Analytical and Bioanalytical Chemistry (v.396, #7)

Forensic and toxicological analysis by Jean-Luc Veuthey (2377-2378).
is a professor at the School of Pharmaceutical Sciences, University of Geneva. He is also acting as Vice Dean of the Faculty of Sciences of the University of Geneva, Switzerland. His research domains is: development of separation techniques in pharmaceutical sciences, and, more precisely, study of the effect of sample-preparation procedures on the analytical process; fundamental studies in liquid chromatography and capillary electrophoresis; separation techniques coupled with mass spectrometry; analysis of drugs and drugs of abuse in different matrices; and analytical tools for the determination of ADME data

Use of liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry for studying the metabolism of paynantheine, an alkaloid of the herbal drug Kratom in rat and human urine by Anika A. Philipp; Dirk K. Wissenbach; Armin. A. Weber; Josef Zapp; Siegfried W. Zoerntlein; Jidapha Kanogsunthornrat; Hans H. Maurer (2379-2391).
The Thai medicinal plant Mitragyna speciosa (Kratom in Thai) is misused as a herbal drug of abuse. During studies on the main Kratom alkaloid mitragynine (MG) in rats and humans, several dehydro analogs could be detected in urine of Kratom users, which were not found in rat urine after administration of pure MG. Questions arose as to whether these compounds are formed from MG only by humans or whether they are metabolites formed from the second abundant Kratom alkaloid paynantheine (PAY), the dehydro analog of MG. Therefore, the aim of the presented study was to identify the phase I and II metabolites of PAY in rat urine after administration of the pure alkaloid. This was first isolated from Kratom leaves. Liquid chromatography–linear ion trap mass spectrometry provided detailed structure information of the metabolites in the MSn mode particularly with high resolution. Besides PAY, the following phase I metabolites could be identified: 9-O-demethyl PAY, 16-carboxy PAY, 9-O-demethyl-16-carboxy PAY, 17-O-demethyl PAY, 17-O-demethyl-16,17-dihydro PAY, 9,17-O-bisdemethyl PAY, 9,17-O-bisdemethyl-16,17-dihydro PAY, 17-carboxy-16,17-dihydro PAY, and 9-O-demethyl-17-carboxy-16,17-dihydro PAY. These metabolites indicated that PAY was metabolized via the same pathways as MG. Several metabolites were excreted as glucuronides or sulfates. The metabolism studies in rats showed that PAY and its metabolites corresponded to the MG-related dehydro compounds detected in urine of the Kratom users. In conclusion, PAY and its metabolites may be further markers for a Kratom abuse in addition of MG and its metabolites. Figure Isolation of paynantheine from kratom leaves; high-resolution mass spectrum of the glucuronide of its 9-O-demethyl metabolite, and paynantheine structure with marked sides of biotransformation.
Keywords: Paynantheine; Mitragynine; Kratom ; Metabolism; LC–MS; Urine

Determination of 19 drugs of abuse and metabolites in whole blood by high-performance liquid chromatography–tandem mass spectrometry by Marie Kjærgaard Bjørk; Marie K. K. Nielsen; Lotte Ø. Markussen; Helene B. Klinke; Kristian Linnet (2393-2401).
A high-performance liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method has been developed and validated for the determination of 19 drugs of abuse and metabolites and used in whole blood. The following compounds were included: amphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, methamphetamine, cocaine, benzoylecgonine, morphine, 6-acetylmorphine, codeine, methadone, buprenorphine, norbuprenorphine, ketobemidone, tramadol, O-desmethyltramadol, zaleplone, zolpidem, and zopiclone. The sample pretreatment consisted of solid-phase extraction using mixed-mode columns (Isolute Confirm HCX). Deuterated analogues were used as internal standards for all analytes, except for ketobemidone and O-desmethyltramadol. The analytes were separated by a methanol/ammonium formate gradient using high-performance LC (Agilent HPLC 1100) with a 3 mm × 100 mm Varian Pursuit 3 C18 column, 3-µm particle size, and were quantified by MS/MS (Waters Quattro micro tandem quadrupole mass spectrometer) using multiple reaction monitoring in positive mode. Two transitions were used for all analytes, except for tramadol and O-desmethyltramadol. The run time of the method was 35 min including the equilibration time. For all analytes, responses were linear over the range investigated, with R 2 > 0.99. One-point calibration was found to be adequate by validation, thereby saving analysis of multiple calibrators. The limits of quantification (LOQs) for the analytes ranged from 0.0005 to 0.01 mg/kg. Absolute recoveries of the analytes were from 34 to 97%, except for zaleplone (6%). Both the interday precision and the intraday precision were less than 15% (20% at the LOQ) for all analytes, except buprenorphine, norburprenorphine, and zaleplone (less than 18%). Accuracy (bias) was within ±15% (±20% at the LOQ) for all analytes, except MDMA and O-desmethyltramadol (within ±19%). No ion suppression or enhancement was seen nor was suppression from coeluted analytes seen. Matrix effects were found to be less than 23% for all analytes, except zopiclone (64%). High-concentration and low-concentration quality control samples gave acceptable values, and the method has been tried in international proficiency test schemes with good results. The present LC-MS/MS method provides a simple, specific, and sensitive solution for the quantification of some of the most frequent drugs of abuse and their metabolites in whole blood. The quantification by LC-MS/MS was successfully applied to 412 forensic cases from October 2008 to mid February 2009, where 267 cases were related to zero-tolerance traffic legislation.
Keywords: Liquid chromatography–tandem mass spectrometry; Whole blood; Drugs of abuse; Zero tolerance

LC-MS/MS screening method for designer amphetamines, tryptamines, and piperazines in serum by Ariane Wohlfarth; Wolfgang Weinmann; Sebastian Dresen (2403-2414).
Since the late 1990s and early 2000s, derivatives of well-known designer drugs as well as new psychoactive compounds have been sold on the illicit drug market and have led to intoxications and fatalities. The LC-MS/MS screening method presented covers 31 new designer drugs as well as cathinone, methcathinone, phencyclidine, and ketamine which were included to complete the screening spectrum. All but the last two are modified molecular structures of amphetamine, tryptamine, or piperazine. Among the amphetamine derivatives are cathinone, methcathinone, 3,4-DMA, 2,5-DMA, DOB, DOET, DOM, ethylamphetamine, MDDMA, 4-MTA, PMA, PMMA, 3,4,5-TMA, TMA-6 and members of the 2C group: 2C-B, 2C-D, 2C-H, 2C-I, 2C-P, 2C-T-2, 2C-T-4, and 2C-T-7. AMT, DPT, DiPT, MiPT, DMT, and 5MeO-DMT are contained in the tryptamine group, BZP, MDBP, TFMPP, mCPP, and MeOPP in the piperazine group. Using an Applied Biosystems LC-MS/MS API 365 TurboIonSpray it is possible to identify all 35 substances. After addition of internal standards and mixed-mode solid-phase extraction the analytes are separated using a Synergi Polar RP column and gradient elution with 1 mM ammonium formate and methanol/0.1% formic acid as mobile phases A and B. Data acquisition is performed in MRM mode with positive electro spray ionization. The assay is selective for all tested substances. Limits of detection were determined by analyzing S/N-ratios and are between 1.0 and 5.0 ng/mL. Matrix effects lie between 65% and 118%, extraction efficiencies range from 72% to 90%.
Keywords: Designer drugs; LC-MS/MS; Screening; Drugs of abuse

Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS by H. Gnann; C. Engelmann; G. Skopp; M. Winkler; V. Auwärter; S. Dresen; N. Ferreirós; F. M. Wurst; W. Weinmann (2415-2423).
Phosphatidylethanol (PEth) is an abnormal phospholipid carrying two fatty acid chains. It is only formed in the presence of ethanol via the action of phospholipase D (PLD). Its use as a biomarker for alcohol consumption is currently under investigation. Previous methods for the analysis of PEth included high-performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD), which is unspecific for the different homologues—improved methods are now based on time of flight mass spectrometry (TOF-MS) and tandem mass spectrometry (MS/MS). The intention of this work was to identify as many homologues of PEth as possible. A screening procedure using multiple-reaction monitoring (MRM) for the identified homologues has subsequently been established. For our investigations, autopsy blood samples collected from heavy drinkers were used. Phosphatidylpropanol 16:0/18:1 (internal standard) was added to the blood samples prior to liquid–liquid extraction using borate buffer (pH 9), 2-propanol and n-hexane. After evaporation, the samples were redissolved in the mobile phase and injected into the LC-MS/MS system. Compounds were separated on a Luna Phenyl Hexyl column (50 mm × 2 mm, 3 µm) by gradient elution, using 2 mM ammonium acetate and methanol/acetone (95/5; v/v). A total of 48 homologues of PEth could be identified by using precursor ion and enhanced product ion scans (EPI).
Keywords: Alcohol; Phosphatidylethanol; Phospholipids; Homologues; LC-MS/MS; Biomarker

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a “scheduled” survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP® hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization–MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography–mass spectrometry, and liquid chromatography–diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).
Keywords: Drug monitoring; Drug screening; Forensics; Toxicology; Mass spectrometry; Liquid chromatography–tandem mass spectrometry

Validation of a fast screening method for the detection of cocaine in hair by MALDI-MS by Susanna Vogliardi; Donata Favretto; Giampietro Frison; Sergio Maietti; Guido Viel; Roberta Seraglia; Pietro Traldi; Santo Davide Ferrara (2435-2440).
The sensitivity and specificity of a novel method of screening for cocaine in hair, based on matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS), have been evaluated. The method entails a rapid extraction procedure consisting of shaking 2.5 mg pulverised hair at high frequency in the presence of an acidic solution (160 µL of water, 20 µL of acetonitrile and 20 µL of 1 M trifluoroacetic acid) and a stainless-steel bullet. Following centrifugation, the supernatant is dried under a nitrogen stream, and the residue is reconstituted in 10 µL of methanol/trifluoroacetic acid (7:3; v/v). One microlitre of the extract is deposed on a MALDI sample holder previously scrubbed with graphite; an α-cyano-4-hydroxycinnamic acid (matrix) solution is electrosprayed over the dried sample surface to achieve a uniform distribution of matrix crystals. The identification of cocaine is obtained by post-source decay experiments performed on its MH+ ion (m/z 304), with a limit of detection of 0.1 ng/mg of cocaine. A total of 304 hair samples were analysed in parallel by MALDI-MS and a reference gas chromatography-MS method. The obtained results demonstrate specificity and sensitivity of 100% for MALDI-MS. Evidence of cocaine presence was easily obtained even when hair samples exhibiting particularly low cocaine levels (<0.5 ng/mg) were analysed.
Keywords: Hair; Cocaine; MALDI; Screening test; Validation; Drug screening; Hair analysis; MALDI-MS

Hair analysis has become a powerful tool for the detection of chronic and past drug consumption. For several years, it has been possible to determine even the intake of ethanol in hair samples by detecting the ethanol metabolites ethyl glucuronide or fatty acid ethyl esters. Recently, new requirements were published for the use of EtG as an abstinence test (c EtG < 7 pg/mg) as well as for heavy-drinking detection (c EtG > 30 pg/mg). In order to perform abstinence tests, a sensitive LC-MS/MS procedure has been developed and fully validated according to the guidelines of forensic toxicology. The nine-point calibration curve showed linearity over the range of concentrations from 2–1,000 pg/mg. Detection and quantification limits were 1 and 4 pg/mg respectively. The intra- and inter-day precision and accuracy were always better than 20%. The validated procedure has successfully been applied to perform abstinence tests and to analyze hair samples from persons in withdrawal treatment. Concentrations between
Keywords: Hair analysis; Abstinence test; Ethyl glucuronide; LC-MS/MS; Driving ability

An anti-ketamine molecularly imprinted polymer (MIP) was synthesized and used as the sorbent in a solid-phase extraction protocol to isolate ketamine and norketamine from human hair extracts prior to LC-MS/MS analysis. Under optimised conditions, the MIP was capable of selectively rebinding ketamine, a licensed anaesthetic that is widely misused as a recreational drug, with an apparent binding capacity of 0.13 μg ketamine per mg polymer. The limit of detection (LOD) and lower limit of quantification (LLOQ) for both ketamine and norketamine were 0.1 ng/mg hair and 0.2 ng/mg hair, respectively, when 10 mg hair were analysed. The method was linear from 0.1 to 10 ng/mg hair, with correlation coefficients (R 2) of better than 0.99 for both ketamine and norketamine. Recoveries from hair samples spiked with ketamine and norketamine at a concentration of 50 ng/mg were 86% and 88%, respectively. The method showed good intra- and interday precisions (<5%) for both analytes. Minimal matrix effects were observed during the LC-MS/MS analysis of ketamine (ion suppression −6.8%) and norketamine (ion enhancement +0.2%). Results for forensic case samples demonstrated that the method successfully detected ketamine and norketamine concentrations in hair samples with analyte concentrations ranging from 0.2 to 5.7 ng/mg and from 0.1 to 1.2 ng/mg, respectively. Figure Illustration of the ketamine-imprinted polymer and its recognition sites.
Keywords: MIPs; SPE; Ketamine; Norketamine; Hair; LC-MS/MS

Analytical evaluation of a rapid on-site oral fluid drug test by An-Sofie Goessaert; Kristof Pil; Jolien Veramme; Alain Verstraete (2461-2468).
There is a need for a reliable rapid on-site oral fluid test that can be used in police controls to detect impaired drivers. We evaluated the Varian Oralab®6 and collected two oral fluid samples from 250 subjects, one with the Varian Oralab®6 and one with the StatSure™ Saliva•Sampler™. The Oralab®6 can detect six drug types: amphetamines, methamphetamine, cocaine, opiates, delta9-tetrahydrocannabinol (THC), and phencyclidine (PCP). On-site results were obtained within 10 to 15 min. The sample collected with StatSure™ was analyzed using liquid chromatography–tandem mass spectrometry after liquid–liquid extraction and these results were used as a reference to determine prevalence, sensitivity, and specificity. Two cut-off values were used in the evaluation. The Varian cut-off values were: amphetamine 50 ng/mL, cocaine 20 ng/mL, opiates 40 ng/mL, and THC 50 ng/mL. The DRUID cut-offs were: amphetamine 25 ng/mL, cocaine 20 ng/mL, opiates 20 ng/mL, and THC 1 ng/mL. Applying the first cut-offs, prevalence, sensitivity, and specificity were: amphetamine 10%, 76%, 100%; cocaine 23%, 34%, 100%; opiates 38%, 83%, 94%; and THC 18%, 41%, 99%. The DRUID cut-off values gave the following results: amphetamine 14%, 56%, 100%; cocaine 28%, 34%, 100%; opiates 49%, 68%, 98%, and THC 45%, 16%, 99%. The specificity of the Oralab®6 is generally good. For both cut-offs, sensitivity was low for cocaine and THC. Therefore, the Varian Oralab®6 test is not sensitive enough to be applied during roadside police controls.
Keywords: Saliva; Point-of-care test; Varian Oralab®6; Sensitivity

Quantification of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in meconium from newborns for detection of alcohol abuse in a maternal health evaluation study by Abdulsallam Bakdash; Pascal Burger; Tamme W. Goecke; Peter A. Fasching; Udo Reulbach; Stefan Bleich; Martin Hastedt; Michael Rothe; Matthias W. Beckmann; Fritz Pragst; Johannes Kornhuber (2469-2477).
Fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) were determined in 602 meconium samples in a maternal health evaluation study for detection of gestational alcohol consumption. A validated headspace solid phase microextraction method in combination with GC-MS was used for FAEE and the cumulative concentration of ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate with a cut-off of 500 ng/g was applied for interpretation. A new and simple method was developed and validated for quantification of EtG from 10–20 mg meconium with D 5-EtG as internal standard consisting of 30 min. extraction with methanol/water (1:1, v/v), evaporation of methanol, filtration of the aqueous solution through a cellulose filter and injection into LC-MS-MS. The limits of detection and quantification for EtG were 10 and 30 ng/g, the recovery 86.6 to 106.4% and the standard deviation of the concentrations ranged from 13% at 37 ng/g to 5% at 46,700 ng/g (N = 6). FAEE above the cut-off were found in 43 cases (7.1%) with cumulative concentrations between 507 and 22,580 ng/g and with one outlier of about 150,000 ng/g (EtG not detected). EtG was detected in 97 cases (16.3%) and concentrations between LOD and 10,200 ng/g with another outlier of 82,000 ng/g (FAEE 10,500 ng/g). Optimal agreement between the two markers was obtained with a cut-off for EtG of 274 ng/g and 547 cases with both FAEE- and EtG-negative, 33 cases with both FAEE- and EtG-positive, nine cases with FAEE-positive and EtG-negative, and seven cases with FAEE-negative and EtG-positive. Differences in physical, chemical, and biochemical properties and in the pharmacokinetic behavior are discussed as reasons for the deviating cases. In none of the 602 cases, serious alcohol consumption was reported by the mothers and no evidence for gestational ethanol exposure was observed in the medical investigation of the newborns. It is concluded that the combined use of FAEE and EtG in meconium as markers for fetal alcohol exposure essentially increases the accuracy of the interpretation and helps to avoid false positive and false-negative results.
Keywords: Ethyl glucuronide; Fatty acid ethyl esters; Fetal alcohol exposure; Headspace solid phase microextraction; Liquid chromatography–mass spectrometry; Meconium

Characterization of two major urinary metabolites of the PPARδ-agonist GW1516 and implementation of the drug in routine doping controls by Mario Thevis; Ines Möller; Andreas Thomas; Simon Beuck; Grigory Rodchenkov; Wolfgang Bornatsch; Hans Geyer; Wilhelm Schänzer (2479-2491).
Since January 2009, the list of prohibited substances and methods of doping as established by the World Anti-Doping Agency includes new therapeutics such as the peroxisome-proliferator-activated receptor (PPAR)-delta agonist GW1516, which is categorized as a gene doping substance. GW1516 has completed phase II and IV clinical trials regarding dyslipidemia and the regulation of the lipoprotein transport in metabolic syndrome conditions; however, its potential to also improve athletic performance due to the upregulation of genes associated with oxidative metabolism and a modified substrate preference that shifted from carbohydrate to lipid consumption has led to a ban of this compound in elite sport. In a recent report, two presumably mono-oxygenated and bisoxygenated urinary metabolites of GW1516 were presented, which could serve as target analytes for doping control purposes after full characterization. Hence, in the present study, phase I metabolism was simulated by in vitro assays employing human liver microsomal fractions yielding the same oxygenation products, followed by chemical synthesis of the assumed structures of the two abundant metabolic reaction products. These allowed the identification and characterization of mono-oxygenated and bisoxygenated metabolites (sulfoxide and sulfone, respectively) as supported by high-resolution/high-accuracy mass spectrometry with higher-energy collision-induced dissociation, tandem mass spectrometry, and nuclear magnetic resonance spectroscopy. Since urine samples have been the preferred matrix for doping control purposes, a method to detect the new target GW1516 in sports drug testing samples was developed in accordance to conventional screening procedures based on enzymatic hydrolysis and liquid–liquid extraction followed by liquid chromatography, electrospray ionization, and tandem mass spectrometry. Validation was performed for specificity, limit of detection (0.1 ng/ml), recovery (72%), intraday and interday precisions (7.7–15.1%), and ion suppression/enhancement effects (<10%). Graphical Abstract The detection of GW1516, a drug candidate potentially enhancing athletic performance, and the identification of major urinary metabolites for doping control purposes is described using liquid chromatography-mass spectrometry.
Keywords: Sport; Doping; Mass spectrometry; GW501516; Exactive; Orbitrap; Gene doping

Since 2004, cannabis has been prohibited by the World Anti-Doping Agency for all sports competitions. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In doping urine analysis, the target analyte is 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), the cutoff being 15 ng/mL. However, the wide urinary detection window of the long-term metabolite of Δ9-tetrahydrocannabinol (THC) does not allow a conclusion to be drawn regarding the time of consumption or the impact on the physical performance. The purpose of the present study on light cannabis smokers was to evaluate target analytes with shorter urinary excretion times. Twelve male volunteers smoked a cannabis cigarette standardized to 70 mg THC per cigarette. Plasma and urine were collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and THC-COOH were determined after hydrolysis followed by solid-phase extraction and gas chromatography/mass spectrometry. The limits of quantitation were 0.1–1.0 ng/mL. Eight puffs delivered a mean THC dose of 45 mg. Plasma levels of total THC, THC-OH, and THC-COOH were measured in the ranges 0.2–59.1, 0.1–3.9, and 0.4–16.4 ng/mL, respectively. Peak concentrations were observed at 5, 5–20, and 20–180 min. Urine levels were measured in the ranges 0.1–1.3, 0.1–14.4, and 0.5–38.2 ng/mL, peaking at 2, 2, and 6–24 h, respectively. The times of the last detectable levels were 2–8, 6–96, and 48–120 h. Besides high to very high THC-COOH levels (245 ± 1,111 ng/mL), THC (3 ± 8 ng/mL) and THC-OH (51 ± 246 ng/mL) were found in 65 and 98% of cannabis-positive athletes’ urine samples, respectively. In conclusion, in addition to THC-COOH, the pharmacologically active THC and THC-OH should be used as target analytes for doping urine analysis. In the case of light cannabis use, this may allow the estimation of more recent consumption, probably influencing performance during competitions. However, it is not possible to discriminate the intention of cannabis use, i.e., for recreational or doping purposes. Additionally, pharmacokinetic data of female volunteers are needed to interpret cannabis-positive doping cases of female athletes. Figure Urine concentration ranges of delta-9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-delta-9-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in athletes tested cannabispositive (15 ng/mL THC-COOH or more; N=81)
Keywords: Cannabis doping; Clinical trial; Plasma and urine levels; Athletes’ samples

This work presents the validation study of the comprehensive two-dimensional gas chromatography (GC×GC)–time-of-flight mass spectrometry method performance in the analysis of the key World Anti-Doping Agency (WADA) anabolic agents in doping control. The relative abundance ratio, retention time, identification and other method performance criteria have been tested in the GC×GC format to confirm that they comply with those set by WADA. Furthermore, tens of other components were identified with an average similarity of >920 (on the 0–999 scale), including 10 other endogenous sterols, and full mass spectra of 5,000+ compounds were retained. The testosterone/epitestosterone ratio was obtained from the same run. A new dimension in doping analysis has been implemented by addressing separation improvement. Instead of increasing the method sensitivity, which is accompanied by making the detector increasingly “blind” to the matrix (as represented by selected ion monitoring mode, high-resolution mass spectrometry (MS) and tandem MS), the method capabilities have been improved by adding a new “separation” dimension while retaining full mass spectral scan information. Apart from the requirement for the mass spectral domain that a minimum of three diagnostic ions with relative abundance of 5% or higher in the MS spectra, all other WADA criteria are satisfied by GC×GC operation. The minimum of three diagnostic ions arises from the need to add some degree of specificity to the acquired mass spectrometry data; however, under the proposed full MS scan method, the high MS similarity to the reference compounds offers more than the required three diagnostic ions for an unambiguous identification. This should be viewed as an extension of the present criteria to a full-scan MS method. Figure Improved separation of AAS in doping control: GC×GC format
Keywords: WADA criteria; GC×GC; Relative abundance ratio; Tolerance window; Deconvolution; Anabolic-androgenic steroids (AAS)

Recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO) and methoxy polyethylene glycol-epoetin beta (PEG-EPO) are synthetic analogues of the endogenous hormone erythropoietin (EPO). These erythropoiesis-stimulating agents have the ability to stimulate the production of red blood cells and are commercially available for the treatment of anaemia in humans. These drugs are understood to have performance-enhancing effects on human athletes due to their stimulation of red blood cell production, thereby improving delivery of oxygen to the muscle tissues. Although their effect on horses has not been proven, these substances were thought to be similarly performance enhancing and have indeed been applied covertly to horses. As such, these protein-based drugs are prohibited by authorities in both human and equine sports. The method officially adopted by the International Olympic Committee (IOC) and World Anti Doping Agency (WADA) for the confirmation of rhEPO and/or DPO (rhEPO/DPO) in human urine is based on electrophoresis in combination with Western blotting. A shortcoming of the WADA method is the lack of definitive mass spectral data for the confirmation of a positive finding. Recently, a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the detection and confirmation of rhEPO/DPO in equine plasma was reported. However, we have not been successful in achieving the reported sensitivity. This paper presents a method for the detection and confirmation of rhEPO/DPO, as well as the newly released PEG-EPO, in equine plasma. The procedures involve immunoaffinity extraction using anti-rhEPO antibody-coated Dynabeads followed by trypsin digestion. The injected extract was further purified and concentrated using an on-line trap column in the nano-LC system. Detection and confirmation were achieved by monitoring a unique peptide segment of rhEPO/DPO/PEG-EPO using nano-liquid chromatography–tandem mass spectrometry equipped with a nanospray ionisation source operated in the selected reaction monitoring mode. rhEPO, DPO and PEG-EPO can be confirmed at 0.1, 0.2 and 1.0 ng/mL, respectively, in equine plasma.
Keywords: Recombinant human erythropoietin; Darbepoetin alfa; Methoxy polyethylene glycol-epoetin beta; Equine; Immunoaffinity extraction; Liquid chromatography–mass spectrometry

The objective of this work was to combine the advantages of the dried blood spot (DBS) sampling process with the highly sensitive and selective negative-ion chemical ionization tandem mass spectrometry (NICI-MS-MS) to analyze for recent antidepressants including fluoxetine, norfluoxetine, reboxetine, and paroxetine from micro whole blood samples (i.e., 10 μL). Before analysis, DBS samples were punched out, and antidepressants were simultaneously extracted and derivatized in a single step by use of pentafluoropropionic acid anhydride and 0.02% triethylamine in butyl chloride for 30 min at 60 °C under ultrasonication. Derivatives were then separated on a gas chromatograph coupled with a triple-quadrupole mass spectrometer operating in negative selected reaction monitoring mode for a total run time of 5 min. To establish the validity of the method, trueness, precision, and selectivity were determined on the basis of the guidelines of the “Société Française des Sciences et des Techniques Pharmaceutiques” (SFSTP). The assay was found to be linear in the concentration ranges 1 to 500 ng mL−1 for fluoxetine and norfluoxetine and 20 to 500 ng mL−1 for reboxetine and paroxetine. Despite the small sampling volume, the limit of detection was estimated at 20 pg mL−1 for all the analytes. The stability of DBS was also evaluated at −20 °C, 4 °C, 25 °C, and 40 °C for up to 30 days. Furthermore, the method was successfully applied to a pharmacokinetic investigation performed on a healthy volunteer after oral administration of a single 40-mg dose of fluoxetine. Thus, this validated DBS method combines an extractive—derivative single step with a fast and sensitive GC-NICI-MS-MS technique. Using microliter blood samples, this procedure offers a patient-friendly tool in many biomedical fields such as checking treatment adherence, therapeutic drug monitoring, toxicological analyses, or pharmacokinetic studies.
Keywords: DBS; Fast GC; NICI-MS-MS; Antidepressants; Pharmacokinetics

The application of the design of experiments to optimize method development in the field of forensic toxicology using the urinary morphine 3-glucuronide acid hydrolysis as an example is described. Morphine and its trideuterated analogue (used as an internal standard) were extracted from urine samples by liquid–liquid extraction (ToxiTubes® A) and derivatized by silylation. Chromatographic analysis was done by gas chromatography–mass spectrometry in the selected ion monitoring mode. Using the peak area ratio (morphine-to-internal standard) as the response, we investigated the independent variables that could influence the acid hydrolysis, including temperature (range 70–130 °C), acid volume (range 500–1,000 µL) and time (range 15–90 min). A 23 full factorial design for the screening and a response surface methodology, including a central composite design for optimization, were applied. The factors which influenced the response to a greater extent were temperature and its interaction both with time and acid volume. By application of a multiple regression analysis to the experimental data, a second-order polynomial equation was obtained. The optimal predicted conditions for morphine 3-glucuronide acid hydrolysis were 115 °C, 38 min and 500 µL for temperature, time and acid volume, respectively. Using design of experiments, instead of the one factor at a time approach, we achieved the optimum combination of all factor values, and this allowed the best results to be obtained, simultaneously optimizing resources. In addition, time and money can be saved, since other approaches are in general more time-consuming and laborious, and do not take into account the interactions between factors.
Keywords: Design of experiments; Forensic toxicology; Morphine 3-glucuronide; Acid hydrolysis

Rapid determination of lithium in serum samples by capillary electrophoresis by Jennifer P. Pascali; Daniela Sorio; Federica Bortolotti; Franco Tagliaro (2543-2546).
Lithium salts are still one of the most popular therapeutic approaches to the treatment of bipolar disorders, notwithstanding the introduction of more modern, less toxic drugs. Because of a narrow therapeutic range, lithium serum concentrations must be strictly monitored during the treatment to avoid life-threatening neurotoxicity. For this purpose, methods based on flame photometry or ion-selective electrodes are usually applied. The aim of the present work was to develop and validate a simple method for the determination of lithium in serum based on capillary zone electrophoresis with indirect detection. A validation of the method was carried out, including a comparison with an automated routine method based on ion-selective electrodes.
Keywords: Lithium; Capillary electrophoresis; Analysis; Serum; Bipolar disorders

Detection of proteases using an immunochemical method with haptenylated–gelatin as a solid-phase substrate by Ramadan A. Abuknesha; Fiona Jeganathan; Rens DeGroot; Dirk Wildeboer; Robert G. Price (2547-2558).
A simplified method for the measurement of proteases utilising solid-phase substrates incorporating an ELISA end-point detection step is described. Gelatin–hapten conjugates adsorbed onto polystyrene surfaces were found to be efficient substrates for proteases. Digestion of the solid-phase protein–hapten complexes resulted in proportional desorption of the attached conjugates and decrease in the detectable hapten species. Gelatin–cholic acid conjugates, affinity-purified sheep anti-cholic acid antibody–HRP and a chromogenic substrate were incorporated into a convenient and highly sensitive solid-phase immunochemical method. The detectable signal is inversely proportional to enzyme activity. Bacterial proteases (alpha-chymotrypsin Type II, Type IX from Bacillus polymyxa, Type XIV from Streptomyces griseus, Type XXIV from Bacillus licheniformens) were assayed. Dose–response curves for enzyme activities were measured within ranges of 0–550 µunits mL−1 for chymotrypsin, 0–12 µunits mL−1 for type IX, 0–35 µunits mL−1 for type XIV and 0–100 µunits mL−1 for type XXIV. The detection limits of the proteases studied were 89 µunits mL−1 for chymotrypsin, 0.26 µunits mL−1 for type IX, 5.8 µunits mL−1 for type XIV and 6.5 µunits mL−1 for type XXIV. It was demonstrated that the two-step immunochemical method combines the simplicity and sensitivity of solid-phase enzyme immunoassays, the broad specificity of gelatin as a protease substrate and the flexibility of the solid-phase format. Effect of increasing hapten density on the hydrolysis of gelatin–hapten conjugates by proteinase
Keywords: Proteases universal substrate; Solid-phase substrate; ELISA

Ligand-protein docking studies of potential HIV-1 drug compounds using the algorithm FlexX by George Patargias; Gary Ewart; Carolyn Luscombe; Wolfgang B. Fischer (2559-2563).
Four compounds are docked to a pentameric bundle representing the transmembrane part of the Vpu protein from HIV-1. Employing the docking algorithm FlexX, their free energy of binding is estimated leading to the conclusion that potential drug candidates need to form H-bonds either with neighbouring or with n + 2 helices at the site of the serines within the bundle.
Keywords: Docking approach; Vpu; HIV-1; Ligand binding; Viral membrane proteins

Cystic fibrosis: a label-free detection approach based on thermally modulated electrochemical impedance spectroscopy by Hany Nasef; Valerio Beni; Veli C. Őzalp; Ciara K. O’Sullivan (2565-2574).
Cystic fibrosis is one of the most common genetically inherited diseases in northern Europe, with the DF508 mutation being the most common, and among the Caucasian population being responsible for almost 70% of cases. In this work, we report on the use of thermally modulated electrochemical impedance spectroscopy for the discrimination of the DF508 mutation from the wild-type sequence. DNA probes (15 and 21 bases long) were immobilised on the surface of gold electrodes and the variation of the charge-transfer resistance was monitored as a function of hybridisation. Two sets of targets were used in this work: synthetic 15-mer sequences and two single-stranded synthetic analogues of PCR products 82 (mutant) and 85 (wild type) bases long. Hybridisation with short targets resulted in very sequence specific charge-transfer-resistance variation with a discrimination factor at room temperature between fully complementary and mismatched sequences of approximately fivefold. However, in the case of the single-stranded synthetic PCR product analogues, a lower discrimination factor was recorded (1.5-fold). The effect of temperature was investigated to improve discrimination and the use of a posthybridisation wash at elevated temperature resulted in a fivefold improvement in the discrimination factor. Using an electrode array with probes immobilised against each of the mutant and wild-type sequences, we achieved an unequivocal detection of the DF508 mutation. Online Abstract Figure Electrochemical impedance spectroscopy response before and after sensor regeneration
Keywords: Electrochemical impedance spectroscopy; Cystic fibrosis; Label-less detection; DNA sensor; DF508

Developments of optical protein sensors with nanostructure based on the noble metals have currently received great attention for their high efficiency and simultaneous analysis of various important biomolecules from proteomics to genetics. In this study, we exploited the absorbance spectra of gold-capped nanoparticles substrate for label-free detections of antigen–antibody reactions using a specific thiolated RNA aptamer. These synthesized RNA aptamers have been optimized to bind to the Fc portion of the human IgG1 subclass, due to their ability to orient antibodies direction on the gold surface. After attaching the anti-fibrinogen antibodies on the surface via these linkers, our thiolated RNA aptamer-based nanostructured sensors were easily applicable to specific detections of fibrinogen with a limit of detection of 0.1 ng/mL. These nanostructured sensor-based models will open a way to display numerous immunosensors as well as to develop other functionally similar sensors which could then be expanded into multi-arrays assay systems.
Keywords: RNA aptamer-based nanostructured sensor; Label-free detection; Antigen–antibody interactions; Gold-capped nanoparticles substrates

A unification of doping-control screening procedures of prohibited small molecule substances—including stimulants, narcotics, steroids, β2-agonists and diuretics—is highly urgent in order to free resources for new classes such as banned proteins. Conceptually this may be achieved by the use of a combination of one gas chromatography–time-of-flight mass spectrometry method and one liquid chromatography–time-of-flight mass spectrometry method. In this work a quantitative screening method using high-resolution liquid chromatography in combination with accurate-mass time-of-flight mass spectrometry was developed and validated for determination of glucocorticosteroids, β2-agonists, thiazide diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation of all the compounds of interest and the necessary purification of the resulting extracts, a generic extraction and hydrolysis procedure was combined with a solid-phase extraction modified for these groups of compounds. All 56 compounds are determined using positive electrospray ionisation with the exception of the thiazide diuretics for which the best sensitivity was obtained by using negative electrospray ionisation. The results show that, with the exception of clenhexyl, procaterol, and reproterol, all compounds can be detected below the respective minimum required performance level and the results for linearity, repeatability, within-lab reproducibility, and accuracy show that the method can be used for quantitative screening. If qualitative screening is sufficient the instrumental analysis may be limited to positive ionisation, because all analytes including the thiazides can be detected at the respective minimum required levels in the positive mode. The results show that the application of accurate-mass time-of-flight mass spectrometry in combination with generic extraction and purification procedures is suitable for unification and expansion of the window of screening methods of doping laboratories. Moreover, the full-scan accurate-mass data sets obtained still allow retrospective examination for emerging doping agents, without re-analyzing the samples.
Keywords: Doping control; Validation; High-resolution liquid chromatography; Accurate-mass time-of-flight mass spectrometry; Quantitative screening; Retrospective data analysis

Interaction of a commercial lipid dispersion and local anesthetics in human plasma: implications for drug trapping by “lipid-sinks” by Jaana Laine; Jana Lokajová; Jevgeni Parshintsev; Juha M. Holopainen; Susanne K. Wiedmer (2599-2607).
Interactions between Intralipid dispersion and local anesthetics (bupivacaine, prilocaine, and lidocaine) were investigated. The amount of bupivacaine (the most cardiotoxic analyte of the local anesthetics studied) entrapped in Intralipid in the presence of plasma was studied using an off-line filtration and solid phase extraction method combined with capillary zone electrophoresis for quantification of free unbound bupivacaine. To confirm interactions between the analytes and Intralipid at lower concentrations, direct injection mass spectrometry was used. The use of immobilized Intralipid chromatography-atmospheric pressure ionization–ion trap mass spectrometry in the study of interactions between drugs and Intralipid dispersion is demonstrated. Finally, interactions between Intralipid dispersion and local anesthetics were investigated by electrokinetic capillary chromatography. The electrophoretic mobility of the Intralipid dispersed phase was calculated using the iterative procedure and a homologous series of alkyl phenyl benzoates (C1–C6), and the retention factors for the analytes were determined.
Keywords: Electrokinetic capillary chromatography; Immobilized Intralipid chromatography; Intralipid; Local anesthetics; Mass spectrometry

Automated mass spectrometric analysis of urinary and plasma serotonin by Wilhelmina H. A. de Jong; Marianne H. L. I. Wilkens; Elisabeth G. E. de Vries; Ido P. Kema (2609-2616).
Serotonin emerges as crucial neurotransmitter and hormone in a growing number of different physiologic processes. Besides extensive serotonin production previously noted in patients with metastatic carcinoid tumors, serotonin now is implicated in liver cell regeneration and bone formation. The aim was to develop a rapid, sensitive, and highly selective automated on-line solid-phase extraction method coupled to high-performance liquid chromatography–tandem mass spectrometry (XLC-MS/MS) to quantify low serotonin concentrations in matrices such as platelet-poor plasma and urine. Fifty microliters plasma or 2.5 μL urine equivalent were pre-purified by automated on-line solid-phase extraction, using weak cation exchange. Chromatography of serotonin and its deuterated internal standard was performed with hydrophilic interaction chromatography. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Serotonin concentrations were determined in platelet-poor plasma of metastatic carcinoid patients (n = 23) and healthy controls (n = 22). Urinary reference intervals were set by analyzing 24-h urine collections of 120 healthy subjects. Total run-time was 6 min. Intra- and inter-assay analytical variation were <10%. Linearity in the 0–7300 μmol/L calibration range was excellent (R2 > 0.99). Quantification limits were 30 and 0.9 nmol/L in urine and plasma, respectively. Platelet-poor serotonin concentrations in metastatic carcinoid patients were significantly higher than in controls. The urinary reference interval was 10–78 μmol/mol creatinine. Serotonin analysis with sensitive and specific XLC-MS/MS overcomes limitations of conventional HPLC. This enables accurate quantification of serotonin for both routine diagnostic procedures and research in serotonin-related disorders.
Keywords: Serotonin; 5-hydroxytryptamine; LC-MS/MS; On-line SPE; Mass spectrometry; Automation

A highly sensitive caffeine immunoassay based on a monoclonal antibody by José João Carvalho; Michael G. Weller; Ulrich Panne; Rudolf J. Schneider (2617-2628).
A new immunoassay has been developed based on a commercially available anti-caffeine monoclonal antibody and a de novo synthesized tracer, using horseradish peroxidase and UV–visible detection. Caffeine, which is frequently found in surface waters, can be quantified with a relative error lower than 20% for concentrations above 0.025 μg L−1 (limit of quantitation, direct analysis). The limit of detection is 0.001 μg L−1 and can be reduced by solid-phase extraction (SPE). Moreover, with minor adaptations, the assay can be used to quantify caffeine in several beverages, shampoo, and caffeine tablets. The results obtained by ELISA correlate well with those from liquid chromatography–tandem mass spectrometry (LC–MS–MS) for the tested matrices. Several surface waters from Berlin were analysed and all tested positive for caffeine, with concentrations higher than 0.030 μg L−1. In one run 66 samples can be analysed within 2 h. Figure A caffeine ELISA is described that allows sensitive and selective analysis of surface water concentrations as well as determination of caffeine in beverages.
Keywords: Caffeine; ELISA; Antibody; LC–MS–MS; Water; Wastewater marker

Simultaneous quantitative determination of α-ketoglutaric acid and 5-hydroxymethylfurfural in human plasma by gas chromatography–mass spectrometry by Bernhard M. Wagner; Fabrizio Donnarumma; Reinhold Wintersteiger; Werner Windischhofer; Hans J. Leis (2629-2637).
α-Ketoglutaric acid (α-KG) and 5-hydroxymethylfurfural (5-HMF) are currently under investigation as promising cancer cell damaging agents. A method for the simultaneous quantitative determination of α-KG and 5-HMF in human plasma was established for screening these compounds in human plasma. Plasma samples were directly treated with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride to form the corresponding oximes, thus facilitating subsequent liquid–liquid extraction. After formation of the trimethylsilyl ethers, samples were analyzed by gas chromatography with electron ionization mass spectrometry. Stable isotope labeled standards were used, the preparation of 13C6-5-HMF is described. Limits of quantitation were set to 0.938 µg/mL for α-KG and 0.156 µg/mL for 5-HMF. Inter-day accuracy was ≤93.7% (α-KG) and ≤92.8% (5-HMF). Inter-day precision was ≤6.0% (α-KG) and ≤4.6% (5-HMF). The method has been successfully applied to pharmacokinetic profiling of the compounds after intravenous application.
Keywords: α-Ketoglutaric acid; 5-Hydroxymethylfurfural; GC-MS

An SPME–GC–MS method using an octadecyl silica fibre for the determination of the potential angiogenesis modulators 17β-estradiol and 2-methoxyestradiol in culture media by Federica Bianchi; Monica Mattarozzi; Maria Careri; Alessandro Mangia; Marilena Musci; Francesca Grasselli; Simona Bussolati; Giuseppina Basini (2639-2645).
A simple and easily automable method based on solid-phase microextraction followed by gas chromatographic–mass spectrometric analysis was developed for the determination of two potential angiogenesis modulators 17β-estradiol (17-BE) and 2-methoxyestradiol (2-MEOE) in culture media. Trifluoroacetic anhydride was used as the derivatising agent. A homemade octadecyl silica coating, characterised by a coating thickness of 72 ± 10 μm and a good thermal stability until 250 °C, was prepared. Experimental design was used to optimise the extraction conditions in terms of derivatisation time, derivatisation temperature and time of extraction. As for method validation, lower limits of quantification of 0.17 and 0.015 µg/l for 17β-estradiol and 2-methoxyestradiol, respectively, were obtained. Finally, the capabilities of the developed fibres were evaluated for the analysis of the investigated analytes developed by granulosa cells in culture media maintained under normoxic, hypoxic and anoxic conditions, in order to better elucidate their possible role in the angiogenic process. An increase of the production of both 17-BE and 2-MEOE in hypoxic and anoxic conditions seems to be related to the effect of oxygen deprivation. Figure 17-βestradiol and 2-methoxyestradiol as potential angiogenesis modulators
Keywords: Angiogenesis; Solid-phase microextraction; 17β-Estradiol; 2-Methoxyestradiol; Derivatisation

New approach to optimize HPLC separations of acid–base compounds with elution order involved, by using combined three-band resolution maps by L. V. Pérez-Arribas; F. J. Manuel de Villena-Rueda; M. E. León-González; R. Gonzalo-Lumbreras; L. M. Polo-Díez (2647-2656).
Some of the optimization methods in reversed phase high-performance liquid chromatography (RP-HPLC) are based on resolution of the critical band pair. Mobile phase composition is changed systematically to establish those conditions giving an acceptable resolution for such a critical band pair, but sometimes the critical pair may change with the separation conditions, which obliges to identify it for each of those conditions. In the case of ionizable compounds, more than two bands may be involved in resolution, showing—in some cases—changes in the elution order when the mobile phase composition was modified. In this paper, an alternative way that does not identify the critical pair after changing experimental conditions is proposed. The relative separation of the three bands involved in two alternating critical band pairs is evaluated as a sort of conjugate or combined resolution, represented as contour maps vs. two variables (content of organic modifier and pH). These maps are obtained from data of chromatograms made under different separation conditions; these conditions were generated by experimental design and data was mathematically processed with a computer program. Analytes of three families that have acid–base properties, triazines, phenoxyacids, and phenols, were used for this purpose. The chromatographic behavior when elution order reversion of ionizable compounds exists is studied.
Keywords: Optimization; RP-HPLC; Triazines; Phenols; Phenoxyacids; Resolution maps; Critical pair change

2-(5-Benzoacridine)ethyl-p-toluenesulfonate (BAETS), a dual-sensitive probe, was reacted with bile acids in the presence of K2CO3 catalyst in dimethyl sulfoxide (DMSO) solvent to give BAETS–bile acid derivatives. Derivatives exhibited intense fluorescence (FL) with an excitation maximum at λ ex 270 nm and an emission maximum at λ em 510 nm. MS analysis using APCI-MS indicated that derivatives had excellent APCI-MS ionizability with percentage ionization δ values changing from 0 to 88.83% in aqueous acetonitrile and from 0 to 89.15% in aqueous methanol. The collision induced dissociation spectra of m/z [M + H]+ contained specific fragment ions at m/z [M + H−H2O]+, [M + H−2H2O]+, [M + H−3H2O]+, 347.3, and 290.1. Repeatability was good for LC separation of BAETS–bile acid derivatives with aqueous acetonitrile as mobile phase. The relative standard deviations (RSDs) of retention time and peak area at 6.6 nmol mL−1 levels with fluorescence detection (FL) were from 0.045 to 0.072% and from 2.16 to 2.73%, respectively. Excellent linear responses were observed, with regression coefficients >0.9995. The FL detection limits (S/N = 3) were in the range of 18.0–36.1 fmol. The online APCI-MS detection limits are in the range of 500–790 fmol (at a signal-to-noise ratio of 3). Figure Schematic diagram of derivatization procedure
Keywords: 2-(5-Benzoacridine)ethyl-p-toluenesulfonate; Bile acids; Column liquid chromatography; Fluorescence detection; Mass spectrometry

A thiol-selective fluorogenic probe based on the cleavage of 4-methylumbelliferyl-2’,4’,6’-trinitropheyl ether by Xiao-Feng Yang; Zhuan Su; Chenghai Liu; Haiping Qi; Minglei Zhao (2667-2674).
A new fluorescent probe 1,4-methylumbelliferyl-2′,4′,6′-trinitropheyl ether (Probe 1) was designed and synthesized. Probe 1 was a nonfluorescent compound and was synthesized via the one-step reaction of 4-methylumbelliferone (4-MU) with 1-chloro-2,4,6-trinitrobenzene. Upon mixing with biothiols under neutral aqueous conditions, the 2,4,6-trinitrophenyl group of 1 was efficiently removed, and the emissive free dye 4-MU was released, hence leading to a dramatic increase in fluorescence emission of the reaction mixture. A good linear relationship was obtained from 0.1 to 4.0 μmol L−1 for cysteine (Cys), from 0.1 to 3.0 μmol L−1 for homocysteine (Hcy), and from 0.2 to 3.0 μmol L−1 for glutathione (GSH), respectively. The detection limits of Cys, Hcy, and GSH were 24.3, 35.6, and 26.8 nmol L−1, respectively. Furthermore, probe 1 was highly selective for biothiols without the interference of some biologically relevant analytes and has been applied to detecting biothiols in human serum samples. Figure Fluorescence response of probe 1 in the presence of thiols
Keywords: 4-Methylumbelliferone; Thiols; 2,4,6-Trinitrophenol; Fluorescent probe

Geostatistical and multivariate statistical analysis of heavily and manifoldly contaminated soil samples by Kristin Schaefer; Jürgen W. Einax; Vasil Simeonov; Stefan Tsakovski (2675-2683).
The surroundings of the former Kremikovtzi steel mill near Sofia (Bulgaria) are influenced by various emissions from the factory. In addition to steel and alloys, they produce different products based on inorganic compounds in different smelters. Soil in this region is multiply contaminated. We collected 65 soil samples and analyzed 15 elements by different methods of atomic spectroscopy for a survey of this field site. Here we present a novel hybrid approach for environmental risk assessment of polluted soil combining geostatistical methods and source apportionment modeling. We could distinguish areas with heavily and slightly polluted soils in the vicinity of the iron smelter by applying unsupervised pattern recognition methods. This result was supported by geostatistical methods such as semivariogram analysis and kriging. The modes of action of the metals examined differ significantly in such a way that iron and lead account for the main pollutants of the iron smelter, whereas, e.g., arsenic shows a haphazard distribution. The application of factor analysis and source-apportionment modeling on absolute principal component scores revealed novel information about the composition of the emissions from the different stacks. It is possible to estimate the impact of every element examined on the pollution due to their emission source. This investigation allows an objective assessment of the different spatial distributions of the elements examined in the soil of the Kremikovtzi region. The geostatistical analysis illustrates this distribution and is supported by multivariate statistical analysis revealing relations between the elements.
Keywords: Source apportionment modeling; Semivariogram analysis; Kriging; Environmental pollution; Chemometrics; Kremikovtzi

Dispersive liquid—liquid microextraction coupled with high-performance liquid chromatography—diode-array detection was applied for the extraction and determination of 11 priority pollutant phenols in wastewater samples. The analytes were extracted from a 5-mL sample solution using a mixture of carbon disulfide as the extraction solvent and acetone as the dispersive solvent. After extraction, solvent exchange was carried out by evaporating the solvent and then reconstituting the residue in a mixture of methanol–water (30:70). The influences of different experimental dispersive liquid—liquid microextraction parameters such as extraction solvent type, dispersive solvent type, extraction and dispersive solvent volume, salt addition, and pH were studied. Under optimal conditions, namely pH 2, 165-µL extraction solvent volume, 2.50-mL dispersive solvent volume, and no salt addition, enrichment factors and limits of detection ranged over 30–373 and 0.01–1.3 µg/L, respectively. The relative standard deviation for spiked wastewater samples at 10 µg/L of each phenol ranged between 4.3 and 19.3% (n = 5). The relative recovery for wastewater samples at a spiked level of 10 µg/L varied from 65.5 to 108.3%.
Keywords: Dispersive liquid–liquid microextraction; Liquid chromatography; Wastewater analysis; Priority pollutant phenols

Fatty acid methyl esters obtained from a fish oil sample were fractionated by non-aqueous reversed-phase high-performance liquid chromatography (RP-HPLC) using three serially connected C18-columns and pure methanol as the eluent. The HPLC fractions were analyzed by gas chromatography–electron ionization mass spectrometry in the selected ion monitoring mode. Data analysis and visualization was performed by the creation of a two-dimensional (2D) contour plot, in which GC retention times were plotted against the HPLC fractions. The 2D contour plot resulted in a full resolution of more than 120 fatty acids. The fatty acids were arranged on predictable lines and curves in dependence of the number of carbons and double bonds. The 2D contour plot enabled both the recognition of unknown fatty acids (which were found off the lines and curves) and the prediction of the coordinates of known fatty acids. Finally, selected HPLC fractions were subjected to further experiments (hydrogenation, silver ion fractionation, specific GC/MS measurements) in order to verify the structural assignments predicted from the 2D contour plot. All in all, the structures of over 100 FAs could be assigned to the peaks detected in the 2D contour plots. Figure Excerpt of the 2D contour plot of the methyl esters of the fatty acids from a fish oil obtained by plotting the GC/EI-MS retention time against the HPLC fraction number showing the polyunsaturated fatty acids with the chain lengths C16 and C22. Unknown compounds are marked with an arrow; dotted ellipses show the different groups of isomers. The color spectrum reflects the signal intensity relative to the internal standard.
Keywords: Non-aqueous reversed-phase HPLC; GC/EI-MS; Two-dimensional contour plot; Fish oil; Fatty acid methyl ester; Predictable elution pattern

Glycol ethers still continue to be a workplace hazard due to their important use on an industrial scale. Currently, chronic occupational exposures to low levels of xenobiotics become increasingly relevant. Thus, sensitive analytical methods for detecting biomarkers of exposure are of interest in the field of occupational exposure assessment. 1-Methoxy-2-propanol (1M2P) is one of the dominant glycol ethers and the unmetabolized urinary fraction has been identified to be a good biological indicator of exposure. An existing analytical method including a solid-phase extraction and derivatization before GC/FID analysis is available but presents some disadvantages. We present here an alternative method for the determination of urinary 1M2P based on the headspace gas chromatography technique. We determined the 1M2P values by the direct headspace method for 47 samples that had previously been assayed by the solid-phase extraction and derivatization gas chromatography procedure. An inter-method comparison based on a Bland–Altman analysis showed that both techniques can be used interchangeably. The alternative method showed a tenfold lower limit of detection (0.1 mg/L) as well as good accuracy and precision which were determined by several urinary 1M2P analyses carried out on a series of urine samples obtained from a human volunteer study. The within- and between-run precisions were generally about 10%, which corresponds to the usual injection variability. We observed that the differences between the results obtained with both methods are not clinically relevant in comparison to the current biological exposure index of urinary 1M2P. Accordingly, the headspace gas chromatography technique turned out to be a more sensitive, accurate, and simple method for the determination of urinary 1M2P.
Keywords: Biological monitoring; 1-Methoxy-2-propanol; Headspace gas chromatography

Hot embossing of electrophoresis microchannels in PMMA substrates using electric heating wires by Zhibing Gan; Zhengyin Yu; Zhi Chen; Gang Chen (2715-2720).
A simple method based on electric heating wires has been developed for the rapid fabrication of poly(methyl methacrylate) (PMMA) electrophoresis microchips in ordinary laboratories without the need for microfabrication facilities. A piece of stretched electric heating wire placed across the length of a PMMA plate along its midline was sandwiched between two microscope slides under pressure. Subsequently, alternating current was allowed to pass through the wire to generate heat to emboss a separation microchannel on the PMMA separation channel plate at room temperature. The injection channel was fabricated using the same procedure on a PMMA sheet that was perpendicular to the separation channel. The complete microchip was obtained by bonding the separation channel plate to the injection channel sheet, sealing the channels inside. The electric heating wires used in this work not only generated heat; they also served as templates for embossing the microchannels. The prepared microfluidic microchips have been successfully employed in the electrophoresis separation and detection of ions in connection with contactless conductivity detection. Figure 1 SEM image illustrating the cross sections of an open microchannel in a PMMA channel plate. Also shown, in the inset, is a 3D image of the open channel
Keywords: Microfluidic chip; Miniaturization; Poly(methyl methacrylate); Electric heating wire; Embossing