Simultaneous determination of amphetamine and its analogs in human whole blood by gas chromatography-mass spectrometry

A sensitive and specific gas chromatography-mass spectrometry (GC-MS) method for the determination of amphetamine (AM), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA) and methylenedioxyethylamphetamine (MDEA) in whole blood was designed, using the respective pentadeuterated analogs of the analytes as internal standards (I.S.). After alkalinisation of blood samples, the amphetamines were extracted using diethyl ether, derivatized with heptafluorobutyric anhydride, then purified by successive washings with deionized water and 4% NH₄OH. Extraction recoveries were 85.2% for AM, 90.9% for MA, 76.5% for MDA, 84.1% for MDMA and 63.6% for MDEA. Chromatographic separation was performed on a non-polar 30 m×0.32 mm HP 5 MS capillary column using a temperature program. Detection was carried out in the electron-impact, selected ion-monitoring mode, using three mass-to-charge ratios for each analyte and one for each I.S. Limits of detection ranged from 0.5 to 8 ng/ml and limits of quantification were 10 ng/ml for AM, MDMA and MDEA; 20 ng/ml for MA; and 50 ng/ml for MDA. The method was linear from this limit up to 1000 ng/ml for all analytes, with good intra-assay precision and good intermediate precision and accuracy over these ranges. There was no interferences from other sympathomimetic drugs such as ephedrine, norephedrine or methoxyphenamine. This method is thus suitable for clinical and forensic toxicology, as well as for doping control.     

Stereoselective method development and validation for determination of concentrations of amphetamine-type stimulants and metabolites in human urine using a simultaneous extraction-chiral derivatization approach

Amphetamine-type stimulants (ATS) are a group of chiral amine drugs which are commonly abused for their sympathomimetic and stimulant properties. ATS are extensively metabolised by hepatic cytochrome P450 enzymes. As metabolism of ATS has been shown to be highly stereospecific, stereoselective analytical methods are essential for the quantitative determination of ATS concentrations for both in vivo and in vitro studies of ATS metabolism. This paper describes a new stereoselective method for the simultaneous determination of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 4-hydroxy-3-methoxyamphetamine (HMA), 3,4-hydroxymethamphetamine (HHMA) and 3,4-hydroxyamphetamine (HHA) in human urine samples validated according to the United States Food and Drug Administration guidelines. In this method, analytes are simultaneously extracted and derivatized with R-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (R-MTPCl) as the chiral derivatization reagent. Following this, the analytes were subjected to a second derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) which targets the hydroxyl groups present in HMMA, HMA, HHMA and HHA. The derivatized analytes were separated and quantified using gas chromatography-mass spectrometry (GC-MS). The method was evaluated according to the established guidelines for specificity, linearity, precision, accuracy, recovery and stability using a five-day protocol. Intra-day precision ranged from 0.89 to 11.23% RSD whereas inter-day precision was between 1.03 and 12.95% RSD. Accuracy values for the analytes ranged from -5.29% to 13.75%. Limits of quantitation were 10 μg/L for AM, MA, MDMA, HMA and HMMA and 2μg/L for MDA, HMA and HHA. Recoveries and stability values were also within accepted values. The method was applied to authentic ATS-positive samples.     

Application of solid-phase microextraction combined with derivatization to the determination of amphetamines by liquid chromatography

This work evaluates the utility of solid-phase microextraction (SPME) in the analysis of amphetamines by liquid chromatography (LC) after chemical derivatization of the analytes. Two approaches have been tested and compared, SPME followed by on-fiber derivatization of the extracted amphetamines, and solution derivatization followed by SPME of the derivatives formed. Both methods have been applied to measure amphetamine (AP), methamphetamine (MA), and 3,4-methylenedioxymethamphetamine (MDMA), using the fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) and carbowax-templated resin (CW-TR)-coated fibers. Data on the application of the proposed methods for the analysis of different kind of samples are presented. When analyzing aqueous solutions of the analytes, both approaches gave similar analytical performance, but the sensitivity attainable with the solution derivatization/SPME method was better. The efficiencies observed when processing spiked urine samples by the SPME/on-fiber derivatization approach were very low. This was because the extraction of matrix components into the fiber coating prevented the extraction of the reagent. In contrast, the efficiencies obtained for spiked urine samples by the solution derivatization/SPME approach were similar to those obtained for aqueous samples. Therefore, the later method would be the method of choice for the quantification of amphetamines in urine.     

Determination of MDMA, MDEA and MDA in urine by high performance liquid chromatography with fluorescence detection

This paper describes the development and validation of analytical methodology for the determination of the use of MDMA, MDEA and MDA in urine. After a simple liquid extraction, the analyses were carried out on a high performance liquid chromatography (HPLC) in an octadecyl column, with fluorescence detection. The mobile phase using a sodium dodecyl sulfate ion-pairing reagent allows good separation and efficiency. The method showed good linearity and precision. Recovery was between 85 and 102% and detection limits were 10, 15 and 20 ng/ml for MDA, MDMA and MDEA, respectively. No interfering substances were detected with fluorescence detection.     

Determination of amphetamines in human urine by headspace solid-phase microextraction and gas chromatography

Solid-phase microextraction (SPME) is under investigation for its usefulness in the determination of a widening variety of volatile and semivolatile analytes in biological fluids and materials. Semivolatiles are increasingly under study as analytical targets, and difficulties with small partition coefficients and long equilibration times have been identified. Amphetamines were selected as semivolatiles exhibiting these limitations and methods to optimize their determination were investigated. A 100- micro m polydimethylsiloxane (PDMS)-coated SPME fiber was used for the extraction of the amphetamines from human urine. Amphetamine determination was made using gas chromatography (GC) with flame-ionization detection (FID). Temperature, time and salt saturation were optimized to obtain consistent extraction. A simple procedure for the analysis of amphetamine (AMP) and methamphetamine (MA) in urine was developed and another for 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methamphetamine (MDMA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) using headspace solid-phase microextraction (HS-SPME) and GC-FID. Higher recoveries were obtained for amphetamine (19.5-47%) and methamphetamine (20-38.1%) than MDA (5.1-6.6%), MDMA (7-9.6%) and MDEA (5.4-9.6%).     

Routine analysis of amphetamine class drugs as their naphthaquinone derivatives in human urine by high-performance liquid chromatography

We describe a simple HPLC method which is suitable for the routine confirmation of immunoassay positive amphetamine urine samples. The precolumn derivisation method employing sodium naphthaquinone-4-sulphonate was found to have adequate sensitivity, selectivity and precision for the measurement of amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxyethylamphetamine (MDEA) at 500 μg/l cutoff level for confirmatory analysis of amphetamines in urine. The specificity of the method is enhanced by detecting the peaks at two different wavelengths. The ratios of the peak heights measured at the two wavelengths were different for each of the 5 amphetamines analysed. There was no interference from other phenylethylamine analogues that are commonly found in “over the counter” preparations. The HPLC method is compared to a commercial TLC system for detecting amphetamines in urine of drug abusers attending drug rehabilitation programmes. The HPLC confirmatory method described is a viable alternative to GC or to the more complex and costly GC–MS techniques for confirming amphetamine, methamphetamine, MDMA, MDA and MDEA in urine of drug abusers especially when used in a clinical care setting     

Sensitive, rapid and validated gas chromatography/negative ion chemical ionization-mass spectrometry assay including derivatisation with a novel chiral agent for the enantioselective quantification of amphetamine-type stimulants in hair

A novel chiral derivatisation agent, (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, was used for the indirect resolution of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers using gas chromatography coupled to mass spectrometry operating in the negative-ion chemical ionization mode (GC/MS-NICI). This new chiral derivatisation reagent was readily obtained in optically pure form after a simple two-step synthesis. Optimal derivatisation was accomplished in 15 min at room temperature in a carbonate buffer and the resulting diastereoisomers were base line separated by GC in 12 min only. No racemization was observed during the derivatisation. The method was applied and fully validated for the enantiomeric quantification of amphetamines and methylenedioxylated amphetamines in hair. The analyses of 24 hair specimens from suspected ATS abusers showed that 24 cases were positive for MA and/or AM enantiomers and that in most cases the concentrations of (S)-MA and (S)-AM exceeded those of the corresponding (R)-enantiomers. One hair specimen was tested positive for both enantiomers of MDMA and MDA.     

Integration of GC/EI-MS and GC/NCI-MS for simultaneous quantitative determination of opiates, amphetamines, MDMA, ketamine, and metabolites in human hair

In this paper, the possibility of using a multiple ionization mode approach of GC/MS was developed for the simultaneous hair testing of common drugs of abuse in Asia, including amphetamines (amphetamine, AP; methamphetamine, MA; methylenedioxy amphetamine, MDA; methylenedioxy methamphetamine, MDMA; methylenedioxy ethylamphetamine, MDEA), ketamine (ketamine, K; norketamine, NK), and opiates (morphine, MOR; codeine, COD; 6-acetylmorphine, 6-AM). This strategy integrated the characteristics of gas chromatography-mass spectrometry (GC-MS) using electron impact ionization (EI) and negative chemical ionization (NCI). Hair samples (25 mg) were washed, cut, and incubated overnight at 25 degrees C in methanol-trifluoroacetic acid (methanol-TFA). The samples were extracted by solid phase extraction (SPE) procedure, derivatized using heptafluorobutyric acid anhydride (HFBA) at 70 degrees C for 30 min, and the derivatives analyzed by GC-MS with EI and NCI. The limit of detection (LOD) with GC/EI-MS analysis obtained were 0.03 ng/mg for AP, MA, MDA, MDMA, and MDEA; 0.05 ng/mg for K, NK, MOR, and COD; and 0.08 ng/mg for 6-AM. The LOD of GC/NCI-MS analysis was much lower than GC/EI-MS analysis. The LOD obtained were 30 pg/mg for AP and MDA in GC/EI-MS and 2 pg/mg in GC/NCI-MS. Therefore, the sensitivity of AP and MDA in GC/NCI-MS was improved from 15-fold compared with EI. The sensitivity of AP, MA, MDA, MDMA, MDEA, MOR, and COD was improved from 15- to 60-fold compared with EI. In addition, the sensitivity of 6-AM increased 8-fold through selection of m/z 197 for the quantitative ion. Moreover, K and NK could dramatically improve their sensitivity at 200- and 2000-fold. The integration of GC/EI-MS and GC/NCI-MS can obtain the high sensitivity and complementary results of drugs of abuse in hair. Six hair samples from known drug abusers were examined by this new strategy. These results show that integrating the characteristics of GC/EI-MS and GC/NCI-MS were not only enhancement of the sensitivity but also avoid wrong results and wrong interpretations of correct results.     

Confirmation of amphetamine,methamphetamine, MDA and MDMA in urine samples using disk solid-phase extraction and gas chromatography-mass spectrometry after immunoassay screening

A method using mixed phase disk solid-phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) was developed for confirmation of amphetamine (AMP), methamphetamine (MET), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples after immunoassay screening. Disk SPE provided hydrophobic (C(18)) and strong cation-exchange (SCX) interactions. The analytes were retained on SCX functional groups in the disk and eluted with ammoniated ethyl acetate after washed with methanol. Confirmation and quantitation was exercised by selected ion monitoring using nikethamide as chromatographic standard. Recoveries of the amphetamines were between 73.0 and 104.6% with RSDs in range of 2.1-6.4% (n=3). The limits of detection were 2 ng/ml for AMP, MET and MDMA, and 4 ng/ml for MDA. Five real urine samples were tested with the method after immunoassay screening, and the results were comparable to those of traditional liquid-liquid extraction (LLE). The method was solvent-saved, simple, rapid and reliable, and the extract was cleaner than that of LLE.     

Development and validation of a disk solid phase extraction and gas chromatography-mass spectrometry method for MDMA, MDA, HMMA, HMA, MDEA, methamphetamine and amphetamine in sweat

We describe the development and validation of a method for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3-hydroxy-4-methoxymethamphetamine (HMMA), 3-hydroxy-4-methoxyamphetamine (HMA), 3,4-methylenedioxyethylamphetamine (MDEA), methamphetamine (MAMP) and amphetamine (AMP) in sweat. Drugs were eluted from PharmChek sweat patches with sodium acetate buffer, extracted with disk solid phase extraction and analyzed using GC/MS-EI with selected ion monitoring. Limits of quantification (LOQ) for MDMA, MDEA, MAMP and AMP were 2.5 ng/patch, and 5 ng/patch for MDA, HMA and HMMA. This fully validated procedure was more sensitive than previously published analytical methods and permitted the simultaneous analysis of multiple amphetamine analogs in human sweat.     

Simultaneous enantioselective determination of amphetamine and congeners in hair specimens by negative chemical ionization gas chromatography-mass spectrometry

Enantioselective quantification of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers in hair using gas chromatography-mass spectrometry (GC-MS) is described. Hair specimens were digested with 1M sodium hydroxide at 100 degrees C for 30 min and extracted by a solid phase procedure using Cleanscreen ZSDAU020. Extracted analytes were derivatised with (S)-heptafluorobutyrylprolyl chloride and the resulting diastereoisomers were quantified by GC-MS operating in the negative chemical ionization mode. Extraction yields were between 73.0 and 97.9%. Limits of detection varied in the range of 2.1-45.9 pg/mg hair, whereas the lowest limits of quantification varied between 4.3 and 91.8 pg/mg hair. Intra- and inter-assay precision and respective accuracy were acceptable. The enantiomeric ratios (R versus S) of AM, MA, MDA, MDMA and MDEA were determined in hair from suspected amphetamine abusers. Only MA and AM enantiomers were detectable in this collective and the quantification data showed in most cases higher concentrations of (R)-MA and (R)-AM than those of the corresponding (S)-enantiomers.     

Simultaneous,quantitative determination of opiates,amphetamines, cocaine and benzoylecgonine in oral fluid by liquid chromatography quadrupole-time-of-flight mass spectrometry

A method using liquid chromatography coupled to tandem mass spectrometry is described for the determination of drugs of abuse in oral fluid. The method is able to simultaneously quantify amphetamines (amphetamine, methamphetamine, MDA, MDMA and MDEA), opiates (morphine and codeine), cocaine and benzoylecgonine. Only 200 micro of oral fluid is spent for analysis. The sample preparation is easy and consists of mixed mode phase solid-phase extraction. Reversed-phase chromatography is carried out on a narrow bore phenyl type column at a flow-rate of 0.2 ml/min. A gradient is applied ranging from 6 to 67.6% methanol with ammonium formate (10 mM, pH 5.0) added to the mobile phase. The column effluent was directed into a quadrupole-time-of-flight instrument by electrospray ionization, without the use of a splitter. A validation study was carried out. Recovery ranged from 52.3 to 98.8%, within-day and between-day precision expressed by relative standard deviation were less than 11.9 and 16.8%, respectively, and inaccuracy did not exceed 11.6%. The limit of quantification was 2 ng/ml (0.66 x 10(-5)-1.48 x 10(-5) M) for all compounds. Internal standards were used to generate quadratic calibration curves (r(2)>0.999). The method was applied to real samples obtained from suspected drug users. An interference was observed from the device used to sample the oral fluid, consequently this was excluded from the method which was validated on oral fluid obtained by spitting in a test-tube.     

A validated gas chromatographic-electron impact ionization mass spectrometric method for methylenedioxymethamphetamine (MDMA), methamphetamine and metabolites in oral fluid

An analytical method to simultaneously quantify amphetamine (AMP), methamphetamine (MAMP), methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), methylenedioxyethylamphetamine (MDEA), 3-hydroxy-4-methoxy-methamphetamine (HMMA) and 3-hydroxy-4-methoxy-amphetamine (HMA) in oral fluid is presented. Four hundred microlitres of oral fluid collected via expectoration was extracted by solid phase extraction. GC/MS-EI with selected ion monitoring (SIM) yielded linear curves 5-250 ng/mL for AMP, MAMP, MDMA and MDEA, 5-500 ng/mL for MDA and 25-1,000 ng/mL for HMA and HMMA. Recoveries were greater than 85%, accuracy 87-104%, and precision less than 8.3% coefficient of variation. This assay will be used to investigate distribution of sympathomimetic amines into human oral fluid following controlled drug administration.     

A demonstration of the use of ultra-performance liquid chromatography-mass spectrometry [UPLC/MS] in the determination of amphetamine-type substances and ketamine for forensic and toxicological analysis

We have recently seen the emergence of ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry as an alternative to traditional high-performance liquid chromatography techniques. The strengths of UPLC technology promote the ability to separate and identify drug compounds with significant gains in resolution and sensitivity and marked reductions in the overall time of analysis. As increased throughput is the desire of the practical toxicology laboratory, the aim of this study was to trial commercially available technology by assessment of the separation of several commonly encountered amphetamine-type substances. From injection of a poly-drug reference standard and whole blood extract, we successfully separated and identified amphetamine, methamphetamine, ephedrine, pseudoephedrine, phentermine, MDA, MDMA, MDEA and ketamine in less than 3 min using the Acquity UPLC-Micromass Quattro Micro API MS instrumentation (Waters Corporation, USA). In addition to this significant reduction in overall run time, all peaks exhibited acceptable resolution using selected ion recording (SIR), with analysis indicating the capability to separate 5-11 peaks in 1.75 min using the current system parameters. From this introductory data, it is therefore indicated that the technological advancements defining ultra-performance liquid chromatography will allow it to serve as a powerful analytical tool for rapid throughput analysis.     

Development and validation of a gas chromatography-mass spectrometry assay for hair analysis of amphetamine, methamphetamine and methylenedioxy derivatives

A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), 3,4-methylenedioxyethylamphetamine (MDE or MDEA) and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair. Hair samples were digested with 1 M sodium sulfide at 37 degrees C (by shaking for 3 h and was kept at room temperature overnight), and extracted with two sequential extraction procedures: liquid-liquid extraction with tert-butyl methyl ether and solid-phase extraction with Bond-Elut Certify columns. Extracted analytes were derivatised with N-methyl-bis(trifluoroacetamide), separated by a 5% phenylmethylsilicone column and determined by a mass spectrometer detector in selected ion monitoring mode. A good reproducibility (intra-assay R.S.D.=1.5-15.7%), accuracy (intra-assay error = 2.0-11.7%) and sensitivity (LOD=0.03-0.08 ng/mg hair) were attained. The method was successfully applied to the analysis of the proximal (1 cm) hair segment to assess recent self-reported use in "ecstasy" consumers. Otherwise, further studies are needed to validate methodology developed in case of amphetamine consumption.     

Analysis of malondialdehyde in biological matrices by capillary gas chromatography with electron-capture detection and mass spectrometry

A gas chromatographic method is described for the quantification of free and total malondialdehyde (MDA) in biological materials. The procedure involves derivatization of the analyte with 2,4,6-trichlorophenylhydrazine, extraction with n-hexane, and separation of the cyclic derivatization product on a OV-5 gas chromatographic column. Concentration of the derivatization reagent, pH, reaction time, and temperature were investigated to determine the optimal derivatization conditions. Under these conditions, the method allows for the selective detection of free and total MDA at femtomole levels in several biological materials without any interferences. The procedure yields relative standard deviation values for the intra- and interassays in the range 3.3 and 3.9%, respectively, for the electron-capture and mass-selective (SIM mode) detection systems. Recoveries of MDA from spiked matrices reached 96%. The present method offers the advantage of the alternative use of either electron-capture or mass-selective detection. Furthermore it avoids overestimation of MDA since it employs mild conditions for sample processing and there is no need for preventing protein separation for the assessment of free MDA.     

Development and validation of an EI-GC/MS method for the determination of sertraline and its major metabolite desmethyl-sertraline in blood

A sensitive and specific GC/MS method for the determination of sertraline and its main metabolite desmethyl-sertraline in whole blood has been developed, optimized and validated. Sample preparation included solid-phase extraction of both analytes and their derivatization with heptafluorobutyric anhydride (HFBA). Protriptyline was used as internal standard for the determination of both analytes. Limits of detection and quantification for both sertraline and desmethyl-sertraline were 0.30 and 1.00 μg/L, respectively. The calibration curves were linear within the dynamic range of each analyte (1.00-500.0 μg/L) with a correlation coefficient (R(2)) exceeding 0.991. Extraction efficiency ranged from 90.1(± 5.8)% to 95.4(± 3.0)% for sertraline, and from 84.9(± 8.2)% to 107.7(± 4.4)% for desmethyl-sertraline. The precision for sertraline and desmethyl-sertraline was between 3.6-5.5% and 4.7-7.2%, respectively, while the accuracy was in the range of -6.67% to 2.20% and -6.33% to 2.88% for sertraline and desmethyl-sertraline, respectively. The method was applied to real blood samples obtained from patients that follow sertraline treatment and also in cases of forensic interest. The developed method can be used in routine every day analysis by clinical and forensic laboratories, for pharmacokinetic studies, for therapeutic sertraline monitoring or for the investigation of forensic cases where sertraline is involved.     

Enantiomeric determination of amphetamines: exploring a novel one-step solid-phase microextraction-based approach

The recent advances in fiber manufacturing technology, solid-phase microextraction (SPME) is now widely studied for its effectiveness for the pretreatment of various categories of samples. This study explores a novel SPME approach for enantiomeric analysis of amphetamines, in which absorption/derivatization are accomplished in one step. Specifically, (S)-(-)-N-(Trifluoroacetyl)-prolyl chloride was adopted as the chiral derivatizing reagent and added directly into the sample matrix. Analytical parameters, such as temperature, absorption/desorption duration, and the amount of derivatizing reagent, were studied to determine their effects on the yield of analytes. The derivatization products resulting from this study show excellent desorption characteristics on the polydimethylsiloxane-coated fiber adopted in this study. Optimal operational parameters (absorption: 70 degrees C for 10 min; injection: 250 degrees C for 5 min) cause minimal negative impact on the fiber, allowing repeated use of the fiber for more than 30 times. For quantitation, data were collected under selected ion monitoring (SIM) mode using m/z 237 and 251 to designate derivatized amphetamine and methamphetamine. This method was evaluated and proved to be effective in (a) quantitative determination of the enantiomeric pairs of amphetamine and methamphetamine--in terms of repeatability, linearity, and limits of detection and quantitation; and (b) generating case-specimen data comparable to those derived from a conventional Liquid-liquid extraction approach. Good linearity for the calibration curves were established in the range of 1000-50 ng/mL for both analytes. The limits of detection for these analytes were 30 ng/mL.     

Determination of drugs of abuse and their metabolites in human plasma by liquid chromatography-mass spectrometry. An application to 156 road fatalities

A method, using 0.2 ml of plasma, was designed for the simultaneous determination of morphine, 6-monoacetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, benzoylecgonine and cocaine. The drugs were analysed by LC-MS, after solid phase extraction in the presence of the deuterated analogues. Reversed phase separation on an Atlantis dC18 column was achieved in 10 min, under gradient conditions. The method was full validated, including linearity (2-250 ng/ml, r2>0.99), recovery (>50%), within-day and between-day precision and accuracy (CV and bias <15%), limit of detection (0.5 and 1 ng/ml) and quantitation (2 ng/ml), relative ion intensities and no matrix effect was observed. The procedure showed to be sensitive and specific, and was applied to 156 real cases from road fatalities (7.1% cases positive to cocaine and 0.6% to designer drugs).     

Quantification of 3,4-methylenedioxymetamphetamine and its metabolites in plasma and urine by gas chromatography with nitrogen–phosphorus detection

A gas chromatographic method with nitrogen–phosphorus detection involving a solid–liquid extraction phase was developed and validated for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in plasma. A modification of this method was validated for the analysis of MDMA, MDA, 4-hydroxy-3-methoxymethamphetamine (HMMA) and, 4-hydroxy-3-methoxyamphetamine (HMA) in urine. Under the analytical conditions described, the limits of detection in plasma and urine were less than 1.6 μg/l and 47 μg/l, respectively, for all the compounds studied. Good linearity was observed in the concentration range evaluated in plasma (5–400 μg/l) and urine (100–2000 μg/l) for all compounds tested. The recoveries obtained from plasma were 85.1% and 91.6% for MDMA and MDA, respectively. Urine recoveries were higher than 90% for MDMA and MDA, 74% for HMMA, and 64% for HMA. Methods have been successfully used in the assessment of plasma and urine concentrations of MDMA and its main metabolites in samples from clinical studies in healthy volunteers.