Separation of isotopologues of amphetamine with various degree of deuteration on achiral and polysaccharide-based chiral columns in high-performance liquid chromatography.

Hydrogen/deuterium (H/D) isotope effects are not unusual in chromatography and such phenomena have been observed in both gas- and liquid-phase separations. Despite the numerous reports on this topic, the understanding of mechanisms and the underlying noncovalent interactions at play remains rather challenging. In our recent study, we reported baseline separation of isotopologoues of some amphetamine (AMP) derivatives on achiral and polysaccharide-based chiral columns, as well as some correlations between the degree of separation of enantiomers and isotopologues on (the same) polysaccharide-based chiral column(s). Following our previous findings on isotope effects in high-performance liquid chromatography, we report herein a comparative study on the isotope effects observed with AMP and methamphetamine (MET). The impact of some pivotal factors such as the number of deuterium atoms part of AMP isotopologues, the structure of its isotopomers, the chemical structure of the achiral and chiral stationary phases used in this study, and the use of methanol- vs acetonitrile-containing mobile phases on the isotope effects was examined and discussed. Quantitative correlations between the observed isotope effects and the enantioselectivity of the chiral columns used are also shortly discussed. Furthermore, considering the chromatographic results as benchmark experimental data, we attempted to elucidate the molecular bases of the observed phenomena using quantum mechanics calculations.

Polyoxometalate-Based Frameworks as Adsorbents for Drug of Abuse Extraction from Hair Samples.

The linkage of molecular components into functional heterogeneous framework materials has revolutionized modern materials chemistry. Here, we use this principle to design polyoxometalate-based frameworks as high affinity adsorbents for drugs of abuse, leading to their application in solid-phase extraction analysis. The frameworks are assembled by the reaction of a Keggin-type polyanion, [SiW12O40]4-, with lanthanoids Dy(III), La(III), Nd(III), and Sm(III) and the multidentate linking ligand 1,10-phenanthroline-2,9-dicarboxylic acid (H2PDA). Their reaction leads to the formation of crystalline 1D coordination polymers. Because of the charge mismatch between the lanthanoids (+3) and the dodecasilicotungstate (-4), we observe incorporation of the PDA2- ligands into crystalline materials, leading to four polyoxometalate-based frameworks where Keggin-type heteropolyanions are linked by cationic {Lnn(PDA)n} groups (Ln = Dy (1), La (2), Nd (3), and Sm (4)). Structural analysis of the polyoxometalate-based frameworks suggested that they might be suitable for surface binding of common drugs of abuse via supramolecular interactions. To this end, they were used for the extraction and quantitative determination of four model drugs of abuse (amphetamine, methamphetamine, codeine, and morphine) by using micro-solid-phase extraction (D-µSPE) and high-performance liquid chromatography (HPLC). The method showed wide linear ranges, low limits of detection (0.1-0.3 ng mL-1), high precision, and satisfactory spiked recoveries. Our results demonstrate that polyoxometalate-based frameworks are suitable sorbents in D-µSPE for molecules containing amine functionalities. The modular design of these networks could in the future be used to expand and tune their substrate binding behavior.

Successful use of a novel lux® i-Amylose-1 chiral column for enantioseparation of "legal highs" by HPLC.

Bath salts, fumigations, cleaners and air fresheners, behind these terms substances are hidden, which count as "Legal Highs". These fancy names are used to pretend Legal Highs as harmless compounds, to circumvent legal regulations for marketing as well as to increase the sales. Besides classic illicit drugs of synthetic origin such as amphetamines, cocaine and MDMA, the trade of these compounds, also known as new psychoactive substances (NPS), is not uncommon today. In many countries, NPS are still not subject to drug control. Among them, there are stimulants such as new amphetamine derivatives or cathinones, which possess a chiral centre. Little is known about the fact that the two possible enantiomers may differ in their pharmacological effect. The aim of this study was to test a novel HPLC column for the enantioseparation of a set of 112 NPS coming from different chemical groups and collected by internet purchases during the years 2010-2018. The CSP, namely Lux® 5 µm i-Amylose-1, LC Column 250 x 4.6 mm, was run in normal phase mode under isocratic conditions, UV detection was performed at 245 nm and 230 nm, injection volume was 10 µl and flow rate was 1 ml/min. With a mobile phase consisting of n-hexane/isopropanol/diethylamine (90:10:0.1), herein, 79 NPS were resolved into their enantiomers successfully, for 37 of them baseline resolution was achieved. After increase of lipophily of the mobile phase to 99:1:0.1, another 27 compounds were baseline separated. It was found that all separated NPS are traded as racemic compounds.

Multi-residue method for enantioseparation of psychoactive substances and beta blockers by gas chromatography-mass spectrometry.

Currently, consumption of illicit drugs and pharmaceuticals has increased significantly. Many of these substances are chiral and can be available as racemates or enantiomerically pure. Determination of the enantiomeric fraction (EF) in wastewater is useful for: i) distinguishing between the consumption of prescribed and illicit drugs; ii) identification of possible local of illegal synthesis; iii) illegal discharge of sewage and estimation of illicit drugs and pharmaceuticals consumption by a community (wastewater-based epidemiology). This work describes the development of an indirect method by gas chromatography-mass spectrometry (GC-MS) for enantiomeric quantification of chiral substances namely psychoactive drugs and ß-blockers based on the formation of diastereomers using (R)-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride ((R)-MTPA-Cl) as chiral derivatization reagent. The developed method presented linearity (R2 > 0.99) for 20 compounds, 9 diastereomer pairs and paroxetine (PAR) and sertraline (SER). Recovery ranged from 80.7 to 114.5% (RSD < 9.1%) and accuracy between 84.6 and 118% (RSD < 9.9%). The limits of detection (LOD) varied from 0.03 and 26.0 ngL-1 and limits of quantification (LOQ) from 0.15 and 104 ngL-1. Results showed the occurrence of amphetamine (AMP), illicit drugs as 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (MAMP), alprenolol (ALP), norfluoxetine (NFLX), (SER), metoprolol (MET) and propranolol (PHO) at concentrations ranging from 21.7 ngL-1 (MDMA) to 622 ngL-1 (PHO). Measured concentrations were used to estimate the drug loads of the target chiral substances in a specific population. The EF was determined providing valuable information about the consumption and origin of the target drugs.

Hybrid Solid-Phase Extraction for Selective Determination of Methamphetamine and Amphetamine in Dyed Hair by Using Gas Chromatography-Mass Spectrometry.

Sample preparation is an important step in the isolation of target compounds from complex matrices to perform their reliable and accurate analysis. Hair samples are commonly pulverized or processed as fine cut, depending on preference, before extraction by techniques such as solid-phase extraction (SPE), liquid-liquid extraction, and other methods. In this study, a method based on hybrid solid-phase extraction (hybridSPE) and gas chromatography-mass spectrometry (GC-MS) was developed and validated for the determination of methamphetamine (MA) and amphetamine (AP) in hair. The hair samples were mechanically pulverized after washing with de-ionized water and acetone. The samples were then sonicated in methanol at 50 °C for 1 h and centrifuged at 50,000× g for 3 min. The supernatants were transferred onto the hybridSPE cartridge and extracted using 1 mL of 0.05 M methanolic hydrogen chloride. The combined solutions were evaporated to dryness, derivatized using pentafluoropropionic anhydride, and analyzed by GC-MS. Excellent linearity (R2 > 0.9998) was achieved in the ranges of 0.05-5.0 ng/mg for AP and 0.1-10.0 ng/mg for MA. The recovery was 83.4-96.8%. The intra- and inter-day accuracies were -9.4% to 5.5% and -5.1% to 3.1%, while the intra- and inter-day precisions were within 8.3% and 6.7%, respectively. The limits of detections were 0.016 ng/mg for AP and 0.031 ng/mg for MA. The validated hybridSPE method was applied to dyed hair for MA and AP extraction and compared to a methanol extraction method currently being used in our laboratory. The results showed that an additional hybridSPE step improved the recovery by 5.7% for low-concentration quality control (QC) samples and by 24.1% for high-concentration QC samples. Additionally, the hybridSPE method was compared to polymeric reversed-phase SPE methods, and the absolute recoveries for hybridSPE were 50% and 20% greater for AP (1.5 ng/mg) and MA (3.0 ng/mg), respectively. In short, the hybridSPE technique was shown to minimize the matrix effects, improving GC-MS analysis of hair. Based on the results, the proposed method proved to be effective for the selective determination of MA and AP in hair samples.

Amphetamine-type stimulants analysis in oral fluid based on molecularly imprinting extraction.

A methamphetamine-based molecularly imprinted polymer (MIP) has been prepared by bulk polymerization to recognize new psychoactive substances (NPS) of the amphetamine, cathinones and 2C families in oral fluid samples, being the first precedent of a synthetized MIP for the extraction and preconcentration 32 NPS including amphetamine type substances and synthetic cathinones from oral fluids. Pre-polymerization complex and resulting materials were appropriately characterized by infrared spectroscopy, scanning electron microscopy, and nitrogen adsorption-desorption isotherms. Appropriateness of the material for the specific recognition of the target analytes was also evaluated through computational calculations and experimentally assessed by solid phase extraction (SPE). The most appropriate SPE conditions were evaluated and recoveries of 32 different NPS were obtained, ranging from 80 to 120% with a relative standard deviation (RSD) in all cases lower than 12%. Amphetamine-related NPS were analyzed by a fast and portable methodology based on ion mobility spectrometry (IMS) and a rearguard procedure based on ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) providing limit of detection values from 10 to 80 µg L-1 and from 0.03 to 1.3 µg L-1, respectively. Oral fluid samples, containing different interferents like caffeine, fluticasone and cetirizine, were spiked with 300 µg L-1 amphetamine and subsequently analyzed, showing recoveries ranging from 81 to 115% using both methodologies. Thus, this paper shows preliminary results to demonstrate the applicability of the developed procedure which could be used with minor modifications as screening technique in on-road drug analysis.

Postmortem analysis of famprofazone and its metabolites, methamphetamine and amphetamine, in porcine bone marrow.

Forensic toxicologists typically work with body fluids, such as blood and urine, or visceral tissues. The analysis of alternative samples, such as bone marrow, can be requested when the commonly used samples are unavailable due to an extended time lapse between the time of death and collection of the material to be analysed. In this study, a method for the analysis of the lipophilic drug famprofazone (FA) and its metabolites, methamphetamine (MA) and amphetamine (AM), in bone marrow was developed, validated and applied to bone marrow from pigs given controlled doses of famprofazone. This method involves enzymatic bone-cleaning, fragmentation of the bones with the assistance of a micro electric motor, optimization of clean-up and LLE (liquid/liquid extraction) conditions and determination by GC/MS. After evaluation through statistical tests, such as Shapiro Wilk for normality and Cochran for homoscedasticity, a linear model was applied in the range of 100 (LOQ) - 2000 ng g-1. Inter-day precision and bias was always < 4.6%. In real sample analysis, bone marrow FA and MA concentrations ranged from 103 to 232 and from 174 to 267 ng g-1, respectively; AM was not detected. The obtained results are useful for application in forensic toxicological protocols (human autopsy cases) and as a starting point for the development of further analytical tools.

Emulsification microextraction of amphetamine and methamphetamine in complex matrices using an up-to-date generation of eco-friendly and relatively hydrophobic deep eutectic solvent.

In this study, a new extraction medium based on a quite bio-compatible and bio-degradable deep eutectic solvent comprising choline chloride and phenylethanol (ChCl: Ph-ETOH) was simply and cheaply synthesized at room temperature. At the next step, it was effectively utilized at the service of air agitated-emulsification microextraction (AA-EME) of two major amphetamine-type stimulants (ATSs) in human plasma and pharmaceutical wastewater pursued by high performance liquid chromatography-ultraviolet detection (HPLC-UV). This safe, effective, and rapid enrichment process based on the new low-density DES was easily practicable via a homemade extraction cell possessing a narrow neck and with no extra demand the emulsifier intermediates. Statistical study of main parameters effects using central composite design (CCD) combined with desirability function (DF) demonstrated that pH 12, 250 µL of extraction solvent, 8 air agitation cycles, and 5% of salt amount resulted in maximum extraction efficiencies (63-66%) with DF value close to 0.98. Under optimal conditions, wide linear dynamic ranges (LDRs) of 15.0-2000 and 8.0-3000 ng mL-1 with the determination coefficients (R2s) close to 0.99 were obtainable for amphetamine and methamphetamine, respectively. Low limits of detection (LODs) as well as relative standard deviations (%RSDs, n = 3) were found to be 2.0-5.0 ng mL-1 and 5.7-7.8%, respectively. Also, enrichment factors (EFs) were quantitative in the span of 47-50. On the other hand, satisfactory and accurate assessment at low levels close to therapeutic and toxic domains in human plasma sample and pharmaceutical wastewater was successfully obtained.

Synthesis of a novel polymeric magnetic solid phase extraction adsorbent for selective extraction of amphetamine from urine samples coupled with high performance liquid chromatography.

A novel pH-responsive block copolymer (Poly ethylene glycol-b-poly (N,N-dimethylaminoethylmethacrylate-co-maleic acid) was designed for the decoration and stabilization of magnetic nanoparticles (MNPs) as an efficient magnetic nano adsorbent for extraction of amphetamine (AM) from biological urine samples to be determined by high performance liquid chromatography-ultra violet detector (HPLC-UV). Full characterization of the synthesized polymeric magnetic nanoparticles (PMNPs) were followed by various techniques like Fourier transform infrared (FT-IR) spectroscopy, powder x-ray diffraction (XRD), scanning electron microscopy (SEM), and vibrating sample magnetometer (VSM). Important extraction parameters including pH, amount of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, agitation rate (rpm), and ionic strength of the extraction medium were studied and optimized. Under optimized extraction conditions, good linearity was observed in the concentration range of 30-2000 ng/mL for AM. The amount of the qe was calculated as 0.18 (mg/g). The method was applied in determination of AM from positive urine samples with the recovery of 99.84%. Results indicated that the proposed method could be applied in clinical and forensic laboratories for simple, selective, and fast determination of AM from urine samples.

Differentiation of ring-substituted regioisomers of amphetamine and methamphetamine by supercritical fluid chromatography.

Chromatographic differentiation of the ring-substituted regioisomers of amphetamine (AMP) and methamphetamine (MA) was performed by supercritical fluid chromatography (SFC). The behaviour of the retention against the changes of column temperature and co-solvent proportion was studied. The obtained information facilitated the optimization of the each regioisomer. As a result, 2-, 3-, and 4-ring-substituted analogues of AMP and MA with methyl, methoxy, fluoro, chloro, and bromo groups were separated, generally within 6 min. In addition, we found that the separation pattern of the examined regioisomers was classified into two, which depended on the electron donating/withdrawing effect of the substituent. Our results indicate that SFC could be used in forensic drug analysis for fast, reliable identification of structurally similar drugs of abuse. Copyright © 2016 John Wiley & Sons, Ltd.

Modifier-assisted differential mobility-tandem mass spectrometry method for detection and quantification of amphetamine-type stimulants in urine.

An advantage of differential mobility spectrometry (DMS) is it provides an orthogonal mechanism to mass spectrometry (MS). The DMS-MS/MS detects analytes in the gas phase on the basis of differences in ion mobility in low and high electric fields, which makes DMS-MS/MS an alternative to chromatographic separation-MS. One drawback of DMS is its limited resolution and sensitivity, especially for detecting small molecules when using a nonpolar inert gas as the carrier gas. The present work has evaluated the effects on peak capacity of adding chemical modifiers to inert carrier gases (nitrogen, helium, argon and carbon dioxide). Use of a methanol-helium mixture gave improvements in both separation and sensitivity. Nine structurally similar amphetamine-type stimulants were determined in urine without pretreatment of the samples before analysis. After optimization of carrier gas, nature and concentration of chemical modifier, and DMS temperature, limits of detection ranging from 1.1 to 2.7 ng mL-1, with a linear range of three orders of magnitude (5-5000 ng mL-1) were achieved. Precision was <15% and the accuracy of the quality control samples was 87.6-113.7%. For the quantitation of urine samples from drug abusers, data obtained using DMS-MS/MS showed reasonable agreement (within ±19.5%) with that obtained using LC-MS/MS. The analysis time for DMS-MS/MS was only 1.1 min and a paired sample t-test between the two methods gave a p-value of 0.0894, which indicates that DMS-MS/MS is a reliable method, with comparable precision and sensitivity to LC-MS/MS.

Analysis of amphetamine and methamphetamine in municipal wastewater influent and effluent using weak cation-exchange SPE and LC-MS/MS.

Amphetamine and methamphetamine are emerging contaminants-those for which no regulations currently require monitoring or public reporting of their presence in our water supply. In this research, a protocol for weak cation-exchange (WCX) SPE coupled with LC-MS/MS was developed for determination of emerging contaminants amphetamine and methamphetamine in a complex wastewater matrix. Gradient LC parameters were adjusted to yield baseline separation of methamphetamine from other contaminants. Methamphetamine-D5 was used as the internal standard (IS) to compensate for sample loss during SPE and for signal loss during MS (matrix effects). Recoveries were 102.1 ± 7.9% and 99.4 ± 4.0% for amphetamine and methamphetamine, respectively, using WCX sorbent. Notably, methamphetamine was determined to be present in wastewater influent at each sampling date tested. Amphetamine was present in wastewater influent on two of four sampling dates. Amphetamine concentrations ranged from undetectable to 86.4 ng/L in influent, but it was undetectable in wastewater effluent. Methamphetamine was detected in influent at concentrations ranging from 27.0-60.3 ng/L. Methamphetamine concentration was reduced but incompletely removed at this facility. Although absent in one post-UV effluent sample, concentrations of methamphetamine ranged from 10.8-14.8 ng/L.

Enantioselective separation of biologically active basic compounds in ultra-performance supercritical fluid chromatography.

The enantioseparation of basic compounds represent a challenging task in modern SFC. Therefore this work is focused on development and optimization of fast SFC methods suitable for enantioseparation of 27 biologically active basic compounds of various structures. The influences of the co-solvent type as well as different mobile phase additives on retention, enantioselectivity and enantioresolution were investigated. Obtained results confirmed that the mobile phase additives, especially bases (or the mixture of base and acid), improve peak shape and enhance enantioresolution. The best results were achieved with isopropylamine or the mixture of isopropylamine and trifluoroacetic acid as additives. In addition, the effect of temperature and back pressure were evaluated to optimize the enantioseparation process. The immobilized amylose-based chiral stationary phase, i.e. tris(3,5-dimethylphenylcarbamate) derivative of amylose proved to be useful tool for the enantioseparation of a broad spectrum of chiral bases. The chromatographic conditions that yielded baseline enantioseparations of all tested compounds were discovered. The presented work can serve as a guide for simplifying the method development for enantioseparation of basic racemates in SFC.

Development and validation of a magnetic solid-phase extraction with high-performance liquid chromatography method for the simultaneous determination of amphetamine and methadone in urine.

The simultaneous determination of amphetamine and methadone was carried out by magnetic graphene oxide nanoparticles, a magnetic solid-phase extraction adsorbent, as a new sample treatment technique. The main factors (the amounts of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, pH, the ionic strength of extraction medium, and agitation rate) influencing the extraction efficiency were investigated and optimized. Under the optimized conditions, good linearity was observed in the range of 100-1500 ng/mL for amphetamine and 100-1000 ng/mL for methadone. The method was evaluated for determination of AM and methadone in positive urine samples, satisfactory results were obtained, therefore magnetic solid-phase extraction can be applied as a novel method for the determination of drugs of abuse in forensic laboratories.

Chiral separation of cathinone and amphetamine derivatives by HPLC/UV using sulfated ß-cyclodextrin as chiral mobile phase additive.

In the last years the identification of new legal and illegal highs has become a huge challenge for the police and prosecution authorities. In an analytical context, only a few analytical methods are available to identify these new substances. Moreover, many of these recreational drugs are chiral and it is supposed that the enantiomers differ in their pharmacological potency. Since nonenantioselective synthesis is easier and cheaper, they are mainly sold as racemic mixtures. The goal of this research work was to develop an inexpensive method for the chiral separation of cathinones and amphetamines. This should help to discover if the substances are sold as racemic mixtures and give further information about their quality as well as their origin. Chiral separation of a set of 6 amphetamine and 25 cathinone derivatives, mainly purchased from various Internet shops, is presented. A LiChrospher 100 RP-18e, 250 x 4 mm, 5 µm served as the stationary phase. The chiral mobile phase consisted of methanol, water, and sulfated ß-cyclodextrin. Measurements were performed under isocratic conditions in reversed phase mode using UV detection. Four model compounds of the two substance classes were used to optimize the mobile phase. Under final conditions (methanol:water 2.5:97.5 + 2% sulfated ß-cyclodextrin) enantiomers of amphetamine and five derivatives were baseline separated within 23 min. In all, 17 cathinones were completely or partially chirally separated. However, as only 3 of 25 cathinones were baseline resolved, the application of this method is limited for cathinone analogs. Additionally, the results were compared with an RP-8e column.

A comprehensive chromatographic comparison of amphetamine and methylamphetamine extracted from river water using molecular imprinted polymers and without the need for sample derivatization.

This work explores the differences between two GCMS instruments for the determination of amphetamine and methylamphetamine extracted from water samples (ultra pure water and river water) without the necessity for derivatization. The instruments contained different generations of gas chromatograph and mass selective detector components and revealed significantly different results when presented with the same samples. The extraction methodology also compared two SPE systems. The extraction efficiency of commercially available molecular imprinted polymers as a sorbent in SPE was compared with commonly used hydrophilic balance sorbent. Molecular imprinted polymers provided excellent recoveries (81 ± 2% and 108 ± 3% at 30 µg L(-1), and 94 ± 2% and 94 ± 2% at 200 µg L(-1) for amphetamine and methylamphetamine, respectively). The best LOD obtained was sufficient for the determination of both drugs extracted from river water (0.029 ± 0.003 and 0.015 ± 0.004 µg L(-1) for amphetamine and methylamphetamine, respectively). These were comparable to literature values obtained through conventional extraction and analysis using LC-MS/MS but had the advantage of being achieved using an underivatized GCMS method.

Molecularly imprinted solid-phase extraction for the selective determination of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs in human whole blood by gas chromatography-mass spectrometry.

A novel method is described for the extraction of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs, such as 3,4-methylenedioxy-methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine, and 3,4-(methylenedioxyphenyl)-2-butanamine, from human whole blood using molecularly imprinted solid-phase extraction as highly selective sample clean-up technique. Whole blood samples were diluted with 10 mmol/L ammonium acetate (pH 8.6) and applied to a SupelMIP-Amphetamine molecularly imprinted solid-phase extraction cartridge. The cartridge was then washed to eliminate interferences, and the amphetamines of interest were eluted with formic acid/methanol (1:100, v/v). After derivatization with trifluoroacetic anhydride, the analytes were quantified using gas chromatography-mass spectrometry. Recoveries of the seven amphetamines spiked into whole blood were 89.1-102%. The limits of quantification for each compound in 200 µL of whole blood were between 0.25 and 1.0 ng. The maximum intra- and inter-day coefficients of variation were 9.96 and 13.8%, respectively. The results show that methamphetamine, amphetamine, and methylenedioxyphenylalkyl-amine designer drugs can be efficiently extracted from crude biological samples such as whole blood by molecularly imprinted solid-phase extraction with good reproducibility. This extraction method will be useful for the pretreatment of human samples before gas chromatography-mass spectrometry.

A new strategy for basic drug extraction in aqueous medium using electrochemically enhanced solid-phase microextraction.

This work describes an electrochemically enhanced solid-phase microextraction (EE-SPME) method using a mild negative potential (-0.6 V) for the enhanced extraction of the selected basic drugs in a pure aqueous matrix and urine samples. The EE-SPME method gave a more effective extraction of drugs (primarily via electrophoresis and complementary charge interaction) compared to that obtained with SPME (without applying a potential, and which is based on passive partitioning). The EE-SPME method eliminated the need for alkalizing, derivatizing the drugs, or modifying the fiber coating before extraction. The analysis of methamphetamine (MA) and amphetamine (AM) was selected as a typical example to demonstrate in detail the advantages of EE-SPME over SPME. Based on the results obtained, 3-min extraction efficiency for both the amphetamines using EE-SPME was better than that of 30-min using SPME. The developed EE-SPME-GC method exhibited wide linear ranges (2-1000 ng mL(-1)) for both the amphetamines with R(2) larger than 0.99, and the method detection limits (MDLs) for AM and MA were 0.26 and 0.12 ng mL(-1), respectively. In addition, the EE-SPME method developed was also successfully applied to enhance the extraction of several other basic drugs (ephedrine, 3,4-methylenedioxyamphetamine (MDA), atropine, methadone, cocaine, codeine, acetylcodeine and papaverine) with preconcentration factors from 157 to 2199, indicating the potential applicability of this method in the field of forensic, clinical and pharmaceutical analysis.

Simultaneous separation and confirmation of amphetamine and related drugs in equine plasma by non-aqueous capillary-electrophoresis-tandem mass spectrometry.

A non-aqueous capillary electrophoresis-mass spectrometry (NACE-MS) method was developed for simultaneous separation and identification of 12 amphetamine and related compounds in equine plasma. Analytes were recovered from plasma by liquid-liquid extraction using methyl tertiary butyl ether (MTBE). A bare fused-silica capillary was used for separation of the analytes. Addition of sheath liquid to the capillary effluent allowed the detection of the analytes by positive electrospray ionization mass spectrometry using full scan data acquisition. The limit of detection (LOD) for the target analytes was 10-200 ng/mL and that of confirmation (LOC) was 50-1000 ng/mL in equine plasma. Capillary electrophoresis (CE) and mass spectrometry (MS) parameters were optimized for full CE separation and MS detection of the analytes. Separation buffer comprised 25 mM ammonium formate in acetonitrile/methanol (20: 80, v/v) plus 1 M formic acid. Sheath liquid was isopropanol-water-formic acid (50:50:0.5, v/v/v). Samples were hydrodynamically injected and separated at 25 kV. Analytes were electrokinetically separated and mass spectrometrically identified and confirmed. This simple, fast, inexpensive and reproducible method was successfully applied to post race equine plasma and research samples in screening for amphetamine and related drugs.

Comparison of molecularly imprinted, mixed-mode and hydrophilic balance sorbents performance in the solid-phase extraction of amphetamine drugs from wastewater samples for liquid chromatography-tandem mass spectrometry determination.

Recent studies have shown that amphetamines and other drugs of abuse residues occur in wastewater. Consequently, several methods have been developed for their determination by solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, a major drawback of these methods is the lack of selectivity during SPE that results in reduced sensitivity, due to matrix effects, and in some cases in low precision and poor accuracy. In order to tackle this problem, three different SPE alternatives have been evaluated in this work for the determination of five amphetamines: common hydrophilic balance (Oasis HLB), mixed-mode (Oasis MCX) and molecularly imprinted polymers (MIPs) sorbents. Among them, Oasis HLB showed the worst performance, as three amphetamines (MDA, MDMA and MDEA) could not be determined because of interfering signals in the LC-MS/MS chromatogram, and amphetamine recoveries could not be corrected by the use of the deuterated analogue internal standard. Oasis MCX permitted the determination of all target analytes, but with still strong signal suppression: ca. 70% signal drop with wastewater samples, which could in this case be corrected by the internal standards providing acceptable trueness (overall recoveries: 101-137%), precision (RSD: 2.0-12%) and limits of detection (LOD: 1.5-4.4 ng/L). Alternatively, MIPs rendered cleaner extracts with less matrix effects (ca. 30% signal drop), and thus lower LODs (0.5-2.7 ng/L) and even better trueness (91-114% overall recovery) and precision (1.5-4.4%RSD). The final application of the method with MIP cartridges showed the presence of MDA and MDMA in the seven analysed wastewaters at the 4-20 ng/L level.