前言 INTRODUCTION

安非他命-典型的興奮劑，是興奮劑藥物的一類，其結構來源于β-苯乙胺。
 

1887年，安非他命作為1-甲基托布津-2-苯乙胺在柏林合成。它是幾種化學物質中的第一個,包括冰毒和3,4-甲基托布津、脫氧麻黃堿、紗羅等,也有類似的結構和生物學性質,被統稱為“安非他命”。自1887年以來,隨著冰毒和德克薩斯州刺槐灌木中的尼古丁、麥司卡林的發現，其復合形式在1997得以正式形成。氨芐青霉素和蛋氨酸是最常見的濫用藥物。他們中心不對稱，存在兩種對映體。
 

在試圖維持厭食癥患者活動的時候，應該及時限制不良的副作用,替換苯丙胺和甲基苯丙胺。其他人已經提高刺激活動或避免生產和使用的藥物的法律限制。安非他命的相關組織衍生品圖3和圖4所示。圖5顯示了另一組前體藥物,促進身體的新陳代謝,形成氨芐青霉素和蛋氨酸。
 

Amphetamine-type stimulants (ATS) are a group of drugs, mostly synthetic in origin, that are structurally derived from β-phenethylamine (Figure 1).
 

Amphetamine (AMP, "Speed") was initially synthesized in Berlin in 1887 as 1-methyl-2-phenethylamine. It was the first of several chemicals, including methamphetamine (MET, "Ice") and 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy"), which have similar structures and biological properties, and are referred to collectively as "amphetamines" (Cody, 2005). Since 1887, amphetamine was thought to be a human invention (Berman et al., 2009), but the compound was found in 1997, along with methamphetamine, nicotine and mescaline, within two species of Texas acacia bushes (Clement, Goff and Forbes, 1998). AMP and MET are most commonly abused drugs. They have asymmetric centre and exists as one of the two possible enantiomers (see Figure 2) (Cody, 2005).
 

In attempt to maintain anorexic activity while limiting undesirable side effects, substitutions have been made to amphetamine and methamphetamine. Others have been made to enhance the stimulatory activity or to avoid legal restrictions on the manufacture and use of the drugs (Cody, 2005). The related groups of amphetamine derivatives are shown in Figures 3 and 4. Figure 5 shows another group of precursor drugs that is metabolized by the body into AMP and MET.
 

Administration and neurotoxicity of amphetamines

Amphetamines are generally administered as oral capsules. This route results in a gradual increase in drug concentration, which peaks in around an hour and maintains effective drug levels for 8 – 12 hours. Amphetamines can also be injected into the circulation (Parrott et al., 2004). Amphetamines readily cross the blood-brain barrier to reach the sites (Berman et al., 2009) of action in the brain. The acute administration of amphetamines produce a wide range of dose-dependent behavioral changes, including increased arousal or wakefulness, anorexia, hyperactivity, perseverative movements, and, in particular, a state of pleasurable affect, elation, and euphoria, which can lead to the abuse of the drug (Berman, 2009).#p#分頁標題#e#


This causes amphetamines to be associated with acts of violence. Acute drug abusers will develop tolerance, where the same dose of drug has diminishing physiological and psychological effects. They need to increase their dosage if they wish to generate the same strength of effect. Cross-tolerance will also occur as tolerance to one drug affects another drug with similar neurochemical profile. As a result, drug abusers will seek for another class of drug and become polydrug users (Parrott et al., 2004). Chronic drug abusers usually take in amphetamines through injection or smoking ice amphetamines. These abusers suffer many health problems and a reduced life expectancy. They are more susceptible to HIV (human immunodeficiency virus), AIDS (acquired immunity deficiency syndrome) and SIDS (sudden infant death syndrome) (Parrott at al., 2004).
 

Clinical uses

In accordance with the Convention on Psychotropic Substances of 1971, amphetamines are enlisted as narcotic compounds in the List of psychotropic substances under international control. The list is prepared by the International Narcotics Control Board. These compounds are prohibited to be imported and exported in countries like Japan, Nigeria, Pakistan, Thailand and etc (International Narcotics Control Board, 2003). Amphetamines and related compounds are clinically used for narcolepsy (sudden day-time onset sleep) and Attention Deficit Hyperactivity Disorder (ADHD) in young children. It was formerly used as a short-term slimming agent, as an antidepressant and to boost athletic performance (Parrott et al., 2004).
 

1.-METHYLENEDIOXYMETHAMPHETAMINE (MDMA, "ECSTASY")
 

History of MDMA abuse

MDMA, also known as "ecstasy", "ETC", or "Adam", is one of the most commonly abused amphetamine derivatives that was re-synthesised by Alexander Shulgin during his research career at the Dow Chemical Company in 1970s. Soon MDMA was being synthesised in illicit laboratories, and became popular as recreational drug since then. As MDMA does not have any clinical/medical use, it is scheduled as Class I illicit drug by the American Drug Enforcement Agency in 1985 (Parrott et al., 2005). Also, MDMA other ring-substituted phenylethylamines were generically classified under the Misuse of Drugs Act as Class A drugs, in United Kingdom (Wikipedia, 2009).
 

Chemical Properties of MDMA

The methylenedioxy analogues of amphetamine (see Figure 3) are series of compounds referred to designer drugs. They include methylenedioxyamphetamine (MDA), methylenedioxyethylamphetamine (MDEA) and MDMA (Hensley and Cody, 1999). The synthesis of N-alkyl-MDA derivatives only produces (±) racemic mixtures. As a results, only racemic forms of (capsules, loose powder or tablets) the compounds are sold in the illicit market and abused (Matsushima, Nagai and Kamiyama, 1998; Fallon et al., 1999).#p#分頁標題#e#
 

MDMA is chiral, possessing two enantiomers, S-(+)-MDMA and R-(-)-MDMA (see Figure 6), with S-(+)-MDMA is more potent than R-(-)-MDMA (Lyon, Glannon and Titeller, 1986; Shulgin 1986). The basic structure of MDMA is ?-phenylisopropylamine group (see Figure 6), with a methylenedioxy group forming a 5-membered ring including C-3 and C-4 of the benzene ring (Cho and Segal, 1994). The empirical formula of MDMA is C11H15NO2 (Shulgin, 1986).
 

MDMA is a phenylisopopylamine derived from safrole, aromatic oil found in sassafras, nutmeg, and other plants. The methyl group on α-carbon (R2) (see Figure 6) of MDMA confers resistance to oxidative deamination of this compound and, therefore, increased its metabolic half-life (Cho and Segal, 1994). According to Cone and his colleague Huestis (2009), S(+) isomer of MDMA is responsible for its psychostimulant and empathic effects and the R(-) isomer for its hallucinogenic properties.
 

Uptake, absorption, metabolism and elimination of MDMA in human body

MDMA is usually formulated in tablets of its racemate (1:1 mixture of its enantiomers) in doses ranging from 50 to 200 mg (Pizarro et al., 2004), which is most commonly sold in batches of 3–5 for ?10 (Wikipedia, 2009). MDMA powder is also found in the market at a higher price, indicating that it has higher purity. MDMA powder is not usually insufflated (snorted) as it causes sneezing, pain and nosebleeds. MDMA cannot be smoked and is very rarely injected intravenously (AMCD, 2008).
 

MDMA is absorbed into the blood streams and distributed in body. Postmortem analysis by Letter et al. (2002) shows that MDMA is distributed in cardiac muscle, both lungs, liver, both kidneys, spleen, the four brain lobes, cerebellum and brainstem, adipose tissue, serum, vitreous humor, urine, hair and bile upon administration. Rapid distribution of MDMA in body is mainly due to its basic property of pKa around 9.9 and low plasma protein binding, MDMA can diffuse across biological matrices that is more acidic than blood (Pichini, 2005). After an oral administration of MDMA, the plasma concentration peaks in within 1.5 to 2 hours (Cone and Huestis, 2009).
 

MDMA is metabolized by multiple pathways (see Figure 7), primarily involving N-demethylation and O-demethylenation. The enzymes involved in the pathway are a group of cytochrome P450 isoenzymes, including CYP1A2, CYP3A4, and CYP2B6.
 

Firstly, MDMA is O-demethylenated to 3,4-dihydroxymethamphetamine (HHMA) followed by O-methylation to 4-hydroxy-3-methoxymethamphetamine (HMMA). The enzymes involved in the metabolic process are CYP2D6 and catechol-methyltransferase respectively. At a lower rate, MDMA is N-demethylated to 3,4-methylenedioxyamphetamine (MDA) (a reaction regulated by CYP2B6), which is further metabolized to the catechol intermediate (3,4-dihydroxyamphetamine) and finally O-methylated to 4-hydroxy-3-methoxyamphetamine (HMA). In the reactions, the α-carbon responsible for stereochemical properties of MDMA is not affected and all the metabolites are chiral compounds that may be presented as a mixture of their enantiomers. In addition to these major compounds, some other minor metabolites derived from the activity of monoamine oxidase on the amine residue are also formed (Kolbrich et al., 2008; Pizarro et al., 2004).#p#分頁標題#e#
 

N-demethylation of MDMA yields 3,4-methylenedioxyamphetamine (MDA), an active metabolite exhibiting similar pharmacological properties as the parent drug. A further O-demethylenation of MDA produces 3,4-dihydroxyamphetamine (HHA) which is mainly regulated by CYP2D6. Additional metabolites are formed by O-methylation of HHMA to 4-hydroxy-3-methoxymethamphetamine (HMMA) and of HHA to 4-hydroxy-3-methoxyamphetamine (HMA), deamination, and conjugation (Cone and Huestis, 2009).
 

The metabolic pathway mainly happens in the liver. Some people with reduced CYP2D6 shows lower metabolic rate of MDMA and thus are more susceptible to MDMA toxicity (O'Donohoe et al., 1998; Schwab et al., 1999).
 

Physiological and psychological effects of MDMA

Berman et al. (2009), Hensley and Cody (1999) and Piper (2008) reported an increased alertness and euphoria, increased heart rate, blood pressure, respiration and body temperature upon administration of MDMA. United Nation Office on Drugs and Crime (2006) conveys that chronic amphetamines abuse causes agitation, tremors, hypertension, memory loss, hallucinations, psychotic episodes, paranoid delusions, and violent behavior. Withdrawal from high doses of amphetamine-type stimulants (ATS) could result in severe depression. MDMA impairs the temperature control by hypothalamus. This causes MDMA users to die of hyperthermia (Piper, 2008) and some die from hyponatraemia, i.e. the dilution of blood due to excessive fluids taken to counteract heat exhaustion (Parrott et al., 2004).
 

Neurotoxicity of MDMA

Nichols (1986) and Vollenweider et al. (1998) categorize MDMA as entactogens, a special class of drug that produce changes in mood, social interactions or feelings of interpersonal closeness and changes in perception. MDMA shares some of the pharmacological effects of stimulants and serotonergic hallucinogens (Cami et al. 2000; Gouzoulis-Mayfrank et al. 1999; Liechti Gamma and Vollenweider, 2001; Tancer and Johanson 2003).
 

MDMA acts an agonists on various neurotransmitters action especially serotonin. Boost in serotonin turnover induced by MDMA tends to generate feelings of contentment, elation, liveliness and intense emotional closeness to others. This causes people to enjoy themselves without their normal concerns and inhibitions. MDMA is classified as neurotoxin. Studies have found evidence for dopaminergic nerve destruction in higher brain regions. As shown in Table 2, the higher brain function such as memory, information processing and storage, complex stimulus analysis and decision making of MDMA users are impaired.
 

CHIRAL DRUG ANALYSIS

Chirality

Chirality is formally defined as the geometric property of a rigid object (like a molecule or drug) of not being superimposable with its mirror image (McConathy and Owen, 2003). Achiral molecules can be superimposed on their mirror images. Molecules that are not superimposable with their mirror images are said to be chiral. Each chiral molecule will have at least one chirality centre or stereogenic centre (Leffingwell, 2003). Chirality centre of an organic molecule is usually a carbon atom, bonded to four different groups of atoms. Chiral molecules with one chirality centre exist in two enantiomeric forms (see Figure 8).#p#分頁標題#e#
 

The two mirror images are termed enantiomers. Both molecules of an enantiomer pair have the same chemical formulae and can be drawn the same way in 2 dimensions but in chiral environments such as the receptors and enzymes in the body, they will behave differently. Enantiomers are identical in all physical properties except for their optical activity, or direction in which they rotate plane-polarized light (McMurry, 2004). Some optically active molecules rotate polarized light to the left (levorotatory) while others to the right (dextrorotatory) (Baker, Prior and Coutts, 2002). A racemate (often called a racemic mixture) is a mixture of 1:1 amount of both enantiomers of (+) and (-) enantiomers and is optically inactive. The optical inactivity results from the rotation caused by one enantiomer canceling out that produced by its complementary enantiomer (Beesley and Scott, 1998). The absolute configuration at a chirality center is designated as R or S to unambiguously describe the 3-dimensional structure of the molecule. R is from the Latin rectus and means to the right or clockwise, and S is from the Latin sinister for to the left or counterclockwise (McConathy and Owen, 2003; Baker, Prior and Coutts, 2002).
 

Pharmacological aspect of chiral drugs

In pharmacology, chirality is an important factor in drug efficacy. About 56% of the drugs currently in use are chiral compounds, and about 88% of these chiral synthetic drugs are used therapeutically as racemates (Leffingwell, 2003). As previously mentioned, MDMA is a chiral drug that exists in two enantiomeric forms as shown in Figure 6. Chemical modification at the positions R1 to R9 (refer to Figure 9) of MDMA results in unlimited number of pharmacologically active compounds, some of which are more potent stimulants than others.
 

Although there are several possibilities for side chain modification, substitution on the aromatic ring contributes the most to substantial qualitative differences in pharmacological effects. Hence, it is important to discriminate between the enantiomers present in the drugs administrated as both the enantiomers of a chiral drug may differ significantly in their bioavailability, rate of metabolism, metabolites, excretion, potency and selectivity for receptors, transporters and/or enzymes, and toxicity (McConathy and Owen, 2003). The difference in interaction between a chiral drug and its chiral binding site is illustrated in Figure 10.
 

The different domain of a drug molecule has different binding affinity towards the active site of biochemical molecules in the body. As shown in Figure 10, it is obvious that the active enantiomer has a 3-dimensional structure that allows drug domain A to interact with binding site domain a, B to interact with b, and C to interact with c. In contrast, the inactive enantiomer cannot be aligned to bind the same 3 sites simultaneously. Due to the difference in 3-dimensional structure, binding of the active enantiomer exerts a biological effect, while the inactive enantiomer does not possess any (McConathy and Owen, 2003).#p#分頁標題#e#
 

The hypothetical interaction of drug enantiomers is supported by the studies done by Matsushima, Nagai and Kamiyama (1998) and Kolbrich et al. (2008) shows that stereoselective cellular transport of MDMA allows the drug to accumulate at different extent in biological matrices. According to O'Donohoe et al. (1998) and Schwab et al. (1999), stereoselectivity also affects genetic differences in the expression of metabolic enzymes that are responsible to metabolize MDMA in the body. For example, CYP2D6 is expressed as 2 phenotypes; one being extensive and another as poor metabolizers. Thus, it is obvious that the stereospecificity of a chiral drug can alter absorption, elimination and cellular transport of the drug itself.
 

Analytical aspect of chiral drugs

Approximately 50% of marketed drugs are chiral, and of these approximately 50% are racemix mixtures of enantiomers rather than single enantiomers (McConathy and Owen, 2003). Differences in pharmacokinetic and pharmacodynamic activities of the enantiomers of drugs administered as racemates are increasingly appreciated (Porter, 1991). Thus, quantification and qualification of drugs of abuse play important roles in the prediction of and protection from the risk to human health (Nakashima, 2006).
 

Two main approaches to chiral drug analysis have been taken. In the indirect approach, the drug enantiomers are derivatized with an optically pure chiral reagent to form a pair of diastereomers, which may then have sufficiently different physical properties for separation to occur on conventional chromatographic columns (UNODC, 2006; Porter, 1991). In the direct approach, the enantiomers form transient rather than covalent diastereomeric complexes with a chiral selector present either in the mobile or the stationary chromatographic phase (Porter, 1991). Each of these analytical approaches has advantages and disadvantages prevail, depending upon factors such as time, purity, chemical processing, and inherent side reactions (Carvalho et al., 2006).
 

Indirect chiral drug analysis

In order to successfully resolve the enantiomers, a stable, optically pure chiral derivatizing reagent (CDR) has to be available for the covalent formation of diastereomeric derivatives (Porter, 1991). Diastereoisomers of amphetamine-type stimulants can be prepared using different reagents such as acylchlorides, alkylsulphonates, isothiocyanates, chloroformates. Mosher's acid [R(+) or S(-)-methoxy(trifluoromethyl)phenylacetic acid], Mosher's acid chloride, and N-trifluoroacetyl-1-prolyl chloride (TPC, also known as TFAP-Cl) are the most popularly used chiral derivatizing agents (UNODC, 2006). The reaction scheme may be illustrated as follows:
 

The purity of the chiral derivatizing agent is vital in the process of separation of the racemic mixture. The resolution of a racemic drug by the R-enantiomer of a CDR contaminated with its S-enantiomer causes an additional pair of diastereoisomers to be formed, each of which is the enantiomer of one of the first pair (Porter, 1991), as shown in Figure 12.#p#分頁標題#e#
 

As a result, the enantiomers R-R', S-S' and S-R', R-S' would coelute in conventional chromatographic systems due to their similar physical properties. Racemization during the reaction would bring about analytical error especially when attempting to quantitate small quantities of one enantiomer in the presence of a large excess of its antipode (Porter, 1991).
 

Methods using chiral derivatization are essentially less expensive and do not require specialized equipment or columns. The use of normal, achiral columns allows easy integration of chiral separations into routine analysis schemes (UNODC, 2006). Thus, considerable flexibility in chromatographic conditions is available to achieve the desired resolution and to eliminate interferences from metabolites and endogenous substances. Moreover, a reasonably good selection of chemically and optically pure CDRs is available for derivatizing various functional groups (Porter, 1991).
 

Direct chiral analysis

Chiral gas chromatography (GC), High Performance Liquid Chromatography (HPLC) or Capillary Electrophoresis (CE) are popular methods in direct analysis of illicit drugs (UNODC, 2006). Direct analysis does not require a CDR for covalent diastereomeric complexation. Instead, separation of chiral drugs occurs via the interaction between the enantiomers and a chiral selector. The chiral selector is an optically active compound that may be present in the mobile phase for use with conventional HPLC columns or it may be incorporated into the stationary phase to provide specialized chiral stationary phases (Porter, 1991). Calvalho (2006) lists the most successful chiral packing materials i.e. amylose, Pirkle type stationary phase, cyclodextrin, proteins, and cellulose ester and carbamate derivatives used in GC.


Sometimes, derivitization may be carried out with a nonchiral reagent, in order for appropriate molecular interactions with the chiral discriminator to occur and/or to impart requisite spectral or fluorescent properties to the molecule (Porter, 1991). HPLC with fluorescence detection method is done by Al-Dirbashi et al. (1999) in attempt for the determination of methamphetamine in human hair. Nakashima (2006) claimed that the use of a chiral stationary phase in GC to separate pairs of enantiomers after suitable derivatization with an achiral reagent is able to achieve a powerful separation.
 

Recently CE has become a highly competitive tool for chiral analysis of many compounds since it allows for the highly efficient separation of enantiomers without derivatization and specialty columns (capillaries) (Porter, 1991; Ramseier, Caslavska and Thormann, 1999). For the separation of amphetamine-type stimulant using CE, chiral additives such as hydroxyl-propyl beta-cyclodextrin are added in the running buffer. This eliminates the need of derivatization in analysis of chiral drugs commonly used (Iio et al., 2005; Ramseier, Caslavska and Thormann, 1999).#p#分頁標題#e#
 

Separation of chiral drugs using gas chromatography

UNODC (2006), Pirnay, Abraham and Huestis (2006) and Rouen, Dolan and Kimber (2001) agree that gas chromatography/mass spectrometry (GC/MS) is the most common instrumental technique for analysis of amphetamines and derivatives. However, GC/MS still has its limitations.
 

Chiral gas chromatography is selected as the separation technique if the materials are volatile and stable at elevated temperatures. In addition, if the solutes can be derivatized to form a sufficiently volatile product without racemizing the enantiomers, or changing their racemic proportion, then GC may be the choice. GC offers much higher efficiencies, much higher peak capacities and significantly higher sensitivities than LC. It follows, that GC can easily contend with multicomponent mixtures, especially mixtures from biological samples. In addition, the columns have short equilibrium times, trace impurities are easily assayed, and the analyses are shorter providing much faster sample throughput (Beesley and Scott, 1998).
 

Prior to analysis by GC, compounds containing functional groups with active hydrogens such as COOH, OH, NH, and SH have to be derivatized. This is because these compounds tend to form intermolecular hydrogen bonds, hence reducing volatility of the compounds in the machine. They are also thermally unstable and can interact with either fused silica or the stationary phase, causing peak broadening (Danielson, Gallgher and Bao, 2000).
 

Most underivatized amphetamine-type stimulants (ATS) have fragment ions of low m/z ratio, low intensity, and only one fragment ion of higher abundance (base peak). Derivatized ATS usually produces fragment ions of higher m/z ratio and higher abundance. Molecular ions with greater molecular mass have greater diagnostic value, due to the reason that they are not affected by interfering background ions such as column bleed or other contaminants (UNODC, 2006).
 

Capillary electrophoresis as a complementary method in the analysis of MDMA

According to Meng et al. (2006), capillary electrophoresis (CE) can be used to complement GC and HPLC methods of amphetamines analysis due to their high efficiency, accuracy, very high resolution, and tolerance to biological matrices. Capillary electrophoresis utilizes the electrical nature of charged molecules and enables the separation of molecules based on charged in an applied electrical field (Landers, 1995). MDMA is an organic compound and so its enantiomers are not charged. Hence, for the separation of enantiomers of MDMA, micellar electrokinetic chromatography (MEKC) is utilized (Beesley and Scott, 1998). This is a modified electrophoresis system in which the chiral selector is added to the electrolyte as additives, or be immobilized on the capillary tube surface as a traditional type of stationary phase (Beesley and Scott, 1998). The applied voltage causes the analytes to migrate through the capillary and being separated (Landers, 1995).#p#分頁標題#e#
 

Figure 13 shows the instrument used for micellar electrokinetic chromatography (MEKC). As seen in the figure, during sample separation, the individual analytes are driven in the appropriate direction by their inherent electrophoretic mobility (neutral species are static, anionic species move towards the anode, and cationic species move towards the cathode) with a magnitude represented by the arrows. Concurrently, the EOF of buffer towards the cathode, with a magnitude greater than the individual electrophoretic mobilitles, results in electrophoretic zone formation as all analytes (neutral, positive, and negative) are swept past the detector (Landers, 1995). The detector produces an electropherogram that is almost the same as the one obtained from the gas chromatography (see Figure 14).
 

The chiral selector used in micellar electrokinetic chromatography is usually beta cyclodextrin. Cyclodextrin is an oligosaccharide with an external hydrophilic surface and a hydrophobic cavity, in which they can include other compounds by hydrophobic interaction (Tagliaro, Turrina and Smith, 1996). This allow for the separation of molecules with different sizes, charges and polarity.
 

The aim of this literature review is to investigate the effectiveness of GC/MS and CE in the analysis of MDMA enantiomers. Not only that, the enantioselective disposition of MDMA in hair and urine is also reviewed. The use of hair and urine as a medium for drug detection is also explored.
 

CASE STUDY

Urine analysis

Urine is the most widely used biological specimen for the analysis of illicit drugs (Nakashima, 2006; Rouen, Dolan and Kimber, 2001). According to Ramseier, Caslavska and Thormann (1999), urinary screening of drugs of abuse is usually performed with immunoassay, whereas GC/MS is the standard approach employed for confirmation of the presence and absence of a specific drug or metabolite. The goal of urine drug testing may be stated as the reliable demonstration of the presence, or absence, of specified drugs or metabolites in the specimen (Chiang and Hawks, 1986). Despite a number of persistent shortcomings, such as its susceptibility to tampering, urinalysis is a well-researched technology in which most of the problems have been identified and addressed, if not resolved. It offers an intermediate window of detection making test scheduling an important issue in many situations (Rouen, Dolan and Kimber, 2001).
 

The Physiology of Urine Production

Blood is drained through the kidney in the rate of 1.5 litres per minute. Ultrafiltration of blood that occurs at the kidney leads to the production of urine continuously. During urine production the kidneys reabsorb essential substances. Excess water and waste products, such as urea, organic substances and inorganic substances, are eliminated from the body. The daily amount and composition of urine varies widely depending upon many factors such as fluid intake, diet, health, drug effects and environmental conditions. The volume of urine produced by a healthy adult ranges from 1-2 litres in a 24 hour period but normal values outside these limits are frequently reported (Rouen, Dolan and Kimber, 2001; Pichini, 2005).#p#分頁標題#e#
 

Incorporation of Drugs into Urine

The possible ways of drug disposition in the human body is shown in Figure 15. When a drug is smoked or injected, absorption is nearly instant and excretion in urine begins almost immediately. According to Pichini (2005), 80% of the drug is metabolized by the liver, leaving 20% of the drug to be excreted unaltered. However, absorption is slower when a drug is orally administered and excretion may be delayed for several hours.
 

Generally, a urine specimen will contain the highest concentration of parent drug and metabolite within 6 hours of administration. As for MDMA, the peak concentration is reached after 2 hours of administration (Cone and Huestis, 2009). As drug elimination usually occurs at an exponential rate, for most illicit drugs a dose will be eliminated almost completely within 48 hours.
 

A number of factors influence the detection times of drugs in urine including the quantity of drug administered, parent drug and its metabolite half-life, cut-off level used, and a number of physiological factors. Fallon et al. (1999) reported that the plasma half-life in humans of (R)-MDMA (5.8 ± 2.2 h) was significantly longer than that of (S)-MDMA (3.6 ± 0.9 h). It is also noted that for many of drugs, frequent, multiple dosing over extended periods of time can cause the drug to accumulate in the body resulting in significantly extended detection times, and leads to the possibility of hair analysis which will be discussed in the later part.
 

The detection times in urine are significantly greater than the detection times in blood because most drugs are rapidly eliminated from blood both by the body's metabolic system and by excretion into urine (DuPont and Baumgartner, 1995). As the bladder is emptied only a few times during the day, the urine becomes a reservoir of drugs and metabolites (AIC Research and Public Policy, 2003). According to DuPont and Baumgartner (1995), most abused drugs, including their metabolites, fall to low levels in the blood within a few hours of last drug use and so urine samples generally have a short surveillance window (SW) of about l-3 days (see Table 3). AIC Research (2003) also reported that longer detection time of drugs is due to high doses and high urine pH.
 

Despite of its small detection time, urine testing is still a reliable and convenient way of investigating whether a person has abused drugs in the past few days. The comparison between commonly used specimens for drug analysis is shown in Table 3.
 

Case Study One: Stereochemical Analysis Of 3,4-Methylenedioxymethamphetamine And Its Main Metabolites In Human Samples Including The Catechol-Type Metabolite (3,4-Dihydroxymethamphetamine)
 

Objectives

This case study aims to determine the enantioselective disposition of MDMA and its major metabolites, 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (HHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA) in human urine. The R versus S enantiomer of MDMA and its metabolites in urine samples after administration of known amount of MDMA is also calculated. Other than that, the use of indirect method in determining concentration of MDMA and its metabolites by chemical derivatization is also illustrated.#p#分頁標題#e#
 

Results and Discussion

Urine samples were obtained from seven healthy recreational users of MDMA. They were given a single 100-mg oral dose of (R,S)-MDMA·HCl (Pizarro et al., 2004). Participants were phenotyped with dextromethorphan for CYP2D6 enzyme activity and all were categorized as extensive metabolizers (Schmid et al., 1985). Urine samples were collected before and after drug administration at 0 to 2, 2 to 6, 6 to 12, 12 to 24, 24 to 48 and 48 to 72 hour time periods, acidified with HCl, and stored at around 20°C until analysis (Pizarro et al., 2004). The samples and standard solutions were analyzed by GC/MS using achiral column with 5% phenyl 95% dimethylpolysiloxane cross link (15 m × 0.25 mm × 0.25 µm film thickness) before and after a chiral derivatization.
 

MDMA in the urine sample was derivatized using (R)-(-)-α-methoxy-α-trifluoromethylphenylacetyl chloride (Figure 16) in ethyl acetate/hexane (50:50) that contained 0.015% triethylamine as described by Pizarro et al. (2003). Derivatization step functions to induce volatility to the sample for GC analysis (Beesley and Scott, 1998). A baseline enantiomeric separation was obtained for all the studied compounds in a single run. Chiral analysis of plasma and urine samples was carried out by combining the extraction procedure developed for the high performance liquid chromatography analysis method for HHMA quantification (Segura et al., 2002) and derivatization steps developed for GC/MS determination of enantiomers of MDMA, MDA, HMMA, and HMA (Pizarro et al., 2003). Extraction and derivatization coupling was not achieved easily because chemical properties of extracted samples make it impossible for the target compounds to be derivatized. The presence of considerable amounts of HCl in the elution mixture was responsible for the formation of the corresponding amine chlorhydrate salts making amine reaction unfeasible.


An attempt using evaporation of extracts to eliminate HCl before the first derivatization step gave rise to the precipitation of antioxidant (metabisulphite) and antichelant (EDTA) reagents required for the extraction. These reagents were insoluble in the mixture of solvents required for derivatization and compounds were still in their chlorohydrate form, preventing the correct reaction. Treatment of the precipitated salts with a mixture of ethyl acetate that contained NH3 (2%) allowed the recovery of the target compounds and made them ready for the derivatization procedure (Pizarro et al., 2004).
 

Calculated pharmacokinetic parameters for MDMA and its main metabolites are presented in Table 4 (Pizarro et al., 2004). Pharmacokinetic data of the achiral analysis of MDMA and its main metabolites are similar to those in previous reports (de la Torre et al., 2000a).
 

To check the appropriate fitting between chiral and achiral approaches, results obtained using the achiral method were compared with data of chiral analysis [sum of the corresponding (R)- and (S)-enantiomers] by correlation analysis. For all compounds for which this analysis was performed (MDMA, MDA, and HMMA), the correlation coefficient was higher than r = 0.92 (Pizarro et al., 2004).#p#分頁標題#e#
 

Urine collection was performed until 72 hour post-ingestion, and recoveries of MDMA and metabolites are reported in Table 5. The variability observed is quite acceptable, taking into account enzymes involved in MDMA disposition in humans. Two enzymes crucial in the metabolic disposition of MDMA, CYP2D6 and COMT, are highly polymorphic in humans. Subjects participating in the study were all phenotypically extensive metabolizers for CYP2D6 but in different degree. A relatively high variability in drugs in which metabolism is regulated by CYP2D6 is expected. The picture is further compounded by the contribution of COMT (Pizarro et al., 2004).
 

Results obtained showed that the retention times for (R)-enantiomers of MDMA, MDA and HMMA are always shorter than their corresponding (S)-enantiomers (Pizarro et al., 2004). The enantiomeric ratio (R)/(S) for MDMA is around 2.9 and this result is very close to the ratio of 2.4 reported in a previous study (Fallon et al., 1999). The elimination half-life of the (R)-enantiomer is 3 times higher than that of the (S)-enantiomer (14.8 h versus 4.8 h) and quite similar to the elimination half-life calculated under achiral conditions (11.8 h). This result confirms that (R)-MDMA is the major component of the calculated racemic MDMA elimination half-life (Pizarro et al., 2004). Moreover, the half-life of the (S)-enantiomer fits very well with the kinetics of subjective effects, psychomotor performance, neuroendocrine-induced changes, and cardiovascular effects observed in humans after the use of MDMA in controlled studies (Mas et al., 1999; Cam?´ et al., 2000). In contrast, the longer half-life calculated for the (R)-enantiomer may explain mood and cognitive effects experienced by MDMA consumers on the next days after ingestion (Curran and Travill, 1997). Pharmacological effects of HMMA are still unknown and further research is yet to be done to investigate chiral aspects of their biological activity and disposition (Pizarro et al., 2004).
 

MDA enantiomeric ratios < 1 are opposed to those observed for MDMA. These results most probably reflect changes in the availability of MDMA enantiomers rather than to an enantioselectivity of this metabolic pathway. Urinary recoveries at 24 h and up to 72 h are close to unity, further confirming the lack of enantioselectivity of this pathway (Pizarro et al., 2004).
 

Enantiomeric ratios for HHMA are, as expected, just the reverse of those observed for MDMA. However, they are lower (around 0.65) than expected. This observation is most likely related to the nonlinearity of MDMA pharmacokinetics (de la Torre et al., 2000b) due to inhibition of the CYP2D6 enzyme (responsible for enantioselectivity) as a result of the formation of an enzyme-metabolite complex (Delaforge, Jaouen and Bouille, 1999). When CYP2D6 becomes inactivated, the enantioselectivity of the pathway is lost because other cytochrome P450 isoenzymes (CYP1A2, CYP3A4, and CYP2B6) that begin to be involved in the reaction (Kreth et al., 2000) probably lack this chiral selectivity. In fact, enantiomeric ratios observed for MDMA should be greater than those observed in the absence of this process of autoinhibition of the enantioselective pathway (Pizarro et al., 2004).#p#分頁標題#e#
 

HMMA enantiomeric ratios should follow the same trend as those of HHMA: (R)/(S) ratio <1. In practice, however, they follow a trend close to that observed for MDMA [(R)/(S) ratio >1]. In a recent report in which enantiomeric ratios of MDMA and HMMA in urine were determined (Pizarro et al., 2002), results confirmed MDMA enantioselective disposition, but HMMA (R)/(S) ratios were also close to 1 (first 24 h). Both results may be explained by the autoinhibition of CYP2D6 that, over time and after several metabolic steps (O-demethylenation and O-methylation), make differences between enantiomers minimal. Studies in humans, in which racemic MDEA was administered and MDEA, MDA, and 4-hydroxy-3-methoxyethylamphetamine (the equivalent compound to HMMA in MDMA metabolism) and enantiomers were measured, found that 4-hydroxy-3-methoxyethylamphetamine ratios were lower than 1, although only the first 0 to 4 h in blood samples were assessed (Brunnenberg and Kovar, 2001). This finding is similar to ratios <1 found in this case study. The shift in the R/S ratios in the last phase of the kinetics and in urinary recoveries may indicate a certain degree of enantioselectivity of the catechol methyl transferase (COMT), an assumption that has to be further substantiated experimentally (Pizarro et al., 2004).
 

Hair Analysis Of MDMA Enantiomers

Hair Analysis

According to Rouen, Dolan and Kimber (2001) hair is becoming recognized as a third fundamental biological specimen for drug testing after urine and blood. Hair analysis and urinalysis has become complementary tests for establishing drug use (DuPont and Baumgartner, 1995) on account of their capacity to expose different patterns of drug use. Tsatsakis and Tzatzarakis (2000) claimed that hair analysis provides long-term information, from months to years, concerning the severity and pattern of drug use. It can be said that hair has become a matrix that is suitable to extend the window of detection when compared to blood and urine (Pujol et al., 2007; Al-Dirbashi, et al., 1999).
 

Anatomy and Physiology of Hair

The structure of a hair follicle is shown in Figure 17. The five components that make up human hair follicle include cuticle, cortex, medulla, melanin granules and cell membrane complex. The number of hair follicle ranges from 80, 000 to 100, 000 and it decreases as human ages. Hair follicles (see figure 17) are embedded within 3 – 4 mm in the dermis layer of skin and is highly vascularised to nourish the growing hair root or bulb. This region has a high rate of cell division. As the hair grows, the hair follicle is being pushed outwards and leads to the formation of different layers of hair shaft (cuticle, cortex and medulla). Keratin is the major component of hair. Melanin present in the cortex layer gives the hair colour black. A relatively small amount of amino acids, lipids, carbohydrates and water are present and this give rise to the strength and durability of hair (Rouen, Dolan and Kimber, 2001; Pichini, 2005).#p#分頁標題#e#
 

Hair does not grow continuously but in phases (see Figure 15). Keratogenesis is the process involved in hair growth. The first stage, i.e. anagen phase is the growth phase of hair and is a time of increased metabolic activity and cell division in the hair bulb. Following anagen phase, where cell division stops, and hair shaft becomes fully keratinized the bulb begins to degenerate. The final phase, the telogen or resting phase is the quiescent period where there is no hair growth, the follicle is short and the hair can be easily removed. The resting phase lasts for approximately ten weeks for scalp hair and two to six years for body hair. The rate of hair growth is approximately 1 cm per month and is affected by sex, race and age (Rouen, Dolan and Kimber, 2001; Pichini, 2005).
 

Mechanism of drugs incorporation into hair

Possible pathways for incorporation of drugs into hair include passive diffusion from blood into the hair follicle, excretion onto the surface of hair from sweat and sebum and from external contamination. Drugs enter the growing hair follicle by passive diffusion from the capillaries at the base of the hair bulb and are then bound to the hair shaft during keratogenesis (Pichini, 2005). In the study done by Martins et al. (2007), there is no correlation between the amounts of drug intake with the concentration of the particular drug in hair. In a study using MET as an AMP substitute, Nakahara, Shimamine and Takahashi (1992) found poor correlations between the drug concentrations in the hair of individuals receiving the same dose, but the location of the drugs along the hair shaft was correlated with the time of ingestion. It can be explained that the difference in concentration of the drugs in hair is due to excretion absorption of drug onto or from the surface of hair from sweat, sebum and external contamination. Furthermore, as mentioned above, since hair grows at an average rate of 1 cm per month, it is theoretically possible to extrapolate a record of eventual drug usage by segmental hair analysis (Rouen, Dolan and Kimber, 2001; Pichini, 2005).
 

Inter-individual differences in hair structure and porosity, hair growth, melanin content, hair hygiene and use of cosmetic hair treatments and bleaching have also been shown to have significant effects on the observed concentrations of drugs in hair further increasing the difficulty of inter-individual comparison. Decontamination by certain chemicals is done in the hair analyisis protocol. It is supported by the study done by Tsanaclics and Wicks (2007), it appears that amphetamine is extracted to a comparatively larger extent from the hair by the washing protocol. Another possibility is that the hair may be damaged by coloring and bleaches. It is now vital to develop a method to eliminate false positive results arise from contamination and to establish a reference material for the analysis (Rouen, Dolan and Kimber, 2001; Pichini, 2005).
 

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To our concern, AMP and their related compounds have been determined in consumers' hair (Rohrich and Kauert, 1997; Uhl, 1997; Nakashima, 2006; Al-Dirbashi, et al., 1999, Martins et al., 2007). The present study would like to investigate the stereoselective disposition of MDMA in hair.
 

Case Study Two: Time-resolved hair analysis of MDMA enantiomers by GC/MS-NCI

Objectives

The objective of this case study is to determine the enantioselective disposition of MDMA in human hair. In order to illustrate this, ratio of R and S enantiomers of MDMA and its metabolites in hair samples after administration of known amount of MDMA is calculated. Also, the use of indirect method in MDMA analysis is illustrated.
 

Results and discussion

In this case study, the authentic hair specimens were obtained from 14 self-declared ecstasy abusers following a double-blind placebo-controlled six-way crossover study realized in Maastricht (The Netherlands) for seven weeks (Martins et al., 2007). The subjects were given different amount of MDMA and/or alcohol at different timing as shown in Table 6.
 

Pretreated hair specimens were digested with 1M sodium hydroxide at 100°C for 30min and extracted by a solid phase procedure using Cleanscreen ZSDAU020 (Martins, et al., 2007). Extraction yields were between 73.0 and 97.9%. Limits of detection varied in the range of 2.1–45.9pg/mg hair, whereas the lowest limits of quantification varied between 4.3 and 91.8pg/mg hair (Martins et al., 2005).
 

Drug enantiomers were converted into their diastereomeric derivatives by the derivatization reagent (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride [(S,R)-HFBOPCl]. This chiral derivatisation reagent was developed by Martins et al. (2006) and is readily obtained in optically pure form after a simple two-step synthesis. Optimal derivitization was accomplished in 15minutes at room temperature in a basic carbonate buffer and the resulting diastereoisomers were base line separated by GC in 12minutes only. An added advantage of using this derivatizing agent is that no racemization was observed during the process (Martins et al., 2006).
 

The gas chromatograph was equipped with a Hewlett-Packard HP-5MS (crosslinked 5% phenyl 95% methylpolysiloxane) capillary column (30 m × 0.25 mm × 0.25 µm film thickness). The mass spectrometer was running in the negative chemical ionization (NCI) mode with methane (standard purity > 99.99%) as reagent gas (flow of 40%) using pulsed splitless injection mode (Martins et al., 2007). Not forgetting to mention, the derivatives were readily ionized in the NCI mode due to the electronegativity of the heptafluorobutyryl moiety. Operating the MS in the selected-ion monitoring mode further increased the high sensitivity of this ionization technique (Martins et al., 2007).
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For calibration, 10 mg of drug-free hair were spiked with ATS (AM, MA, MDA, MDMA and MDEA), each enantiomer covering the range from 0.002 to 20 ng/mg. The internal standards (IS) ATS were added at a fixed concentration of 2.5 ng/mg. Assuming a 1:1 ratio between the enantiomers of each analyte, the calibration curves were obtained for each enantiomer by plotting the peak-area ratios of the spiked calibrations standards versus their concentrations. The enantiomers of MDMA, MDA and of the other ATS tested (AM, MA and MDEA) in abusers' hair were quantitated by comparison of their peak-area ratios (enantiomer of analyte versus corresponding enantiomers of IS) to calibration curves (Martins et al., 2007).
 

A total of 47 hair segments of 2 cm length were analyzed for the presence of MDMA and MDA enantiomers as well as for the optical isomers of AM, MA and MDEA (Martins et al., 2007). Relative concentration of MDMA, MDA and AM enantiomers determined after segmental hair analysis is shown in Table 7. For all 3 analytes, the derivatives corresponding to the S-enantiomers were found to elute after those of the corresponding R-enantiomers (see Figure 18). The enantiomeric ratios of each of the compound are shown in Table 8 (Martins et al., 2007).
 

As the growth rate of hair is 1–1.3 cm/month (Rouen, Dolan and Kimber, 2001; Pichini, 2005), both enantiomers of MDMA have been detected in the proximal 2 cm segment, which in major part corresponded to the time when these volunteers participated in the study. Median concentrations of (R)-MDMA and (S)-MDMA in the proximal segment were 0.54 and 0.24 ng/mg, respectively, ranging from 0.14 to 10.12 ng/mg for (R)-MDMA and from 0.06 to 9.97 ng/mg for (S)-MDMA. No correlation could be established between the doses of MDMA ingested under controlled conditions and the concentration of MDMA found in the proximal hair segment. One reason may be that the 2 cm proximal segment collected did not cover the whole period of the controlled study and that the MDMA concentrations observed could also be the result of the ''uncontrolled use'' before the study (Martins et al., 2007). Mieczkowski (1996) mentioned that it is estimated that approximately 5 to 7 days is needed for a drug ingested to be detectable in the scalp hair shaft. Furthermore, although urine tests for the most frequently used illicit drugs had only been performed at each administration day, an undeclared consumption of MDMA cannot be excluded during the controlled study (Martins et al., 2007).
 

In total, 39 of the 47 hair segments analyzed (all hair segments) contained MDMA enantiomers and the total concentration of MDMA ranged from 0.2 to 20.1 ng/mg. In each hair segment, the concentration of (R)-MDMA exceeded those of the (S)-MDMA with ratios varying from 1.02 to 2.75 (Martins et al., 2007).
 

MDA enantiomers were also detected during segmental hair analysis, but their concentrations were lower than those of the non-metabolized MDMA (metabolite/parent drug MDA/MDMA ratio < 0.09), which suggests that MDA is principally the result of the metabolism of MDMA rather than the result of its consumption as a parent drug (Rothe et al., 1997; Cooper et al., 2000). The enantiomeric ratios of MDA varied from 0.60 to 1.60 in the hair segments tested positive for MDA; most MDA positive hair segments presented comparable enantiomeric concentrations. Furthermore, most MDA positive hair segments from one individual presented comparable enantiomeric ratios (Martins et al., 2007).#p#分頁標題#e#
 

It is found that both enantiomers of amphetamines are present in the proximal hair segment and/or in the distal hair segments of four subjects. This indicated that they had consumed amphetamine before the period of studies (Martins et al., 2007).
 

Figure 18 represents the chromatograms of two hair segments from subject 9, who has been tested positive for both enantiomers of MDMA, MDA and AMP. The R/S ratios of AMP determined during this segmental hair analysis ranged from 1.00 to 1.47 with higher concentrations of (R)-AMP in most hair segments. Although the subjects were tested by means of a urine screening just before the administration, the presence of AMP enantiomers in the proximal hair segment of some volunteers may be the result of an undeclared consumption of AMP between the administration sessions and/or of AMP ingestion before the controlled study (Martins, et al., 2007).
 

The stereoselective disposition of MDMA enantiomers (R/S > 1) observed in the hair segments is in accordance with those determined in other human matrices like plasma or urine (Peters et al., 2005; Pizarro et al., 2004; Pichini, 2005). Inter-individual differences observed for the disposition of the MDA enantiomers were also reported by some authors for other human matrixes (Pichini, 2005; Cone and Huestis, 2007).
 

The higher concentration of (R)-AMP determined in our study also confirmed the study done by Nyström et al. (2005). These results seem also to reinforce the assumption that the mechanism of incorporation of ATS in hair is not enantioselective, implying that no inversion of the R/S ratio was observed during the sequestration into the keratin hair matrix. Thus, the disposition of ATS enantiomers may be the consequence of the hepatic stereoselective metabolism, the faster metabolism and/or the faster renal elimination of the (S)-isomers (Paul et al., 2004).
 

The enantiomeric ratios of MDMA, MDA and AMP remained relatively stable in all the segments of one single hair bundle (see Table 7 and 8). Even when a ''1 cm'' segmentation (see Figure 19 and Table 9) was done for the hair specimen of volunteer 1, the enantiomeric disposition of the MDMA, MDA and AMP followed the same trend from the proximal hair segment to the distal hair segment and no inversion of the enantiomeric ratios was observed, which may indicate that the corresponding (R)- and (S)-enantiomers present the same stability after incorporation in the hair specimens (Martins et al., 2007).
 

Case Study Three: Chiral separation of MDMA and related compounds in clandestine tablets and urine samples by capillary electrophoresis/fluorescence spectroscopy
 

Objectives

To compare the effectiveness of capillary electrophoresis and GC/MS in analysis of MDMA enantiomers
 

Results and Discussion

The R-(-)- and S-(+)-isomers of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolite 3,4-methylenedioxyamphetamine (MDA) were prepared, identified by gas chromatography/mass spectrometry (GC/MS) and then used as standards in a series of capillary electrophoresis (CE) experiments. One mL of urine sample was made alkaline by the addition of an excess of K2CO3. The free bases were then extracted into 2 mL of a hexane/CH2Cl2 solution (3:1 v/v) by mixing for 1 minute. After centrifugation, the upper layer was collected and this organic phase was then evaporated to dryness. The residue was dissolved in 10 µL of methanol for the subsequent CE separation.#p#分頁標題#e#
 

Identification of S-(+)/(R)-(-)-MDA and S-(+)/(R)-(-)-MDMA

Figure 20(A) shows the total ion chromatogram (TIC) of a mixture of R-MDA-S-TPC and S-MDA-S-TPC derivatives (upper). The detected peak, with a retention time of 21.7 min permitted the specific characterization of MDA-TPC and its mass fragmentation spectrum is shown at the bottom. Thus, the derivative was sufficiently pure to be used for the subsequent column separation and hydrolysis to give the R-(-)- or S-(+)-MDA. Figure 20(B) shows the TIC of S-(+)-MDA (upper) and the mass fragmentation spectrum (bottom). The presence of specific fragments, such as m/z 44 and 136, permitted this characterization. Following this, the product was checked by polarimetry to confirm the optical rotation. Polarimetry result is consistent with the findings in TIC.
 

Figure 21(A) shows typical fluorescence (?ex/?em = 280/320 nm) electropherograms for (RS)-MDA before (electropherogram a) and after spiking with S-(+)-MDA (electropherogram b). The peak that appeared at 29 min corresponded to S-(+)-MDA; whereas the peak at 28.5 min corresponded to R-(-)- MDA. The inset shows the CE electropherogram of S-(+)-MDA under similar CE conditions. Only one major peak appeared and this is sufficient to proof that S-(+)-MDA synthesized was pure.
 

Similarly, Figure 21(B) shows the fluorescence electropherograms of (R,S)-MDMA before (electropherogram c) and after spiking with R-(-)-MDMA (electropherogram d), respectively. The peak corresponding to R-(-)-MDMA verifies that pure R-MDMA was also successful prepared. Thus, we conclude that the S-(+)/(R)-(-)-MDA and S-(+)/(R)-(-)-MDMA were synthesized successfully and were sufficiently pure to be used as standards in the experiments described.
 

Optimization of the separation of (R,S)-MDMA and related designer drugs

In the case of the CE separation, β-cyclodextrin (β-CD) is the most commonly used chiral additive. However, its solubility in water (1.8 g/100 mL at 25?C) is low (Szejtli, 1991) and highly sulphated cyclodextrins have been developed to improve solubility. Although modified β-cyclodextrins provide unique advantages for routine analyses, the use of conventional β-cyclodextrin can also be used for the separation of MDMA enantiomers, by using optimized CE buffer and separation conditions. The optimum conditions for the separation were investigated, by changing several parameters, such as the concentration of β-cyclodextrinss, the length of the capillary, the concentration of surfactant the organic solvents used.
 

The typical CE chromatograms of the model mixture of MDMA and related drugs are shown in Figure 22.

As shown in Figure 22(A), without using β-CD, the migration order was: MDA < DMMDA < MDMA < BDB < MBDB. These compounds basically migrated in the order of mass per charge. Figure 22(B) (with 50 mM β-CD) shows that these enantiomers can be completely separated when a longer capillary (total: 97 cm; effective length: 92 cm) was used and with β-CD added to the buffer.#p#分頁標題#e#
 

In order to investigate the effects of organic solvents, various methanol- acetonitrile-water (M:A:W) solutions such as M:A:W = 16:4:80; 10:4:86; 8:4:88 were examined, but in all cases the separation was poorer. Other than that, other experimental data show that 50 mM of β-CD was better than 20 mM. The sodium chlorate surfactant (10 mM) and a longer capillary (total 92 cm in length) were found to be necessary for complete separation.
 

Separation and identification of MDA and MDMA in suspect urine samples

As in the previous case studies, the concentration of R-(-)-MDMA was always slightly higher than that of S-(+)-MDMA. In this case study, urine extract from an abuser (No. III in Table 10) was analysed by CE and the results are shown in Figure 23 (frame A: without β-CD; frame B: with 50 mM β-CD).
 

The MDA and MDMA peaks are marked on the figure by spiking with the standards, whereas peaks a–c represents unknown compounds. Comparing Figure 23(A) and 23(B), one of the unknown peak is split into two (a' and a'') after the addition of β-CD. This indicates the presence of enantiomers, possibly of MBDB. Also, addition of β-CD causes MDA and MDMA peaks to be split into two, for their R and S enantiomers respectively.
 

Using the same procedures, ten suspect urine samples were investigated and the results are shown in Table 10. R-(-)-MDMA was found in higher concentrations than the S-(+)-MDMA for all specimens analyzed. These data are in agreement with the previous case studies. Not only that, the concentration of R-(-)-MDA in urine was also found to be lower than that of S-(+)-MDA. It is also found that there are certain discrepancies between the two isomers of the MDA, as shown in Table 10 for ten forensic cases. This is possibly due to the selective disposition of MDA in the liver, as discussed in Case Study One.
 

Fluorescence detection rather than UV is utilized in this study. Studies by Fang et al. (2002) found that in typical human urine samples, only a few native fluorescent compounds are present which fluoresce in the wavelength range of 320 ± 2 nm. MDMA is naturally fluorescent and thus derivatization is unnecessary. This provides an added advantage that with this fluorescence detection, the electropherogram was much simpler than UV detection and can be established in a shorter time. This is due to the UV-absorption of numerous organic compounds in urine sample would produce multiple peaks in the spectrum that would interferes with results interpretation. Thus, CE with fluorescence detection provides a simple way to observe non-natural compounds in urine, such as MDMAs.
 

Last but not least, it is found that the R/S ratio of MDMA in Case Study One is 2.9 while that in Case Study Three is approximately 1.5. The inconsistency of both data arises due to the difference in method of analysis. Perhaps derivatization rather than liquid-liquid extraction allows MDMA enantiomers to be extracted more efficiently. This may need future analysis and support.#p#分頁標題#e#
 

CONCLUSION

This project deals with the simultaneous monitoring of the two optical isomers of MDMA and their excretion kinetics in human hair and urine. Determination of enantiomeric ratio of the compound is vital in the studies of pharmacology and pharmacokinetics of the designer drug.
 

An analytical method has been developed by Pizarro et al. (2004) to determine MDMA enantiomers and those from its major metabolites, 3,4-methylenedioxyamphetamine (MDA), 3,4-dihydroxymethamphetamine (HHMA), and 4-hydroxy-3-methoxymethamphetamine (HMMA). It has been applied to the analysis of plasma and urine samples from healthy recreational users of MDMA who participated voluntarily in a clinical trial and received 100 mg (R, S) - MDMA?HCl orally. (R)/(S) ratios both in plasma (0–48 h) and urine (0–72 h) for MDMA and MDA were >1 and <1, respectively. The short elimination half-life of (S)-MDMA (4.8 h) is consistent with the subjective effects and psychomotor performance reported in subjects exposed to MDMA, whereas the much longer half-life of the (R)-enantiomer (14.8 h) correlates with mood and cognitive effects experienced on the next days after MDMA use.
 

A study was done by Martins et al. (2007) to determine the enantioselective disposition of 3,4-methylenedioxymethamphetamine (MDMA) and other amphetamine-type stimulants (ATS) in segmented hair specimens of self-declared ecstasy abusers, who took part in a double-blind placebo controlled six-way crossover study during approximately 7 weeks, during which they received a 75 and a 100 mg dose of racemic MDMA twice. Hair specimens were washed and cut into pieces of 2 cm length.

After digestion and solid phase extraction, the enantiomers were derivatized with a chiral agent (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, developed at the authors laboratory and quantified by gas chromatography coupled to mass spectrometry operating in the negative chemical ionization mode. Most of the hair specimens that were tested positive for MDMA showed a predominance of the (R)-enantiomer. When segmental analysis was performed on single hair specimens, no inversion of the R versus S ratios of MDMA and MDA was observed. The predominance of (R)-MDMA in hair was in accordance with those already published for other matrices.


Furthermore, both enantiomers of amphetamine (AMP) were also detected in hair segments of four volunteers and the R/S ratios ranged from 1.00 to 1.47. The collected data have shown a stereoselective disposition of MDMA, MDA and AMP in hair similar to other biological specimens. Furthermore, the quite stable distribution of the enantiomeric ratios during segmental analysis of single hair shafts showed that both enantiomers were comparably stable after their incorporation into the hair matrix (Martins et al., 2007).
 

Last but not least, it is also found that CE with fluorescence detection will not be able to replace GC/MS in forensic analysis, as the fluorescence spectrum lacks the specificity of MS that is unequivocally required in forensic analysis in order to hold up in court, this sensitive and rapid method could serve as a reliable complementary method to GC/MS for routine use. The separation of MDMA enantiomers in drug analysis is still relying on the use of GC/MS.#p#分頁標題#e#
 

REFERENCES

AIC Research and Public Policy Series. n.d. Drug Testing Methods. America: AIC Research and Public Policy Series.

Al-Dirbashi, O., Kuroda, N., Inuduka, S., Menichini, F. and Nakashima, K. 1999. HPLC with fluorescence detection of methamphetamine and amphethamine in segmentally analyzed human hair. Analyst, 124, pp. 493-497.

Baker, G.B., Prior, P.I. and Coutts, R.T. 2002. Chirality and drugs used to treat psychiatric disorders. J. Psychiatry Neurosci. 27(6), pp. 401-3.

Beesley, T.E. and Scott, R.T.W. (eds). 1998. Chiral Chromatography. West Sussex, England: John Wiley & Sons.

Berman, S.M., Kuczenski, R., McCracken, J.T. and London, E.D. 2009. Potential Adverse Effects of Amphetamine Treatment on Brain and Behavior: A Review. Mol Psychiatry. 14(2), pp. 123–142.

Brunnenberg, M. and Kovar, K.A. 2001. Stereospecific analysis of ecstasy-like N-ethyl-3,4- methylenedioxyamphetamine and its metabolites in humans. J Chromatogr B. 751, pp. 9–18.

Camí, J., Farre, M., Mas, M., Roset, P.N., Poudevida, S., Mas, A., San, L. and de la Torre, R. 2000. Human pharmacology of 3,4-methylenedioxymethamphetamine ("ecstasy"): psychomotor performance and subjective effects. J Clin Psychopharmacol. 20, pp. 455-466.

Carvalho, P. O., Cass, Q. B., Calafatti, S. A., F. J. Contesini, and, Bizaco, R. 2006. Review- Alternatives For The Separation Of Drug Enantiomers: Ibuprofen As A Model Compound. Brazilian Journal of Chemical Engineering.23(1), pp. 291 – 300.

Chiang, C. N. and Hawks, R. 1986. Implications of drug levels in body fluids: Basic concepts. In: Hawks, R. & Chiang, C. N. (eds.), Urine Testing for Drugs of Abuse. National Institute on Drug Abuse Research Monograph 73, Department of Health and Human Services, Public Health Service and Alcohol, Drug Abuse, and Mental Health Administration, Maryland.

Cho, A.K. and Segal, D.S. (ed.) 1994. Amphetamine and its analogs. San Diego: CA Academic Press.

Clement, B.A., Goff, C.M., Forbes, T.D.A. 1997. Toxic amines and alkaloids from Acacia berlandieri. Phytochemistry , 46(2), pp. 249–254.

Clement, B.A., Goff, C.M., Forbes, T.D.A. 1998. Toxic amines and alkaloids from Acacia rigidula. Phytochemistry, 49(5), pp. 1377–1380.

Cody, J.T. 2005. Amphetamines: Methods of Forensic Analysis. In Frederick P. Smith. (eds). Handbook of Forensic Drug Analysis. London: Elsevier Academic Press.

Cone, E. J. 1997. New developments in biological measures of drug prevalence. NIDA Monograph 167, pp. 108-129.

Cone, E.J. and Huestis, M.A. 2009. Interpretation of Oral Fluid Tests for Drugs of Abuse. Ann N Y Acad Sci.1098, pp.51-103.

Cooper, G.A. Allen, D.L. Scoot, K.S. Oliver, J.S., Ditton, J. and Smith, I.D. 2000. Hair analysis: self-reported use of ''speed'' and ''ecstasy'' compared with laboratory findings, J. Forensic Sci. 45, pp. 400–406.#p#分頁標題#e#

Curran, H.V. and Travill, R.A. 1997. Mood and cognitive effects of ±3,4-methylenedioxymethamphetamine (MDMA, ecstasy): week-end "high" followed by mid-week low. Addiction. 92, pp. 821–831.

Danielson, N.D., Gallagher, P.A. and Bao, J.J. 2000. 'Chemical Reagents and Derivatization Procedures in Drug Analysis' In Meyers, R.A. (Ed.). Encyclopedia of Analytical Chemistry. Chichester: John Wiley & Sons, pp. 7042–7076.

Delaforge, M., Jaouen, M. and Bouille, G. 1999. Inhibitory metabolite complex formation of methylenedioxymethamphetamine with rat and human cytochrome P450. Particular involvement of CYP2D. Environ Toxicol Pharmacol. 7, pp. 153–158.

de la Torre, R., Farré, M., Roset, P.N., Pizarro, N., Segura, J. and Camí, J. 2000a. Pharmacology of MDMA in humans. Ann NY Acad Sci. 914, pp. 225–237.

de la Torre, R., Farré, M., Ortunõ, J., Mas, M., Brenneisen, R., Segura, J. and Camí, J. 2000b. Non-linear pharmacokinetics of MDMA ("ecstasy") in humans. British J. Clinical Pharmacol. 49, pp. 104–109.

De Letter, E.A., Clauwaert, K.M., Lambert, W.E., Van Bocxlaer, J.F., De Leenheer, A.P. and Piette, M.H.A. 2002. Distribution Study of 3,4-Methylenedioxymethamphetamine and 3,4-Methylenedioxyamphetamine in a Fatal Overdose. Journal of Analytical Toxicology. 26, pp. 113-118.

DuPont, R. L. and Baumgartner, W. A. 1995. Drug testing by urine and hair analysis: Complementary features and scientific issues. Forensic Science International. 70, pp. 63-76.

Fang, C., Chung, Y.L., Liu, J.T. and Cheng, H.L. 2002. Rapid analysis of 3,4-methylenedioxymethamphetamine: a comparison of nonaqueous capillary electrophoresis/fluorescence detection with GC/MS. Forensic Science International. 125, pp. 142-148.

Fallon, J.K., Kicman, A.T., Henry, J.A., Milligan, P.J., Cowan, D.A. and Hutt, A.J. 1999. Stereospecific Analysis and Enantiomeric Disposition of 3,4-Methylenedioxymethamphtamine (Ecstasy) in Humans. Clinical Chemistry. 45(7), pp. 1058-1069.

Freudenmann, R. W., Öxler, F. and Bernschneider-Reif, S. 2006. The origin of MDMA (ecstasy) revisited: the true story reconstructed from the original documents. Addiction. 101, pp. 1241-1245.

Gouzoulis-Mayfrank, E., Thelen, B., Habermeyer, E., Kunert, H.J., Kovar, K.A., Lindenblatt, H., Hermle, L., Spitzer, M. and Sass, H. 1999. Psychopathological, neuroendocrine and autonomic effects of 3,4-methylenedioxyethylamphetamine (MDE), psilocybin and d-methamphetamine in healthy volunteers. Results of an experimental double-blind placebo-controlled study. Psychopharmacology . 142, pp. 41-50.

Hensley, D and Cody, J.T. 1999. Simultaneous Determination of Amphetamine, Methamphetamine, Methylenedioxyamphetamine (MDA), Methylenedioxymethamphetamin(MDMA) and Methylenedioxyethylamphetamine(MDEA) Enantiomers by GC-MS*. Journal of Analytical Toxicology. 23, pp. 518-523.

International Narcotics Control Board. 2003. List of psychotropic substances under international control. Vienna: International Narcotics Control Board.#p#分頁標題#e#

Iio, R., Chinaka, S., Takayama, N. and Hayakawa, K. 2005. Simultaneous CHiral Analysis of Methamphetamine and Related Compounds by Capilary Electrophoresis/Mass Spectroscopy Using Anionic Cyclodextrin. Analytical Sciences.25, pp. 11-19.

Kolbrich , E.A., Goodwin, R.S., Gorelick D.A. Hayes, R.J., Stein, E.A. and Heustis, M.A. 2008. Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine After Controlled Oral Administration to Young Adults. Ther Drug Monit. 30(3), pp. 320-332.

Kreth, K., Kovar, K., Schwab, M. and Zanger, U.M. 2000. Identification of the human cytochrome P450 involved in the oxidative metabolism of "Ecstasy" related designer drugs. Biochem Pharmacol. 59, pp. 1563–1571.

Landers, J.P. 1995. Clinical Capillary Electrophoresis. Clin. Chem. 41(4), pp. 495 – 509.

Leffingwell, J.C. 2003. Chirality & Bioactivity I.: Pharmacology. Leffingwell Reports. 3(1), pp. 1-27.

Liechti, M.E., Gamma, A., and Vollenweider, F.X. 2001. Gender differences in the subjective effects of MDMA. Psychopharmacology. 154, pp. 161-168.

Lyon RA, Glennon RA, Titeler M (1986) 3,4-Methylenedioxymethamphetamine (MDMA): stereoselective interactions at brain 5-HT1 and 5-HT2 receptors. Psychopharmacology (Berl) 88: 525-6

Maier, N.M., Franco, P., and Lindner, W. 2001. Separation of Enantiomers: Needs, Challenges, Perspectives, J. Chromatography A, 906 (1-2), pp. 3-33.

Martins, L.F., Yegles, M., Samyn, N. Ramaekers, J.G. and Wennig, R. 2007. Time-resolved hair analysis of MDMA enantiomers by GC/MS-NCI. Forensic Science International. 172, pp. 150-155.

Martins, L.F., Yegles, M., Chung, H.S. and Wennig, R. 2005. Simultaneous enantioselective determination of amphetamine and congeners in hair specimens by negative chemical ionization gas chromatography-mass spectrometry. J. Chromatogr. B. 825, pp. 57–62.

Martins, L.F., Yegles, M., Chung, H.S. and Wennig, R. 2006. Sensitive, rapid and validated gas chromatography/negative ion chemical ionization-mass spectrometry assay including derivatization with a novel agent for the enantioselective quantification of amphetamine-type stimulants in hair, J. Chromatogr. B. 842, pp. 98–105.

Mas, M., Farré, M., de la Torre, R., Roset, P.N., Ortunõ J., Segura, J. and Camí, J. 1999. Cardiovascular and neuroendocrine effects and pharmacokinetics of 3,4-methylenedioxymethamphetamine in humans. J Pharmacol Exp Ther. 290, pp. 136–145.

Matsushima, K., Nagai, T. and Kamiyama, S. 1998. Optical Isomer Analysis of 3,4-Methylenedioxyamphetamine Analogues and their Stereoselective Disposition in Rat. Journal of Analytical Toxicology. 22, pp. 33-39.

McConathy, J. and Owen, M.J. 2003. Stereochemistry in Drug Action. J. Clin Psychiatry. 5(2), pp. 70-73.

McMurry, J. (eds). 2004. Organic Chemistry. CA: Thomson Learning.

Meng, P., Fang, N., Wang, M., Liu, H., Chen, D.D.Y. 2006. Analysis of amphetamine, methamphetamine and methylenedioxy-methamphetamine by micellar capillary electrophoresis using cation-selective exhaustive injection. Electrophoresis. 27, pp. 3210–3217.#p#分頁標題#e#

Nakahara Y., Shimamine, M. and Takahashi, K. 1992. Hair analysis for drugs of abuse. III. Movement and stability of methoxyphenamine (as a model compound of methamphetamine) along hair shaft with hair growth. Journal of Analytical Toxicology, 16, pp. 253-257.

Nakashima, K. 2006. Development and application of highly sensitive high performance liquid chromatography methods for analysis of drugs of abuse. Chromatograph. 27(1).

Nystro¨m, I., Trygg, T., Woxler, P., Ahlner, J., Kronstrand, R. 2005. Quantitation of R-(-)- and S-(+)-amphetamine in hair and blood by gas chromatography mass spectrometry: an application to compliance monitoring in adult attention deficit hyperactivity disorder treatment. J. Anal. Toxicol. 29, pp. 682–688.

Nichols, D.E. 1986. Differences between the mechanism of action of MDMA, MBDB, and the classic hallucinogens. Identification of a new therapeutic class: entactogens. J Psychoactive Drugs. 18, pp. 305-313.

O'Donohoe, A., O'Flynn, K., Shields, K., Hawi, Z. and Gill, M. 1998, MDMA toxicity: no evidence for a major influence of metabolic genotype at CYP2D6, Addiction Biology. 3, pp. 309–314.

Parrott, A., Morinan, A., Moss, M. and Scholey, A. 2004. Understanding Drugs and Behavior. West Sussex: John Wiley and Sons.

Paul, B.D., Jemionek, J., Lesser, D. and Jacobs, A. 2004. Enantiomeric separation and quantification of (±)-amphetamine, (±)-methamphetamine, (±)- MDA, (±)-MDMA and (±)-MDEA in urine specimens by GC-EI-MS after derivatization with (R)-(-)- or (S)-(+)-a-methoxy-a-(trifluromethy) phenylacetyl cholride (MTPA). J. Anal. Toxicol. 28, pp. 449–455.

Pavlova, V. and Petrovska-Jovanovic', S. 2007. Analytical Study Of Amphetamine And Methamphetamine By Reversed-Phase High-Performance Liquid Chromatography Method. Chemistry. 16(3), pp. 226-238.

Peter, F.T., Samyn, N., Lamers, C.T.J., Riedel, W.J., Kraemer, T., de Boeck, G. and Maurer, H.H. 2005. Drug Testing in Blood: Validated Negative-Ion Chemical Ionization Gas Chromatographic–Mass Spectrometric Assay for Enantioselective Measurement of the Designer Drugs MDEA, MDMA, and MDA and Its Application to Samples from a Controlled Study with MDMA. Clinical Chemistry. 51(10), pp. 1811–1822.

Pichini, S. 2005. Distribution of 3,4-methylenedioxymethamphetamine (MDMA) in nonconventional matrices and its applications in clinical toxicology. Unpublished doctorate thesis. Doctorate Program in University Autonoma de Barcelona.

Piper B.J. 2008. A Developmental Comparison of The Neurobehavioral Effects Of Ecstasy (MDMA). Neurotoxicol Teratol. 29(2), pp.288–300.

Pirnay, S.O., Abraham, T.T. and Huestis, M.A. 2006. Sensitive Gas Chromatography-Mass Spectrometry Method for Simultaneous Measurement of MDEA, MDMA, and Metabolites HMA, MDA, and HMMA in Human Urine. Clin Chem.52(9), pp. 1728-1734.

Pizarro, N., Ortunõ, J., Farré, M., Hernández-López, C., Pujadas, M., Llebaria, A., Joglar, J., Roset, P.N., Mas, M. and Segura, J. 2002. Determination of MDMA and its metabolites in blood and urine by gas chromatography-mass spectrometry and analysis of enantiomers by capillary electrophoresis. J Anal Toxicol. 26, pp. 157–165.#p#分頁標題#e#

Pizarro, N., Llebaria, A., Cano, S., Farré, M., Segura, J. and de la Torre, R. 2003. Method development for stereochemical analysis of MDMA and its main metabolites by GC-MS. Rapid Commun Mass Spectrom. 17, pp. 330–336.

Pizarro, N., Farré M., Pujadas, M., Peiró, A.M., Roset, P.N., Joglar, J. and de la Torre, R. 2004. Stereochemical Analysis Of 3,4-Methylenedioxymethamphetamine And Its Main Metabolites In Human Samples Including The Catechol-Type Metabolite (3,4-Dihydroxymethamphetamine). Drug Metabolism and Disposition. 32(9), pp. 1001-1007.

Meng, P., Fang, N., Wang, M., Liu, H. and Chen, D.D.Y. 2006. Analysis of amphetamine, methamphetamine and methylenedioxy-methamphetamine by micellar capillary electrophoresis using cation-selective exhaustive injection. Electrophoresis, 27, pp. 3210–3217.

Porter, W.H. 1991. Resolution of chiral drugs. Pure & Appl. Chem. 63(8), pp. 1119-1122.

Pujol, M. L., Cirimele, V., Tritsch, J.P., Villain, M. and Kintz, P. 2007. Evaluation of the IDS One-StepTM ELISA kits for the detection of illicit drugs in hair. Forensic Science International. 170, pp. 189-192.

Ramseier, A., Caslavska, J. and Thormann, W. 1999. Stereoselective Screening for and confirmation of urinary enantiomers of amphetamine, methamphetamine and designer drugs, methadone and selected metabolites by capillary electrophoresis. Electrophoresis. 20, pp. 2726-2738.

Rohrich, J. and Kauert, G. 1997. Determination of amphetamine and methylenedioxy-amphetamine derivatives in hair. Forensic Science International. 84, pp. 179-188.

Rothe, M., Pragst, F., Spiegel, K., Harrach, T., Fischer, K. and Kunkel, J. 1997. Hair concentrations and self-reported abuse history of 20 amphetamine and ecstasy users. Forensic Sci. Int. 89, pp. 111–128.

Rouen, D., Dolan, K. & Kimber, J. 2001. A review of drug detection testing and an examination of urine, hair, saliva and sweat. Technical Report No. 120, National Drug and Alcohol Research Centre. University of New South Wales: Sydney.

Schmid B, Bircher J, Preisig R, and Kupfer A (1985) Polymorphic dextromethorphan metabolism: co-segregation of oxidative O-demethylation with debrisoquin hydroxylation. Clin Pharmacol Ther. 38, pp. 618–624.

Schwab, M., Seyringer, E., Brauer, R.B., Hellinger, A. and Griese, E.U. 1999. Fatal MDMA intoxication, Lancet. 353(9152), pp. 593–594.

Segura, M., Ortuno, J., McLure, J.A., Pujadas, M., Pizarro, N., Farre, M., Llebaria, A., Joglar, J., Segura, J. and de la Torre, R. 2002. High-performance liquid chromatography with electrochemical detection applied to the analysis of 3,4-dihydroxymethamphetamine in human plasma and urine. J Chromatogr B. 769, pp. 313–321.

Shulgin, A. 1986. The Background and Chemistry of MDMA. Journal of Psychoactive Drugs. 18(4), pp. 291-304.

Szejtli, J. 1991. Cyclodextrin in Drug Formulations: Part II. J. Pharm.Tech.Int. 3, pp. 24 – 38.#p#分頁標題#e#

Tancer, M. and Johanson, C.E. 2003. Reinforcing, subjective, and physiological effects of MDMA in humans: a comparison with d-amphetamine and mCPP. Drug Alcohol Depend. 72, pp. 33-44.

Tagliaro, F., Turrina, S. and Smith, F.P. 1996. Capillary electrophoresis: principles and applications in illicit drug analysis. Forensic Science International. 77, pp. 211-229.

Tsanaclis, L. and Wicks, J.F.C. 2007. Differentiation between drug use and environmental contamination when testing for drugs in hair. Forensic Science International. 176, pp. 19–22.

Tsatsakis, A. M. and Tzatzarakis, M. 2000. Sectional hair testing. Judicial and clinical applications*. Pure Appl. Chem. 72(6), pp. 1057–1066.

Uhl, M. 1997. Determination of drugs in hair using GC/MS/MS. Forensic Science International. 84, pp. 281-294.

United Nations Office on Drugs and Crime (UNODC). 2006. Recommended Methods For The Identification And Analysis Of Amphetamine, Methamphetamine And Their Ring-Substituted Analogues In Seized Materials. United Nations Office on Drugs and Crime, Vienna.

Vollenweider, F.X., Gamma, A., Liechti, M., and Huber, T. 1998. Psychological and cardiovascular effects and short-term sequelae of MDMA ("ecstasy") in MDMA naïve healthy volunteers. Neuropsychopharmacology. 19, pp. 241-251.

Wikipedia. 2009. MDMA. [Online] (Updated 14 December 2009) Available at http://en.wikipedia.org/w/index.php?title=MDMA&oldid=331637232. [Accessed 15 December 2009].