Screening of Astec® CHIRALDEX™ G-PN and LIPODEX™ D gas chromatography columns for enantioseparation of amphetamine derivatives.

Among different substance classes, New Psychoactive Substances (NPS) comprise chiral amphetamines for stimulant and empathic effects. There is little knowledge in terms of clinical studies about possibly different effects of the two enantiomers of novel amphetamine derivatives. For this reason, there is a big demand for enantioseparation method development of this new substance class. Regarding gas chromatography, cyclodextrins proved to be effective for enantioseparation of NPS. In our attempt, an Astec® Chiraldex™ G-PN column containing 2,6-di-O-pentyl-3-propionyl-γ-cyclodextrin and a Lipodex™ D column containing heptakis-(2,6-di-O-pentyl-O-acetyl)-ß-cyclodextrin as chiral selector served as stationary phases in a Shimadzu GCMS-QP2010 SE system. Because of the special coating, maximum temperature is limited to 200 °C isothermal or 220 °C in programmed mode. To ensure detection, trifluoroacetic anhydride (TFAA) was used to increase sample volatility.1 As a result, 35 amphetamines were tested as their TFAA-derivatives. A screening method with a temperature gradient from 140 °C to 200 °C at a heating ramp of 1 °C per minute and final time of 5 min, showed baseline separation for seven and partial separations for 16 trifluoro acetylated amphetamines using the Chiraldex™ G-PN column. Six baseline and nine partial separations were observed with the Lipodex™ D column, respectively.

[Research progress on chiral separation of amphetamines, ketamine, cathinones].

Enantiomers are ubiquitous in nature, and they are especially important in the field of pharmaceutical chemistry. Although the enantiomers of chiral drugs have identical chemical structures, they differ notably in their pharmacological, toxicological, pharmacokinetic, metabolic, and other biological activities. The same is true for amphetamines, ketamine, and cathinones, as the chiral separation of these three drugs is representative of drugs. Gas chromatography (GC), high performance liquid chromatography (HPLC), and capillary electrophoresis (CE) are widely used for the chiral separation of these three kinds of drugs. There are some similarities among the three methods for the chiral separation of amphetamines, ketamine, and cathinones: n-trifluoroacetyl-L-prolinyl chloride and (+)R-α-methoxy-α-trifluoromethylphenylacetic acid are the two typical chiral derivatization reagents used in GC. In HPLC, three kinds of chiral stationary phases are used: proteins, polysaccharides, and macrocyclic antibiotics. Cyclodextrin and its derivatives are most commonly used in CE. However, these three methods have inherent shortcomings. In the case of GC, impurities produced during chiral derivatization may interfere with the analysis, and high reaction temperatures affect the efficiency of chiral separation. HPLC has limited application scope and is expensive. In CE, there has no established process to determine the appropriate chiral selector. In recent years, research into application of the chiral separation of the above-mentioned three kinds of drugs has its own characteristics in forensic toxicology. The chiral separation of amphetamine drugs is mostly used to infer the prototype and synthesis route of drugs on the market. The chiral separation of ketamine involves a variety of biological samples. For cathinones, chiral separation methods emphasize their wide applicability. In this review, 66 reports published in professional local and overseas magazines during the past decade are collated. The characteristics of the enantiomers of amphetamines, ketamine, and cathinones as well as the mechanism of chiral recognition are briefly introduced. The commonness of the research and the application of chiral separation in forensic toxicology are reviewed. This paper proposes that the chiral separation of drugs can be further investigated from the following three aspects: 1) the use of computer technology to establish a molecular model for exploring the mechanism of chiral recognition; 2) developing new technologies for chiral separation and carrying out commercial research on the supercritical fluid method; 3) applying chiral separation to judicial practice, pharmaceutical research and development, and other practical fields.

Rapid Analysis of Four Amphetamines in Urine by Self-Made Pipette-Tip Solid-Phase Extraction Followed by GC-MS/MS.

A simple and rapid pipette-tip solid-phase extraction (PT-SPE) procedure with derivatization prior to gas chromatography triple quadrupole mass spectrometry analysis is developed for the simultaneous determination of amphetamine (AMP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples. The PT-SPE procedure using self-made sorbent could extract drugs within 6 min from 100-µL urine samples, requiring low solvent-consumption (<2.0 mL). Besides, the self-made pipette tip could be reused at least five times. Under optimized conditions, the recoveries of four amphetamines at spiked levels (low, medium and high) ranged from 87.7 to 110.4%, with relative standard deviations < 9.5%. The limit of detections and limit of quantifications for AMP, MA, MDA and MDMA were in the range of 2.52-8.25 ng⋅mL-1 and 8.4-27.5 ng⋅mL-1, respectively. Validation results show that the proposed method is suitable for the quantitation of amphetamines and has been successfully applied in the urine samples of suspected drug abusers.

Hollow-fibre liquid-phase microextraction and gas chromatography-mass spectrometric determination of amphetamines in whole blood.

Here, we present a fully validated method using a hollow-fibre liquid-phase microextraction technique for the determination by gas chromatography-mass spectrometry (GC-MS) of amphetamine (AMP), methamphetamine (MET), fenproporex (FEN), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxyethylamphetamine (MDEA) in whole blood. The validation parameters presented successful values within those recommended by the Scientific Working Group for Forensic Toxicology (SWGTox) in the Standard Practices for Method Validation in Forensic Toxicology. The limits of detection ranged from 1 to 3 ng/mL, and the limits of quantification ranged from 2 to 5 ng/mL. The determination coefficients (r2) ranged from 0.990 to 0.997, and the method presented good intraday and interday accuracy (from 90.4% to 97.2%) and satisfactory recovery (from 68% to 110%). No carryover was observed. The heteroscedasticity was tested, and only AMP presented homoscedasticity. Weighting factors were applied to correct the linearity of MET (1/x2), MDA (1/x), FEN (1/x1/2), MDMA (1/x2) and MDEA (1/y). Dilution integrity was tested at ratios of 1:2, 1:5 and 1:10, and all maintained intraday precision (from 94.9% to 99.3%) and interday precision (from 89.4% to 94.9%). The validated method was applied to six real whole blood samples from individuals suspected of consuming ecstasy, and MDMA, MDA and amphetamine were successfully identified and quantified.

Developed nano carbon-based coating for simultaneous extraction of potent central nervous system stimulants from urine media by stir bar sorptive extraction method coupled to high performance liquid chromatography.

A nano graphene oxide sol-gel composite (NGO/sol-gel) applied as a coating of a capillary glass tube stir bar to develop a novel stir bar sorptive extraction (SBSE) method for simultaneous extraction of amphetamine (AMP) and methamphetamine (MET) from biological urine sample. Lab-synthesized NGO was applied with methyltrimethoxysilane (MTMOS) and Tetraethoxysilane (TEOS) as sol-gel precursor. NGO/sol-gel was deposited on the surface of a capillary glass tube to prepare stir bar sorptive extraction adsorbent by a simple and fast method. The scanning electron micrograph images showed a three dimensional structure of lab-made device suitable for SBSE method for simultaneous extraction of AMP and MET. Effective extraction parameters were investigated. Through studied suitable extraction conditions, satisfactory linearity was achieved in the concentration range of 50-2000 ngmL-1 for AMP and 40-2500 ngmL-1 for MET. The relative recovery of the analytes were 99.5 and 99.7% for AMP and MET, respectively for positive urine samples were studied by novel introduced method. The results cleared that NGO/sol-gel composite could be used as practical method in laboratories as an efficient SBSE adsorbent for drugs determination in urine matrix.

Salt-Assisted Liquid-Liquid Extraction of Meconium for Analysis of Cocaine and Amphetamines by Liquid Chromatography-Tandem Mass Spectrometry.

Meconium, the first stool of a newborn, can be analyzed to identify prenatal exposure to drugs of abuse. Meconium accumulates in a fetus during the second and third trimesters of pregnancy providing a wide window of exposure. Identification of in utero drug exposure is essential for the diagnosis and treatment of infants for dependency/withdrawal caused from the exposure. However, testing of meconium samples is often cumbersome and time-consuming. Unlike liquid samples, meconium is a viscous, semisolid, tar-like substance that needs to be individually weighed prior to extraction. Additionally, the meconium matrix is not homogeneous and not easily mixed or extracted. A method for analyzing cocaine and metabolites as well as amphetamines in meconium utilizing ceramic homogenizers prior to salt-assisted liquid-liquid extraction and liquid chromatography tandem-mass spectrometry (LC-MS/MS) is presented.

Analysis of Drugs in Oral Fluid Using LC-MS/MS.

Oral fluid analysis for drugs is increasingly used in a variety of testing areas: pain management and medication monitoring, parole and probation situations, driving under the influence of drugs (DUID), therapeutic drug monitoring, and testing for drugs in the workplace. The sample collection itself is straightforward, rapid, observable, and noninvasive, requiring no special facilities (compared to urine) or medical personnel (compared to blood). The pH of saliva is slightly acidic relative to blood; therefore, drugs which are more basic tend to be present in higher concentration in oral fluid than in blood: cocaine, amphetamines, oxycodone, tramadol, buprenorphine, methadone, and fentanyl. Conversely, acidic drugs and drugs which are strongly protein bound have lower concentrations in oral fluid than in blood: examples include benzodiazepines, barbiturates, and carisoprodol. Because of the low volume of specimen available for analysis and the drug concentrations present (generally much lower than those in urine), efficient extraction methods and sensitive confirmation procedures are necessary for routine analysis of drugs in oral fluid. In this chapter, solid-phase extraction methods are described for a variety of drugs with liquid chromatography-tandem mass spectrometry detection.

Separation of ortho, meta and para isomers of methylmethcathinone (MMC) and methylethcathinone (MEC) using LC-ESI-MS/MS: Application to forensic serum samples.

Separation and identification of positional isomers is an important issue in forensic toxicology, particularly in the context of new psychoactive substances (NPS). Despite the structural similarity, positional isomers often show different pharmacological properties and thus can exhibit dramatic differences with respect to their toxicity. Additionally, besides these pharmacological and toxicological effects, the legal status is also of great importance. We present a sensitive and selective LC-MS/MS method to separate the ortho, meta and para isomers of methylmethcathinone (MMC) and methylethcathinone (MEC) using a core-shell biphenyl analytical column. Reliability of the method was confirmed under consideration of the validation parameters selectivity, linearity, accuracy and precision, analytical limits, processed sample stability, matrix effects and recovery. Linearity was demonstrated over the entire calibration range from 5 to 250ng/ml with the use of a 1/x2 weighting. Appropriate quantification and detection limits (LLOQ=5ng/ml, LOD<2ng/ml) could be achieved. Application of the method to real serum samples collected between June 2014 and August 2016 revealed the proof of a recent MMC or MEC consumption, respectively, in eight cases. Isomers of MMC could be detected in three of these eight cases, of which two were positive for 3-MMC and one was positive for 2-MMC. The other samples were tested positively for 3-MEC. In none of the samples 4-MMC, 2-MEC or 4-MEC could be detected. Only substances that were not governmentally controlled at that time could be detected, reflecting the rapid response of the recreational drug market to newly enacting drug laws.

Combination of counter current salting-out homogenous liquid-liquid extraction and dispersive liquid-liquid microextraction as a novel microextraction of drugs in urine samples.

The counter current salting-out homogenous liquid-liquid extraction (CCSHLLE) joined with the dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) has been developed as a high preconcentration technique for the determination of different drugs in urine samples. Amphetamines were employed as model compounds to assess the extraction procedure and were determined by high performance liquid chromatography-ultraviolet detection (HPLC-UV). In this method, initially, NaCl as a separation reagent is filled into a small column and a mixture of urine and acetonitrile is passed through the column. By passing the mixture, NaCl is dissolved and the fine droplets of acetonitrile are formed due to salting-out effect. The produced droplets go up through the remained mixture and collect as a separated layer. Then, the collected acetonitrile is removed with a syringe and mixed with 30.0µL 1-undecanol (extraction solvent). In the second step, the 5.00mLK2CO3 solution (2% w/v) is rapidly injected into the above mixture placed in a test tube for further DLLME-SFO. Under the optimum conditions, calibration curves are linear in the range of 1-3000µgL(-1) and limit of detections (LODs) are in the range of 0.5-2µgL(-1). The extraction recoveries and enrichment factors ranged from 78 to 84% and 157 to 168, respectively. Repeatability (intra-day) and reproducibility (inter-day) of method based on seven replicate measurements of 100µgL(-1) of amphetamines were in the range of 3.5-4.5% and 4-5%, respectively. The method was successfully applied for the determination of amphetamines in the actual urine samples. The relative recoveries of urine samples spiked with amphetamine and methamphetamine are 90-108%.

QuEChERS sample preparation prior to LC-MS/MS determination of opiates, amphetamines, and cocaine metabolites in whole blood.

Modern LC-MS/MS instruments have sensitivity and scanning velocity high enough to analyze many different compounds in single runs. Consequently, the sample preparation procedure has become the bottleneck for developing efficient, rapid, and cheap multi-compound methods. Here, we examined one-step sample preparation based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) salts to set up and validate a LC-MS/MS method for the simultaneous determination of 35 drugs of abuse and their metabolites in whole blood. Despite large differences in physicochemical properties, this simplified QuEChERS extraction method yielded satisfactory recoveries (until 96%) for the 35 molecules. The amounts of QuEChERS salts had no influence on extraction yield. Chromatographic separation was obtained in less than 6 min. LLOD and LLOQ were 3 and 5 ng/mL, respectively. The procedure was successfully validated and then applied to 253 cases of driving under the influence of drugs (DUID), collected over a 6-month period.

An amphetamine isomer whose efficacy and safety in humans has never been studied, ß-methylphenylethylamine (BMPEA), is found in multiple dietary supplements.

The amphetamine isomer ß-methylphenylethylamine (BMPEA) was first synthesized in the early 1930s, but its efficacy and safety in humans has not been studied. Recently, the United States Food and Drug Administration (FDA) detected BMPEA in dietary supplements labelled as containing Acacia rigidula. Over a year after the FDA reported its findings, we analyzed Acacia rigidula dietary supplements to determine if BMPEA had been removed. Supplements were analyzed using liquid chromatography-quadrupole time-of-flight mass spectrometry. Diluted methanolic extract from each supplement was run three times and each data set obtained was analyzed using Agilent MassHunter Qualitative Analysis. The presence of BMPEA was confirmed by accurate mass, retention time and mass spectra match against a reference standard. Quantification of BMPEA was determined using an eight-point calibration curve of spiked standard to a matrix blank. Twenty-one brands of Acacia rigidula supplements were analyzed. More than half (11/21; 52.4%) of the Acacia rigidula supplement brands contained BMPEA. The stimulant was present at quantities such that consumers following recommended maximum daily servings would consume a maximum of 93.7 mg of BMPEA per day. Consumers of Acacia rigidula supplements may be exposed to pharmacological dosages of an amphetamine isomer that lacks evidence of safety in humans. The FDA should immediately warn consumers about BMPEA and take aggressive enforcement action to eliminate BMPEA in dietary supplements. Copyright © 2015 John Wiley & Sons, Ltd.

Screening determination of four amphetamine-type drugs in street-grade illegal tablets and urine samples by portable capillary electrophoresis with contactless conductivity detection.

A simple and inexpensive method for the identification of four substituted amphetamines, namely, 3,4-methylenedioxy methamphetamine (MDMA), methamphetamine (MA), 3,4-methylenedioxy amphetamine (MDA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) was developed using an in-house constructed semi-automated portable capillary electrophoresis instrument (CE) with capacitively coupled contactless conductivity detection (C(4)D). Arginine 10mM adjusted to pH4.5 with acetic acid was found to be the optimal background electrolyte for the CE-C(4)D determination of these compounds. The best detection limits achieved with and without a sample preconcentration process were 10ppb and 500ppb, respectively. Substituted amphetamines were found in different seized illicit club drug tablets and urine samples collected from different suspected users. Good agreement between results from CE-C(4)D and those with the confirmation method (GC-MS) was achieved, with correlation coefficients for the two pairs of data of more than 0.99.

Validation of a fast UPLC-MS/MS method for quantitative analysis of opioids, cocaine, amphetamines (and their derivatives) in human whole blood.

BACKGROUND: Conventional methods for analysis of drugs of abuse require multiple assays which can be both expensive and time-consuming. This work describes a novel, rapid, simple and sensitive method for the quantification of 14 illicit drugs and their metabolites in whole blood. Results/methodology: This method employed a rapid liquid-liquid sample extraction of whole blood followed by UPLC-MS/MS analysis. Calibration curves were validated for analysis of appropriate concentrations. Inter- and intra-assay variations were <14.8%. Deviation of accuracy was <14.9% from target concentration for each quality control level. CONCLUSION: This work described the development and the full validation of a precise, sensitive and accurate assay. After validation, this new assay was successfully applied to routine toxicological analysis.

Comparison of hollow fiber liquid-phase microextraction and ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction for the determination of drugs of abuse in biological samples by gas chromatography-mass spectrometry.

Two microextraction techniques based on hollow fiber liquid-phase microextraction (HF-LPME) and ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) had been applied for the determination of drugs of abuse (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, methcathinone, ketamine, meperidine, and methadone) in urine and blood samples by gas chromatography-mass spectrometry. Parameters affecting extraction efficiency have been investigated and optimized for both methods. Under the optimum conditions, linearities were observed for all analytes in the range 0.0030-10 µg/ml with the correlation coefficient (R) ranging from 0.9985 to 0.9995 for HF-LPME and in the range 0.0030-10 µg/ml with the R ranging from 0.9985 to 0.9994 for DLLME. The recovery of 79.3-98.6% with RSDs of 1.2-4.5% was obtained for HF-LPME, and the recovery of 79.3-103.4% with RSDs of 2.4-5.7% was obtained for DLLME. The LODs (S/N=3) were estimated to be in the range from 0.5 to 5 ng/ml and 0.5 to 4 ng/ml, respectively. Compared with HF-LPME, the UA-LDS-DLLME technique had the advantages of less extraction time, suitability for batches of sample pretreatment simultaneously, and higher extraction efficiency, while HF-LPME has excellent sample clean-up effect, and is a robust and suitable technique for various sample matrices with better repeatability. Both methods were successfully applied to the analysis of drugs of abuse in real human blood sample.

Comparison of the performance of different silicon-based SALDI substrates for illicit drug detection.

Surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) is an emerging technique used for the detection of small molecules (<700 Da) such as illicit drugs. In recent times, this technique has been employed for the detection of illicit drugs in various body fluids including saliva. Three common SALDI techniques, desorption ionization on porous silicon (DIOS), nanostructure-initiator mass spectrometry (NIMS) and nanostructured laser desorption ionization (NALDI(™)) are compared for the detection of four drug classes, amphetamines, benzodiazepines, opiates and tropane alkaloids. We focus in our comparison on structural and chemical characteristics, as well as analytical performance and longevity.

Forensic electrochemistry applied to the sensing of new psychoactive substances: electroanalytical sensing of synthetic cathinones and analytical validation in the quantification of seized street samples.

The electrochemical sensing of new psychoactive substance(s) (NPSs), synthetic cathinone derivatives also termed "legal highs", are explored with the use of metallic modified screen-printed electrochemical sensors (SPES). It is found that no significant electrochemical enhancement is evident with the use of either in situ bismuth or mercury film modified SPES compared to the bare underlying electrode substrate. In fact, the direct electrochemical reduction of the cathinone derivatives mephedrone (4-methylmethcathinone; 4-MMC) and 4'-methyl-N-ethylcathinone (4-methylethcathinone; 4-MEC) is found to be possible for the first time, without heavy metal catalysis, giving rise to useful voltammetric electroanalytical signatures in model aqueous buffer solutions. This novel electroanalytical methodology is validated toward the determination of cathinone derivatives (4-MMC and 4-MEC) in three seized street samples that are independently analyzed with high-performance liquid chromatography (HPLC) wherein excellent agreement between the two analytical protocols is found. Such an approach provides a validated laboratory tool for the quantification of synthetic cathinone derivatives and holds potential for the basis of a portable analytical sensor for the determination of synthetic cathinone derivatives in seized street samples.

A quantitative, selective and fast ultra-high performance liquid chromatography tandem mass spectrometry method for the simultaneous analysis of 33 basic drugs in hair (amphetamines, cocaine, opiates, opioids and metabolites).

Forensic testing for drugs of abuse in hair has become a useful diagnostic tool in determining chronic drug use as well as examining long-term drug history thorough segmental analysis. However, sensitive and specific analytical methods are needed. A simple, rapid and highly sensitive and specific method for the extraction and quantification of 33 opioids, opiates, cocaine, and amphetamines is presented. The method was fully validated according to international guidelines. Twenty milligrams of hair sample was pulverized and then incubated in the same disposable tube with methanol (under sonication at 45°C) during 4h. After centrifugation the supernatant was evaporated up to about 100 µL and a solid phase extraction (SPE) followed by separation and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UHLC-MS/MS) were carried out. Chromatographic separation was achieved using a BEH phenyl column eluted with 0.1% formic acid: methanol (0.1% formic acid). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. Good intra-assay and inter-assay precision (relative standard deviations (RSDs) were observed (<15%) for most of the compounds. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments and it ranged from 0.006 to 0.063 ng/mg. No instability was observed in processed samples. Extraction efficiency varied from 37 to 107% (except for EDDP with a recovery of 5%) and matrix effects ranged from 52 to 160%, and for most of the compounds it was compensated by the internal standard (IS). The method was subsequently applied to authentic hair samples obtained from forensic and toxicology cases and to proficiency test (obtaining z-scores lower than 1 for most of the compounds). The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well-suited for routine hair analysis.

A high-throughput method based on microwave-assisted extraction and liquid chromatography-tandem mass spectrometry for simultaneous analysis of amphetamines, ketamine, opiates, and their metabolites in hair.

A total sample-preparation and analysis time of 50 min is required for the high-throughput method of hair analysis proposed in this paper. The method is applicable to analysis of drugs commonly used in Asia, and their metabolites--methamphetamine (MA), amphetamine (AMP), methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), ketamine (K), norketamine (NK), dehydronorketamine (DHNK), 6-acetylmorphine (6-AM), morphine (MOR), and codeine (COD). Cut and weighed hair (10 mg) was incubated for 3 min with methanol-trifluoroacetic acid (TFA) during microwave-assisted extraction (MAE) at 700 W. The incubation solution was evaporated, the residue was reconstituted in deionized water-methanol, 99:1 (v/v), and 20 µL was injected on to a core-shell column (50 × 4.6 mm, 2.6 µm particle size) for liquid chromatographic-tandem mass spectrometric (LC-MS-MS) analysis. Gradient elution separation was performed in 8 min at a flow rate of 1 mL min(-1). No signal interfering with any of the analytes was found in fourteen blank hair samples from different sources. The limits of detection and quantification were 0.5 pg mg(-1) and 2.0 pg mg(-1), respectively, for MA, AMP, MDMA, MDA, K, NK, and DHNK, and 2.0 pg mg(-1) and 5.0 pg mg(-1), respectively, for 6-AM, MOR and COD. The linear range was between the LOQ and 1000 pg mg(-1), and the correlation coefficients were all greater than 0.999. Investigation of matrix effects revealed that all the analytes were suppressed by less than 20% and the standard deviation (SD) was always less than 7%. Recovery was always greater than 90% and the SD for each compound was less than 6%. Precision and accuracy for each analyte were within 15%. Eight authentic hair specimens from known drug abusers were successfully analyzed. Compared with traditional overnight incubation methods, the rapid 3-min extraction time achieved similar or greater extraction yields. Sample preparation by MAE was a reliable procedure for extraction of the analytes from hair but substantially simpler and faster than other methods.

Hollow-fiber liquid-phase microextraction of amphetamine-type stimulants in human hair samples.

A fast method was optimized and validated in order to quantify amphetamine-type stimulants (amphetamine, AMP; methamphetamine, MAMP; fenproporex, FPX; 3,4-methylenedioxymethamphetamine, MDMA; and 3,4-methylenedioxyamphetamine, MDA) in human hair samples. The method was based in an initial procedure of decontamination of hair samples (50 mg) with dichloromethane, followed by alkaline hydrolysis and extraction of the amphetamines using hollow-fiber liquid-phase micro extraction (HF-LPME) in the three-phase mode. Gas chromatography-mass spectrometry (GC-MS) was used for identification and quantification of the analytes. The LoQs obtained for all amphetamines (around 0.05 ng/mg) were below the cut-off value (0.2 ng/mg) established by the Society of Hair Testing (SoHT). The method showed to be simple and precise. The intra-day and inter-day precisions were within 10.6% and 11.4%, respectively, with the use of only two deuterated internal standards (AMP-d5 and MDMA-d5). By using the weighted least squares linear regression (1/x²), the accuracy of the method was satisfied in the lower concentration levels (accuracy values better than 87%). Hair samples collected from six volunteers who reported regular use of amphetamines were submitted to the developed method. Drug detection was observed in all samples of the volunteers.

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.