development of new color presumptive test...

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

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 co-

advisor, 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


Fac. of Grad. Studies, Mahidol Univ. Thesis / iv

DEVELOPMENT OF NEW COLOR PRESUMPTIVE TEST FOR AMPHETA-MINE/ 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. Tetrabromo-phenolphthalein 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


Fac. of Grad. Studies, Mahidol Univ. Thesis / v

การพัฒนาชุดน้ํายาชนดิสีเพือ่การตรวจสอบยาบาในปสสาวะ DEVELOPMENT OF NEW COLOR PRESUMPTIVE TEST FOR AMPHETAMINE/ METHAMPHETAMINE IN URINE ศุภณัฐ พนมนพธรรม 4936103 SCFS/M วท.ม. (นิตวิิทยาศาสตร) คณะกรรมการที่ปรึกษาวิทยานิพนธ : ประพิณ วไิลรัตน Ph.D. (Physical Chemistry), นภดล ไชยคาํ Ph.D. (Geochemistry)

บทคัดยอ ในปจจุบนัชุดตรวจสอบสีในประเทศไทยมีสารสําคัญคือ Tetrabromophenolphthalein ethyl ester (TBPE) ซ่ึงนํามาใชตรวจสอบสารแอมเฟตามีนและเมทแอมเฟตามีนในปสสาวะของผูเสพ อยางไรก็ตามชุดตรวจสอบนี้กอผลบวกลวงจํานวนมาก การศึกษานีจ้งึตองการหาสารเคมีที่สามารถทําปฏิกิริยากับสารดังกลาวและกอใหเกิดผลบวกลวงนอยลง โดยนําปสสาวะทีไ่ดรับการตรวจพบวามีสารดังกลาวอยูมาใส borax และ NaOH เพื่อปรับ pH ใหเหมาะสมแลวหลังจากนั้นจงึใส benzene เพื่อสกัดเมทแอมเฟตามีนออกมา แยก benzene ที่สกัดออกมาแลวและนํามาแยกผสมกับสีสามชนิดคือ bromophenol blue (BPB), bromocresol purple (BCP) และ bromothymol blue (BTB) BPB สามารถเกิดสารเชิงซอนกับเมทแอมเฟตามนีได โดยที่สารละลายเปลี่ยนจากสีเหลืองเปนสีน้ําเงิน ซ่ึงพบวาคาดูดกลืนแสงของสารเชิงซอนนี้อยูที ่ 570 นาโนเมตร สวนผลจากสีอีกสองชนิดคือ BCP และ BTB จะปรากฏแคสีเหลืองเทานั้น เมื่อเปรยีบเทียบผลของ BPB กับ TBPE พบวาสีทั้งสองชนิดใหผลเหมือนกันในตัวอยางปสสาวะที่มีเมทแอมเฟตามีน แตจะใหผลบวกลวงไมเทากัน โดยที่ BPB จะแสดงผลบวกลวงนอยกวา TBPE อยู 2 ตัวอยาง จากตวัอยางปสสาวะที่มีสารชนิดอ่ืนที่ไมใชเมทแอมเฟตามีนอยู 14 ตัวอยาง วิธีที่แสดงในการศึกษานีส้ามารถนํามาประยุกตใชเปนทางเลือกในการตรวจสอบยาบาไดอีกทางหนึ่งแตควรศกึษาเพิ่มเติมกอนนํามาใชจรงิ 56 หนา


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 methamphe-

tamine………………………………………. 5

2.1.4 Routes of administration……………………. 6

2.1.5 Metabolism and excretion of amphetamine

and methamphetamine……………………… 6

2.2 Color tests for detection of amphetamine or metham-

phetamine in urine………………………………….. 9

2.2.1 Spot tests for detection of amphetamine or

Methamphetamine………………………….. 9

2.2.2 Color tests for amphetamine or methampheta-

mine detection in urine……………………… 12


vii

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 tetrabromophe-

nolphthalein ethyl ester……………………... 14

2.4 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

3.1 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

3.2 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

3.3 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

3.4 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

4.1 Preliminary dye selection…………………………..... 30


viii

CONTENTS (cont.)

Page

4.1.1 Dye selection 1……………………………...... 31

4.1.2 Dye selection 2……………………………….. 31

4.1.3 Dye selection 3……………………………….. 35

4.2 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


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


x

LIST OF FIGURES

Figure Page

2.1 The schemes of metabolic pathways of amphetamine and meth-

amphetamine…………………………………………………… 7

2.2 Drugs metabolizing to amphetamine or methamphetamine…… 8

2.3 Scheme of purposed reactions when amphetamine and meth-

amphetamine are tested by Marquis reagent…………………… 11

2.4 Scheme of purposed reactions when methamphetamine is tested

by Simon’s reagents giving a blue color product………………. 11

2.5 Proposed color reaction of amines (R3N) with TBPE molecule

in organic phase………………………………………………... 15

2.6 The structures of some drugs which can form charge transfer

complexes with TBPE cause false positive results for ampheta-

mine 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

wavelengths to create the perceived reflected color (middle)…. 18

2.9 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)…………………………………….. 20

2.11 (A) General structural formula for sulfonephthalein indicators:

colorless lactone (sultone) form and (B) example of changing

from bromophenol blue lactone to its color quinoid form……. 22

3.1 Flow chart of the methods for “Dye Selection” …..………….. 28


xi

LIST OF FIGURES (cont.)

Figure Page

4.1 Four control samples tested by TBPE solution………………… 30

4.2 The 1,2-napthoquinone-4-sulphonate (NQS) was tested for the

four test samples using method A (left) and N (right)………… 31

4.3 Results of dyes number 1-9 tested with the four test samples (1.

distilled water, 2.negative human urine, 3.positive methamphe-

tamine 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 bromo-

cresol purple tested with four test samples using method C….. 35

4.5 Reaction between NQS and the primary amine in alkaline solu-

tion causing a color compound……………………………….. 36

4.6 UV-visible spectra for BPB, BCP, and BTB in benzene at

approximately 2.5x10-4 M ……………………………………... 38

4.7 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)……………………………………………………. 39

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…………………………………. 42

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……………………………. 43

4.10 Chemical structures of TBPE, BPB, BCP and BTB, and the

general structural formula for phenolsulfonephthalein indicators

(A) (upper right)……………………………………………….... 45


xii

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


xiii

LIST OF ABBREVIATIONS (cont.)

rpm Round per minute

TBPE Tetrabromophenolphthalein ethyl ester

UV Ultraviolet

VIS Visible light

λmax Maximum absorption

μl Microlitre

μg Microgram


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 cross-

reactivity 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.


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.

.



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.


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]

Structure

Amphetamine

Methamphetamine

Chemical formula

Molecular weight

Functional group

Boiling point

Melting point

pKa

C9H13N

135.2

Primary amine

203 °C

156.5-158.5 °C

9.8

C10H15N

149.2

Secondary amine

214 °C

170-175 °C [9]

10.1



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 Long-term effects

- increased attention and

decreased fatigue

- increased activity and wakefulness

- loss of appetite

- Euphoria and rush

- increased respiration

- rapid/irregular heartbeat

- hyperthermia

- addiction

- psychosis, including:

paranoid

hallucinations

repetitive motor activity

- changes in brain structure and function

- memory loss

- aggressive or violent behavior

- mood disturbances

- severe dental problems

- weight loss



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 meth-

amphetamine 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].



Figure 2.1: The schemes of metabolic pathways of amphetamine (upper) and

methamphetamine (lower)



Figure 2.2: Drugs metabolizing to amphetamine or methamphetamine [13]



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].



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-methylene-

dioxy-N-ethylamphetamine (MDE or MDEA). Hence, it can be used to distinguish

between MDA and amphetamine [9].



Figure 2.4: Scheme of purposed reactions when methamphetamine is tested by

Simon’s reagents giving a blue color product [16]

Figure 2.3: Scheme of purposed reactions when amphetamine and methamphe-

tamine are tested by Marquis reagent [16]

Blue

Methamphetamine



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.



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 Henderson-

Hasselbalch equations, and some relationships between pH and pKa

Acid A Base B

-

a

H AK

HA

+⎡ ⎤ ⎡ ⎤⎣ ⎦ ⎣ ⎦=⎡ ⎤⎣ ⎦

a

H BK

BH

+

+

⎡ ⎤ ⎡ ⎤⎣ ⎦ ⎣ ⎦=⎡ ⎤⎣ ⎦

pH > pKa; [Ionized] > [Un-inonized]

pH < pKa; [Ionized] < [Un-inonized]

pH = pKa; [Ionized] = [Un-inonized]

pH > pKa; [Un-ionized] > [Inonized]

pH < pKa; [Un-ionized] < [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].

HA H A+ −+ BH H B+ + +

Inonized Water soluble

Organic insoluble Un-inonized Organic soluble Water insoluble

Un-inonized Organic soluble Water insoluble



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 donor-

electron 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.



Figure 2.5: Proposed color reaction of amines (R3N) with TBPE molecule in

organic phase (subscript “o”) [20, 23]

R3N + HTBPE ↔ R3N•HTBPE colorless yellow red-violet



Figure 2.6: The structures of some drugs which can form charge transfer

complexes with TBPE cause false positive results for amphetamine and meth-

amphetamine color screening

Chlorpheniramine Brompheniramine Ephedrine

Phenylpropanolamine

Fenfluramine Phentermine

Dextromethorphan

Codiene

Quinine

Amitriptyline

Chlorpromazine

Erythromycin

Diphenhydramine

Theophylline

Strychnine



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]



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]



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]



Figure 2.10: the effect of auxochromes on absorbance (upper) and summary of the

λmax shifting (lower) [16]

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).

2.4.2 Dyes

Colorants are substances or materials that can absorb or emit electro-

magnetic 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.



Table 2.5 Dyes categorized by chromophore [16]

Chromophore Structure Example

Antraquinone

Alizarin

Acridine Acridine yellow

Azo

-N=N-

Methyl orange

Carbonyl

Conjugated C=O system

Indigo

Nitro and

nitroso

-NO2 (nitro functional group)

-N=O (nitroso functional group)

Para red

Polymethine

Long chain of conjugated double

bonds by e-donor and e-acceptor

Triarylmethine subcategory:

Phenolphthalein



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.

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].

A

B



CHAPTER III

MATERIALS AND METHODS

3.1 Materials

3.1.1 Instrumentation

Table 3.1 List of instruments

Equipment/ Instrument Model Company

1. Micropipette Pipetteman Gilson

2. Microcentrifuge Denville 260D Denville Scientific Inc.

3. Balance TE 1535 Sartorius

4. Vortex VTX-3000L LMS

5. Spectrophotometer NanoDrop 1000 Thermo Scientific


Supanat Panomnoptham Materials and Methods / 24

3.1.2 Reagents

Table 3.2 List of reagents

Chemical Supplier Grade

1. Sodium tetraborate (borax) Ajax -

2. Distilled water - -

3. Dichloromethane or methylene chloride Fisher Scientific AR

4. Hydrochloric acid BDH AR

5. Sodium hydroxide Merck AR

6. Benzene Merck AR

Dye

1. Alizarine S sodium salt Merck AR

2. Methylene blue Merck AR

3. Methyl orange Merck AR

4. Methyl red Merck AR

5. Xylene cyanol FF Bio Basic AR

6. Cresol red Merck AR

7. Bromophenol blue Bio-Rad Laboratories Laboratory

8. Bromothymol blue Merck AR

9. Bromocresol purple Merck AR

10. 3,4-Dihydro-3,4-dioxo-1-napthalene-

sulfonic acid sodium salt

Fluka chemica AR

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.



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



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.



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.


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


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.




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-1,2-napthoquinone-4-sulphonate (NQS)

Method N

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 B Method C

Figure 3.1: Flow chart of the methods for “Dye Selection”



3.4 Study on acid-base reaction in organic solvent

3.4.1 General Procedure

Extraction


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


Supanat Panomnoptham Results and Discussion / 30

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 charge-

transfer 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

Figure 4.1: Four control samples tested by TBPE solution.

Upper urine layer

Lower organic layer




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].


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.

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)



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.

(2) Methylene blue

(1) Alizarin red S 1A 2A 3A 4A 1B 2B 3B 4B

1A 2A 3A 4A 1B 2B 3B 4B

(4) Methyl red 1A 2A 3A 4A 1B 2B 3B 4B

1B 2B 3B 4B (3) Methyl orange 1A 2A 3A 4A



Figure 4.3 (cont.):

(9) Bromocresol purple (BCP) 1B 2B 3B 4B 1A 2A 3A 4A

(8) Bromothymol blue (BTB) 1A 2A 3A 4A 1B 2B 3B 4B

(7) Bromophenol blue (BPB) 1A 2A 3A 4A 1B 2B 3B 4B

(6) Cresol red 1A 2A 3A 4A 1B 2B 3B 4B

(5) Xylene cyanol FF 1A 2A 3A 4A 1B 2B 3B 4B



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 Method B

Dye 1A 2A 3A 4A 1B 2B 3B 4B

1. Alizarin red S - - - - - - - -

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 - - - -

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.




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.

Bromothymol blue (BTB)

1C 2C 3C 4C

Figure 4.4: Results for bromophenol blue, bromothymol blue and bromocresol

purple tested with four test samples using method C

Bromophenol blue (BPB)

1C 2C 3C 4C

Bromocresol purple (BCP)

1C 2C 3C 4C



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.

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,

Figure 4.5: Reaction between NQS and the primary amine in alkaline solution

causing a color compound [27]



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 charge-

transfer 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.



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

(B) BCP (C) BTB

Figure 4.6: UV-visible spectra for BPB, BCP and BTB in benzene at

approximately 2.5x10-4 M



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.

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).

Volume ratio

Sample: BPB

(A) 10:10

(B) 10:2

(C) 30:10

(D) 30:5



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.



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 BCP BTB

Yellow Blue Yellow Blue Yellow Blue

(1) Positive methamphetamine urine

1.1 Meth (horse)

1.2 Meth (human)

1.3 Meth + others

-

-

6

1

7

3

1

7

9

-

-

-

1

7

9

-

-

-

(2) Negative methamphetamine urine

2.1 MDMA or MDEA

2.2 Negative control

2.3 Other drugs

-

1

11

3

-

3

3

1

14

-

-

-

1

1

14

-

-

-

Total 18 17 35 0 35 0

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).



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

Negative urine sample

Negative urine sample + BPB

λmax = 409 nm

A = 0.073

Negative urine sample + BTB λmax = 409 nm

A = 0.086

Negative urine sample + BCP λmax = 402 nm

A = 0.090



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

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



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 TBPE (kit)

Neg Pos Neg Pos

(1) Positive methamphetamine urine

1.1 Meth (horse)

1.2 Meth (human)

1.3 Meth + others

-

-

6

1

7

3

-

-

6

1

7

3

(2) Negative methamphetamine urine

2.1 MDMA or MDEA

2.2 Negative control

2.3 Other drugs

-

1

11

3

-

3

-

1

9

3

-

5

Total 18 17 16 19

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.



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].

BPB BTB BCP

TBPE A

Figure 4.10: Chemical structures of TBPE, BPB, BCP and BTB, and the general

structural formula for phenolsulfonephthalein indicators (A) (upper right)


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.



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


Supanat Panomnoptham References / 48

REFERENCES

1. Rouen D, Dolan K, Kimber J. A review of drug detection testing and an

examination of urine, hair, saliva and sweat National Drug and Alcohol

Research Centre, University of New South Wales, Sydney; 2001.

2. วงศววิัฒน ทัศนียกุล. Screening & Identification of DRUG ABUSE: ภาควิชาพิษวิทยา คณะเภสัชศาสตร มหาวิทยาลัยขอนแกน.

3. Chairat Jai-Ob-Orm PC. Confirmatory test of urine methamphetamine from

dichloromethane layer of chemical reaction test kit by GC/MS [Master's

Thesis]: Mahidol University; 2004.

4. United Nations Office on Drugs and Crime Regional Centre for East Asia and the

Pacific. Patterns and Trends of Amphetamine-Type Stimulants (ATS) and

Other Drugs of Abuse in East Asia and the Pacific 2006; June 2007.

5. Couper FJ, Logan BK. Drugs and Human Performance Fact Sheets: National

Highway Traffic Safety Administration; 2004.

6. United Nations Office on Drugs and Crime (UNODC). Terminalogy and

Information on Drugs. 2003.

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

University; 2006.

9. United Nations Office on Drugs and Crime (UNODC). Recommended Methods for

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.



11. National Institute on Drug Abuse (NIDA). NIDA Research Report:

Methamphetamine: Abuse and Addiction. 2006.

12. National Institute on Drug Abuse (NIDA). NIDA InfoFacts: Methamphetamine.

2006.

13. United Nations Office on Drugs and Crime (UNODC). Recommended Methods

for the detection and Assay of Heroin, Cannabinoids, Cocaine,

Amphetamine, Methamphetamine and Ring-Substituted Amphetamine

Derivatives in Biological Specimens. 1995.

14. Heit HA, Gourlay DL. Urine drug testing in pain medicine. J Pain Symptom

Manage. 2004 Mar;27(3):260-7.

15. Rouen D, Dolan K, Kimber J. A review of drug detection testing and an

examination of urine, hair, saliva and sweat National Drug and Alcohol

Research Centre, University of New South Wales, Sydney; 2001.

16. Bell S. Forensic chemistry. Upper Saddle River, N.J. Pearson Prentice Hall, 2006:

Upper Saddle River N.J. : Pearson Prentice Hall 2006; 2006.

17. Legua CM, Campins Falco P, Sevillano Cabeza A. Determination of

methamphetamine in urine samples with sodium 1, 2-naphthoquinone-4-

sulphonate. 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 1,2-

naphthoquinone 4-sulphonate. Analytica chimica acta. 1993.

19. Frings CS, Queen C, Foster LB. Improved colorimetric method for assay of

amphetamines in urine. Clin Chem. 1971 Oct;17(10):1016-9.

20. Sakai T, OhNo N. Spectrophotometric Determination of Stimulant Drugs in Urine

by Color Reaction with Tetrabromophenolphthalein Ethyl Ester.

Analytical Sciences. 1986;2.

21. วีรวรรณ เล็กสกุลไชย. ปจจยัตอผลการตรวจยาบาดวยชุดน้ํายาตรวจยาบาในปสสาวะ. วารสารวิชาการสาธารณสุข 2548;14.

22. Reichardt C. Solvent effects in organic chemistry. Weinheim Verlag Chemie,

1979: Weinheim : Verlag Chemie 1979; 1979.


Supanat Panomnoptham References / 50

23. Sakai T, Watanabe S, Yamamoto S. Thermospectrophotometric Analysis of

Alkylamines Utilizing Ion Association with Tetrabromophenolphthalein

Ethyl Ester. Analytical Chemistry. 1997;69(9):1766-70.

24. Hornback JM, Young PR. Organic chemistry. Pacific Grove, Calif. Brooks/Cole,

c1998: Pacific Grove Calif. : Brooks/Cole c1998; 1998.

25. Davis MM, Schuhmann PJ, Lovelace ME. Acid-Base Reactions in Organic

Solvents. Behavior of Some Halogenated Derivatives of

Phenolsulfonephthalein with Different Classes of Organic Bases in

Benzene. Journal of Research of the National Bureau of Standards.

1948;41.

26. Ashour S, Chehna MF, Bayram R. Spectrophotometric Determination of

Alfuzosin HCl in Pharmaceutical Formulations with some

Sulphonephthalein Dyes. International Journal of Biomedical Science.

2006;2.

27. Khummueng W. Determination of amphetamine and related compounds in urine

by high performance liquid chromatography with sodium 1,2-

naphthoquinone-4-sulphonate as derivatizing agent [Master's Thesis]:

Mahidol University; 2001.



APPENDIX


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

Phenethylamine H H H H H H H H

Amphetamine

Methamphetamine

CH3

CH3

H

H

H

H

H

H

H

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

MDMA

CH3

CH3

H

H

H

H

-O-CH2-O-

-O-CH2-O-

H

H

H

H

H

CH3

Cathine

PPA

CH3

CH3

OH

OH

H

H

H

H

H

H

H

H

H

H

H

H

Ephedrine

Pseudoephedrine

CH3

CH3

OH

OH

H

H

H

H

H

H

H

H

H

H

CH3

CH3

Dopamine

Norephinephrine

Ephinephrine

H

H

H

H

OH

OH

H

H

H

OH

OH

OH

OH

OH

OH

H

H

H

H

H

H

H

H

CH3



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).


Supanat Panomnoptham 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).



Result

TableA.2 Result of five color tests for amphetamine, methamphetamine, MDA,

MDMA, and ephedrine (and pseudoephedrine)

Note: NR = no reaction

* The color of reagent should be considered as negative

Compound Marquis Simon’s Mandelin Chen’s Gallic

Amphetamine Orange to

brown

NR* (dark) green NR* NR

Methamphetamine Orange to

brown

Deep blue (dark) green NR* NR

MDA Dark blue/

black

NR* Bluish black NR* Bright to

dark green

MDMA Dark blue/

black

Deep blue Bluish black NR* Bright to

dark green

Ephedrine

Pseudoephedrine

NR NR* NR Purple NR


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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