Determination of four thiophenethylamine designer drugs (2C-T-series) in human plasma by capillary electrophoresis with mass spectrometry detection

In recent years, the frequent appearance of phenethylamine designer drugs on the illicit drug market has been a matter of concern for all authorities involved. New phenethylamine drugs are being introduced because these compounds are not covered by existing legislation. Therefore, the new drugs cannot be considered illicit drugs until their names are officially recognized. This paper describes a method to screen for and quantify four 2,5-methylenedioxy-derivatives of 4-thio-phenethylamine (2C-T-series) in human plasma, using capillary electrophoresis coupled with electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple liquid extraction was used for sample cleanup. The method was validated according to international guidelines.     

Confirmation testing of amphetamines and designer drugs in human urine by capillary electrophoresis-ion trap mass spectrometry

Monitoring of amphetamines and designer drugs in human urine is a timely topic in clinical toxicology, surveillance of drug substitution, forensic science, drug testing at the workplace, and doping control. Confirmation testing of urinary amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and 3,4-methylenedioxyamphetamine (MDA) by capillary electrophoresis (CE) combined with atmospheric pressure electrospray ionization and ion trap mass spectrometry (MS) is described. Using an aqueous pH 4.6 buffer composed of ammonium acetate/acetic acid, CE-MS and CE-MS2 provided data that permitted the unambiguous confirmation of these drugs in external quality control urines. Furthermore, other drugs of abuse present in alkaline urinary extracts, including methadone and morphine, could also be monitored. The data presented illustrate that the sensitivity achieved with the benchtop MS is comparable to that observed by CE with UV absorption detection. CE-MS2 is further shown to be capable of identifying comigrating compounds, including the comigration of amphetamine with nicotine.     

Multi‐residue analysis of eight thioamphetamine designer drugs in human urine by liquid chromatography/tandem mass spectrometry

An analytical procedure for the simultaneous determination in human urine of several thioamphetamine designer drugs (2C‐T and ALEPH series) is reported. The quantitative analysis was performed by liquid chromatography/tandem mass spectrometry and has been fully validated. The mass spectrometer was operated in positive‐ion, selected reaction monitoring (SRM) mode. In order to minimize interferences with matrix components and to preconcentrate target analytes, solid‐phase extraction was introduced in the method as a clean‐up step. The entire method was validated for selectivity, linearity, precision and accuracy. The method turned out to be specific, sensitive, and reliable for the analysis of amphetamine derivatives in urine samples. The calibration curves were linear over the concentration range of 1 to 100 ng mL^?1 for all drugs with correlation coefficients that exceeded 0.996. The lower limits of detection (LODs) and quantification (LOQs) ranged from 1.2 to 4.9 ng mL^?1 and from 3.2 to 9.6 ng mL^?1, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of new thioamphetamine designer drugs in plasma by capillary electrophoresis coupled with mass spectrometry

A simple method for the simultaneous identification and quantification of four 2,5-methylenedioxy derivatives of 4-thioamphetamine (ALEPH series) in plasma samples was developed. The method consists of solid-phase extraction (SPE) using a Bond Elut C(18) cartridge and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE/ESI-MS). The SPE method used required only simple steps and provided a clean extract from which identification of each drug was feasible, even at low concentrations. The method was validated according to international guidelines. The calibration curves were linear over the concentration range of 50 to 1000 ng/mL for all drugs with correlation coefficients that exceeded 0.998. The lower limits of detection of the drugs were 23-43 ng/mL. The absolute recoveries for the drugs were 64-92% and 75-96% at concentrations of 100 and 500 ng/mL, respectively. The validation data (precision, accuracy, and recovery) show the reproducibility and selectivity of the method. This clean and simple method allows the routine detection of designer drugs such as thioamphetamines which may become a serious problem in the control of illegal drugs.     

Determination of chlorine species by capillary electrophoresis – mass spectrometry

The applicability of CZE with mass spectrometric detection for the determination of four chlorine species, namely chloride and three stable chlorine oxyanions, was studied. The main aspects of the proper selection of BGE and sheath liquid for the CE‐MS determinations of anions with high mobility were demonstrated, pointing out the importance of pH and the mobility of the anion in the BGE. The possibility of using uncoated fused silica capillary and common electrolytes for the separation was shown and the advantage of using extra pressure at the inlet capillary end was also presented. The linear range was found to be 1–100 µg/mL for ClO₃^? and ClO₄^?, 5–500 µg/mL for ClO₂^?, and 25–500 µg/mL for Cl^?, but the sensitivity can be greatly improved if larger sample volume is injected and electrostacking effect is utilized. The LOD for ClO₃^? in drinking water was 6 ng/mL, when very large sample volume was injected (10 000 mbar·s was applied).     

Determination of eight illegal drugs in human urine by combination of magnetic solid‐phase extraction with capillary zone electrophoresis

Using magnetite/silica/poly(methacrylic acid‐co‐ethylene glycol dimethacrylate) (Fe₃O₄/SiO₂/poly(MAA‐co‐EDMA)) magnetic microspheres, a rapid and high‐throughput magnetic solid‐phase extraction coupled with capillary zone electrophoresis (MSPE‐CZE) method was developed for the determination of illegal drugs (ketamine, amphetamines, opiates, and metabolites). The MSPE of target analytes could be completed within 2 min, and the eight target analytes could be baseline separated within 15 min by CZE with 30 mM phosphate buffer solution (PBS, pH 2.0) containing 15% v/v ACN as background electrolyte. Furthermore, hydrodynamic injection with field‐amplified sample stacking (FASS) was employed to enhance the sensitivity of this MSPE‐CZE method. Under such optimal conditions, the limits of detection for the eight target analytes ranged from 0.015 to 0.105 μg/mL. The application feasibility of MSPE‐CZE in illegal drugs monitoring was demonstrated by analyzing urine samples, and the recoveries of target drugs for the spiked sample ranging from 85.4 to 110.1%. The method reproducibility was tested by evaluating the intra‐ and interday precisions, and relative standard deviations of <10.3 and 12.4%, respectively, were obtained. To increase throughput of the analysis, a home‐made MSPE array that has potential application to the treatment of 96 samples simultaneously was used.     

Determination of 8-hydroxy-2'-deoxyguanosine in untreated urine by capillary electrophoresis with UV detection

A capillary electrophoresis method with UV detection was developed for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in untreated urine samples. The calibration graph for 8-OHdG in urine is linear in the concentration range 10-500 mg/l. and the detection limit is 5 mg/l (17 microM). 8-OHdG was determined in urine from oncological patients treated by radiation therapy. Its concentrations relative to creatinine were found to be in the range 10-47 microg 8-OHdG/l mg creatinine (4-19 micromol 8-OHdG/mmol creatinine). The overall time of the analysis of a urine sample was less than 15 min.     

Determination of drugs used as anti-Parkinson's disease drugs in urine and serum by capillary electrophoresis

A new capillary electrophoresis method to determine simultaneously eight of the most important anti-Parkinson's disease compounds has been developed. The generic names of the drugs studied are benactyzine (BA), trihexyphenidyl (TP), fenpiverin (FP), diphemin (DF), scopolamine (BL), adiphenine (TS), diethylaminoethylester 1-phenylcyclopentane-1-carboxylate (EKK), and diethylaminoethylester tetramethoxydiphenylacetate (EKO). An untreated fused-silica capillary tube (75 microns i.d., 57 cm total length, 49.5 cm length to the detector) was used with detection at 190 nm. The optimal separation conditions were 50 mM phosphate buffer (pH 2.7) with 7 mM-beta-cyclodextrin, electrokinetic injection for 15 sec at 5 kV, temperature 25 degrees C, and 15-20 kV separation voltage. Complete separation of all compounds was achieved in less than 16 min. The procedure was applied for the determination in urine and serum. The limits of detection (LOD, S/N = 3) for serum were 209 (FP), 234 (EKO), 168 (DF), 182 (BA), 168 (TP), 220 (BL), 174 (TS), and 163 (EKK) ppb. The method can be used for the therapeutic drug monitoring of these central active cholinolytics in clinical laboratories.     

Determination of phytochelatins by capillary zone electrophoresis with electrospray tandem mass spectrometry detection (CZE-ES MS/MS)

The coupling of capillary zone electrophoresis with electrospray mass spectrometry was optimized for the direct determination of phytochelatins (PCs) in extracts obtained from cells and plants that had been exposed to metal stress. Gluthathione and phytochelatins belonging to the different families (gamma Glu-Cys)nGly (n-PC), (gamma Glu-Cys)nSer, (gamma Glu-Cys)n beta Ala and (gamma Glu-Cys)n were separated in an uncoated capillary at pH 4 using a 5 mM ammonium acetate buffer, and detected by electrospray (ES) MS in the full scan mode (300-1100 u). The use of on-line tandem MS detection in the product ion scan mode of putative protonated molecules of PCs allowed the unambiguous confirmation of the identity of the compounds detected by ES MS. The operational conditions were optimized and the figures of merit were evaluated using n-PC2, n-PC3 and n-PC4 standards purified from a mixture obtained after the reaction of glutathione in the presence of Cd2+ and the enzyme PC-synthase. The method was applied to the characterization of bioinduced ligands in cell cultures of soybeans (Glycine max) and in rice (Oryza sativa) roots without the need for a preliminary sample cleanup by size-exclusion and/or reversed phase chromatography.     

Determination of gamma-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ, respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ.     

Determination of hormones in human urine by ultra‐high‐performance liquid chromatography/triple‐quadrupole mass spectrometry

Steroid hormones play a critical role in maintaining the homeostasis of human metabolism. Urine as a noninvasive sample has been extensively used in clinical diagnosis for hormones homeostasis. In this study, the simultaneous characterization of fourteen hormones in urine was performed based on ultra‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPHLC/ESI(+)‐MS/MS) with multiple reaction monitoring in the positive ionization mode. The target hormones were cortisone, cortisol, 11‐deoxycortisol, aldosterone, corticosterone, 11‐deoxycorticosterone, progesterone, 17‐OH‐progesterone, pregnenolone, estrone, estradiol, estriol, testosterone and dehydreopiandrosterone. β‐Glucuronidase/sulfatase deconjugation and liquid–liquid extraction (LLE) were conducted for the determination of urinary hormones (free + conjugated forms). The limits of detection (LODs) ranged from 0.2 ng/mL (11‐deoxycortisol and testosterone) to 1 ng/mL (cortisone). The extraction recovery of the targeted compounds ranged from 87% to 127%, indicating sufficient extraction efficiency for the LLE process. Intraday precision was below 10% and the accuracy ranged from 84% to 122%. The profiling analysis of hormones in urine samples helps to understand the metabolic state of biological systems and can be employed as a diagnostic tool in diseases developed by endocrine‐disrupted systems.     

Double‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis

Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined with various detection methods provided remarkable developments for high‐sensitivity glycan profiling. The coating of the capillary is needed for highly polar molecules from complex biosamples. A poly(vinyl alcohol)‐coated capillary is commonly utilized in the capillary electrophoresis separation of saccharides sample due to the high‐hydrophilicity properties. A modified facile coating workflow was carried out to acquire a novel multiple‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable analysis of glycans. The migration time fluctuation was used as index in the optimization of layers and a double layer was finally chosen, considering both the effects and simplicity in fabrication. With migration time relative standard deviation less than 1% and theoretical plates kept stable during 100 consecutive separations, the method was presented to be suitable for the analysis of glycosylation with wide linear dynamic range and good reproducibility. The glycan profiling of enzymatically released N‐glycans from human serum was obtained by the presented capillary electrophoresis method combined with mass spectrometry detection with acceptable results.     

Multidrug analysis of pharmaceutical and urine matrices by on‐line coupled capillary electrophoresis and triple quadrupole mass spectrometry

The present work illustrates potentialities of CE hyphenated with MS/MS for the simultaneous determination and identification of a mixture of simultaneously acting drugs in pharmaceutical and biological matrices. Here, the hyphenation was provided by ESI interface, while the MS/MS technique was based on the triple quadrupole configuration. Three drugs, namely pheniramine, phenylephrine, and paracetamol were determined and identified with high reliability due to their characterization in three different dimensions, i.e. electrophoresis and MS/MS, that prevented practically any interference. Appropriately selected transitions of the analytes (parent ion‐quantifier product ion‐qualifier product ion) provided their selective determination at maximum S/N. The proposed CE‐MS/MS method was validated (LOD/LOQ, linearity, precision, recovery, accuracy) and applied for (i) the multidrug composition pharmaceuticals, namely Theraflu®, and (ii) human urine taken after per‐oral administration of the same pharmaceutical preparation. The method was applied also for the investigation of potential weak associates of the drugs and monitoring of predicted (bio)degradation products of the drugs. Successful validation and application of the proposed method suggest its routine use in highly effective and reliable advanced drug control and biomedical research.     

In silico and in vitro metabolism studies support identification of designer drugs in human urine by liquid chromatography/quadrupole-time-of-flight mass spectrometry

Human phase I metabolism of four designer drugs, 2-desoxypipradrol (2-DPMP), 3,4-dimethylmethcathinone (3,4-DMMC), α-pyrrolidinovalerophenone (α-PVP), and methiopropamine (MPA), was studied using in silico and in vitro metabolite prediction. The metabolites were identified in drug abusers’ urine samples using liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/Q-TOF/MS). The aim of the study was to evaluate the ability of the in silico and in vitro methods to generate the main urinary metabolites found in vivo. Meteor 14.0.0 software (Lhasa Limited) was used for in silico metabolite prediction, and in vitro metabolites were produced in human liver microsomes (HLMs). 2-DPMP was metabolized by hydroxylation, dehydrogenation, and oxidation, resulting in six phase I metabolites. Six metabolites were identified for 3,4-DMMC formed via N-demethylation, reduction, hydroxylation, and oxidation reactions. α-PVP was found to undergo reduction, hydroxylation, dehydrogenation, and oxidation reactions, as well as degradation of the pyrrolidine ring, and seven phase I metabolites were identified. For MPA, the nor-MPA metabolite was detected. Meteor software predicted the main human urinary phase I metabolites of 3,4-DMMC, α-PVP, and MPA and two of the four main metabolites of 2-DPMP. It assisted in the identification of the previously unreported metabolic reactions for α-PVP. Eight of the 12 most abundant in vivo phase I metabolites were detected in the in vitro HLM experiments. In vitro tests serve as material for exploitation of in silico data when an authentic urine sample is not available. In silico and in vitro designer drug metabolism studies with LC/Q-TOF/MS produced sufficient metabolic information to support identification of the parent compound in vivo.

Top‐down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

The original article to which this Erratum refers was published in Rapid Commun. Mass Spectrom. 2006; 20 : 1932–1938.     

Determination of paramethoxyamphetamine and other amphetamine-related designer drugs by liquid chromatography/sonic spray ionization mass spectrometry

Paramethoxyamphetamine (PMA) is an amphetamine-like designer drug that has emerged recently on the European illicit drug market. This drug has a wicked reputation, as a number of lethal intoxications have occurred. A method using high-performance liquid chromatography coupled to ion trap based mass spectrometry (LC/MS) is described for the determination of this compound together with 3,4-methylenedioxymethamphetamine (XTC or MDMA), amphetamine and 3,4-methylenedioxyamphetamine (MDA) in human matrices. A liquid/liquid extraction (LLE) was applied to whole blood, urine and postmortem tissues. Reversed-phase liquid chromatography was performed on a narrow-bore phenyl-type column at a flow rate of 0.3 mL/min. A switch box allowed disposal of early-eluting irrelevant material to waste, protecting the mass spectrometer from contamination. The column effluent was directed into an ion trap mass spectrometer by a sonic spray ionization (SSI) interface. The method was validated for all three matrices, proving the applicability of SSI even when dealing with complex biological matrices. The within-and between-day precisions were less than 17.5% and accuracy was below 16.2%. Weighted (1/x) quadratic calibration curves were generated ranging from 10 to 1000 ng/mL (blood and urine) or 20 to 2000 ng/g (tissue) and correlation coefficients (r(2)) always exceeded 0.995. In addition, the mass spectrum of PMA is given together with a proposed fragmentation pattern for the obtained LC/MS spectrum. This information can be useful for future identification of PMA with LC/MS in biological matrices as well as in confiscated powders or tablets.     

Determination of norfloxacin in human urine by capillary electrophoresis with electrochemiluminescence detection

A fast and sensitive approach that can be used to detect norfloxacin in human urine using capillary electrophoresis with end-column electrochemiluminescence (ECL) detection of is described. The separation column was a 75-μm i.d. capillary. The running buffer was 15 mmol L⁻¹ sodium phosphate (pH 8.2). The solution in the detection cell was 50 mmol L⁻¹ sodium phosphate (pH 8.0) and 5 mmol L⁻¹ The ECL intensity varied linearly with norfloxacin concentration from 0.05 to 10 μmol L⁻¹. The detection limit (S/N=3) was 0.0048 μmol L⁻¹, and the relative standard deviations of the ECL intensity and the migration time for eleven consecutive injections of 1.0 μmol L⁻¹ norfloxacin (n=11) were 2.6% and 0.8%, respectively. The method was successfully applied to the determination of norfloxacin spiked in human urine without sample pretreatment. The recoveries were 92.7–97.9%.      

Determination of growth hormone secretagogue pralmorelin (GHRP‐2) and its metabolite in human urine by liquid chromatography/electrospray ionization tandem mass spectrometry

GHRP‐2 (pralmorelin, D‐Ala‐D‐(β‐naphthyl)‐Ala‐Ala‐Trp‐D‐Phe‐Lys‐NH₂), which belongs to a class of growth hormone secretagogue (GHS), is intravenously used to diagnose growth hormone (GH) deficiency. Because it may be misused in expectation of a growth‐promoting effect by athletes, the illicit use of GHS by athletes has been prohibited by the World Anti‐Doping Agency (WADA). Therefore, the mass spectrometric identification of urinary GHRP‐2 and its metabolite D‐Ala‐D‐(β‐naphthyl)‐Ala‐Ala‐OH (AA‐3) was studied using liquid chromatography/electrospray ionization tandem mass spectrometry for doping control purposes. The method consists of solid‐phase extraction using stable‐isotope‐labeled GHRP‐2 as an internal standard and subsequent ultra‐performance liquid chromatography/tandem mass spectrometry, and the two target peptides were determined at urinary concentrations of 0.5–10 ng/mL. The recoveries ranged from 84 to 101%, and the assay precisions were calculated as 1.6–3.8% (intra‐day) and 1.9–4.3% (inter‐day). Intravenous administration of GHRP‐2 in ten male volunteers was studied to demonstrate the applicability of the method. In all ten cases, unchanged GHRP‐2 and its specific metabolite AA‐3 were detected in urine. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of antidepressants in human urine extracted by magnetic multiwalled carbon nanotube poly(styrene‐co‐divinylbenzene) composites and separation by capillary electrophoresis

Poly(styrene‐co‐divinylbenzene)‐coated magnetic multiwalled carbon nanotube composite synthesized by in‐situ high temperature combination and precipitation polymerization of styrene‐co‐divinylbenzene has been employed as a magnetic sorbent for the solid phase extraction of antidepressants in human urine samples. Fluoxetine, venlafaxine, citalopram and sertraline were, afterwards, separated and determined by capillary electrophoresis with diode array detection. The presence of magnetic multiwalled carbon nanotubes in native poly(styrene‐co‐divinylbenzene) not only simplified sample treatment but also enhanced the adsorption efficiencies, obtaining extraction recoveries higher than 89.5% for all analytes. Moreover, this composite can be re‐used at least ten times without loss of efficiency and limits of detection ranging from 0.014 to 0.041 μg/mL were calculated. Additionally, precision values ranging from 0.08 to 7.50% and from 0.21 to 3.05% were obtained for the responses and for the migration times of the analytes, respectively.