DEVELOPMENT OF NEW COLOR PRESUMPTIVE TEST FOR
AMPHETAMINE / METHAMPHETAMINE IN URINE
SUPANAT PANOMNOPTHAM
A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR
THE DEGREE OF MASTER OF SCIENCE
(FORENSIC SCIENCE)
FACULTY OF GRADUATE STUDIES
MAHIDOL UNIVERSITY
2009
COPYRIGHT OF MAHIDOL UNIVERSITY
Copyright by Mahidol University
Copyright by Mahidol University
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iii
ACKNOWLEDGEMENTS
The success of this thesis can be attributed to the extensive support and
assistance from my major advisor, Assoc. Prof. Dr. Prapin Wilairat, and my coadvisor, Asist. Prof. Dr. Nopphadol Chaikum. I respectfully thank them for their
valuable advice, time, and guidance in this research.
I would like to thank Department of Chemistry, Faculty of Science, Mahidol
University, for laboratory support, and the National Doping Control Centre (NDCC)
of Mahidol University for providing the samples.
I wish to thank Dr. Nathinee Panvisavas, Director of the Forensic Science
Graduate Programme. In addition, thanks to all the lectures, friends, and staffs of the
Forensic Science Department for their love and care.
Finally, I am grateful to my family for their entire support. The usefulness of this
thesis, I dedicate to my family, friends, and all the teachers whose support have
nurtured my life and knowledge.
Supanat Panomnoptham
Copyright by Mahidol University
Fac. of Grad. Studies, Mahidol Univ.
Thesis / iv
DEVELOPMENT OF NEW COLOR PRESUMPTIVE TEST FOR AMPHETAMINE/ METHAMPHETAMINE IN URINE
SUPANAT PANOMNOPTHAM
4936103
SCFS/M
M.Sc. (FORENSIC SCIENCE)
THESIS ADVISORY COMMITTEE: PRAPIN WILAIRAT, Ph.D. (PHYSICAL
CHEMISTRY), NOPADOL CHAIKUM, Ph.D. (GEOCHEMISTRY)
ABSTRACT
Amphetamine and methamphetamine, called “YABA” in Thai, are prevalent
worldwide for the last decades causing social and health problems. Tetrabromophenolphthalein ethyl ester (TBPE) is the main reagent used in the Thai color kit for
methamphetamine detection in urine. A false positive result is a major problem of the
presumptive test. This study aimed to find a chemical reagent which provided less
false positive outcomes. Borax and sodium hydroxide solution were added to urine
samples and then benzene was added in order to extract the drug. The oraganic layer
was separated into new tubes and three dyes were added: bromophenol blue (BPB),
bromocresol purple (BCP), and bromothymol blue (BTB), dissolved in benzene.
In this work, BPB showed a better sensitivity to methamphetamine than the
others. BPB was the only dye to give a blue color due to the charge transfer complex
between BPB and methamphetamine, absorbing at 570 nm. Of all the results, BCP
and BTB were yellow in color. When BPB was compared with the commercial TBPE
kit, results for urine samples containing methamphetamine were the same, but the
number of false positives from BPB was less than for TPBE. BPB can thus detect
methamphetamine in urine and may be a possible alternative reagent. However,
further study is required before it can be applied to real cases.
KEY WORDS: COLOR TEST /AMPHETAMINE / METHAMPHETAMINE /
URINE / TBPE / BPB
56 pages
Copyright by Mahidol University
Fac. of Grad. Studies, Mahidol Univ.
Thesis / v
การพัฒนาชุดน้ํายาชนิดสีเพือ่ การตรวจสอบยาบาในปสสาวะ
DEVELOPMENT OF NEW COLOR PRESUMPTIVE TEST FOR AMPHETAMINE/
METHAMPHETAMINE IN URINE
ศุภณัฐ พนมนพธรรม
4936103 SCFS/M
วท.ม. (นิตวิ ิทยาศาสตร)
คณะกรรมการที่ปรึกษาวิทยานิพนธ : ประพิณ วิไลรัตน Ph.D. (Physical Chemistry),
นภดล ไชยคํา Ph.D. (Geochemistry)
บทคัดยอ
ในปจจุบนั ชุดตรวจสอบสีในประเทศไทยมีสารสําคัญคือ Tetrabromophenolphthalein ethyl
ester (TBPE) ซึ่งนํามาใชตรวจสอบสารแอมเฟตามีนและเมทแอมเฟตามีนในปสสาวะของผูเสพ
อยางไรก็ตามชุดตรวจสอบนี้กอผลบวกลวงจํานวนมาก การศึกษานีจ้ งึ ตองการหาสารเคมีที่สามารถ
ทําปฏิกิริยากับสารดังกลาวและกอใหเกิดผลบวกลวงนอยลง
โดยนําปสสาวะทีไ่ ดรับการตรวจ
พบวามีสารดังกลาวอยูมาใส borax และ NaOH เพื่อปรับ pH ใหเหมาะสมแลวหลังจากนั้นจึงใส
benzene เพื่อสกัดเมทแอมเฟตามีนออกมา แยก benzene ที่สกัดออกมาแลวและนํามาแยกผสมกับสี
สามชนิดคือ bromophenol blue (BPB), bromocresol purple (BCP) และ bromothymol blue (BTB)
BPB สามารถเกิดสารเชิงซอนกับเมทแอมเฟตามีนได โดยที่สารละลายเปลี่ยนจากสีเหลืองเปน
สีน้ําเงิน ซึ่งพบวาคาดูดกลืนแสงของสารเชิงซอนนี้อยูที่ 570 นาโนเมตร สวนผลจากสีอีกสองชนิด
คือ BCP และ BTB จะปรากฏแคสีเหลืองเทานั้น เมื่อเปรียบเทียบผลของ BPB กับ TBPE พบวาสีทั้ง
สองชนิดใหผลเหมือนกันในตัวอยางปสสาวะที่มีเมทแอมเฟตามีน แตจะใหผลบวกลวงไมเทากัน
โดยที่ BPB จะแสดงผลบวกลวงนอยกวา TBPE อยู 2 ตัวอยาง จากตัวอยางปสสาวะที่มีสารชนิดอื่น
ที่ไมใชเมทแอมเฟตามีนอยู 14 ตัวอยาง วิธีที่แสดงในการศึกษานีส้ ามารถนํามาประยุกตใชเปน
ทางเลือกในการตรวจสอบยาบาไดอีกทางหนึ่งแตควรศึกษาเพิ่มเติมกอนนํามาใชจริง
56 หนา
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vi
CONTENTS
Page
ACKNOWLEDGEMENTS……………………………………………….
iii
ABSTRACT (IN ENGLISH)……………………………………………...
iv
ABSTRACT (IN THAI)…………………………………………………...
v
LIST OF TABLES………………………………………………………....
ix
LIST OF FIGURES………………………………………………………..
x
LIST OF ABBREVIATIONS……………………………………………..
xii
CHAPTER I INTRODUCTION………………........................................
1
1.1
Introduction………………………………………….
1
1.2
Aim of Study………………………………………...
2
CHAPTER II LITERATURE REVIEW………………………………....
3
2.1
Amphetamine and Methamphetamine………………
3
2.1.1
Synonym…………………………..…………
3
2.1.2 General properties…………………………...
4
2.1.3
The effects of amphetamine and methamphetamine……………………………………….
5
2.1.4
Routes of administration…………………….
6
2.1.5
Metabolism and excretion of amphetamine
and methamphetamine………………………
2.2
6
Color tests for detection of amphetamine or methamphetamine in urine…………………………………..
2.2.1
Spot tests for detection of amphetamine or
Methamphetamine…………………………..
2.2.2
9
9
Color tests for amphetamine or methamphetamine detection in urine………………………
12
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CONTENTS (cont.)
Page
2.3
The color test kit available in Thailand……………...
12
2.3.1 Partition with liquid phases…………………
13
2.3.2
Charge transfer complex with tetrabromophenolphthalein ethyl ester……………………...
14
Color and Colorants…………………………………
17
2.4.1
Color………………………………………...
17
2.4.2 Dyes…………………………………………
20
2.4.3 Sulfonephthalein indicators…………………
22
CHAPTER III MATERIALS AND METHODS………...……………...
23
2.4
3.1
3.2
Materials…………………………………………….
23
3.1.1
Instrumentation………………………………
23
3.1.2
Reagents……………………………………..
24
3.1.3
Urine samples ……………………………….
24
3.1.4
The kit for methamphetamine test in urine….
25
Procedure of the kit for methamphetamine test in urine
26
3.2.1
Method K: testing guide of the kit…………..
26
3.2.2
Interpretation………………………………...
26
Preliminary dye selection……………………………
26
3.3.1
Dye selection 1……………………………....
26
3.3.2
Dye selection 2………………………………
27
3.3.3
Dye selection 3………………………………
28
Study on acid-base reaction in organic solvent……...
29
3.4.1
General Procedure…………………………...
29
3.4.2
Urine samples………………………………..
29
CHAPTER IV RESULTS AND DISCUSSION…………………………..
30
3.3
3.4
4.1
Preliminary dye selection………………………….....
30
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CONTENTS (cont.)
Page
4.2
4.1.1
Dye selection 1……………………………......
31
4.1.2
Dye selection 2………………………………..
31
4.1.3
Dye selection 3………………………………..
35
Study on acid-base reaction in organic solvent…….....
38
4.2.1
UV-visible spectrum of the 3 dyes in benzene..
38
4.2.2
Effect of volume………………………………
39
4.2.3
Testing on samples by BPB, BCP, and BTB.....
40
4.2.4
Comparison of BPB and TBPE test kit……….
44
CHAPTER V CONCLUSION……………………………………………..
46
REFERENCES……………………………………………………………..
48
APPENDIX………………………………………………………………....
51
BIOGRAPHY……………………………………………………………....
56
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ix
LIST OF TABLES
Table
Page
2.1 Some properties of amphetamine and methamphetamine…………
4
2.2 Short- and long-term effects of amphetamine/methamphetamine
abuse………………………………………………………………
5
2.3 Acidic dissociation equations of the acid A and the basic B,
Henderson-Hasselbalch equations, and some relationships between
pH and pKa………………………………………………………..
13
2.4 Relationship between color absorbed and color observed………...
18
2.5 Dyes categorized by chromophore………………………………...
21
3.1 List of instruments…………………………………………………
23
3.2 List of reagents…………………………………………………….
24
4.1 The colors of lower organic layers resulting of dyes number 1-9
tested on the four samples using method A and B……………….
34
4.2 Number of color samples when tested by three dyes ……………..
41
4.3 Number of positive and negative results for urine samples tested
with BPB and the TBPE test kit…………………………………...
44
A.1 Structures of amphetamine, methamphetamine, and some related
compounds………………………………………………………...
52
A.2 Result of five color tests for amphetamine, methamphetamine,
MDA, MDMA, and ephedrine (and pseudoephedrine)……………
55
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LIST OF FIGURES
Figure
2.1
Page
The schemes of metabolic pathways of amphetamine and methamphetamine……………………………………………………
7
2.2
Drugs metabolizing to amphetamine or methamphetamine……
8
2.3
Scheme of purposed reactions when amphetamine and methamphetamine are tested by Marquis reagent……………………
2.4
Scheme of purposed reactions when methamphetamine is tested
by Simon’s reagents giving a blue color product……………….
2.5
11
Proposed color reaction of amines (R3N) with TBPE molecule
in organic phase………………………………………………...
2.6
11
15
The structures of some drugs which can form charge transfer
complexes with TBPE cause false positive results for amphetamine and methamphetamine color screening…………………..
16
2.7
The electromagnetic spectrum………………………………....
17
2.8
The RGB additive colors (left), The CMY subtractive colors
(right), and the surface interacts with white light and subtracts
2.9
wavelengths to create the perceived reflected color (middle)….
18
The effect of conjugation is to reduce energy gaps…………….
19
2.10 the effect of auxochromes on absorbance (upper) and summary
of the λmax shifting (lower)……………………………………..
2.11
20
(A) General structural formula for sulfonephthalein indicators:
colorless lactone (sultone) form and (B) example of changing
3.1
from bromophenol blue lactone to its color quinoid form…….
22
Flow chart of the methods for “Dye Selection” …..…………..
28
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LIST OF FIGURES (cont.)
Figure
Page
4.1
Four control samples tested by TBPE solution…………………
4.2
The 1,2-napthoquinone-4-sulphonate (NQS) was tested for the
four test samples using method A (left) and N (right)…………
4.3
30
31
Results of dyes number 1-9 tested with the four test samples (1.
distilled water, 2.negative human urine, 3.positive methamphetamine horse urine, and 4.positive human urine) using Method
A (left) and Method B (right); the structures of each dye are
also shown……………………………………………………..
32
4.3
(cont.) ………………………………………………………….
33
4.4
Results for bromophenol blue, bromothymol blue and bromocresol purple tested with four test samples using method C…..
4.5
Reaction between NQS and the primary amine in alkaline solution causing a color compound………………………………..
4.6
36
UV-visible spectra for BPB, BCP, and BTB in benzene at
approximately 2.5x10-4 M ……………………………………...
4.7
35
38
UV-visible spectra when 2.5x10-4 M BPB was added to the
extract benzene of urine with methamphetamine in a variety of
volumes (µl)…………………………………………………….
4.8
39
UV-visible spectra of the benzene extract of negative control
urine sample and the spectra after the benzene extract were
added to BPB, BCP, and BTB………………………………….
4.9
42
UV-visible spectra of the benzene extract of urine sample
with methamphetamine and the spectra after the benzene extract
were added to BPB, BCP, and BTB…………………………….
4.10
43
Chemical structures of TBPE, BPB, BCP and BTB, and the
general structural formula for phenolsulfonephthalein indicators
(A) (upper right)………………………………………………....
45
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LIST OF ABBREVIATIONS
AR
Analytical reagent
BCP
Bromocresol purple
BPB
Bromophenol blue
BTB
Bromothymol blue
CMYK
Cyan, magenta, yellow, and key (black)
°C
Degree celsius
EPD-EPA complex
Electron pair donor-electron pair acceptor complex
etc.
Et cetera
e.g.
Exempli gratia
GC
Gas chromatography
g
Gram
IUPAC
International Union of Pure and Applied Chemistry
M
Molarity
Meth
Methamphetamine
MDA
3,4-Methylenedioxyamphetamine
MDMA
3,4-Methylenedioxy- N-methylamphetamine
MDE or MDEA
3,4-Methylenedioxy-N-ethylamphetamine
min
Minute
ml
Milliliter
MS
Mass spectrometer
N
Normality
NQS
1,2-Napthoquinone-4-sulphonate
nm
Nanometre
PPA
Phenylpropanolamine
RGB
Red, green, and blue
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LIST OF ABBREVIATIONS (cont.)
rpm
Round per minute
TBPE
Tetrabromophenolphthalein ethyl ester
UV
Ultraviolet
VIS
Visible light
λmax
Maximum absorption
μl
Microlitre
μg
Microgram
Copyright by Mahidol University
Fac. of Grad. Studies, Mahidol Univ.
M.Sc. (Forensic Science) / 1
CHAPTER I
INTRODUCTION
1.1 Introduction
Many detection techniques have been developed for a variety of drugs for a long
time. Each procedure has its advantages and disadvantages. Two main types of drug
detection are classified as presumptive and confirmatory tests. The presumptive test,
such as immunoassay and thin layer chromatography, is quick and inexpensive; while,
the confirmatory test, such as GC-MS, has higher specificity and lower crossreactivity than the former [1]. Color tests are one of the screening methods suitable
for mass screening. In Thailand, there is a “kit for methamphetamine test in urine”
used as a color test for YABA (amphetamine or methamphetamine) detection.
Tetrabromophenolpthalein ethyl ester (TBPE) solution, a main chemical of the color
kit, is able to form a reddish complex with the amine group of amphetamine or
methamphetamine. However, many drugs also contain amino functional groups
leading possibly to false positive results. When TBPE solution was tested on urine
samples of students in Chiang Mai (1995), Lopburi (1996), and Phitsanulok (1998),
provinces of Thailand, true positive outcomes were 43.5%, 25.8%, and 38.5%
respectively [2, 3]. In addition, TBPE solution tested on human urine samples from
the Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital and
Scientific Crime Detection Division gave only 30% true positive results [3].
The high rate of false positive is always the problem of screening technique. The
true and false positives from the screening test must be followed by a confirmatory
test. The more accurate the screening test, the less will be the cost of detection.
Copyright by Mahidol University
Supanat Panomnoptham
Introduction / 2
1.2 Aim of this study
The aim of this study is to find a chemical which reacts more specifically with
amphetamine or methamphetamine than that used in the available test kit.
.
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M.Sc. (Forensic Science) / 3
CHAPTER II
LITERATURE REVIEW
2.1 Amphetamine and Methamphetamine
Amphetamine and methamphetamine are one group of the most popular
stimulants, synthetic in origin (first synthesized in Germany in 1887 and in Japan in
1919). They continue decades-long impact, causing significant health and social
problems to nations worldwide [4]. Medically, they are used in the treatment of
narcolepsy1, attention deficit disorder (ADD), attention deficit hyperactivity disorder
(ADHD), obesity, and overeating disorders. However, they were abused to increase
alertness, relieve fatigue, control weight, treat mild depression, and feel physical and
mental well-being [5, 6]. The substances were abused differently in each region:
amphetamine is more prevalent in Europe, while methamphetamine is a more
common drug in United States of America and a region of East Asia and the Pacific
[7].
2.1.1 Synonym
Amphetamine:1-phenylpropan-2-amine (IUPAC name); dextroamphetamine;
Dexedrine®, Adderall®, Benzedrine®, Dextrostat®, Biphetamine®, Gradumet®
Methamphetamine: (2S)-N-methyl-1-phenylpropan-2-amine (IUPAC name);
chalk, chrissy, crank, crystal, go, glass, hydro, ice, meth, rock candy, speed, whiz;
Desoxyn®
In addition, some local names of methamphetamine used in the East Asia
and the Pacific are shown below [4];
1
Narcolepsy is a chronic neurological disorder caused by the brain’s inability to regulate sleep-wake
cycles normally.
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Supanat Panomnoptham
Literature Review / 4
Crystal methamphetamine: Yaba or yama chakk (injectable) in Cambodia
Bingdu in China
Shabu in Indonesia, Japan and Philippines
Anpon, philopon (liquid) and speed in Japan
Sha and siopao in Philippines
Ice in Cambodia, Japan and Thailand
Methamphetamine pills: Yama in Cambodia, Lao PDR and Myanmar
Yaba in Cambodia, Lao PDR and Thailand
Bingdu pian in China
Seik kwya say and myin say in Myanmar
2.1.2 General properties
Some properties of amphetamine and methamphetamine are included below.
Table 2.1 Some properties of amphetamine and methamphetamine [8]
Amphetamine
Methamphetamine
Chemical formula
C9H13N
C10H15N
Molecular weight
135.2
149.2
Functional group
Primary amine
Secondary amine
Boiling point
203 °C
214 °C
Melting point
156.5-158.5 °C
170-175 °C [9]
9.8
10.1
Structure
pKa
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M.Sc. (Forensic Science) / 5
2.1.3 The effects of amphetamine and methamphetamine
Amphetamine and methamphetamine are highly addictive stimulants. They
are sympathomimetic drugs, meaning that they mimic endogenous transmitters in the
sympathetic nervous system by interaction with their receptors due to their structural
similarity [5, 10]. They block breakdown and reuptake of the neurotransmitters;
moreover, they increase synaptic levels of the neurotransmitters dopamine, serotonin
(5-HT), and norepinephrine. The brain cells are stimulated, therefore, enhancing mood
and body movement. However, the high concentrations of the neurotransmitters can
be toxic to the nerve terminals [11, 12].
Amphetamine and methamphetamine have similar actions; however, at
comparable doses, the effects of methamphetamine are much more potent, longer
lasting, and more harmful to the central nervous system (CNS) [12].
Table 2.2 Short- and long-term effects of amphetamine/methamphetamine abuse [11]
Short-term effects
- increased attention and
decreased fatigue
Long-term effects
- addiction
- psychosis, including:
- increased activity and wakefulness
paranoid
- loss of appetite
hallucinations
- Euphoria and rush
repetitive motor activity
- increased respiration
- changes in brain structure and function
- rapid/irregular heartbeat
- memory loss
- hyperthermia
- aggressive or violent behavior
- mood disturbances
- severe dental problems
- weight loss
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Literature Review / 6
2.1.4 Routes of administration
Amphetamine and methamphetamine are found in many forms (powder,
crystal, tablets and capsules) and can be smoked, snorted (sniffed), injected or orally
ingested [6, 11]. The abuse patterns affect some differences in mood alteration of
users. Immediately after smoking or injecting, the user experiences an intense rush or
“flash” (lasting only a few minutes) and is described as extremely pleasurable while
snorting or oral ingestion produces euphoria (a high rush): sniffing generates effects
within 3 to 5 minutes, and oral ingestion produces effects within 15 to 20 minutes [11].
2.1.5 Metabolism and excretion of amphetamine and methamphetamine
Following oral administration, peak amphetamine concentrations in plasma
has occurred in around 2 hours and the plasma elimination half-life range from 8 to 12
hours [13], and peak methamphetamine concentrations are seen in 2.6-3.6 hours and
the elimination half-life range is 6.4-15 hours [5].
Amphetamine and methamphetamine begin to appear in the urine within 20
minutes of administration. Amphetamine is excreted as the unchanged drug, typically
20-30% of the dose, and as deaminated (hippuric acid and benzoic acid) and
hydroxylated metabolites, partly as conjugates, typically adding up to 25% of the dose.
Methamphetamine is eliminated as the unchanged drug (44%) and as its
major metabolites amphetamine (6-20%) and 4-hydroxymethamphetamine (10%).
However, the rate of excretion and the fraction of dose excreted as unchanged drug
vary according to the pH of the urine. In acidic urine both the rate of excretion and the
percentage of unchanged drug excreted increase; on the other hand, in alkaline urine
both decrease. In addition, other drugs can metabolize to amphetamine and methamphetamine in urine (Figure2.2) [13].
After
chronic
administration,
abusers
have
shown
amphetamine
concentrations in urine of 1-90 μg/ml and methamphetamine concentrations of 25-300
μg/ml [13].
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M.Sc. (Forensic Science) / 7
Figure 2.1: The schemes of metabolic pathways of amphetamine (upper) and
methamphetamine (lower)
Copyright by Mahidol University
Supanat Panomnoptham
Literature Review / 8
Figure 2.2: Drugs metabolizing to amphetamine or methamphetamine [13]
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M.Sc. (Forensic Science) / 9
2.2 Color tests for detection of amphetamine or methamphetamine in urine
Forensic testing of drug misuse has the purpose of identifying illegal substances
in biological samples such as urine [14]. It is a two-step process: a screening test and
a confirmatory test. The presumptive test, the former, is fast screening procedures
designed to provide an indication of the presence or absence of drug classes in the test
samples. If the samples are found to be positive, they are followed by the
confirmatory test for the specific drug and/or metabolites present and to ensure that
the samples are truly positive for the targeted drug [9, 15].
2.2.1 Spot tests for detection of amphetamine or methamphetamine
Color tests are developed as a presumptive test. The color tests arose from
organic qualitative analysis dating back to the 1800s. The appearance of color or a
change in color is a sign that a chemical reaction has occurred. Since color tests target
the type of compound and functional groups: aromatic rings, amines, etc., and many
drugs have more than one active moiety, the color tests are more complicated than the
simple identification of the drug’s functional groups [16].
Several different reagents are typically employed for color testing of
amphetamine and methamphetamine. The most important for these substances and
some related compound are the Marquis, the Simon’s and the Chen’s tests [9].
Marquis test
The Marquis test is the most versatile and widely used color test in drug analysis even
if its chemistry is complex and not completely understood. The Marquis reagent
reacts with amphetamine and methamphetamine to produce the orange-red products
shown in Figure 2.3. It allows the distinction between amphetamine and its ring
substituted analogues [9, 16].
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Literature Review / 10
Simon’s test
The Simon’s test is a variation of the sodium nitroprusside test that has been utilized
in organic qualitative analysis for decades. It is generally used as a test for secondary
amines, such as methamphetamine and secondary ring-substituted amphetamines,
including MDMA. Methamphetamine gives a blue color when treated with this test,
while amphetamine has no reaction because of its primary amine [9, 16].
Chen’s test
The Chen’s test is used to discriminate ephedrine, pseudoephedrine, norephedrine,
phenylpropanolamine and methcathinone from amphetamine and methamphetamine,
which do not react with the Chen’s test reagents [9].
Moreover, there are other available reagents such as the Mandelin test and
the Gallic acid test (see Appendix II). The Gallic acid reagent reacts specifically with
methylenedioxy-substituted aromatic compounds: 3,4-Methylenedioxyamphetamine
(MDA), 3,4-methylenedioxy- N-methylamphetamine (MDMA), and 3,4-methylenedioxy-N-ethylamphetamine (MDE or MDEA). Hence, it can be used to distinguish
between MDA and amphetamine [9].
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Figure 2.3: Scheme of purposed reactions when amphetamine and methamphetamine are tested by Marquis reagent [16]
Methamphetamine
Blue
Figure 2.4: Scheme of purposed reactions when methamphetamine is tested by
Simon’s reagents giving a blue color product [16]
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2.2.2 Color tests for amphetamine or methamphetamine detection in urine
Several kinds of biological specimens can be applied to test the drugs
including urine, blood, hair, saliva, and sweat, which have difference in advantages
and disadvantages; nevertheless, urine is the most widely used matrix. Urine has an
intermediate window of detection (1-3 days) [1]. Although many methods of
spectrophotometric determination of pharmaceutical amines in biological specimens
have been developed, there is little works in the literature about amphetamine and
methamphetamine determination in urine samples by UV spectrophotometry [17].
Most UV/VIS spectrophotometric methods for the determination of
compounds in biological samples require extraction of the analyte with an organic
solvent and reaction with a chromogenic reagent [18]. Methyl orange [19] and sodium
1,2-napthoquinone 4-sulphonate [17, 18] are examples of chromogens for
colorimetric determination of amphetamine and methamphetamine in urine. In field
testing, a simple and rapid method is preferred; in addition, the results should be
interpreted by the naked eyes. In Thailand, Division of Narcotics, Department of
Medical Science, Ministry of Public Health has modified a technique using
tetrabromophenolphthalein ethyl ester (TBPE) by Tadao Sakai [20].
2.3 The color test kit available in Thailand
In Thailand at present, the color test kit for methamphetamine in urine uses two
main important chemicals: sodium tetraborate (borax) and tetrabromophenolphthalein
ethyl ester solution (TBPE in CH2Cl2). Urine samples are added to tubes containing
borax to adjust pH to around 9 before TBPE solution is added to react with
amphetamine or methamphetamine, causing a color change from yellow to violet.
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2.3.1 Partition with liquid phases [16]
Liquid –liquid extraction is one of the separation techniques which can be
used to distinct a target compound due to solubility in two different fluids. For
example, acidic dissociation equations of an acid A and a base B are demonstrated as
following:
Table 2.3 Acidic dissociation equations of the acid A and the base B, the HendersonHasselbalch equations, and some relationships between pH and pKa
Base B
Acid A
ZZX H + + B
BH + YZZ
ZZX H + + A−
HA YZZ
Inonized
Water soluble
Organic insoluble
Un-inonized
Organic soluble
Water insoluble
⎡ H + ⎤ ⎡ A- ⎤
Ka = ⎣ ⎦ ⎣ ⎦
⎡⎣ HA ⎤⎦
Un-inonized
Organic soluble
Water insoluble
⎡H + ⎤ ⎡B ⎤
K a = ⎣ ⎦ ⎣+ ⎦
⎡⎣ BH ⎤⎦
pH > pKa; [Ionized] > [Un-inonized]
pH > pKa; [Un-ionized] > [Inonized]
pH < pKa; [Ionized] < [Un-inonized]
pH < pKa; [Un-ionized] < [Inonized]
pH = pKa; [Ionized] = [Un-inonized]
pH = pKa; [Un-ionized] = [Inonized]
Note: Ka is the acid dissociation equilibrium constant.
Amphetamine and methamphetamine are classified as weak bases with pKa
of 9.8 and 10.1, respectively. Humans normally excrete them in ionized forms
including the human urine which has a pH around 5-8. Borax is used to adjust the pH
of the solutions to 9.2-9.5 [21]. The drugs in water can be extracted at a maximum pH
of 11 [20].
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2.3.2 Charge transfer complex with tetrabromophenolphthalein ethyl ester
Charge-transfer (CT) complex is an association between two or more
molecules, electron donor and electron acceptor, also called electron pair donorelectron pair acceptor complex (EPD-EPA complex). They attach to form the
complex, but not a stable chemical bond, and with weaker than covalent forces. In
principle, any donor is able to form a complex with any acceptor. The fundamental
difference between EPD-EPA bonding interaction and a normal chemical bond is that
in an ordinary chemical bond each atom supplies the pair of electrons, while the
second molecule (the acceptor) provides the vacant molecular orbital. It is generally
accepted that the characteristic long-wavelength absorptions of these EPD-EPA
complexes are associated with an electron transfer from the donor to the acceptor
molecule [22].
Tetrabromophenolphthalein ethyl ester (TBPE), bromophthalein magenta E,
react with primary, secondary, and tertiary alkylamines to form reddish charge
transfer complexes in the organic layer. Maximum absorbance of the complexes with
primary, secondary, and tertiary amines occur at 560, 570, and 580 nm respectively
[20, 23]. Amphetamine and methamphetamine, therefore, form charge transfer
complexes with TBPE giving red-violet colors.
Since TBPE is not specific to amphetamine and methamphetamine, other
amino containing drugs can react with TBPE leading to false positive result. False
positive outcome occurs when the drug is detected by the test, but in fact that drug is
not present in the sample [1]. Examples of these substances are as follows (structures
given in Figure 2.6) [3]:
Psychoactive drug: MDA, MDMA.
Anti-tussive (cough suppressant): dextromethorphan, codeine.
Anti-malariae drug: quinine, quinidine.
Anti-depressant: imipramine, amitrityline.
Anti-obesity / appetite suppressant: fenfluniramine, phentermine.
Anti-histamine: chlorpheniramine, brompheniramine, diphenhydramine.
Decongestant: phenylpropanolamine, ephedrine and pseudoephedrine.
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R3N + HTBPE ↔ R3N•HTBPE
colorless
yellow
red-violet
Figure 2.5: Proposed color reaction of amines (R3N) with TBPE molecule in
organic phase (subscript “o”) [20, 23]
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Ephedrine
Brompheniramine
Chlorpheniramine
Phenylpropanolamine
Dextromethorphan
Diphenhydramine
Amitriptyline
Quinine
Codiene
Theophylline
Strychnine
Erythromycin
Chlorpromazine
Phentermine
Fenfluramine
Figure 2.6: The structures of some drugs which can form charge transfer
complexes with TBPE cause false positive results for amphetamine and methamphetamine color screening
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2.4 Color and Colorants [16]
2.4.1 Color
Human eyes can detect electromagnetic spectrum at wavelengths from 380
to 750 nm, called visible light. Each color has its own specific range of spectrum (see
Figure 2.6). Light from the sun emits a mix of the visible wavelengths and is referred
to it as white light. If a substance or surface reflects the light (or the substance does
not absorb any visible wavelengths), the light appears white. By contrast, if the
material absorbs all wavelengths, it will appear black. If all wavelengths are partially
absorbed in equal proportion, observers will perceive gray. White, gray, black are
referred to as achromatic-literally, lacking color. A specific color is perceived when
each spectrum of visible lights is absorbed unequally.
Figure 2.7: The electromagnetic spectrum [16]
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White light consists of all wavelengths, which can be divided into three
primary color bands: red, green, and blue. The red-green-blue or RGB model can
explain the addition of colors; for example, red and green colors are combined to
produce a yellow one, called a complementary color (figure 2.7). Three
complementary colors, cyan, magenta, and yellow, are the main colors of subtractive
system, used in printers. Subtractive colors are produced by reflection interactions.
For example, yellow objects can subtract blue color and reflect red and green;
therefore, they’re seen as yellow color. Moreover, cyan and yellow surfaces can
absorb red and blue color ranges and reflect green remainder, so they appear green.
Table 2.4 Relationship between color absorbed and color observed [24]
Wavelength absorbed (nm)
Color absorbed
Color observed
400
425
450
490
510
530
550
590
640
730
Violet
Blue-violet
Blue
Blue-green
Green
Yellow-green
Yellow
Orange
Red
Purple
Yellow-green
Yellow
Orange
Red
Purple
Violet
Blue-violet
Blue
Blue-green
Green
Figure 2.8: The RGB additive colors (left), The CMYK subtractive colors (right),
and the surface interacts with white light and subtracts wavelengths to create the
perceived reflected color (middle) [16]
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An ultraviolet (UV) region of the spectrum covers the range from 200 to
400 nm, and the visible region covers the range from 400 to 800 nm. The amount of
energy available in this radiation is enough to cause an electronic transition in a
molecule, exciting an electron from an occupied molecular orbital (MO) to an
antibonding MO. Two kinds of electronic transitions, σ→ π* and π→ π*, are possible
to absorb the light in UV/VIS ranges. Since the UV/VIS light is relatively low energy,
and most simple organic compounds have energy gaps too large for the visible
absorption, the compounds are colorless. It has to note that an organic compound
must have π electrons if there is to be any possibility of absorption of a UV/VIS
photon.
To generate color, the transitions require lower energy photons
corresponding to smaller energy gaps. One way to decrease the gap size is through
conjugation. The more conjugation in the system, the longer is the wavelength of light
absorbed (figure 2.8). Another method of altering transition is via the addition of
other functional groups. An auxochrome is a group that can alter the wavelength or
intensity of the chromophore (the part of molecule that is responsible for the
absorption of ultraviolet or visible light), but itself is not the chromophore. For
example, oxygen and nitrogen contain unshared electrons and posses π electrons that
are available to interact with aromatic system (figure 2.9). This property decreases the
energy gap and increases the wavelength of absorbed light. Furthermore, when lone
pair electrons are removed as in the case of anilinium ion, the wavelength of
maximum absorption (λmax) drops back to the value it has for unsubstituted benzene.
Figure 2.9: The effect of conjugation is to reduce energy gaps [16]
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When the wavelength of absorption increase, the energy of light absorbed decrease, or
becomes redder occurring to a “blueshift” in the observed color: called bathochromic
shift. On the other hand, a shift to absorbance of bluer light (the wavelength decrease
or the energy increase) causing “redshift” in appearance is called a hypsochromic shift.
In addition, increasing and decreasing the intensity correlate with a hyperchromic
shift and hypochromic shift, respectively (figure 2.9).
Figure 2.10: the effect of auxochromes on absorbance (upper) and summary of the
λmax shifting (lower) [16]
2.4.2 Dyes
Colorants are substances or materials that can absorb or emit electromagnetic energy in visible range; two types of colorants (dyes and pigments) are of
particular interest in forensic chemistry. The fundamental difference between dyes
and pigments is solubility: dyes are soluble in solvent, whereas pigments are
suspensions of insoluble materials in solvent. A number of presumptive tests generate
color through dye formation. Some groups of dyes are showed in table 2.5.
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Table 2.5 Dyes categorized by chromophore [16]
Chromophore
Structure
Example
Antraquinone
Alizarin
Acridine
Acridine yellow
Azo
Methyl orange
-N=N-
Carbonyl
Indigo
Conjugated C=O system
Nitro and
nitroso
Para red
-NO2 (nitro functional group)
-N=O (nitroso functional group)
Polymethine
Triarylmethine subcategory:
Long chain of conjugated double
Phenolphthalein
bonds by e-donor and e-acceptor
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2.4.3 Sulfonephthalein indicators [25]
The sulfonephthaleins were developed for use in aqueous media. The
sulfonic acid group increases their solubility in water and decreases in hydrocarbons
and other inert solvents. The general structure formula is showed in figure 2.11 (A).
Solutions of the indicators in benzene are completely or nearly colorless (lactone
form), but in most cases, the solution becomes a pale yellow color on standing. It is
probably caused by humidity or alkaline reaction on glass containers. The yellow
color indicates partial conversion to the quinoid structure (B). Water is sufficiently
basic to convert the lactone into the quinoid sulfonic acid, and the lactone is therefore
not detected in aqueous solutions.
A
B
Figure 2.11: (A) General structural formula for sulfonephthalein indicators:
colorless lactone (sultone) form [25] and (B) example of changing from
bromophenol blue lactone to its color quinoid form [26].
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CHAPTER III
MATERIALS AND METHODS
3.1 Materials
3.1.1 Instrumentation
Table 3.1 List of instruments
Equipment/ Instrument
Model
Company
Pipetteman
Gilson
Denville 260D
Denville Scientific Inc.
3. Balance
TE 1535
Sartorius
4. Vortex
VTX-3000L
LMS
NanoDrop 1000
Thermo Scientific
1. Micropipette
2. Microcentrifuge
5. Spectrophotometer
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Materials and Methods / 24
3.1.2 Reagents
Table 3.2 List of reagents
Chemical
Supplier
Grade
Ajax
-
-
-
Fisher Scientific
AR
4. Hydrochloric acid
BDH
AR
5. Sodium hydroxide
Merck
AR
6. Benzene
Merck
AR
1. Alizarine S sodium salt
Merck
AR
2. Methylene blue
Merck
AR
3. Methyl orange
Merck
AR
4. Methyl red
Merck
AR
Bio Basic
AR
Merck
AR
7. Bromophenol blue
Bio-Rad Laboratories
Laboratory
8. Bromothymol blue
Merck
AR
9. Bromocresol purple
Merck
AR
Fluka chemica
AR
1. Sodium tetraborate (borax)
2. Distilled water
3. Dichloromethane or methylene chloride
Dye
5. Xylene cyanol FF
6. Cresol red
10. 3,4-Dihydro-3,4-dioxo-1-napthalenesulfonic acid sodium salt
3.1.3 Urine samples
Urine samples (n =35), kept at -20 0C until used, were obtained from the
National Doping Control Centre (NDCC), Mahidol University. The urine samples had
been analyzed by GC-MS.
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3.1.4 The kit for methamphetamine test in urine
The available kit was developed by Division of Narcotics, Department of
Medical Science, Ministry of Public Health, Thailand. This kit can be bought from the
Government Pharmaceutical Organization, Ministry of Public Health, Thailand.
The kit is composed of the following:
1. Tetrabromophenolpthalein ethyl ester solution (C22H14Br4O4)
[0.1%TBPE in dichloromethane (CH2Cl2)]
2. Plastic test tubes containing sodium tetraborate (Na2B4O7) or borax 100 mg
3. An operation manual
4. A color band paper for interpretation
5. Urine droppers
6. A TBPE reagent dropper
7. Plastic bottles for collecting urine specimen
8. Specimen labels
9. A pair of gloves
10. A paper rack for microcentrifuge tubes
11. A paper stage for the bottle of TBPE solution
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3.2 Procedure of the kit for methamphetamine test in urine
3.2.1 Method K: testing guide of the kit
1. Add 1.0 ml of urine to the microcentrifuge tube containing borax.
2. Shake well.
3. Add about 5 drops TBPE solution, and then shake 10-15 times.
4. Allow the layer to separate, and then observe color in the lower layer.
3.2.2 Interpretation
The color of the bottom layer should be interpreted within 2 minutes after
the layer separation. The cut-off level for methamphetamine is 3 µg/ ml.
Negative result: olive-green to brownish green
Positive result: red-violet to blue-violet
However, many chemicals may react in this test to give false positive results
such as pseudoephedrine, chlorpheniramine, dextromethorphan, quinine, and
amitriptyline. The presumptive positive sample has to be tested with other techniques
to confirm the result.
3.3 Preliminary dye selection
Four samples (1. distilled water; 2. negative control human urine; 3. positive
methamphetamine horse urine; 4. positive methamphetamine human urine) were
tested with a variety of dyes using methods given below.
3.3.1 Dye selection 1
3,4-Dihydro-3,4-dioxo-1-napthalene-sulfonic acid sodium salt (also called
sodium-1,2-napthoquinone-4-sulphonate: NQS) was used with methods, A and N.
The method A is described in the “Dye selection 2”. One drop of 1%NQS was
employed in both methods.
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Method N (NQS)
1. Pipette 1.0 ml of samples into microcentrifuge tubes.
2. Add 2 large drops of 10% sodium bicarbonate and shake.
3. Add 1 drop of 1% NQS in water, shake and wait 10 min.
4. Add 400 µl of CH2Cl2, and shake.
5. Centrifuge at 13,000 rpm 1 min, and observe the color in the lower
organic layer.
3.3.2 Dye selection 2
Nine dyes were used:
1. Alizarin red S
2. Methylene blue
3. Methyl orange
4. Methyl red
5. Xylene cyanol FF
6. Cresol red
7. Bromophenol blue
8. Bromothymol blue
9. Bromocresol purple
The 9 dyes were tested using methods A and B.
Method A
1. Pipette 1.0 ml of samples into microcentrifuge tubes.
2. Add 80 µl of 1N HCl and then shake.
3. Add 100 µl of 0.1% dye in water (ethanol for methyl red), shake and
wait 10 min.
4. Add 400 µl of CH2Cl2 and shake.
5. Centrifuge at 13,000 rpm 1 min and then observe the color in the lower
organic layer.
Method B
1. Pipette 1.0 ml of samples to microcentrifuge tubes.
2. Add 0.045 g of borax or sodium tetraborate and shake.
3. Add 100 µl of 0.1% dye in water (ethanol for methyl red), shake and
then wait 10 min.
4. Add 400 µl of CH2Cl2 and then shake.
5. Centrifuge at 13,000 rpm 1 min and then observe the color in the lower
oraganic layer.
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3.3.3 Dye selection 3
The 9 dyes in “Dye selection 2” were tested with method C.
Method C
1. Pipette 1.0 ml of samples to microcentrifuge tubes.
2. Add 0.045 g of borax or sodium tetraborate and shake.
3. Add 100 µl of 0.1% dye in CH2Cl2 and shake.
4. Allow the layer to separate and then observe the color in the lower
organic layer.
Four samples:
1. Distilled water
2. Negative control human urine
3. Positive methamphetamine horse urine
4. Positive methamphetamine human urine
Dye: Sodium-1,2napthoquinone-4sulphonate (NQS)
Method A
Nine dyes:
1. Alizarin red S
6. Cresol red
2. Methylene blue
7. Bromophenol blue
3. Methyl orange
8. Bromothymol blue
4. Methyl red
9. Bromocresol purple
5. Xylene cyanol FF
Method A
Method N
Method B
Method C
Figure 3.1: Flow chart of the methods for “Dye Selection”
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3.4 Study on acid-base reaction in organic solvent
3.4.1 General Procedure
Extraction
1. Pipette 1.0 ml of samples into microcentrifuge tubes.
2. Add 0.050 g Borax and 200 µl of 1N NaOH and mix.
3. Add 200 µl of benzene and shake.
4. Centrifuge.
5. Pipette at least 100 µl of benzene (upper) to new microcentrifuge tubes.
Acid-base reaction
6. Separate the extract benzene into three tubes (30 µl of each tube).
7. Add 5 µl of 2.5x10-4 M dyes to each tube.
(The three dyes used were bromophenol blue, bromothymol blue, and
bromocresol purple)
8. Observe the color and measure the absorbance.
3.4.2 Urine samples
The 35 urine samples were divided into two groups: (1) positive
methamphetamine urines and (2) negative methamphetamine urines. Subgroups are as
following.
(1) Positive methamphetamine urine, 17 samples
1.1 Horse urine with methamphetamine, 1 sample
1.2 Human urine with amphetamine and methamphetamine, 7 samples
1.3 Human urine with methamphetamine with other drugs (such as
pholedrine), 9 samples
(2) Negative methamphetamine urine, 18 samples
2.1 Human urine with MDMA or MDEA, 3 samples
2.2 Negative human control urine, 1 sample
2.3 Human urine with other drugs such as ephedrine, pseudoephedrine, cathine, and
PPA, 14 samples
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CHAPTER IV
RESULTS AND DISCUSSION
Amphetamine or methamphetamine solutions are colorless. Therefore, color
tests were designed for visual ion through formations of ion-association or chargetransfer complex.
4.1 Preliminary dye selection
The different dyes were tested with the same set of test samples (distilled water,
negative control human urine, positive methamphetamine horse urine, and positive
human urine) to compare results between negative and positive samples and also
between dyes. The expected results are color appearance or change of color in the
lower organic phase. Figure 4.1 shows the results.
1. Blank sample (distilled water)
2. Negative human urine sample
3. Positive methamphetamine horse urine sample
4. Positive methamphetamine human urine sample
A, B, C, K, or N denote the method that was used
1K
2K
3K
4K
Upper urine layer
Lower organic layer
Figure 4.1: Four control samples tested by TBPE solution.
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4.1.1 Dye selection 1
1A
2A
3A
4A
1N
2N
3N
4N
Figure 4.2: The 1,2-napthoquinone-4-sulphonate (NQS) was tested on the four
test samples using methods A (left) and N (right)
The samples were adjusted to acidic solutions in method A and basic
solutions in method N.
Method A:
The lower organic layers of all the samples were colorless.
Method N:
The color of the lower organic layers of negative and positive samples (2N, 3N and
4N) appeared as light brown. NQS reacts with primary and secondary amines in
alkaline solution to form colored compounds [27].
4.1.2 Dye selection 2
Similarly to “Dye selection1”, the samples were adjusted to acidic solutions
in method A and basic solutions in method B. Nine dyes were tested and the color of
lower organic layers was observed.
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Results and Discussion / 32
(1) Alizarin red S
1A
2A
3A
4A
1B
2B
3B
4B
4A
1B
2B
3B
4B
3A
4A
1B
2B
3B
4B
3A
4A
1B
2B
3B
4B
(2) Methylene blue
1A
2A
3A
(3) Methyl orange
1A
2A
(4) Methyl red
1A
2A
Figure 4.3: Results of dyes number 1-9 tested with the four test samples
(1.distilled water, 2.negative human urine, 3.positive methamphetamine horse
urine, and 4.positive human urine) using Method A (left) and Method B (right); the
structures of each dye are also shown.
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(5) Xylene cyanol FF
1A
2A
3A
4A
3A
4A
1B
2B
3B
4B
(6) Cresol red
1A
2A
1B
2B
3B
4B
1B
2B
3B
4B
1B
2B
3B
4B
1B
2B
3B
4B
(7) Bromophenol blue (BPB)
1A
2A
3A
4A
(8) Bromothymol blue (BTB)
1A
2A
3A
4A
(9) Bromocresol purple (BCP)
1A
2A
3A
4A
Figure 4.3 (cont.):
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Results and Discussion / 34
Table 4.1 The colors of lower organic layers resulting from dyes number 1-9 tested
on the four samples using method A and B
Method A
Dye
Method B
1A
2A
3A
4A
1B
2B
3B
4B
-
-
-
-
-
-
-
-
2. Methylene blue
L-Bl
L-Bl
Bl
Bl
P
Bl
Bl
Bl
3. Methyl orange
-
-
-
-
-
-
-
-
4. Methyl red
O
O
O
O
O
O
O
O
5. Xylene cyanol
-
-
-
-
-
-
-
-
6. Cresol red
-
-
-
-
-
-
-
-
7. BPB
-
-
-
-
-
-
-
-
8. BTB
-
P-Y
Y
Y
-
-
Y
Y
9. BCP
-
-
Y
Y
-
-
-
-
1. Alizarin red S
Note: - : colorless; Bl: Blue; L-Bl: Light Blue; P: purple; O: Orange;
Y: Yellow; P-Y: Pale Yellow
Bromothymol blue:
A yellow color appeared in the organic layers (bottom) of the positive samples in both
methods (3A, 4A, 3B and 4B). In addition, for the negative sample using method A
(2A) a pale yellow color could be observed.
Bromocresol purple:
A yellow color appeared in the organic layers (bottom) of the positive samples using
method A (3A and 4A).
Methylene blue and methyl red:
A color appeared in the organic layers (bottom) of the positive samples using methods
A and B but a color also appeared in the organic layers of water and the negative
samples.
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4.1.3 Dye selection 3
In “Dye selection 1 and 2”, the dyes were dissolved in water, while in this
section the dyes were first dissolved in organic solution. However, the following dyes
were not solute in organic solvent: alizarin red S, methyl orange, xylene cyanol FF
and cresol red. The five remaining dyes, methylene blue, methyl red, bromophenol
blue, bromothymol blue and bromocresol purple, were tested using Method C.
The results of methylene blue and methyl red are the same as the results for
Method B. Bromophenol blue, bromothymol blue and bromocresol purple showed
different results, as shown in the Figure 4.4.
Bromophenol blue (BPB)
1C
2C
3C
4C
Bromothymol blue (BTB)
1C
2C
3C
4C
Bromocresol purple (BCP)
1C
2C
3C
4C
Figure 4.4: Results for bromophenol blue, bromothymol blue and bromocresol
purple tested with four test samples using method C
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Results and Discussion / 36
In “Dye selection 1 and 2”, the aqueous dyes were added to the samples which
were adjusted to acidic (method A) or basic (method B and N). Since human urines
have pH 5-8 normally [1], amphetamine and methamphetamine are excreted as
charged states. In acid medium, both drugs are in charged states. They will become
uncharged in basic solution. Unlike basic compounds, acidic dyes will be in charged
states in alkaline solution and become uncharged in acidic solution. The expected
result is the color occurring or changing in organic solvent from the ion-pair
formation or ion-association between the positive charge of amphetamine or
methamphetamine and the negative charge of acid dyes.
In “Dye selection 1”, one dye was tested, NQS. The organic layers of the four
samples using method A were colorless. However, in method N, a light brown color
appeared in the organic layers of negative and positive samples because NQS can
react with primary and secondary amines in alkaline solution to form colored
compounds [27]. The reaction between NQS and primary amine are shown in Figure
4.5.
Figure 4.5: Reaction between NQS and the primary amine in alkaline solution
causing a color compound [27]
In “Dye selection 2”, nine dyes were tested. Methylene blue and methyl red gave
no change in color of the organic layers of the positive samples; while, bromothymol
blue and bromocresol purple were yellow in the organic solvent of the positive
samples. In method A, drugs and acid dyes are in the ionized forms so that it is
possible to form ion-pair complex in acidic solution. However, in the basic solution
(method B), the result from bromothymol blue showed a yellow color at the bottom
(in less intensity). Borax adjusts solutions to about pH 9, and amphetamine and
methamphetamine have pKa 9.8 and 10.1 respectively. At the pH equal to pKa,
Copyright by Mahidol University
Fac. of Grad. Studies, Mahidol Univ.
M.Sc. (Forensic Science) / 37
substances exist in charged and uncharged forms equally; so that the drugs have
ionized form remaining in the solution of pH 9. The yellow color of the organic layer
in method B showed a lower intensity because the amount of the ions of the drugs in
base solution is less than in an acidic solution.
In “Dye selection 3”, the dyes (in the organic solvent: dichloromethane) were
tested on the samples adjusted to a basic range. Both drugs are uncharged in a basic
solution. The expected outcome is the color appearance in organic solvent resulting
from charge-transfer complex of the dyes and the drug molecules. The dark blue color
in the organic layers was found in bromophenol blue (negative and positive human
samples) and bromocresol purple (positive human sample). In addition, the positive
horse urine sample gave a pale blue color in the lower organic layer when tested by
bromophenol blue. Bromophenol blue, bromothymol blue and bromocresol purple are
in the sulfonephthalein dye group. The purple-blue color is due to their chargetransfer complex of this dye group [25]. However, result of bromothymol blue was
yellow.
These are simply methods in order to screen roughly the reactions between dyes
and methamphetamine in urine. Bromothymol blue and bromocresol purple reacted
with methamphetamine forming ion-pair and charge transfer complexes. Thus both
dyes are of interest for further study. However, the reactions are not specific to
amphetamine or methamphetamine molecules but their amino group, and urine
usually contains many other inorganic and organic substances [20]. In ion-pair
method, dyes are added to the urine directly; while, in charge transfer complex
method, methamphetamine is separated and reacted with dyes in another layer
(organic phase). So, charge transfer complexes of bromophenol blue, bromocresol
purple, and bromothymol blue were studied.
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Results and Discussion / 38
4.2 Study on acid-base reaction in organic solvent
Borax and NaOH were used to adjust pH of solutions to 10 approximately. Then
benzene was added to extract amphetamine and methamphetamine in the aqueous
samples. The benzene extract were divided to the new three tubes. Three dyes,
bromophenol blue (BPB), bromothymol blue (BTB) and bromocresol purple (BCP) in
benzene, were added to the extract and the UV-visible spectra of the solutions were
measured.
4.2.1 UV-visible spectrum of the 3 dyes in benzene
Figure 4.6 shows UV-VIS spectra of three dyes (BPB, BCP, and BTB) in
benzene. The concentration was 2.5x10-4 M. The vertical lines at 380 nm separated
roughly between UV (left) and visible (right) wavelengths. All three dye solutions had
yellow colors because the three dyes absorbed in the visible range around 400 nm.
(A) BPB
(C) BTB
(B) BCP
Figure 4.6: UV-visible spectra for BPB, BCP and BTB in benzene at
approximately 2.5x10-4 M
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M.Sc. (Forensic Science) / 39
4.2.2 Effect of volume
Bromophenol blue (BPB) at 2.5x10-4 M concentration was added to the
extract of positive methamphetamine human sample of various volumes. At first, the
sample was tested with BPB at the same volume (10 µl:10 µl) resulting in a yellow
color. When the volume of the sample was increased 3 times of BPB’s volume (30
µl:10 µl), it was still a yellow color. However, when the sample’s volume was more
than 5-6 folds BPB’s volume (10 µl:2 µl and 30 µl:5 µl), the yellow color changed to
blue. A peak at around 570 nm was found because of the charge transfer complex of
the drug and the dye (Figure 4.7).
BPB in benzene is pale yellow and the benzene extract of positive sample
urine is colorless. A blue color appeared after the two components mix together.
Volume ratio
Sample: BPB
(A) 10:10
(B) 10:2
(C) 30:10
(D) 30:5
Figure 4.7: UV-visible spectra when 2.5x10-4 M BPB was added to the extract
benzene of positive methamphetamine urine in a variety of volumes (µl).
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Results and Discussion / 40
4.2.3 Testing on samples by BPB, BCP, and BTB
The 35 urine samples were categorized to two main types: (1) positive
methamphetamine urine and (2) negative methamphetamine urine. Their subcgroups
are as follows.
(1) Positive methamphetamine urine, 17 samples
1.1 Horse urine with methamphetamine, 1 sample
1.2 Human urine with amphetamine and methamphetamine, 7 samples
1.3 Human urine with methamphetamine with other substances (such as
pholedrine), 9 samples
(2) Negative methamphetamine urine, 18 samples
2.1 Human urine with MDMA or MDEA, 3 samples
2.2 Negative human control urine, 1 sample
2.3 Human urine with other drugs such as ephedrine, pseudoephedrine, cathine, and
PPA, 14 samples
To the urine samples were added borax and NaOH to extract basic drugs
into benzene. The benzene extract was divided into 3 tubes equally (30 µl). Three
dyes (BPB, BCP, and BTB) at 2.5x10-4 M were added to the benzene extract in the
volume ratio 30 µl:5 µl (sample: dye) and then the solution mixture was measured on
a spectrophotometer.
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Fac. of Grad. Studies, Mahidol Univ.
M.Sc. (Forensic Science) / 41
The colors resulted from three dyes are shown in the Table 4.1.
Table 4.2 Number of color samples when tested by three dyes
BPB
Yellow
BCP
BTB
Blue
Yellow
Blue
Yellow
Blue
(1) Positive methamphetamine urine
1.1 Meth (horse)
-
1
1
-
1
-
1.2 Meth (human)
-
7
7
-
7
-
1.3 Meth + others
6
3
9
-
9
-
(2) Negative methamphetamine urine
2.1 MDMA or MDEA
-
3
3
-
1
-
2.2 Negative control
1
-
1
-
1
-
2.3 Other drugs
11
3
14
-
14
-
18
17
35
0
35
0
Total
The observed colors were classified as yellow or blue. The yellow and blue
solutions absorbed light at around 400 and 570 nm, respectively. Bromocresol purple
and bromothymol blue were found only in the yellow tone; while bromophenol blue
was found in both color tones. All the yellow solutions had absorbances at 400 nm
approximately. In addition, all the blue solution resulting from BPB had maximum
peak in the visible region at around 570 nm. In case of the urines with
methamphetamine (group1) and with MDMA (group2.1), their maximum absorbances
were 570 nm, but maximum absorbance at wavelengths slightly more or less than 570
nm were found in cases of the urines with other drugs (e.g. ephedrine; group 2.3).
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Results and Discussion / 42
Negative urine sample
Negative urine sample + BPB
λmax = 409 nm
A = 0.073
Negative urine sample + BCP
λmax = 402 nm
A = 0.090
Negative urine sample + BTB
λmax = 409 nm
A = 0.086
Figure 4.8: UV-visible spectra of the benzene extract of negative control urine
sample and the spectra after the benzene extract were added to BPB, BCP, and
BTB
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M.Sc. (Forensic Science) / 43
Human urine sample with methamphetamine
Urine sample with methamphetamine + BPB
λmax = 387 nm
A = 0.071
λmax = 570 nm
A = 0.776
Urine sample with methamphetamine + BCP
λmax = 393 nm
A = 0.173
λmax = 570 nm
A = 0.041
Urine sample with methamphetamine + BTB
λmax = 405 nm
A = 0.128
Figure 4.9: UV-visible spectra of the benzene extract of urine sample with
methamphetamine and the spectra after the benzene extract were added to BPB,
BCP, and BTB
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Results and Discussion / 44
4.2.4 Comparison of BPB and TBPE test kit
The urine samples were tested by the TBPE test kit (method K: method of
the kit) and compared with the results using BPB. The blue color with BPB was
denoted as positive and the yellow color as negative.
Table 4.3 Number of positive and negative results for urine samples tested with BPB
and the TBPE test kit
BPB
Neg
TBPE (kit)
Pos
Neg
Pos
(1) Positive methamphetamine urine
1.1 Meth (horse)
-
1
-
1
1.2 Meth (human)
-
7
-
7
1.3 Meth + others
6
3
6
3
(2) Negative methamphetamine urine
2.1 MDMA or MDEA
-
3
-
3
2.2 Negative control
1
-
1
-
2.3 Other drugs
11
3
9
5
18
17
16
19
Total
Note: Neg: negative result; Pos: positive result
Both horse and human urines contains only methamphetamine (1.1 and 1.2)
were found to be positive, while urines with methamphetamine and other chemicals
(1.3) such as pholedrine resulted in positive only for some samples.
All urines with MDMA and MDEA (2.1) were found positive and the
negative human urine sample (2.2) was found negative. Results of BPB and TBPE for
samples 1.1 to 2.2 were the same but urines with other drugs such as ephedrine gave
different results. BPB gave lower positive results than TBPE when urines with other
drugs (2.3) were tested.
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M.Sc. (Forensic Science) / 45
Structure of BPB is closer to the structure of TBPE than BTB and BCP
(Figure 4.10). Moreover, BPB showed better reaction with bases than BTB and BCP.
TBPE does not have a lactoid structure and dissolves in organic solvents. It
is yellow in benzene and forms charge transfer complexes with bases.
Sulfonephthalein dye, such as BPB, has the lactone form (Figure 4.10). It is colorless
or pale yellow in benzene. When the bases were added, the solution becomes deeper
yellow. Sulfonephthalein dye, therefore, need more base to produce a deep color such
as blue (from the charge transfer complex); moreover, the charge transfer complex
from sulfonephthalein has a lower solubility in the organic layer than the complex
from TBPE due to the salt of the sulfonephthalein [25].
TBPE
BPB
A
BCP
BTB
Figure 4.10: Chemical structures of TBPE, BPB, BCP and BTB, and the general
structural formula for phenolsulfonephthalein indicators (A) (upper right)
Copyright by Mahidol University
Supanat Panomnoptham
Conclusion / 46
CHAPTER V
CONCLUSION
The commercial test kit for methamphetamine detection in urine in Thailand has
TBPE as the reagent. This kit results in many false positives because TBPE is not
specific to the methamphetamine molecule [25]. Urine contain many compounds [1].
The sulfonephthaleins such as BPB, BCP, and BTB have sulfonic acid group and
easily dissolve in water but has limited solubilities in organic solvents; moreover,
their acid salts also possess limited solubilities in organic solvents [25]. This is one
reason why they are less suitable than TBPE for the detection of amines [25].
The sulfonephthalein dye can be used to react with amphetamine and
methamphetamine. Each dye has its own suitable condition.
A sulfonephthalein analog of the TBPE is bromophenol blue [25]. In this work,
under the same condition, BPB showed better reaction with bases than BCP and BTB.
The charge transfer complex band in benzene of BPB with methamphetamine is
at 570 nm. The result from BPB showed slightly less sensitivity to bases than TBPE,
but it still reacts with methamphetamine like TBPE. Urines with methamphetamine
resulted in 100% positive results. Urines with methamphetamine and other
components, such as pholedrine, were tested positive for both TBPE and BPB.
BPB in this work could detect the methamphetamine in urine and may be a
possible alternative reagent. However, further studies are required before it can be
applied to real cases.
Copyright by Mahidol University
Fac. of Grad. Studies, Mahidol Univ.
M.Sc. (Forensic Science) / 47
Suggestion for future work
The following are suggestion for future work:
•
Study using the pure methamphetamine and also known concentration of the
methamphetamine to test with BPB
•
Study on BCP or others
Copyright by Mahidol University
Supanat Panomnoptham
References / 48
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examination of urine, hair, saliva and sweat National Drug and Alcohol
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คณะเภสัชศาสตร มหาวิทยาลัยขอนแกน.
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7. United Nations Office on Drugs and Crime (UNODC). World Drug Report 2007;
2007.
8. Sukkwan J. Separation and detection of stereoisomers of methamphetamine and
amphetamine in forensic samples by GC/MS [Master's Thesis]: Mahidol
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the Identification and Analysis of Amphetamine, Methamphetamine and
their Ring-substituted Analogues in Seized Materials. 2006.
10. Logan BK. Methamphetamine -Effects on Human Performance and Behavior.
Forensic Science Review. 2002;14.
Copyright by Mahidol University
Fac. of Grad. Studies, Mahidol Univ.
M.Sc. (Forensic Science) / 49
11. National Institute on Drug Abuse (NIDA). NIDA Research Report:
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for the detection and Assay of Heroin, Cannabinoids, Cocaine,
Amphetamine, Methamphetamine and Ring-Substituted Amphetamine
Derivatives in Biological Specimens. 1995.
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17. Legua CM, Campins Falco P, Sevillano Cabeza A. Determination of
methamphetamine in urine samples with sodium 1, 2-naphthoquinone-4sulphonate. Fresenius' journal of analytical chemistry. 1994.
18. Legua CM, Campins Falco P, Sevillano Cabeza A. Extractive-spectrophotometric
determination of amphetamine in urine samples with sodium 1,2naphthoquinone 4-sulphonate. Analytica chimica acta. 1993.
19. Frings CS, Queen C, Foster LB. Improved colorimetric method for assay of
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by Color Reaction with Tetrabromophenolphthalein Ethyl Ester.
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วีรวรรณ เล็กสกุลไชย. ปจจัยตอผลการตรวจยาบาดวยชุดน้ํายาตรวจยาบาในปสสาวะ.
วารสารวิชาการสาธารณสุข 2548;14.
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23. Sakai T, Watanabe S, Yamamoto S. Thermospectrophotometric Analysis of
Alkylamines Utilizing Ion Association with Tetrabromophenolphthalein
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24. Hornback JM, Young PR. Organic chemistry. Pacific Grove, Calif. Brooks/Cole,
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25. Davis MM, Schuhmann PJ, Lovelace ME. Acid-Base Reactions in Organic
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Benzene. Journal of Research of the National Bureau of Standards.
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26. Ashour S, Chehna MF, Bayram R. Spectrophotometric Determination of
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Sulphonephthalein Dyes. International Journal of Biomedical Science.
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27. Khummueng W. Determination of amphetamine and related compounds in urine
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naphthoquinone-4-sulphonate as derivatizing agent [Master's Thesis]:
Mahidol University; 2001.
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Fac. of Grad. Studies, Mahidol Univ.
M.Sc. (Forensic Science) / 51
APPENDIX
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Supanat Panomnoptham
Appendix / 52
APPENDIX A
STRUCTURE
TableA.1 Structures of amphetamine, methamphetamine, and some related
compounds
Phenethylamine as a main skeleton structure:
Common name
Rα
Rβ
R2
R3
R4
R5
R6
RN
H
H
H
H
H
H
H
H
Amphetamine
CH3
H
H
H
H
H
H
H
Methamphetamine
CH3
H
H
H
H
H
H
CH3
Pholedrine
CH3
H
H
H
OH
H
H
CH3
Phentermine
2CH3
H
H
H
H
H
H
H
MDA
CH3
H
H
-O-CH2-O-
H
H
H
MDMA
CH3
H
H
-O-CH2-O-
H
H
CH3
Cathine
CH3
OH
H
H
H
H
H
H
PPA
CH3
OH
H
H
H
H
H
H
Ephedrine
CH3
OH
H
H
H
H
H
CH3
Pseudoephedrine
CH3
OH
H
H
H
H
H
CH3
Dopamine
H
H
H
OH
OH
H
H
H
Norephinephrine
H
OH
H
OH
OH
H
H
H
Ephinephrine
H
OH
H
OH
OH
H
H
CH3
Phenethylamine
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M.Sc. (Forensic Science) / 53
APPENDIX B
SPOT TEST
A. Marquis test
A.1 Reagent
Carefully add 100 ml of concentrated sulfuric acid to 5 ml of 40%
formaldehyde.
B. Simon’s test
B.1 Reagent
Reagent 1B: Dissolve 0.9 g of sodium nitroprusside in 90 ml of distilled
water, then add 10 ml of acetaldehyde.
Reagent 2B: Dissolve 2.0 g of sodium carbonate in 100 ml of water
(= 2% aqueous sodium carbonate solution).
B.2 Procedure
1. Place small amount of the suspected material on a spot plate.
2. Add 1 drop of reagent 1B.
3. Add 2 drops of reagent 2B.
C. Mandelin test
C.1 Reagent
Dissolve 1.0 g of ammonium vanadate in 100 ml of concentrated
sulfuric acid (=1% (w/v) solution).
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Appendix / 54
D. Chen’s test
D.1 Reagent
Reagent 1D: Add 1 ml of glacial acetic acid to 100 ml of water
(=1% (v/v) aqueous acetic acid solution).
Reagent 2D: Dissolve 1.0 g of copper (II) sulphate in 100 ml of distilled
water (=1% (w/v) aqueous CuSO4 solution).
Reagent 3D: Dissolve 8.0 g of sodium hydroxide in 100 ml of distilled
water (=2N aqueous sodium hydroxide solution).
D.2 Procedure
1. Place small amount of the suspected material on a spot plate.
2. Add 2 drops of reagent 1D.
3. Add 2 drops of reagent 2D, then add 2 drops of reagent 3D and stir.
E. Gallic acid test
E.1 Reagent
Dissolve 0.1 g of gallic acid in 20 ml of concentrated sulphuric acid
(=0.5% (w/v) solution).
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M.Sc. (Forensic Science) / 55
Result
TableA.2 Result of five color tests for amphetamine, methamphetamine, MDA,
MDMA, and ephedrine (and pseudoephedrine)
Compound
Amphetamine
Marquis
Simon’s
Mandelin
Chen’s
Gallic
Orange to
NR*
(dark) green
NR*
NR
Deep blue
(dark) green
NR*
NR
NR*
Bluish black
NR*
brown
Methamphetamine
Orange to
brown
MDA
Dark blue/
black
MDMA
dark green
Dark blue/ Deep blue
Bluish black
NR*
black
Ephedrine
NR
Bright to
Bright to
dark green
NR*
NR
Purple
NR
Pseudoephedrine
Note: NR = no reaction
* The color of reagent should be considered as negative
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Supanat Panomnoptham
Biography / 56
BIOGRAPHY
NAME
Mr. Supanat Panomnoptham
DATE OF BIRTH
18 October 1984
PLACE OF BIRTH
Songkhla, Thailand
INSTITUTIONS ATTENDED
Mahidol University, 2002:
Bachelor of Science
(Biology)
Mahidol University, 2006:
Master of Science
(Forensic Science)
HOME ADDRESS
91/67 Home Place Rangsit, Bangpoon, Muang,
Pathumthani 12000, Thailand.
E-mail: [email protected]
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