The determination of stimulating addictive substances (amphetamine, ephedrine, ecstasy) by LC-MS, GC-MS and CE-MS

Transcription

1 The determination of stimulating addictive substances (amphetamine, ephedrine, ecstasy) by LC-MS, GC-MS and CE-MS Stimulating amines Stimulating amines belong among the compounds (stimulants) increasing the levels of norepinephrine, serotonin, and dopamine in the brain. They are used in the treatment of the attention deficit hyperactivity disorder [ADHD] especially in children, and also in the treatment of brain injury, narcolepsy and chronic fatigue syndrome. Some stimulating amines were originally used as an anorectic agent to suppress hunger and weight loss. Stimulating amines belong among the drugs. Drug can be defined as any substance that after introduced into a living organism may modify one or more of its functions and induce the dependence. Except the stimulant amines belong among stimulants also e.g. caffeine and thein, nicotine and cocaine, and partly ecstasy. Chemical structures of drugs belonging to the group of amphetamines (phenethylamines of amphetamine type) and their precursors are illustrated in Fig. 1 Fig. 1: Structures of amphetamines and their precursors

2 Stimulating drugs have generally these effects on the human body: 1) increase internal tension and fear, causing anxiety and psychosis, 2) increase the risk of aggressive behavior, 3) lead to the changes of behavior (e.g. increase self-confidence, lead to an overestimation of own abilities, 4) discontinuation of the use of stimulating amines leads to sleepiness, tiredness, increased dreaming, dysphoria and depression, 5) the use leads to psychological dependence, physical dependence arises. Amphetamine and methamphetamine Amphetamine (methylphenethylamine) and methamphetamine (N-methyl-α-methylphenetylamine). The free base is available in liquid form. Amphetamine is mostly distributed as sulfate, hydrochloride or phosphate in the form of white to yellowish crystalline solid. The purity of distributed amphetamine in illegal street sale is usually less than 20%. Amphetamine is sometimes available also in the form of tablets. The synthesis of amphetamine and methamphetamine There are many methods of the synthesis of amphetamine. The simplest synthesis of amphetamine is Leuckart synthesis. Except Leuckart synthesis there are also for example Birch method (used by the Nazis during the II. World War) and method using hydrogen iodide. Both synthetic ways are shown in Fig. 2. (I.) (II.)

3 (III.) Fig. 2: The reaction scheme leading to synthesis of methamphetamine. (I.) Leuckart synthesis, (II.) method using hydrogen iodide, (III.) Birch synthesis. Amphetamine is usually diluted by caffeine to be masked the low levels of amphetamine in a dose and to enhance the stimulatory effect of amphetamine. Amphetamine and methamphetamine are also optically active compounds, both occurring in the two optical isomers L- a D-. D-enantiomers are more physiologically effective forms, but they are eliminated from the body more rapidly than the corresponding L-isomers. Metabolism of amphetamine and methamphetamine A substantial part of the applied methamphetamine is excreted unchanged in urine (about 45% during 24 hours after application). Excretion is strongly dependent on ph. In acidic urine is excreted in unchanged form up to 76%, whereas in alkaline urine it is only about 2% of the administered dose. About 15% of the dose is metabolized in the liver by hydroxylation to hydroxymethamphetamine. Approximately 7% of the dose is metabolized by N-demethylation to amphetamine, from which is then formed hydroxyamphetamine (2-4%) and norephedrine (2%) (from which hydroxynorefedrin (0.3%) is formed), and finally phenylacetone (0.9%) further metabolized to benzoic acid, respectively. hippuric acid. One of the metabolites, namely amphetamine is active but has weaker effects than methamphetamine. Schematic illustration of metabolism of methamphetamine and amphetamine is shown on Fig. 3 Fig. 3: Schematic illustration of metabolism of methamphetamine and amphetamine

4 MDMA Methylendioxymethamfetamine (MDMA) belongs to a group of drugs with stimulant effects commonly referred as the phenethylamine. Except MDMA there are several structural similar substances having similar effects on the human body (MDA - methylendioxyamphetamine, MDEA - methylendioxyethylamphetamine, MBDB - N-methyl-1-(1,3-benzodioxol-5-yl)-butanamine. MDMA is the most common and most frequently used as a component of tablets known as ecstasy (dance drug). The tablets have a typical dimension of about 10 mm, flat or possibly biconvex shape and usually weigh in the range from 200 to 300 mg. The tablets have engraved distinctive logo or structure. Characteristic design of tablet is not only linked to ecstasy but also to other drugs e.g. amphetamine. The logo on tablets says anything about the content and the chemical structure of the drug. Hundreds of different logos on tablets containing the drug have been recorded until today. One of many ways how can tablets containing ecstasy look like is shown in Fig. 4. Fig. 4: The example of tablet containing MDMA The main pharmacological effect of MDMA is to increase the secretion and the inhibition of serotonin, norepinephrine and dopamine in the brain. MDMA causes euphoria, increased empathy, the feeling of increased energy in the body and also enhance tactile sensations. The use of MDMA is also often associated with transient hypertension, hyperthermia and dehydration, while long-term exposure leads to depression due to reduced production of serotonin in the CNS. Synthesis of MDMA Several synthetic methods have been described, while two the most known are shown in the following reaction scheme (Fig. 5) Fig. 5: Reaction scheme of MDMA synthesis The Identification of reaction intermediates and byproducts of the synthesis allows uniquely identifying the specific synthetic routes and detecting the origin of drugs.

5 The proof of MDMA by color reaction Marquis reagent gives a deep blue color with MDMA. The reaction is not specific, Marquis reagent reacts with other medications or drugs (e.g. phenothiazines, beta blockers and alkaloids). Marquis reagent: To 5 ml of 37-40% formaldehyde solution is carefully added 100 ml of concentrated sulfuric acid (18 M). The analysis of stimulating amines by chromatographic methods Chromatographic methods (GC, LC) and capillary electrophoresis (CE) are the most suitable to distinguish and identify the individual stimulating amines in biological materials or in materials retained in the illegal distribution networks. Especially in the case of the analysis of the biological material is necessary to do a pretreatment and extraction of stimulant amines. Stimulating amines in the body are subject to only very minimally conjugation and therefore the hydrolysis of metabolites (conjugates) is not necessary before extraction. As extraction method is most commonly used extraction liquid-liquid (L-L) extraction and solid phase extraction (SPE). For subsequent analysis of stimulant amines by GC is required the derivatization of amines, while derivatization of amines prior analysis by LC is not necessary. In many cases the derivatization of stimulating amines before LC analysis is performed. Derivatization is not necessary in the case of subsequent analysis by CE. Derivatization of amphetamines for GC In the case of GC the peaks of stimulating amines are very often spread out and distorted ("called tailing") and therefore the derivatization is carried out to improve the shape of the peaks, and reduce unwanted sorption of analytes to the stationary phase. The derivatization of amphetamines before GC analysis provides the increasing of the selectivity of the determination and the reduction of adverse interferents. The derivatization can be performed as an achiral or chiral. The achiral derivatization by derivatizing reagents is used more frequently, and especially in cases where the separation of the individual optical isomers of stimulating amines is not required. If the ratio of individual enantiomers of stimulant amines in the sample is studied, the derivatization with chiral derivatizing reagents has to be performed. Another advantage of the derivatization may be the increasing of the sensitivity and selectivity of detection by mass spectrometry. Stimulating amines do not provide a high sensitivity of mass spectrometry using electron ionization. By the introducing of electronegative residue in the molecule of stimulating amines significantly increases the ionization efficiency and thus detection sensitivity compared to underivatized analytes. Additionally MS spectra of derivatives of stimulating amine are much more unique than MS spectra of underivatized amines which contain nonspecific fragments and thus their identification is very complex. The same non-specific fragments are also commonly found in MS spectra of biological samples, in which the stimulating amines are present. The most frequent derivatization reactions for the derivatizing of stimulating amines include silylation, acylation and alkylation. As derivatizing agents are used trifluoroacetic acid anhydride (TFA) pentafluorpropionic acid anhydride (PFPA), heptafluorobutanoic acid anhydride (HFBA) and also perfluorooctanoylchlorid, carbethoxyhexafluorobutyrylchlorid (CB) and N-methyl-N-tbutyldimethylsilyltrifluoroacetamide (MTBSTFA). For chiral derivatization, trifluoroacetyl-lprolylchlorid (L-TPC), pentafluoropropionyl-l-prolylchloride (L-PPC) and L-heptafluorobutyryl prolylchlorid (L-HPC) are used.

6 Except the GC-MS connection for detecting of stimulating amine derivatives, flame ionization detector (FID), nitrogen-phosphorus detector (NPD), and electron capture detector (ECD) are also used. Fig. 6: The example of acetylation of amphetamines and their derivatives Derivatization of amphetamines for LC The retention of stimulating amines in LC is generally better than in the case of GC analysis. However, there is the derivatization performed for improving retention characteristics and increasing the detection sensitivity. For example, amphetamine absorbs UV light, but its molar decadic absorption coefficient is low. By introducing of another chromophore in a molecule of stimulating amine will increase the absorption of UV radiation and thus the detection sensitivity. Some derivatizing agents allow performing sensitive fluorescence detection and electrochemical detection. As the derivatizing agents for LC are used 3,5 dinitrobenzylchlorid (DNB), phenyl isothiocyanate, 4- (N,N-dimethylaminosulphonyl), 7-fluoro-1,2,3-benzoxadiazole (DBD-F), fluorosceinisothiocyanate, 1,2 -naftochinon-4-sulfonate, fluorenylmethylchloroformiate (FMOC-Cl) and dansyl chloride. For chiral derivatization is used Marfy s reagent (1-fluoro-2,4-dinitrophenyl-5-L-anilinamide) and (-)-1-(9-fluorenyl) ethyl chloroformate (FLEC) and fluorenylmethylchloroformiate-l-prolyl chloride (FMOC ). L-L extraction of stimulating amines L-L presents the simplest method for the extraction of stimulating amines from biological material. Stimulating amines have pk of about 10. They are basic compounds which pass to an organic solvent from the aqueous phase with basic ph. Neutral substances are extracted with basic compounds, so that the extract is usually purged with reextraction of the aqueous phase and finally back into the organic phase. NaOH is typically used as an agent for adjusting the ph of the aqueous sample prior to extraction with an organic agent. Task: Do the isolation of amphetamines from submitted a urine sample and identify each stimulating amines in the sample and make their determination. Chemicals: standard solutions of methamphetamine, amphetamine and ephedrine with the concentration of 10 mg/l in water, 50% (w/v) solution of NaOH, 1-chlorobutane, dilute sulfuric acid (1:1), deionized water, heptafluorobutanoic acid anhydride, methanol, Tools: microtubes, vials for GC, LC and CE, ultrasound, beakers, graduated cylinder, termovap for evaporation under nitrogen stream, SPE manifold, SPE columns BondElut RP/katex

7 Workflow: L-L extraction a) To 2 ml of sample in a test tube is added 50% (w / v) NaOH solution, the resulting ph should be about 10. b) To the sample is further added 5 ml of 1-chlorobutane and shaken 5 minutes. After constitutional balance the organic phase is transferred to a clean tube. c) To the organic phase in a clean test tube is added dilute sulfuric acid (5 ml) and the tube is again sealed and shaken for 5 min. d) The aqueous phase after reextraction is transferred to another clean tube and stimulating amines are back extracted with 5 ml of 1-chlorobutane. The organic phase after extraction is transferred to a clean tube and allowed to evaporate at room temperature under a stream of nitrogen. e) The extract is derivatised using HFBA for the analysis by GC-MS. f) For the analysis by LC and CE is not necessary to derivatize the sample. Evaporated extract is reconstituted in 200 ul of methanol and charged into the liquid chromatography or capillary electrophoresis. SPE extraction For SPE extraction is recovered SPE columns containing a mixture of cation-exchange and reverse phase (SPE BondElute Certify RP/Katex). 1. Place 3 SPE columns into the SPE extractor and perform their gradual conditioning. First condition the columns with 2 ml of methanol and then with 2 ml of buffer with 100 mm sodium phosphate ph Subsequently apply 5 ml of urine sample and 5 ml of the standard mixture of amphetamine and methamphetamine and a blank (deionized water) onto the columns and the columns washed again with 2 ml of 100 mm sodium phosphate ph Then wash all three columns with 2 ml of 1 M acetic acid and dry the columns with a stream of nitrogen and wash them with 6 ml of methanol. 4. Perform the elution of captured analytes with 2 ml of mixture of dichloromethane/isopropanol/ammonium hydroxide (78/20/2). 5. Eluted samples evaporate to dryness under a nitrogen stream; reconstitute the extract in 50 ul mobile phase or appropriate buffer for CE and perform the analysis. 2. The analysis Gradually measure the extract after derivatization by GC-MS and extracts without derivatisation by LC-ESI-MS and CE-ESI-MS with the help of the tutor. Questions: 1. Which other analytical techniques could be used for the detection of amphetamines and their metabolites in urine and blood? 2. Design the fragmentation pathways for EI-MS amphetamine. Literature: 1. S. Bell, Forensic Chemistry, Prentice Hall, 2 nd edition, D.G. Barceloux, Medical Toxicology of Drug Abuse, Wiley, B. Levine ed., Principles of Forensic Toxicology, AACCPress, 4 edition, W.R. Kuelpmann ed., Clinical Toxicology Analysis Vol.1,2, Wiley J. Bogusz: Forensic Science, Wiley, 2000.