Frison G.. Tedeschi L., Favretto D., Ferrara S.D.
Forensic Toxicology and Antidoping, University Hospital, ViaFalloppio 50,1-35121 Padova, Italy A new analytical approach, based on liquid/liquid extraction, derivatization with 2,2,2 trichloroethyl chlo-roformate and gas chromatography-mass spectrometry, has been developed for the quali-quantitative ana-lysis of amphetamine analogs and ephedrines in plasma, urine and hair samples.
Analytes under study were amphetamine, methamphetamine, 2,5DMA (2,5-dimethoxyamphetamine), MDA (3,4-methyIenedioxyamphetamine), MDMA (3,4-methylenedioxymethamphetamine), DOM (2,5dimethoxy-4-methylamphetamine), MDEA (3,4-methylenedíoxyethylamphetamine), MBDB (N-methyl-l-(3,4-methylenedioxyphenyl)-2-butanamine), BDB (3,4-(methylenedioxyphenyl)-2-butanami-ne), TMA (3,4,5-trimethoxyamphetami(3,4-(methylenedioxyphenyl)-2-butanami-ne), DOB (4-bromo-2,5-dimethoxyamphetami(3,4-(methylenedioxyphenyl)-2-butanami-ne), ephedrine, pseudoephedrine, and phenylpropanolamine.
Sample preparation involves a basic extraction of the analytes, using Extrelut columns, from plasma, urine or aqueous supernatants obtained from hair samples incubated with acid methanol, after the addition of the internal standard MDPA (3,4-methylenedioxypropylamphetamine), and a subsequent derivatization to pro-duce 2,2,2 trichloroethyl carbamate derivatives.
GC-MS analysis is carried out by means of quadrupole or ion trap instruments, injecting 2-ul aliquots at 250° C under splitless conditions, using a 30-m slightly polar capillary column with an oven temperature program from 50° C to 300° C and a carrier gas (He) flow of 1 ml/min. MS acquisition mode is Selected Ion Monitoring (SIM) under EI ionization (quadrupole) or multiple mass spectrometry under CI ioniza-tion (ion trap).
EI mass spectra of 2,2,2 trichloroethyl carbamates show weak but characteristic high-mass molecular ions as well as intense diagnostic fragment ions for each analyte, characterized by complex ion clusters due to the isotope effect of three chlorine atoms in the derivatized molecules. CI mass spectra show strong pro-tonated molecular ions which can be selectively stored in the ion trap, and collided with gas to obtain cha-racteristic product ions.
Using the quadrupole mass spectrometer, linearity for most analytes can be obtained over a range of 10 -2000 ng/ml (plasma and urine) and 0.20 - 20 ng/mg (hair). Corresponding limits of detection are in the range 2 - 5 ng/ml and 0 . 1 0 - 0 . 1 5 ng/mg. Main advantages of this analytical approach are its specificity and sensitivity. Applications deal with clinical and forensic toxicology as well as antidoping control.
Annales de Toxicologie Analytique, vol. XIV, n° 3, 2002
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Contribution of the Raman spectroscopy in the characterization of ecstasy derivatives
Belhadj-Tahar H.">. MolnarY.G.(2), Payoux R<», Coulais Y.f,), Costes J.P. <2>, Robert L.(l>, Esquerre J.P.( , ), Bousseksou A/2'(1) CHU Purpan, Toulouse, France
(2) Laboratoire de Chimie de Coordination du CNRS 8241, Toulouse, France
The Raman Spectroscopy, which is an in situ and non destructive method, analyses the chemical bindings of a molecule through its intramolecular vibrations. This is an attractive alternative method in order to cha-racterize Ecstasy tablets, witch are principally used by young people. Indeed this recreation synthetic drug is a real health society problem.
The aim of this work is first to synthesize a series of 3-4 methylenedioxymetamphetamine derivatives and to characterize them by HPLC and spectroscopic (RMN et IR) methods and then to develop a new cha-racterization method using the Raman effect.
40 DO
The condensation of piperonal (1) with ammonium acetate and an excess of nitroethane leads to 3,4 methylendioxyphenyl-2-nitropropene (2). The 3,4-methylenedioxy-amphetamine (3, the Love) is obtained by reduction of (2) by the lithium aluminum hydride ( L Í A I H 4 ) .
The chemical compounds are analyzed by HPLC and by RMN, IR and Raman spectroscopies.
The use of the 514 nm wave length allowed to get high quality Raman signal for the component (I) bet-ween 100 et 3200 cm"1 with different polarisations. The component (2) appeared very damageable with this wavelength and has been destroyed even with a low power level (~ 2 mW), This leads us to change the excitation wavelength (red: 647 nm). The results will be compared and discussed with the component (1).
The component (3) gave a high fluorescence signal with 514 nm. In order to get the Raman spectra, we used a new Raman differential spectroscopy witch is developed in our laboratory, and which enables us to reject the fluorescence.
Our recent development of the Raman differential spectroscopy permitted the extraction of Raman signals even in the presence of fluorescence. This in situ technique permits to analyse efficiently the pharmaceu-tical or illegal products. The preliminary results by using this method will be presented.
25
Detection of quaternary amines by capillary electrophoresis-UV Scott F.J.<'>. Miller M.L.{2>
(1) The George Washington University, Washington, DC, 20052 (2) FBI Laboratory, FBI Academy, Quantico, VA 22135
A universal method for detection of quaternary ammonium neuromuscular blocking agents is needed, as these compounds are suspected agents in homicide cases, particularly those involving hospital personnel.
Quaternary ammonium compounds are inherently difficult to extract, as they are highly water soluble.
They are designed to break down rapidly at physiological pH and temperature. Capillary electrophoresis (CE) with ultra-violet (UV) detection is a simple, rapid detection method ideally suited to this class of drugs due to their charged character. Due to their similar structures and chemical activity, a chiral reco-gnition agent, beta-cyclodextrin is needed in the run buffer to ensure adequate separation. Direct and indi-rect detection are used simultaneously because several of the compounds have weak UV chromophores, which necessitate the inclusion of an indirect detection agent, para-aminopyridine. The nine quaternary ammonium drugs in recent use in the United States, as well as two primary metabolites can be separated and detected in a single run at 10 to 100 ppm levels
26
NMR spectroscopy as a useful tool for diagnosis of poisonings
Imbenotte M.( 1 >. Azaroual N.<2>, Cartigny B.( 3 ), Vermeersch G.(2), Lhermitte M.n 3 )(1) Laboratoire de Toxicologie, Faculté des Sciences Pharmaceutiques et Biologiques, Lille, (2) Laboratoire de Physique et d'Application RMN UPRESA CNRS 8009 (Université de Lille 2), (3) Laboratoire de Biochimie et de Biologie Moléculaire, Hôpital Calmette, CHRU Lille, France.
In order to analyse a wide range of xenobiotics and their metabolites present in biological fluids, such as urine, serum and plasma, NMR spectroscopy can be used. Due to the high content of water in biological specimens and the possible presence of macromolecules, specific 'H NMR acquisition sequences (selecti-ve presaturation and TOCSY) had to be elaborated and optimized.
A large variety of xenobiotics (therapeutic agents, pesticides, solvents, alcohols) can be characterized and quantified directly without preparation of the sample. NMR investigations were applied to acute poiso-nings by drugs, such as salicylates, chloroquine, and valproic acid. For salicylate poisoning, the three major metabolites of acetylsalicylic acid have been assigned in crude urine, and rapid identification of lysi-ne revealed the origin of the intoxication, namely lysilysi-ne acetylsalicylate (Aspegic®). An urilysi-ne sample from a 41-year-old man attempting suicide with chloroquine was analysed by one-dimensional and TOCSY 'H NMR spectroscopy, revealing chloroquine (462 mg/L) and monodesethylchloroquine (140 mg/L). Valproic acid as its glucuronide was identified in urine samples from two poisoned patients.
Concerning pesticides, !H NMR and 3 ,P NMR spectroscopies were used to diagnose acute poisoning with the organophosphorous glyphosate. Plasma concentrations (7.7 and 7.8 mrnol/L, respectively) were quite similar to chromatographic determinations (7,0 mmol/L), and the isopropyl resonances were used as mar-kers for the herbicide formulation (Roundup®) implicated in this case. Paraquat (Gramoxone®) was iden-tified and quaniden-tified by its two aromatic signals at 8.49 and 9.02 ppm, for two acutely poisoned patients (183 and 93 mg/L).
An intentional poisoning with tetrahydrofuran (THF) was also investigated. Serum and urine samples were collected consecutively to the ingestion of this solvent. THF was characterized by its resonances at 1.90 and 3.76 ppm, and quantified at 813 and 850 mg/L in the two biological fluids, respectively. Moreover, two
Annales de Toxicologie Analytique, vol. XIV, n° 3, 2002