenas, de Orbe-Paya, & Capitan-Vallvey,
2010) using the RGB filter of the digital camera which captures
the image. This filtered photon is detected and recorded as an indi-
vidual RGB value by an image sensor such as a charge-coupled
 Quantitative Color Test for Methamphetamine Chapter | 34  353

FIGURE 5  The relationships of RGB values (intensity and calculated absorbance) and the concentration of MA from Marquis and Simon’s
test products. The relationships between MA concentration and (A) RGB intensity from Marquis test, (B) RGB intensity from Simon’s test, (C) RGB
calculated absorbance from Marquis test, and (D) RGB calculated absorbance from Simon’s test. Each point is the mean ± SD of six replicate analysis and
all lines are point-to-point. Data are from Choodum and Nic Daeid (2011a), with permission from publisher.

device or complementary metal oxide semiconductor (CMOS).
After compensating for the variations in the conditions of capture,
the additive recorded data from the three filters are combined to
produce a final color for the digital image.
Analysis of the digital image can be achieved using various
commercial or open source programs such as the image process-
ing tool box in Matlab (Goddijn & White, 2006; Lopez-Molinero,
Linan, Sipiera, & Falcon, 2010), Kylix (Gaiao et al., 2006; Silva Lyra
et al., 2009), Visual basic (Maleki, Safavi, & Sedaghatpour, 2004;
Suzuki, Endo, Jin, Iwase, & Iwatsuki, 2006), or Adobe Photoshop
(Choodum, Kanatharana, Wongniramaikul, & Nic Daeid, 2013;
Choodum, Kanatharana, Wongniramaikul, & NicDaeid, 2012;
Choodum & Nic Daeid, 2011a; Choodum & Nic Daeid, 2011b;
Thongprajukaew, Choodum, Sa-E, & Hayee, 2014). Such analysis
can provide the individual RGB values within the 0–255 range
(Byrne, Barker, Pennarun-Thomas, Diamond, & Edwards, 2000;
Gaiao Eda et al., 2006; Lopez-Molinero et al., 2010; Maleki et al.,
2004) where black images return an RGB value of 0, 0, 0 and
white images have an RGB value of 255, 255, 255.
The new scientific leap is in functionalizing the RGB values
obtained from digital images of the chemical reaction products of
colorimetric presumptive tests for illicit drugs to generate robust
quantitative analytical results.
Using the Marquis and Simon’s tests as an example, the analy-
sis of digital images taken of the reaction products using Adobe
Photoshop provides the RGB values associated with the test result.
This can be related to the concentration of MA within a sample as
shown in Figure 5. Orange–brown products from the Marquis test
exhibit a higher intensity within the red channel (Figure 5(A)),
while the blue products from the Simon’s test reveal the highest
intensity within the blue channel (Figure 5(B)). This is not unex-
pected as the color of the product will dictate the intensity of the
individual RGB values in an image of the sample.
As such, DIC calibration curves can be created for each of
the red, green, and blue values derived from digital images of the
chemical test products using known concentrations of analyte
(in this case MA) to generate the colorimetric products and subse-
quent digital images. When such curves are evaluated, they reveal
354  PART | I Stimulants

regions exhibiting a linear response to the analyte concentration
which facilitates their use as calibration curves. When such an
approach was used for MA, the detection limit of the DIC calibra-
tion method was to be below 2.5 mg/ml for the Marquis test, and
between 2.0 and 6.0 mg/ml for the Simon’s test.
The intensity of the RGB color can be directly converted to the
absorbance using Eqn (1):
(IX − IX , b ) (IX )C
AX = − log = − log = − log RX
(IX , w − IX , b ) (IX , w )C
where AX is the absorbance of X, IX is the intensity of X,
IX,b = 0, IX,w = 255, and RX is the reflectance of light X and C
is the concentration of X (R, B, G) (Choodum & Nic Daeid,
2011a; Kompany-Zareh, Mansourian, & Ravaee, 2002). A lin-
ear portion of the relationship between the calculated absor-
bance and concentration of the target analyte (Figure 5(C) and
(D) for MA) can also be used as the calibration graph for quan-
titative analysis.
In addition to the intensity and calculated absorbance derived
for the digital image of the colorimetric test, the total intensity and/
or total absorbance can also be used to establish calibration curves
for quantitative analysis. The values derived from the combination
of the total RGB data (total intensity (ITOTAL) = IR + IG + IB; total
absorbance (ATOTAL) = AR + AG + AB) may provide additional valu-
able information above that obtained from the individual RGB
values (Choodum & Nic Daeid, 2011b; Choodum et al., 2014;
Lopez-Molinero et al., 2010). The relationships of the total values
of the Simon’s test product of MA are shown as an example in
(1) Figure 6(A) and (B). Illicit drug products, particularly in tablet
form, often appear as colored materials which may affect the RGB
values of the colorimetric product. In general a technique such
as blank subtraction can be used to address this issue, where the
linear portion of the relationship from the blank subtracted RGB
values used for quantification of MA (Figure 6(C) and (D)).
DIC has also been validated for the quantification of MA pro-
viding excellent accuracy and precision (Choodum & Nic Daeid,
2011a; Choodum et al., 2014). A controlled sample of 0.75 mg/ml
was analyzed using DIC (Marquis test) and a concentration range of

FIGURE 6  The relationships of total RGB values and the blank subtracted RGB values and the concentration of MA from Simon’s test products.
The relationships between MA concentration and (A) total intensity, (B) total calculated absorbance, (C) RGB intensity with blank subtracted, and
(D) RGB calculated absorbance with blank subtracted from Simon’s test and their linear portions. Each point is the mean ± SD of three replicate analysis
and all lines are point-to-point, except linear portions which were regression lines. Data are from Choodum et al. (2014) with permission from publisher.

0.69–0.77 mg/ml determined with percentage relative standard devia-
tion (%RSD) of 0.28–2.30% (n = 6) (Choodum & Nic Daeid, 2011a).
Similar results were obtained for the Simon’s test (5.0 mg/ml MA
sample determined to have a concentration range of 4.67–4.90 mg/
ml with a %RSD of 1.5–4.2%) when the digital single-lens reflex
(DSLR) camera was used (n = 6) (Choodum & Nic Daeid, 2011a).
While standard digital cameras (such as DSLR cameras) can be
used to produce the color images, the use of built-in digital cameras
of smart phones such as an iPhone coupled with a phone application
(app) has been used for color analysis of the Simon–Awe product of
MA (Choodum et al., 2014). This facilitated a more rapid approach
to the quantification of MA using DIC as the connection to an exter-
nal computer was no longer necessary. When the iPhone and associ-
ated app was used, %RSD values in the range of 2.27–4.49% for
intraday precisions (n = 3) and 2.65–5.62% for interday (n = 3) were
obtained for MA using the Simon’s test (Choodum et al., 2014)
again demonstrating the value of this approach.
The quantification of MA in Yaba in forensic casework sam-
ples using a Simon’s test and the iPhone and associated app also
revealed an excellent correlation with conventional chromato-
graphic analysis (Table 1) revealing no negative interference
effects from any other potential impurities or reacting products.
Because a colorimetric assay is a rapid, simple, portable, and
inexpensive method for indication of the presence of MA in a
sample, the use of DIC can extend the potential value of such tests
by generating accurate quantitative analysis. Moreover, the use of
digital cameras within smart phones together with an associated
app can facilitate the development of a portable rapid quantita-
tive chemical analysis system deployable as a field device with
the potential to link to global positioning system (GPS) and other
information inherent within such devices.
APPLICATIONS TO OTHER ADDICTIONS
AND SUBSTANCE MISUSE
Quantitative colorimetric assays have also been used for other
illicit drugs in addition to MA such as AM, ephedrine, methy-
lephedrine, and opiates.
Quantification of AM has been reported using colorimetric
tests and spectrophotometric methods (Mohr, 2003; Mohr et al.,
2002) where the red hemiaminal product obtained from the
TABLE 1  Analysis of Yaba Samples
Methamphetamine in Yaba Samples (%)
Sample No. Digital Image Method
1 20.0 ± 1.0%
2 16.0 ± 1.0%
3 20.1 ± 0.9%
4 19.0 ± 2.0%
5 19.0 ± 1.0%
The table reveals very good correlation between the data (mean ± SD of five Yaba Samples) derived from DIC (Simon’s Test) and conventional GC–FID.
ans = no significant difference.
Data are from Choodum et al. (2014) with permission from the publisher.
Quantitative Color Test for Methamphetamine Chapter | 34  355
chemical reaction of AM and the blue bisazo dye in trifluoroace-
tyl form (4-[4-(4-trifluoroacetyl-phenylazo)-1-naphthylazo]-N,N-
dioctylaniline) was detected at 630 nm. This method provided a
linear relation of the AM concentration with absorbance in the
range of 0.3–30 mmol/l with a detection limit of 0.1 mmol/l.
Ephedrine and methylephedrine have also been quantified using
colorimetric assays and spectrophotometry (Sakai & Ohno, 1986).
Both drugs were reacted with TBPE⋅H to form a red product with
maximum absorption at 555 and 550 nm for ephedrine and methy-
lephedrine, respectively. A determination limit of 0.165 μg/cm3 was
obtained for ephedrine and 0.179 μg/cm3 for methylephedrine.
Marquis and nitric acid tests have also been used for quantifi-
cation of opiates, i.e., morphine, codeine, and diamorphine hydro-
chloride (heroin) using DIC (Choodum & Nic Daeid, 2011b).
Violet products (see Figure 3) were obtained from the reaction
of opiates and Marquis reagents due to the formation of oxo-
nium–carbenium salts (Choodum & Nic Daeid, 2011b; Kovar &
Laudszun, 1989). Different colored nitro products were produced
from the reaction of concentrated nitric acid and the various opi-
ates. An orange product rapidly changing to red and then slowly
to yellow indicated the presence of morphine, while the absence
of the red product suggested the presence of codeine and a further
change from the yellow product to a light green product indicated
the presence of diamorphine. Photography of these products
and analysis of their RGB values makes use of the relationship
between the intensity of color product and concentration of opiates
(Figures 7 and 8). The linear portions of these relationships can be
used for quantification of the opiates. The analysis of heroin street
samples revealed that the relationship between the intensity of the
red (R) channel of the violet products from the Marquis test and
the concentration of diamorphine (y = −(496 ± 34)x + (398 ± 11))
provided the most reliable qualitative results with good correla-
tion with data derived from GC–MS analysis of the same samples.
CONCLUSIONS
Developments in the use of digital images of the products from
colorimetric tests of controlled substances have revealed robust
correlations between the RGB values of the images and the con-
centration of the target analytes within photographed samples.
These correlations have been validated for a number of controlled
GC–FID Relative Error (%) P = 0.05
19.9 ± 0.4% +0.2% nsa
17.4 ± 0.1% −7.3% ns
18.7 ± 0.2% +7.1% ns
19.3 ± 0.3% −1.2% ns
18.1 ± 0.5% +4.3% ns
356  PART | I Stimulants

FIGURE 7  The relationships of RGB intensity and concentration of opiates from Marquis test products. The relationships between opiate concen-
trations and RGB intensity from Marquis test for (A) morphine, (B) codeine, and (C) diamorphine. Each point is the mean ± SD from six replicate analysis.
Data are from Choodum and Nic Daeid (2011b), with permission from the publisher.

substances including MA. Digital images produced either on a
conventional digital camera or by using a smart phone using a
designed app hosted on the phone reveal these quantitative results.
Moreover, the use of mobile phone technologies facilitates the
geotagging of the data providing further functionality and oppor-
tunities for the development of field deployable devices.
DEFINITION OF TERMS
Accuracy  It is the closeness of a quantified value to a true value.
Calibration curve  It is the curve for quantifying the concentration
of an analyte. The quantification is achieved by comparing an
unknown response to a set of responses obtained from standards of
known concentration.
Colorimetry  It is the simplest and quickest method for indicating the
presence or absence of an analyte of interest where a specific colored
product is obtained from the chemical reaction between the analyte
and an appropriate reagent.
Digital image colorimetry  DIC is an analysis method for the quan-
tification of an analyte using its colorimetric products. This is
performed by analyzing the intensity of images taken of the colori-
metric product photographed by a digital camera. The RGB values
obtained from the analysis of the digital image can be used to estab-
lish a calibration graph for quantification.
Marquis test  It is a commonly used colorimetric test for indicating the
presence or absence of various compounds. It could be used to dif-
ferentiate AMs from other ring-substituted analogs and opiates.
Precision  It is the closeness of two or more quantified values to each
other.
Presumptive tests  It is a fast screening procedure for indicating the pres-
ence or absence of controlled drug compounds. There are three types
of presumptive test (color tests, anion tests, and microcrystal tests).
Qualitative analysis  It is the analysis of a sample to determine the
nature of its chemical constituents.
Quantitative analysis  It is the analysis of a sample to determine an
amount and proportion of its chemical constituents.
RGB color system  It is a color system which three additive primary
colors (red, green, and blue) are combined together to reproduce a
broad array of colors. It is used for display of images in electronic
systems including digital cameras.
Simon’s test  It is a commonly used colorimetric test for indicating a
presence or absence of secondary amines, including MA and its
ring-substituted analogs such as MDMA. It could be used to dif-
ferentiate MAs from AM which is a primary amine.
 Quantitative Color Test for Methamphetamine Chapter | 34  357
FIGURE 8  The relationships of RGB intensity and concentration of opiates from nitric acid test products The relationships between opiate con-
centrations and RGB intensity from nitric acid test for (A) morphine, (B) codeine, and (C) diamorphine. Each point is the mean ± SD from six replications.
Data are from Choodum and Nic Daeid (2011b) with permission from the publisher.
KEY FACTS
Key Facts of MA
l 
N-methyl-1-phenylpropan-2-amine is the IUPAC name for MA.
l It is a secondary amine with molecular weight 149.2337 g/mol
and its chemical formula is C10H15N.
l It can be easily synthesized by the reduction of ephedrine or
pseudoephedrine using red phosphorus and iodine.
l It is a highly addictive stimulant drug that affects the central
nervous system and is thought to be responsible for a range of
health problems.
l It causes the release of the monoamines dopamine, serotonin,
and norepinephrine.
Key Facts of Quantitative Analysis
l Quantitative analysis is an analysis of a sample to determine
an amount and proportion of its chemical constituents.
l It can be performed in tandem with qualitative analysis which
determines the nature of chemical constituents of the sample.
l It can be achieved by establishing a calibration curve using
three commonly used mechanisms (internal standard, external
standard, and standard addition).
l The external standard method is the simplest method where a
calibration curve is constructed by analyzing a series of known
concentrations of standard and plotting the response (y-axis)
against the concentration of analyte (x-axis).
l For the internal standard method, an internal standard of
known concentration and which should have chemical prop-
erties similar to interested analyte is added to the sample to
compensate for any analytical errors.
l The calibration curve is constructed by analyzing a series of
known concentrations of analyte with constant amount of inter-
nal standard added in each case and plotting the ratio of the
analytes to internal standard response (y-axis) against concen-
tration of analyte (x-axis).
l The standard addition method is usually performed when an
analyte-free sample is not available and/or in cases where
the sample matrix affects the analytical response of the
analyte.
l Standard addition can be achieved by analyzing the sample
followed by analyzing a series of samples to which a known
concentration of standard has been added.
l The quantification is achieved by plotting the response of ana-
lyte (y-axis) against the added concentration (x-axis), and the
value of the intercept of the x-axis provides the value of the
concentration of the analyte in the sample.
358  PART | I Stimulants
Key Facts of Colorimetry
l Colorimetry is a simple, quick, and easy method for the analy-
sis of a sample.
l It is based on a chemical reaction between the analyte and an
appropriate reagent to produce a visible colored product.
l It is commonly used as qualitative analysis to indicate
the presence or absence of an analyte of interest within a
sample.
l A negative result indicates the absence of the analyte.
l A positive result indicates the possible presence of the analytes
within the sample and additional laboratory testing is required
for confirmation.
l Color is subjective and depends on individual perception
of the observer and the use of reference standards before
analyzing the test sample can provide an indication of the
expected colored product should the analyte be present in
the sample.
SUMMARY POINTS
l This chapter focuses on the developments and use of colori-
metric assays for the quantitative analysis of MA.
l The Marquis and Simon’s tests are commonly used to indicate
the presence or absence of MA within a drug sample.
l An orange–brown product from the Marquis test indicates the
possible presence of MA and/or AM.
l A blue product from the Simon’s test indicates the possible
presence of secondary amine including MA.
l Colorimetric assays, i.e., Marquis and Simon’s test, are used
as qualitative analysis and an additional method is required to
convert these tests into quantitative analytical methods.
l Spectrophotometric methods such as UV–Vis can be used for
the quantification of MA from its colorimetric product.
l DIC has been reported as a means of producing quantitative
data from colorimetric tests of MA.
l The results from DIC revealed very good correlation with
accurate instrumental quantification methods such as GC-FID
or GC–MS.
l DIC has considerable potential as a rapid, simple, portable,
and inexpensive colorimetric assay method.
REFERENCES
Anderson, C. (2005). Presumptive and confirmatory drug tests. Journal of
Chemical Education, 82(12), 1809–1810.
Bell, S. (2006). Forensic chemistry. New Jersey: Pearson Prentice Hall.
Byrne, L., Barker, J., Pennarun-Thomas, G., Diamond, D., & Edwards, S.
(2000). Digital imaging as a detector for generic analytical measure-
ments. TrAC Trends in Analytical Chemistry, 19(8), 517–522.
Cai, Z., Lin, Z., Chen, X., Jia, T., Yu, P., & Chen, X. (2010). Electrochemi-
luminescence detection of methamphetamine based on a Ru(bpy)32+-
doped silica nanoparticles/nafion composite film modified electrode.
Luminescence, 25, 367–372.
Cantrell, K., Erenas, M. M., de Orbe-Paya, I., & Capitan-Vallvey, L. F.
(2010). Use of the hue parameter of the hue, saturation, value color
space as a quantitative analytical parameter for bitonal optical sensors.
Analytical Chemistry, 82, 531–542.
Choe, S., Heo, S., Choi, H., Kim, E., Chung, H., & Lee, J. (2013). Analysis
of pharmaceutical impurities in the methamphetamine crystals seized
for drug trafficking in Korea. Forensic Science International,
227(1–3), 48–51.
Choodum, A., Kanatharana, P., Wongniramaikul, W., & Nic Daeid, N.
(2013). Using the iPhone as a device for a rapid quantitative analy-
sis of trinitrotoluene in soil. Talanta, 115, 143–149. http://dx.doi.org/
10.1016/j.talanta.2013.04.037.
Choodum, A., Kanatharana, P., Wongniramaikul, W., & NicDaeid, N. (2012).
Rapid quantitative colourimetric tests for trinitrotoluene (TNT) in soil.
Forensic Science International, 222(1–3), 340–345. http://dx.doi.org/
10.1016/j.forsciint.2012.07.014.
Choodum, A., & Nic Daeid, N. (2011a). Digital image-based colourimet-
ric tests for amphetamine and methamphetamine. Drug Testing and
Analysis, 3, 277–282.
Choodum, A., & Nic Daeid, N. (2011b). Rapid and semi-quantitative pre-
sumptive tests for opiate drugs. Talanta, 86, 284–292. http://dx.doi.org/
10.1016/j.talanta.2011.09.015.
Choodum, A., Parabun, K., Klawach, N., NicDaeid, N., Kanatharana, P., &
Wongniramaikul, W. (2014). Real time quantitative colourimetric test
for methamphetamine detection using digital and mobile phone tech-
nology. Forensic Science International, 235, 8–13. http://dx.doi.org/
10.1016/j.forsciint.2013.11.018.
Dai, H., Wang, Y., Wu, X., Zhang, L., & Chen, G. (2009). An electroche-
miluminescent sensor for methamphetamine hydrochloride based on
multiwall carbon nanotube/ionic liquid composite electrode. Biosen-
sors and Bioelectronics, 24(5), 1230–1234. http://dx.doi.org/10.1016/
j.bios.2008.07.025.
Gaiao Eda, N., Martins, V. L., Lyra Wda, S., de Almeida, L. F., da Silva,
E. C., & Araujo, M. C. (2006). Digital image-based titrations.
Analytica Chimica Acta, 570(2), 283–290.
Goddijn, L. M., & White, M. (2006). Using a digital camera for water
quality measurements in Galway Bay. Estuarine, Coastal and Shelf
Science, 66(3–4), 429–436.
James, R. E., Saferstein, R., & Meloan, C. E. (2001). Lab manual, crimi-
nalistics: An introduction to forensic science (7th ed.). Upper Saddle
River, New Jersey: Prentice-Hall, Inc.
Khajeamiri, A. R., Faizi, M., Sohani, F., Baheri, T., & Kobarfard, F. (2012).
Determination of impurities in illicit methamphetamine samples
seized in Iran. Forensic Science International, 217(1–3), 204–206.
Kompany-Zareh, M., Mansourian, M., & Ravaee, F. (2002). Simple method
for colorimetric spot-test quantitative analysis of Fe(III) using a com-
puter controlled hand-scanner. Analytica Chimica Acta, 471(1), 97–104.
Kovar, K.-A., & Laudszun, M. (1989). Chemistry and reaction mechanisms
of rapid tests for drugs of abuse and precursors chemicals. In Phar-
mazeutisches Institut der Universitat Tubingen, Germany (pp. 8–10).
Kuwayama, K., Inoue, H., Phorachata, J., Kongpatnitiroj, K., Puthaviriyakorn,
V., Tsujikawa, K., … Kishi, T. (2008). Comparison and classification of
methamphetamine seized in Japan and Thailand using gas chromatogra-
phy with liquid-liquid extraction and solid-phase microextraction. Foren-
sic Science International, 175(2–3), 85–92.
Laboratory and Scientific Section. (2006). Recommended methods for
the identification and analysis of amphetamine, methamphetamine
and their ring-substituted analogues in seized materials. The United
Nations Office on Drugs and Crime, 17–21.
Laboratory and Scientific Section. (2009). Screening colour test and
specific colour tests for the detection of methylendioxyamphetamine
and amphetamine type stimulants. The United Nations Office on
Drugs and Crime.
 Quantitative Color Test for Methamphetamine Chapter | 34  359
Lee, J. S., Chung, H. S., Kuwayama, K., Inoue, H., Lee, M. Y., & Park,
J. H. (2008). Determination of impurities in illicit methamphetamine
seized in Korea and Japan. Analytica Chimica Acta, 619(1), 20–25.
Lopez-Molinero, A., Linan, D., Sipiera, D., & Falcon, R. (2010). Chemomet-
ric interpretation of digital image colorimetry. Application for titanium
determination in plastics. Microchemical Journal, 96(2), 380–385.
Maleki, N., Safavi, A., & Sedaghatpour, F. (2004). Single-step calibration,
prediction and real samples data acquisition for artificial neural net-
work using a CCD camera. Talanta, 64(4), 830–835.
del Mar Ramírez Fernández, M., Wille, S. M. R., di Fazio, V., Gosselin,
M., & Samyn, N. (2010). Analysis of amphetamines and metabolites
in urine with ultra performance liquid chromatography tandem mass
spectrometry. Journal of Chromatography B, 878(19), 1616–1622.
http://dx.doi.org/10.1016/j.jchromb.2010.03.048.
Mohr, G. J. (2003). New chromoreactands for the detection of aldehydes,
amines and alcohols. Sensors and Actuators B: Chemical, 90(1–3),
31–36. http://dx.doi.org/10.1016/S0925-4005(03)00018-2.
Mohr, G. J., Wenzel, M., Lehmann, F., & Czerney, P. (2002). A chro-
moreactand for optical sensing of amphetamines. Analytical and
Bioanalytical Chemistry, 374(3), 399–402. http://dx.doi.org/
10.1007/s00216-002-1519-0.
Nakahara, Y., & Sekine, H. (1984). A high selective screening test for
methamphetamine in human urine. Forensic Science International,
26(4), 277–282. http://dx.doi.org/10.1016/0379-0738(84)90032-X.
Nieddu, M., Boatto, G., Pirisi, M. A., & Baralla, E. (2009). Multi-residue
analysis of eight thioamphetamine designer drugs in human urine by
liquid chromatography/tandem mass spectrometry. Rapid Communi-
cations in Mass Spectrometry, 23, 3051–3056.
O’Neal, C. L., Crouch, D. J., & Fatah, A. A. (2000). Validation of
twelve chemical spot tests for the detection of drugs of abuse.
Forensic Science International, 109(3), 189–201. http://dx.doi.org/
10.1016/S0379-0738(99)00235-2.
Pin, T. Y., Chin, L. S., Hin, L. S., & Lim, L. B. (2010). Presumptive testing
of amphetamine-type stimulants via color tests. ASEAN Journal for
Science and Technology Development, 27, 66–75.
Sakai, T., & Ohno, N. (1986). Spectrophotometric determination of stimu-
lant drugs in urine by color reaction with tetrabromophenolphthalein
ethyl ester. Analytical Sciences, 2, 275–279.
Silva Lyra, W., dos Santos, V. B., Dionizio, A. G. G., Martins, V. L.,
Almeida, L. F., Nobrega Gaiao, E., … Araujo, M. C. U. (2009).
Digital image-based flame emission spectrometry. Talanta, 77(5),
1584–1589.
Suzuki, S., Inoue, T., & Niwaguchi, T. (1983). Rapid screening method for
methamphetamine in urine by color reaction in a sep-pak C18 cartridge.
Journal of Chromatography A, 267(2), 381–387. http://dx.doi.org/
10.1016/S0021-9673(01)90857-X.
Suzuki, Y., Endo, M., Jin, J., Iwase, K., & Iwatsuki, M. (2006). Tristimulus
colorimetry using a digital still camera and its application to deter-
mination of iron and residual chlorine in water samples. Analytical
Sciences, 22, 411–414.
Thongprajukaew, K., Choodum, A., Sa-E, B., & Hayee, U. (2014). Smart
phone: a popular device supports amylase activity assay in fisheries
research. Food Chemistry, 163, 87–91. http://dx.doi.org/10.1016/
j.foodchem.2014.04.080.
Tsujikawa, K., Kuwayama, K., Miyaguchi, H., Kanamori, T., Iwata, Y. T.,
& Inoue, H. (2013). Chemical profiling of seized methamphetamine
putatively synthesized from phenylacetic acid derivatives. Forensic
Science International, 227(1–3), 42–44.
United Nations International Drug Control Programme. (1994). Rapid
testing methods of drugs of abuse: Manual for use by national law
enforcement and narcotics laboratory personnel. Vienna: United
Nation.