Simultaneous enantiomeric determination of MDMA and its phase I and phase II metabolites in urine by liquid chromatography–tandem mass spectrometry with chiral derivatization

A liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedure was developed for the simultaneous determination of enantiomers of the prevalent designer drug 3,4-methylenedioxymethamphetamine (MDMA) and its phase I and phase II metabolites in urine with chiral derivatization. The analytes in urine were directly derivatized with chiral Marfey’s reagent, N ^α- (5-fluoro-2,4-dinitrophenyl)-d-leucinamide, without extraction. The diastereomers of the N ^α-(2,4-dinitrophenyl)-d-leucinamide derivatives generated were determined by LC-MS/MS. Satisfactory chromatographic separation was achieved for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfate on a semimicro octadecylsilane column using linear gradient elution. With use of multiple reaction monitoring mode, the limits of detection of these analytes ranged from 0.01 to 0.03?μg/mL. Linear calibration curves were obtained for all enantiomers from 0.1 to 20?μg/mL in urine. The method showed sufficient reproducibility and quantitative ability. This is the first report of a simple LC-MS/MS-based analytical procedure with direct chiral derivatization in aqueous media that allows simultaneous enantiomeric determination of drugs and their metabolites, including glucuronide and sulfate derivatives.     

New designer drug 1-(3,4-methylenedioxybenzyl) piperazine (MDBP): studies on its metabolism and toxicological detection in rat urine using gas chromatography/mass spectrometry

Studies are described on the metabolism and toxicological analysis of the piperazine-derived designer drug 1-(3,4-methylenedioxybenzyl)piperazine (MDBP) in rat urine using gas chromatography/mass spectrometry (GC/MS). The identified metabolites indicated that MDBP was metabolized by demethylenation and subsequent methylation to N-(4-hydroxy-3-methoxybenzyl)piperazine followed by partial glucuronidation or sulfation. Additionally, degradation of the piperazine moiety to N-(3,4-methylenedioxybenzyl)ethylenediamine and 3,4-methylenedioxybenzylamine and N-dealkylation to piperazine were observed. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid/liquid extraction and microwave-assisted acetylation allowed the detection of MDBP and its above-mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of MDBP by analysis of human urine.     

Sensitive determination of methylenedioxylated amphetamines by liquid chromatography

Different strategies for the liquid chromatographic determination of methylenedioxylated amphetamines were evaluated: separation and detection of underivatized analytes by (i) UV or (ii) fluorescence, (iii) derivatization with 3,5-dinitrobenzoyl chloride followed by separation and UV detection of the derivatives formed and (iv) derivatization with 9-fluorenylmethyl chloroformate (FMOC) and subsequent separation and fluorimetric detection of the derivatives. The compounds tested were 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDE). On the basis of these studies, a new procedure for the chromatographic determination of MDA, MDMA and MDE is proposed, based on derivatization with FMOC. The described procedure allows the quantification of the tested compounds with adequate linearity, reproducibility and accuracy in the concentration interval 0.5-20.0 micrograms mL-1. The limits of detection were 0.01 microgram mL-1 for MDA and 0.025 microgram mL-1 for MDMA and MDE. The utility of the described assay was tested by determining methylenedioxylated amphetamines in plasma and urine.     

Sensitive determination of MDMA and its metabolite MDA in rat blood and brain microdialysates by HPLC with fluorescence detection

Simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in rat blood and brain microdialysates by high-performance liquid chromatography with fluorescence detection (HPLC-FL) was developed. Microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The DIB-derivatives of MDMA, MDA and the internal standard, 1-methyl-3-phenylpropylamine (MPPA), were isocratically separated on an ODS column using a mixture of 50 mm phosphate buffer (pH 7.0)-acetonitrile-methanol-2-propanol (50:45:5:2, v/v/v/v %) as an eluent at a flow rate of 1.5 mL/min. The calibration curves of MDA and MDMA spiked to blood and brain microdialysates were linear over the ranges 2.5-500 and 5.0-1000 ng/mL, respectively. The detection limits of MDA and MDMA were 1.2 and 4.2 for blood and 1.3 and 4.8 ng/mL for brain, respectively. Additionally, the intra- and the inter-assay precisions were lower than 5.6% for the blood and brain microdialysates (n = 4). The proposed method was successfully applied for the monitoring of MDMA and its metabolite MDA in rat blood and brain microdialysates, and the pharmacokinetic parameters of MDMA and MDA in the microdialysates after administration of MDMA (5 mg/kg, i.p.) with or without caffeine (20 mg/kg, i.p.) were evaluated.     

Electrochemical and spectroscopic characterisation of amphetamine-like drugs: application to the screening of 3,4-methylenedioxymethamphetamine (MDMA) and its synthetic precursors

A complete physicochemical characterisation of MDMA and its synthetic precursors MDA, 3,4-methylenedioxybenzaldehyde (piperonal) and 3,4-methylenedioxy-β-methyl-β-nitrostyrene was carried out through voltammetric assays and Raman spectroscopy combined with theoretical (DFT) calculations. The former provided important analytical redox data, concluding that the oxidative mechanism of the N-demethylation of MDMA involves the removal of an electron from the amino-nitrogen atom, leading to the formation of a primary amine and an aldehyde. The vibrational spectroscopic experiments enable to afford a rapid and reliable detection of this type of compounds, since they yield characteristic spectral patterns that lead to an unequivocal identification.Moreover, the rational synthesis of the drug of abuse 3,4-methylenedioxymethamphetamine (MDMA or “ecstasy”) from one of its most relevant precursors 3,4-methylene-dioxyamphetamine (MDA), is reported. In addition, several approaches for the N-methylation of MDA, a limiting synthetic step, were attempted and the overall yields compared.     

Development and validation of LC-HRMS and GC-NICI-MS methods for stereoselective determination of MDMA and its phase I and II metabolites in human urine

3,4-Methylenedioxymethamphetamine (MDMA) is a racemic drug of abuse and its R- and S-enantiomers are known to differ in their dose-response curve. The S-enantiomer was shown to be eliminated at a higher rate than the R-enantiomer most likely explained by stereoselective metabolism that was observed in various in vitro experiments. The aim of this work was the development and validation of methods for evaluating the stereoselective elimination of phase I and particularly phase II metabolites of MDMA in human urine. Urine samples were divided into three different methods. Method A allowed stereoselective determination of the 4-hydroxy-3-methoxymethamphetamine (HMMA) glucuronides and only achiral determination of the intact sulfate conjugates of HMMA and 3,4-dihydroxymethamphetamine (DHMA) after C18 solid-phase extraction by liquid chromatography-high-resolution mass spectrometry with electrospray ionization. Method B allowed the determination of the enantiomer ratios of DHMA and HMMA sulfate conjugates after selective enzymatic cleavage and chiral analysis of the corresponding deconjugated metabolites after chiral derivatization with S-heptafluorobutyrylprolyl chloride using gas chromatography-mass spectrometry with negative-ion chemical ionization. Method C allowed the chiral determination of MDMA and its unconjugated metabolites using method B without sulfate cleavage. The validation process including specificity, recovery, matrix effects, process efficiency, accuracy and precision, stabilities and limits of quantification and detection showed that all methods were selective, sensitive, accurate and precise for all tested analytes.     

Determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxymethamphetamine in urine by online solid-phase extraction and ion-pairing liquid chromatography with detection by electrospray tandem mass spectrometry

A method using an online solid-phase extraction (SPE) and ion-pairing liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MS/MS) was developed for determination of amphetamine (Amp), methamphetamine (mAmp), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples. A SPE cartridge column with both hydrophilic and lipophilic functions was utilized for online extraction. A reversed-phase C18 LC column was employed for LC separation and MS/MS was used for detection. Trifluoroacetic acid was added to the mobile phase as an ion-pairing reagent. This method was fully automated and the extraction and analysis procedures were controlled by a six-port switch valve. Recoveries ranging from 85-101% were measured. Good linear ranges (10-500 ng/mL) for Amp and mAmp were determined. For MDA, MDMA and MDEA, dual linear ranges were obtained from 5-100 and 100-500 ng/mL, respectively. The detection limit of each analytical compound, based on a signal-to-noise ratio of 3, ranged from 1-3 ng/mL. The applicability of this newly developed method was examined by analyzing several urine samples from drug users. Good agreement was obtained between the results from this method and a literature GC/MS method.     

Rapid drug-screening of clandestine tablets by MALDI-TOF mass spectrometry

A novel method for the rapid screening of clandestine tablets for drugs by MALDI-TOF mass spectrometry is described. In this method, cetrimonium bromide (CTAB), a surfactant, is added to the conventional α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution used in preparing the MALDI samples. This procedure allows very clean mass spectra to be collected for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), caffeine, ketamine and tramadol. The method was used successfully in the rapid drug-screening of some actual clandestine tablets, which had been seized from the illicit market, and can serve as a good complementary method to GC/MS for use in forensic analysis.     

Chiral separation of 3,4-methylenedioxymeth- amphetamine and related compounds in clandestine tablets and urine samples by capillary electrophoresis/fluorescence spectroscopy

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. Using these R-(-)- and S-(+)-isomers, the distribution of (RS)-MDA and (RS)-MDMA stereoisomers in clandestine tablets and suspect urine samples were identified. Several electrophoretic parameters, such as the concentration of beta-cyclodextrin used in the electrophoretic separation and the amount of organic solvents required for the separation, were optimized.     

Determination of 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine enantiomers in whole blood

A method for the determination of the enantiomeric content of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in microsamples (200 microliters) of whole blood is described. The method involves liquid-liquid extraction of MDA and MDMA from blood and derivatization with the chiral reagent N-trifluoroacetyl-L-prolyl chloride. Separation, identification and quantitation of diastereomeric derivatives is by gas chromatography-mass spectrometry. The analytical range of the assay is from 0.12 ng to 48 ng injected on-column. Details for the synthesis of the enantiomers of MDMA are also provided.     

3,4-Methylenedioxymethamphetamine (MDMA) and metabolites disposition in blood and plasma following controlled oral administration

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit phenethylamine ingested for entactogenic and euphoric effects. Although blood is more commonly submitted for forensic analysis, previous human MDMA pharmacokinetics research focused on plasma data; no direct blood–plasma comparisons were drawn. Blood and plasma specimens from 50 healthy adult volunteers (33 males, 17 females, 36 African-American) who ingested recreational 1.0 and 1.6 mg/kg MDMA doses were quantified for MDMA and metabolites 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-methylenedioxyamphetamine (MDA), and 4-hydroxy-3-methoxyamphetamine (HMA) by two-dimensional gas chromatography–mass spectrometry. Specimens were collected up to 3 h post-dose and evaluated for maximum concentration (C _max), first detection time (t _first), time of C _max (t _max), and 3-h area under the curve (AUC_0–3 h); as well as blood metabolite ratios and blood/plasma ratios. Median blood MDMA and MDA C _max were significantly greater (p?<?0.0005) than in plasma, but HMMA was significantly less (p?<?0.0005). HMA was detected in few blood specimens, at low concentrations. Nonlinear pharmacokinetics were not observed for MDMA or MDA in this absorptive phase, but HMMA C _max and AUC_0–3 h were similar for both doses despite the 1.6-fold dose difference. Blood MDA/MDMA and MDA/HMMA significantly increased (p?<?0.0001) over the 3-h time course, and HMMA/MDMA significantly decreased (p?<?0.0001). Blood MDMA C _max was significantly greater in females (p?=?0.010) after the low dose only. Low-dose HMMA AUC_0–3 h was significantly decreased in females’ blood and plasma (p?=?0.027) and in African-Americans’ plasma (p?=?0.035). These data provide valuable insight into MDMA blood–plasma relationships for forensic interpretation and evidence of sex- and race-based differential metabolism and risk profiles.

安非他明类毒品的手性对映体气相色谱-质谱分析

采用手性衍生化试剂：（S）（－）N－三氟乙酰－1－脯胺酰氯（TPC）和（R）（＋）－α-甲氧基－α－三氟甲基苯乙酸（MTPA）与安非他明类对映体衍生化产物,通过常规的GC／MS方法将其分离,本文较系统地考察了这两种手性试剂衍生化反应中溶剂、手性试剂用量、加热温度、反应时间等因素对安非他明类在体衍生化结果的影响。实现了Am、MAm、MDA、MDMA、MDEA、MBDB等几种毒品对映体间的良好分离。     

Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites in urine by LC‐MS after derivatization and its application for differentiation between positional isomers of prilocaine

The demand for clinical toxicology analytical methods for identifying drugs of abuse and medicinal drugs is steadily increasing. Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites by GC‐EI‐MS and LC‐ESI‐MS/MS is of great analytical challenge. These compounds exhibit only/mostly fragments/product ions representing the amine‐containing residue, while the aromatic amide moiety remains unidentified. This task becomes even more complicated when discrimination between positional isomers of such compounds is required. Here, we report the development of a derivatization procedure for the differentiation and structural elucidation of a mixture of local anesthetic drugs and their metabolites that possess tertiary and secondary amines in water and urine. A method based on two sequential “in‐vial” instantaneous derivatization processes at ambient temperature followed by LC‐ESI‐MS/MS analysis was developed. 2,2,2‐Trichloro‐1,1‐dimethylethyl chloroformate (TCDMECF) was utilized to selectively convert the secondary amines into their carbamate derivatives, followed by hydrogen peroxide addition to produce the corresponding tertiary amine oxides. The resulting derivatives exhibited rich fragmentation patterns, enabling improved structural elucidation of the original compounds. The developed method was successfully applied to the differentiation and structural elucidation of prilocaine and its four positional isomers, which all possess similar GC and LC retention times and four of them exhibit almost identical EI‐MS and ESI‐MS/MS spectra, enabling their structural elucidation in a single LC‐ESI‐MS/MS analysis. The developed technique is fast and simple and enables discrimination between isomers based on different diagnostic ions/fragmentation patterns.     

Simultaneous determination of psychotropic phenylalkylamine derivatives in human hair by gas chromatography/mass spectrometry

A gas chromatography/mass spectrometric (GC/MS) method was developed and validated for the determination of thirteen psychotropic phenylalkylamine derivatives (amphetamine; AP, phentermine; PT, methamphamine; MA, cathinone; Khat, methcathinone; MCAT, fenfluramine; FFA, desmethylselegiline; DSEL, 3,4-methylenedioxyamphetamine; MDA, 3,4-methylenedioxymethamphetamine; MDMA, 3,4-methylenedioxyethylamphetamine; MDEA, norketamine; NKT, mescaline; MES, 4-bromo-2,5-dimethoxyphenethylamine; 2CB) in human hair. Hair samples (20 mg) were washed with distilled water and acetone, cut into small fragments (<1 mm), and incubated in 0.25 M methanolic HCl under ultrasonication at 50 degrees C for 1 h. The resulting solutions were evaporated to dryness, derivatized using trifluoroacetic anhydride (TFAA) at 70 degrees C for 30 min, and analyzed by GC/MS. The linear ranges were 0.02-25.0 ng/mg for AP, PT, Khat, FFA, DSEL, MDMA, and 2CB; 0.05-25.0 ng/mg for MA, MCAT, and MES; 0.05-12.5 ng/mg for MDA; and 0.1-25.0 ng/mg for MDEA and NKT, with good correlation coefficients (r(2) > 0.9985). The intra-day, inter-day, and inter-person precisions were within 12.7%, 14.8%, and 16.8%, respectively. The intra-day, inter-day, and inter-person accuracies were between -10.7 and 13.4%, -12.7 and 11.6%, and -15.3 and 11.9%, respectively. The limits of quantifications (LOQs) for each compound were lower than 0.08 ng/mg. The recoveries were in the range of 76.7-95.6%. The method proved to be suitable for the simultaneous qualification and quantification of phenylalkylamine derivatives in hair specimens.     

Rapid,simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05 − 5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.     

Measurement of catecholamines, their precursor and metabolites in human urine and plasma by solid-phase extraction followed by high-performance liquid chromatography with fluorescence derivatization

A high-performance liquid chromatographic method is described for the determination in human urine and plasma of catecholamines, their precursor and metabolites [amino compounds (norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, 3-methoxytyramine and L-DOPA), acidic compounds (3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, vanillylmandelic acid and homovanillic acid) and alcoholic compounds (3,4-dihydroxyphenylethyleneglycol and 4-hydroxy-3-methoxyphenylethyleneglycol)]. Urine (0.5 ml) containing 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid (internal standards) is deproteinized with perchloric acid, and the resulting solution is fractionated by solid-phase extraction on a strong cation-exchange resin cartridge (Toyopak IC-SP S) into two fractions (amine fraction and acid-alcohol fraction), which include 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid, respectively. Plasma (0.7 ml) is deproteinized in the presence of 3,4-dihydroxybenzylamine (internal standard) in the same manner, and the resulting solution is directly used as an acid-alcohol fraction, while an amine fraction is obtained as for urine. Each fraction is subjected to the previously established ion-pair reversed-phase chromatography with post-column derivatization involving coulometric oxidation followed by fluorescence reaction with 1,2-diphenylethylenediamine. The detection limits, at a signal-to-noise ratio of 5, of the compounds measured in urine are 300 pmol/ml for the two mandelic acids, 2-7 pmol/ml for the other acidic and alcoholic compounds, 12 pmol/ml for L-DOPA and 0.6-2 pmol/ml for the other amino compounds; the corresponding values for plasma samples are 80, 0.5-3, 10 and 0.6-3 pmol/ml, respectively.     

微波萃取-气相色谱法测定尿液中的苯丙胺类毒品

建立了人体尿液中甲基苯丙胺(MA)、3,4-亚甲二氧基苯丙胺(MDA)、3,4-亚甲二氧基甲基苯丙胺(MDMA)的微波萃取-气相色谱(GC)测定方法。分别考察了萃取溶剂种类、用量、pH值以及萃取温度、时间等因素对萃取率的影响。实验结果表明,尿液中MA,MDA,MDMA的最佳提取条件为:调节尿样pH为12,以环己烷为萃取溶剂,于40 ℃下微波提取10 min。在此条件下MA,MDA,MDMA的平均回收率分别为92.25%,85.94%和91.50%,相对标准偏差分别为5.5%,5.5%和6.1% (n=5),提取液经气相色谱-氢火焰离子化检测器(GC-FID)检测,3种药物与基体得到了很好的分离,对尿液中MA,MDA,MDMA的最低检测限分别为10,20和20 ng/mL。该方法未对药物进行衍生化,是一种快速、准确、灵敏度高的同时测定尿液中MA,MDA,MDMA的方法。     

Analysis of hydroxylated polybrominated diphenyl ether metabolites by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

Hydroxylated polybrominated diphenyl ether (OH‐PBDEs) metabolites have the potential to cause endocrine disruption as well as other health effects. Currently, gas chromatography/mass spectrometry (GC/MS) after derivatization is used for the analysis of OH‐PBDEs. However, there is a need for the direct analysis of OH‐PBDEs at relatively low concentrations in environmental and biological samples. Liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI‐MS/MS) was evaluated for the analysis of nine OH‐PBDEs, ranging from tri‐ to hexabrominated. Separation of the nine isomeric metabolites was achieved with reversed‐phase liquid chromatography, followed by detection by APCI‐MS in negative mode. Notably, a significant decrease in ionization was observed in 6‐hydroxyl‐substituted PBDE metabolites in the presence of an ortho‐substituted bromine, relative to the other hydroxylated metabolites. This is probably due to the formation of dioxins in the source as a result of the high‐temperature conditions, which prevented ionization by hydrogen abstraction. The MS/MS experiments also provided evidence of the neutral losses of HBr and Br₂, indicating the possible use of neutral loss scanning and selected reaction monitoring (SRM) for the screening of brominated metabolites in samples. The applicability of LC/APCI‐MS/MS was demonstrated for the analysis of metabolites of BDEs 47 and 99 formed in human liver microsomes. The LC/APCI‐MS/MS method was able to detect metabolites that had previously been identified by GC/MS following derivatization. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass spectrometric characterization of toremifene metabolites in human urine by liquid chromatography-tandem mass spectrometry with different scan modes

The metabolism and excretion of toremifene were investigated in one healthy male volunteer after a single oral administration of 120 mg toremifene citrate. Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were carried out for the characterization of the metabolites in human urine for doping control purposes. The potential characteristic fragmentation pathways of toremifene and its major metabolites were presented. An approach for the metabolism study of toremifene and its analogs by liquid chromatography-tandem mass spectrometry was established. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 72.2, 58.2, 44.2, 45.2, 88.2 relative to five metabolic pathways) in positive ion mode were assessed to recognize the metabolites. Based on product ion scan and precursor ion scan techniques, the metabolites were proposed to be identified as 4-hydroxy-toremifene (m/z 422.4), 4'-hydroxy-toremifene (m/z 422.4), α-hydroxy-toremifene (m/z 422.4), 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2), 3-hydroxy-4-methoxy-toremifene (m/z 456.2), dihydroxy-dehydro-toremifene (m/z 440.2), 3,4-dihydroxy-toremifene (m/z 438.2), N-demethyl-4-hydroxy-toremifene (m/z 408.3), N-demethyl-3-hydroxy-4-methoxy-toremifene (m/z 438.3). In addition, a new metabolite with a protonated molecule at m/z 390.3 was detected in all urine samples. The compound was identified by LC/MS/MS as N-demethyl-4,4'-dihydroxy-tamoxifene. The results indicated that 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2) and N-demethyl-4,4'-dihydroxy-tamoxifene (m/z 390.3) were major metabolites in human urine.     

Enantioselective Analysis of Amphetamine-Related Designer Drugs in Body Fluids Using Liquid Chromatography and Solid-Phase Derivatization

A method for the enantioselective determination of the amphetamine-derived designer drugs 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxyethylamphetamine (MDE) based on their derivatization with (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC) is described. The proposed procedure entails preconcentration and derivatization of the analytes into C₁₈-packed solid-phase extraction cartridges, chromatographic separation of the diastereomers originated in a C₁₈ column under gradient elution, and UV detection at 265 nm. Compared with the solution derivatization approach the described procedure increased analyte responses by factors of 28–58. The reliability of the method has been tested by analysing plasma and urine samples spiked with the analytes in the 0.015–1.0 μg mL^?1 concentration interval. The proposed conditions provided adequate linearity, and coefficients of variation ranging from 5% to 14% in plasma, and from 3% to 12% in urine. The recoveries of the analytes were of 78%–126% and 78%–128% in plasma and urine, respectively. The limits of detection (LODs) obtained for all the analytes were 5 ng mL^?1 in both biological matrices.