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Analytical and Bioanalytical Chemistry (v.400, #1)

Mario Thevis: Mass spectrometry in sports drug testing. Characterization of prohibited substances and doping control analytical assays by Marco Vincenti (pp. 1-2).

Shimshon Belkin, Man Bock Gu (Eds.): Whole cell sensing systems I and II by Elisa Michelini (pp. 3-4).

Alain Berthod (Ed.): Chiral recognition in separation methods. Mechanisms and applications by Caroline West (pp. 5-6).

Forensic toxicology by Frank T. Peters; Hans H. Maurer; Frank Musshoff (pp. 7-8).

is Head of Toxicology at the Institute of Forensic Medicine at the University Hospital Jena. He has published over 60 original papers and five invited reviews on his main areas of research: analytical toxicology, method validation, and metabolism of new designer drugs. He received young scientist awards from The International Association of Forensic Toxicologists (2003), the Society of Toxicological and Forensic Chemistry (2007), and the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (2009). is Head of the Department of Experimental and Clinical Toxicology of Saarland University in Homburg. He has published over 200 original papers and over 20 invited reviews on his main two areas of research, analytical toxicology (GC–MS, LC–MS) and metabolism of xenobiotics. He has received several international scientific awards for his outstanding scientific work, and in 2007 was awarded the title of Doctor Honoris Causa by the University of Ghent. is a forensic toxicologist at the Institute of Forensic Medicine of the University Hospital in Bonn. He has published over 130 original papers and his main topics are drugs and driving, post mortem toxicology, and analytical toxicology. He has received several national and international scientific awards for his outstanding scientific work, and since 2007 he has been the president of the German-speaking Society of Toxicological and Forensic Chemistry (GTFCh).

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry by Stefan König; Beat Aebi; Stephan Lanz; Martina Gasser; Wolfgang Weinmann (pp. 9-16).

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure — in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step.

Keywords: THC; LC-MS/MS; On-line SPE; Quantification

Rapid quantification of tilidine, nortilidine, and bisnortilidine in urine by automated online SPE-LC-MS/MS by Christoph Köhler; Thomas Grobosch; Torsten Binscheck (pp. 17-23).

The opioid tilidine is a prodrug which is hepatically metabolized to active nortilidine and bisnortilidine. Due to the increasing abuse of tilidine by drug users and the lack of a specific immunoassay, we developed an analytical method for the quantification of tilidine, nortilidine, and bisnortilidine in urine suitable for screening. In a following step, this method was used to establish data on excretion kinetics of the substances in order to evaluate the time window of detection after a single oral dose of tilidine/naloxone and also was applied to authentic urine samples from correctional facilities. Urine samples were mixed with internal standard solution and extracted on a weak cation exchanger at pH 6 using a Symbiosis Pico system. The chromatographic separation was achieved within a 3.5-min run time on a Phenylhexyl column (50 × 2.0 mm, 5 μm) via gradient elution (methanol and 0.2% formic acid) at a flow rate of 0.50 mL/min. The ESI-MS/MS was performed on a QTrap 3,200 in positive multiple reaction monitoring mode using two mass transitions per analyte. Validating the method resulted in a lower limit of quantification of 1.0 μg/L followed by a linear calibration range to 100 μg/L for each analyte (r ² > 0.99). The analytical method allowed the detection of a single dose of a commercially available tilidine solution up to 7 days after administration. Using this highly sensitive method, 55 of 3,665 urine samples were tested positive.

Keywords: Tilidine; Nortilidine; Bisnortilidine; Online SPE; LC-MS/MS

Monolithic spin column extraction and GC-MS for the simultaneous assay of diquat, paraquat, and fenitrothion in human serum and urine by Takeshi Saito; Tomokazu Fukushima; Yuko Yui; Shota Miyazaki; Akihiro Nakamoto; Akira Namera; Sadaki Inokuchi (pp. 25-31).

We present a method based on monolitic spin column extraction and gas chromatography–mass spectrometry as an analytical method for screening diquat (DQ), paraquat (PQ), and fenitrothion in serum and urine. This method is useful for clinical and forensic toxicological analyses. Recovery of DQ, PQ, and fenitrothion from serum and urine, spiked at concentrations between 0.1, 2.5, 20, and 45 μg/ml, ranged from 51.3% to 106.1%. Relative standard deviation percentages were between 3.3% and 14.8%. Detection and quantitation limits for serum and urine were 0.025 and 0.05 μg/ml, respectively, for DQ, 0.1 and 0.1 μg/ml, respectively, for PQ, and 0.025 and 0.05 μg/ml, respectively, for fenitrothion. Therefore, these compounds can be detected and quantified in the case of acute poisoning.

Keywords: Diquat; Paraquat; Fenitrothion; Monolitic spin column extraction; GC-MS

Simultaneous identification and validated quantification of 11 oral hypoglycaemic drugs in plasma by electrospray ionisation liquid chromatography–mass spectrometry by Cornelius Hess; Frank Musshoff; Burkhard Madea (pp. 33-41).

The detection of diabetic metabolism disorders raises problems in forensic practice and sudden death with a subsequent negative autopsy finding is a common problem. In the case of an unclear hypoglycaemia, the detection of oral antidiabetics allows the differentiation of hypoglycaemia due to oral antidiabetics from that due to other reasons (insulin-induced, insulinoma). The development of an electrospray ionisation (ESI) liquid chromatography–tandem mass spectrometry (LC-MS/MS) procedure for the simultaneous identification and quantification of oral antidiabetics of the sulfonylurea, the glinide, the thiazolidinedione and the gliptin types in human plasma is desired. The following analytes were included: glimepiride, glibenclamide, gliquidone, glibornuride, glisoxepide, glipizide and gliclazide (sulfonylurea type), nateglinide and repaglinide (glinide type), rosiglitazone and pioglitazone (thiazolidinedione type) and the dipeptidyl peptidase inhibitors vildagliptin, sitagliptin and saxagliptin. After a liquid–liquid extraction with tert-butyl methyl ether at two pHs, the oral antidiabetics were separated with fast gradient elution over a C₈ column. Identification of the oral antidiabetics was achieved by two specific ion transitions of each analyte in multiple reaction monitoring mode. Quantification was performed by referring the most intense ion transition peak areas to peak areas of the ion transitions of deuterated oral antidiabetics (hydroxytolbutamide-d ₉ for the sulfonylureas, repaglinide-ethyl-d ₅ for the glinides, pioglitazone-d ₄ for the thiazolidinediones and vildagliptin-d ₃ for the gliptins). The assay was validated according to international guidelines. The LC-MS/MS assay allows the simultaneous identification of 14 oral antidiabetics and quantification of 11 oral antidiabetics in plasma in the ESI mode in a single run. Linearity is shown up to overdose concentrations. The limits of detection with a signal-noise-ratio greater than 3 were below 1 ng/ml for all analytes. Recoveries ranged from 78 to 105%; for vildagliptin and saxagliptin recoveries were worse (45%) owing to their hydrophilic character. Intraday and interday precision and accuracy were below 20% for 11 drugs at three concentrations. For the gliptins, several validation parameters were out of range and, therefore, quantitatively this method is inappropriate.

Keywords: Oral antidiabetics; Liquid chromatography; Mass spectrometry

Quantitative determination of 43 common drugs and drugs of abuse in human serum by HPLC-MS/MS by David M. Bassan; Freidoon Erdmann; Ralf Krüll (pp. 43-50).

An analytical procedure for the simultaneous determination in human serum of 43 common drugs of abuse and their metabolites belonging to the different chemical and toxicological classes of amphetamines, benzodiazepines, dibenzazepines, cocaine, lysergic acid diethylamide, opioids, phencyclidine, tricyclic antidepressants, and zolpidem, using 33 deuterated standards, is presented. The sample treatment was developed to be a very simple protein precipitation and filtration. All analyses were performed with a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry in positive ionization mode. All analytes were calibrated up to 550 μg/L. The limit of detection ranged from 0.6 ng/mL (EDDP) to 13.7 ng/mL (flunitrazepam). The method has been validated according to the guidelines of the Gesellschaft für Toxikologische und Forensische Chemie, using three multiple reaction mode (MRM) transitions and retention time for positive compound identification, instead of two MRMs, in anticipation of the new guidelines for January 2011.

Keywords: Drugs of abuse; HPLC-MS/MS; Serum; Forensic analytical chemistry

Simultaneous LC-HRMS determination of 28 benzodiazepines and metabolites in hair by Susanna Vogliardi; Donata Favretto; Marianna Tucci; Giulia Stocchero; Santo Davide Ferrara (pp. 51-67).

A liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for the simultaneous identification and quantification of 28 benzodiazepines, including 6 metabolites, in 50 mg of hair has been validated. Positive ion electrospray ionization and HRMS determination in full-scan mode were realized on an Orbitrap mass spectrometer at a nominal resolving power of 60,000. In-source collisional experiments were conducted to obtain additional information for a more reliable identification of the investigated drugs. HRMS in full-scan mode allowed the exact determination of molecular masses of all analytes eluting in the HPLC run, so that both the immediate and retrospective screening of results for drugs and their metabolites were available. Sample preparation consisted of an overnight incubation in phosphate buffer pH 8.4 and a subsequent liquid/liquid extraction with methylene chloride/diethyl ether (90:10). Gradient elution was performed on a Luna C18 analytical column and four deuterated analogues were used as internal standards (IS). Validation was performed using both spiked hair samples and hair samples from subjects treated with benzodiazepines. Selectivity was evaluated by analysis of 20 certified blank hair samples. Extraction efficiency and matrix effects were evaluated by analysis of true positive samples. The lowest limits of quantification (LLOQs) ranged from 1 to 10 pg/mg. Linearity was investigated in the range from LLOQ to 1,000 pg/mg, for each compound (R ² 0.998–0.999). Mean relative errors, calculated at three concentration levels, ranged from 1 to 20% (absolute value). Precision, at concentrations higher than the LLOQs, was always less than 15% expressed as percentage relative standard deviation. After validation, the procedure was applied to real samples collected for clinical and forensic toxicology purposes from subjects who were assumed to have taken benzodiazepines.

Keywords: Benzodiazepines; Hair analysis; HRMS; Orbitrap

Simultaneous analysis of buprenorphine, methadone, cocaine, opiates and nicotine metabolites in sweat by liquid chromatography tandem mass spectrometry by Marta Concheiro; Diaa M. Shakleya; Marilyn A. Huestis (pp. 69-78).

A liquid chromatography tandem mass spectrometry method for buprenorphine (BUP), norbuprenorphine (NBUP), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine, benzoylecgonine, ecgonine methyl ester (EME), morphine, codeine, 6-acetylmorphine, heroin, 6-acetylcodeine, cotinine, and trans-3′-hydroxycotinine quantification in sweat was developed and comprehensively validated. Sweat patches were mixed with 6 mL acetate buffer at pH 4.5, and supernatant extracted with Strata-XC-cartridges. Reverse-phase separation was achieved with a gradient mobile phase of 0.1% formic acid and acetonitrile in 15 min. Quantification was achieved by multiple reaction monitoring of two transitions per compound. The assay was a linear 1–1,000 ng/patch, except EME 5–1,000 ng/patch. Intra-, inter-day and total imprecision were <10.1%CV, analytical recovery 87.2–107.7%, extraction efficiency 35.3–160.9%, and process efficiency 25.5–91.7%. Ion suppression was detected for EME (−63.3%) and EDDP (−60.4%), and enhancement for NBUP (42.6%). Deuterated internal standards compensated for these effects. No carryover was detected, and all analytes were stable for 24 h at 22 °C, 72 h at 4 °C, and after three freeze/thaw cycles. The method was applied to weekly sweat patches from an opioid-dependent BUP-maintained pregnant woman; 75.0% of sweat patches were positive for BUP, 93.8% for cocaine, 37.5% for opiates, 6.3% for methadone and all for tobacco biomarkers. This method permits a fast and simultaneous quantification of 14 drugs and metabolites in sweat patches, with good selectivity and sensitivity.

Keywords: Buprenorphine; Methadone; Cocaine; Cotinine; Sweat; Drug monitoring

Development of the first metabolite-based LC-MS n urine drug screening procedure-exemplified for antidepressants by Dirk K. Wissenbach; Markus R. Meyer; Daniela Remane; Armin A. Weber; Hans H. Maurer (pp. 79-88).

In contrast to GC-MS libraries, currently available LC-MS libraries for toxicological detection contain besides parent drugs only some main metabolites limiting their applicability for urine screening. Therefore, a metabolite-based LC-MS n screening procedure was developed and exemplified for antidepressants. The library was built up with MS² and MS³ wideband spectra using an LXQ linear ion trap with electrospray ionization in the positive mode and full-scan information-dependent acquisition. Pure substance spectra were recorded in methanolic solution and metabolite spectra in urine from rats after administration of the corresponding drugs. After identification, the metabolite spectra were added to the library. Various drugs and metabolites could be sufficiently separated. Recovery, process efficiency, matrix effects, and limits of detection for selected drugs were determined using protein precipitation. Automatic data evaluation was performed using ToxID and SmileMS software. The library consists of over 700 parent compounds including 45 antidepressants, over 1,600 metabolites, and artifacts. Protein precipitation led to sufficient results for sample preparation. ToxID and SmileMS were both suitable for target screening with some pros and cons. In our study, only SmileMS was suitable for untargeted screening being not limited to precursor selection. The LC-MS n method was suitable for urine screening as exemplified for antidepressants. It also allowed detecting unknown compounds based on known fragment structures. As ion suppression can never be excluded, it is advantageous to have several targets per drug. Furthermore, the detection of metabolites confirms the body passage. The presented LC-MS n method complements established GC-MS or LC-MS procedures in the authors’ lab.

Keywords: Urine; Screening; LC-MS; Library; Metabolite; Antidepressants

Development of a fully automated toxicological LC-MSⁿ screening system in urine using online extraction with turbulent flow chromatography by Daniel M. Mueller; Bénédicte Duretz; Francois A. Espourteille; Katharina M. Rentsch (pp. 89-100).

In clinical toxicology, fast and specific methods are necessary for the screening of different classes of drugs. Therefore, an online extraction high-performance liquid chromatography coupled to mass spectrometry (LC-MSⁿ) screening method using a MS² and MS³ spectral library for the identification of xenobiotic substances has been developed and validated. Samples were run twice, once native and once after enzymatic hydrolysis. Internal standards and buffer were added to the urine samples. Following centrifugation, the supernatant was injected into the system. Extraction was performed by online turbulent flow chromatography. The chromatographic separation was achieved using a Phenyl/Hexyl column. For detection, a linear ion trap, equipped with an APCI interface, was used and the different compounds were identified using a MS² and MS³ spectral library containing 356 compounds. The turnaround time to report the results of the screening including hydrolysis was approximately 2 h. About 92% of the 356 substances could be identified with a limit of identification below 100 ng/ml. The recovery and matrix effect experiments showed suitable results, and in six drug-free urine samples of healthy volunteers analyzed for selectivity, no substances have been identified. Carryover could be well controlled, and the method had a good reproducibility. The comparison of the results of 103 real patient urine samples showed a good agreement between the existing GC-MS and LC-MS methods with offline extraction and the new online extraction LC-MSⁿ screening method. The presented method allows a fast and sensitive analysis of a broad range of compounds.

Keywords: LC-MS; Turbulent flow chromatography; Online extraction; Toxicological screening; Clinical toxicology

Development and practical application of a library of CID accurate mass spectra of more than 2,500 toxic compounds for systematic toxicological analysis by LC–QTOF-MS with data-dependent acquisition by Sebastian Broecker; Sieglinde Herre; Bernhard Wüst; Jerry Zweigenbaum; Fritz Pragst (pp. 101-117).

A library of collision-induced dissociation (CID) accurate mass spectra has been developed for efficient use of liquid chromatography in combination with hybrid quadrupole time-of-flight mass spectrometry (LC–QTOF-MS) as a tool in systematic toxicological analysis. The mass spectra (Δm < 3 ppm) of more than 2,500 illegal and therapeutic drugs, pesticides, alkaloids, other toxic chemicals and metabolites were measured, by use of an Agilent 6530 instrument, by flow-injection of 1 ng of the pure substances in aqueous ammonium formate–formic acid–methanol, with positive and negative electrospray-ionization (ESI), selection of the protonated or deprotonated molecules [M+H]⁺ or [M−H]⁻ by the quadrupole, and collision induced dissociation (CID) with nitrogen as collision gas at CID energies of 10, 20, and 40 eV. The fragment mass spectra were controlled for structural plausibility, corrected by recalculation to the theoretical fragment masses and added to a database of accurate mass data and molecular formulas of more than 7,500 toxicologically relevant substances to form the “database and library of toxic compounds”. For practical evaluation, blood and urine samples were spiked with a mixture of 33 drugs at seven concentrations between 0.5 and 500 ng mL⁻¹, prepared by dichloromethane extraction or protein precipitation, and analyzed by LC–QTOF-MS in data-dependent acquisition mode. Unambiguous identification by library search was possible for typical basic drugs down to 0.5–2 ng mL⁻¹ and for benzodiazepines down to 2–20 ng mL⁻¹. The efficiency of the method was also demonstrated by re-analysis of venous blood samples from 50 death cases and comparison with previous results. In conclusion, LC–QTOF-MS in data-dependent acquisition mode combined with an accurate mass database and CID spectra library seemed to be one of the most efficient tools for systematic toxicological analysis. Figure LC-QTOF-MS file measured in auto-MS-MS mode from a blood sample of a poisoning case after application of the tool “File Compounds”

Keywords: Accurate mass spectra library; Collision-induced dissociation; Liquid chromatography; Time of flight mass spectrometry; Peak identification; Systematic toxicological analysis

Comparison of “herbal highs” composition by Dariusz Zuba; Bogumila Byrska; Martyna Maciow (pp. 119-126).

Popularity of new psychoactive substances, known as legal highs or herbal highs, is continuously growing. These products are typically sold via internet and in so-called head shops. The aim of this study was to identify active ingredients of herbal highs and to compare their chemical composition. Twenty-nine various products seized by the police in one of the “head shops” were analysed. Herbal mixtures (0.2 g) were prepared by ultrasonic-assisted extraction with 2.0 ml of ethanol for 2 h. The extracts were analysed by gas chromatography coupled to mass spectrometry (GC/MS). The main active compounds of the herbal mixtures were synthetic cannabinoids: JWH-018, JWH-073 and cannabicyclohexanol (CP-47,497-C8-homolog). Their content differed between the products; some contained only one cannabinoid whereas the others contained two or more. Cluster analysis and principal component analysis revealed that chemical composition of many products was very similar. The similarity was connected with their flavour and not the common name. This statement was true for the synthetic cannabinoids, other potential agonists of cannabinoid receptors (amides of fatty acids) and ingredients of natural origin and confirms that herbal highs are a threat to human health because the purchaser has no information on their real composition. Figure

Keywords: “Legal highs”; Cannabinoids; GC–MS; Chemometrics

Monitoring of kratom or Krypton intake in urine using GC-MS in clinical and forensic toxicology by Anika A. Philipp; Markus R. Meyer; Dirk K. Wissenbach; Armin A. Weber; Siegfried W. Zoerntlein; Peter G. M. Zweipfenning; Hans H. Maurer (pp. 127-135).

The Thai medicinal plant Mitragyna speciosa (kratom) is misused as a herbal drug. Besides this, a new herbal blend has appeared on the drugs of abuse market, named Krypton, a mixture of O-demethyltramadol (ODT) and kratom. Therefore, urine drug screenings should include ODT and focus on the metabolites of the kratom alkaloids mitragynine (MG), paynantheine (PAY), speciogynine (SG), and speciociliatine (SC). The aim of this study was to develop a full-scan gas chromatography–mass spectrometry procedure for monitoring kratom or Krypton intake in urine after enzymatic cleavage of conjugates, solid-phase extraction, and trimethylsilylation. With use of reconstructed mass chromatography with the ions m/z 271, 286, 329, 344, 470, 526, 528, and 586, the presence of MG, 16-carboxy-MG, 9-O-demethyl-MG, and/or 9-O-demethyl-16-carboxy-MG could be indicated, and in case of Krypton, with m/z 58, 84, 116, 142, 303, 361, 393, and 451, the additional presence of ODT and its nor metabolite could be indicated. Compounds were identified by comparison with their respective reference spectra. Depending on the plant type, dose, administration route, and/or sampling time, further metabolites of MG, PAY, SG, and SC could be detected. The limits of detection (signal-to-noise ratio of 3) were 100 ng/ml for the parent alkaloids and 50 ng/ml for ODT. As mainly metabolites of the kratom alkaloids were detected in urine, the detectability of kratom was tested successfully using rat urine after administration of a common user’s dose of MG. As the metabolism in humans was similar, this procedure should be suitable to prove an intake of kratom or Krypton.

Keywords: Kratom ; Mitragynine; Krypton; Metabolism; Gas chromatography–mass spectrometry; Urine

Validation of a GC/MS method for the detection of two quinolinone-derived selective androgen receptor modulators in doping control analysis by E. Gerace; A. Salomone; F. Fasano; R. Costa; D. Boschi; A. Di Stilo; M. Vincenti (pp. 137-144).

Selective androgen receptor modulators (SARMs) represent an emerging class of drugs likely to be abused in sport. For clinical applications, these substances provide a promising alternative to testosterone-replacement therapies and their advantages include oral bioavailability, androgen receptor specificity, tissue selectivity, and the absence of steroid-related side effects. Although not yet commercially available, since January 2008 SARMs have been included on the prohibited list issued yearly by the World Anti-Doping Agency (WADA), so control laboratories need to update their procedures to detect either the parent drugs or their metabolites. Within this context, two quinolinone SARM models were synthesized and automatically characterized to update the existing routine screening procedures. The conditions for the new target analytes are compatible with the existing laboratory protocols used for both in-competition and out-of-competition controls and can be included in them. Validation parameters according to ISO 17025 and WADA guidelines were successfully determined. For analytical determinations, spiked urine samples were hydrolyzed and extracted at pH 9.6 with 10 mL of tert-butyl methyl ether. Then, the analytes were subsequently converted into trimethylsilyl derivatives and detected by gas chromatography–mass spectrometry. The absence of interferents, together with excellent repeatability of both retention times and the relative abundances of diagnostic ions, allowed proper identification of all SARM analytes. The analytes’ quantification was linear up to 500 ng/mL and precision criteria were satisfied (coefficient of variation less than 25% at 10 ng/mL). The limits of detection were 1 ng/mL for both SARMs, whereas recovery values were between 95.5 and 99.3%. The validated method can be efficiently used for urine screening of the 2-quinolinone-derived SARMs tested. Figure Chemical structures of the two quinolinone-derived SARMs investigated

Keywords: Gas chromatography–mass spectrometry; Doping control; Selective androgen receptor modulators; Urine screening; Validation

Influence of ethanol on cannabinoid pharmacokinetic parameters in chronic users by Stefan W. Toennes; Kirsten Schneider; Gerold F. Kauert; Cora Wunder; Manfred R. Moeller; Eef L. Theunissen; Johannes G. Ramaekers (pp. 145-152).

Cannabis is not only the most widely used illicit drug worldwide but is also regularly consumed along with ethanol. In previous studies, it was assumed that cannabis users develop cross-tolerance to ethanol effects. The present study was designed to compare the effects of ethanol in comparison to and in combination with a cannabis joint and investigate changes in pharmacokinetics. In this study, 19 heavy cannabis users participated and received three alcohol dosing conditions that were calculated to achieve steady blood alcohol concentrations (BAC) of about 0, 0.5 and 0.7 g/l during a 5-h time window. Subjects smoked a Δ⁹-tetrahydrocannabinol (THC) cigarette (400 μg/kg) 3 h post-onset of alcohol dosing. Blood samples were taken between 0 and 4 h after smoking. During the first hour, samples were collected every 15 min and every 30 min thereafter. Mean steady-state BACs reached 0, 0.36 and 0.5 g/l. The apparent elimination half-life of THC was slightly prolonged (1.59 vs. 1.93 h, p < 0.05) and the concentration 1 h after smoking was slightly lower (24 vs. 17 ng/ml, p < 0.05) with the higher ethanol dose. The prolonged THC elimination might be explained by a small ethanol-mediated change in distribution to and from deep compartments. Concentrations and pharmacokinetics of 11-hydroxy-THC and 11-nor-9-carboxy-THC (THCA) were not significantly influenced by ethanol. However, THCA concentrations appeared lower in both ethanol conditions, which might also be attributable to changes in distribution. Though not significant in the present study, this might be relevant in the interpretation of cannabinoid concentrations in blood.

Keywords: Forensics/toxicology; Drug monitoring/drug screening; Kinetics; Cannabis; Ethanol; Interaction

Reduction of temazepam to diazepam and lorazepam to delorazepam during enzymatic hydrolysis by Shanlin Fu; Anna Molnar; Peter Bowron; John Lewis; Hongjie Wang (pp. 153-164).

It has been previously reported that treatment of urinary oxazepam by commercial β-glucuronidase enzyme preparations, from Escherichia coli, Helix pomatia and Patella vulgata, results in production of nordiazepam (desmethyldiazepam) artefact. In this study, we report that this unusual reductive transformation also occurs in other benzodiazepines with a hydroxyl group at the C3 position such as temazepam and lorazepam. As determined by liquid chromatography-mass spectrometry analysis, all three enzyme preparations were found capable of converting urinary temazepam into diazepam following enzymatic incubation and subsequent liquid–liquid extraction procedures. For example, when H. pomatia enzymes were used with incubation conditions of 18 h and 50 °C, the percentage conversion, although small, was significant—approximately 1% (0.59–1.54%) in both patient and spiked blank urines. Similarly, using H. pomatia enzyme under these incubation conditions, a reductive transformation of urinary lorazepam into delorazepam (chlordesmethyldiazepam) occurred. These findings have both clinical and forensic implications. Detection of diazepam or delorazepam in biological samples following enzyme treatment should be interpreted with care.

Keywords: β-Glucuronidase; Temazepam; Diazepam; Lorazepam; Delorazepam; Mass spectrometry

Chiral analyses of dextromethorphan/levomethorphan and their metabolites in rat and human samples using LC-MS/MS by Ruri Kikura-Hanajiri; Maiko Kawamura; Atsuko Miyajima; Momoko Sunouchi; Yukihiro Goda (pp. 165-174).

In order to develop an analytical method for the discrimination of dextromethorphan (an antitussive medicine) from its enantiomer, levomethorphan (a narcotic) in biological samples, chiral analyses of these drugs and their O-demethyl and/or N-demethyl metabolites in rat plasma, urine, and hair were carried out using LC-MS/MS. After the i.p. administration of dextromethorphan or levomethorphan to pigmented hairy male DA rats (5 mg/kg/day, 10 days), the parent compounds and their three metabolites in plasma, urine and hair were determined using LC-MS/MS. Complete chiral separation was achieved in 12 min on a Chiral CD-Ph column in 0.1% formic acid–acetonitrile by a linear gradient program. Most of the metabolites were detected as being the corresponding O-demethyl and N, O-didemethyl metabolites in the rat plasma and urine after the hydrolysis of O-glucuronides, although obvious differences in the amounts of these metabolites were found between the dextro and levo forms. No racemation was observed through O- and/or N-demethylation. In the rat hair samples collected 4 weeks after the first administration, those differences were more clearly detected and the concentrations of the parent compounds, their O-demethyl, N-demethyl, and N, O-didemethyl metabolites were 63.4, 2.7, 25.1, and 0.7 ng/mg for the dextro forms and 24.5, 24.6, 2.6, and 0.5 ng/mg for the levo forms, respectively. In order to fully investigate the differences of their metabolic properties between dextromethorphan and levomethorphan, DA rat and human liver microsomes were studied. The results suggested that there might be an enantioselective metabolism of levomethorphan, especially with regard to the O-demethylation, not only in DA rat but human liver microsomes as well. The proposed chiral analyses might be applied to human samples and could be useful for discriminating dextromethorphan use from levomethorphan use in the field of forensic toxicology, although further studies should be carried out using authentic human samples.

Keywords: Levomethorphan; Dextromethorphan; Chiral analysis; Biological samples; LC-MS/MS; Enantioselective metabolism

Comparison of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) concentrations in hair for testing abstinence by M. E. Albermann; F. Musshoff; B. Madea (pp. 175-181).

Hair analysis is a powerful tool for retrospective drug analysis. By determining the minor ethanol metabolites ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) in hair, even a previous consumption of alcohol is detectable. However, previous studies showed a lack of correlation if both parameters are determined simultaneously. A further study was conducted to confirm or refute these results. One hundred and sixty hair samples were analyzed for EtG and FAEE in the context of driving ability. In 109 cases, alcohol abstinence was clearly proven and was excluded in 15 cases. In 36 cases, ambiguous results were found. Possible reasons for the deviating results are discussed. It is recommended, that in context of driving ability diagnostics the EtG result is determinant. In critical cases FAEE concentrations can be determined for checking purposes, but a negative FAEE result cannot refute a determined EtG concentration >7 pg/mg. Figure Comparative illustration of EtG and FAEE concentrations in 160 hair samples analyzed in the context of driving ability diagnostics. The horizontal lines indicate the cut-off values. Color coding: green → teetotalers, red → social or excessive chronic drinkers, yellow and blue → ambiguous results.

Keywords: Hair analysis; Abstinence test; Ethyl glucuronide; Fatty acid ethyl esters; Comparative analysis

Impact of hair-care products on FAEE hair concentrations in substance abuse monitoring by Joey Gareri; Brice Appenzeller; Paula Walasek; Gideon Koren (pp. 183-188).

Previous studies have indicated that the use of high-ethanol-content (>65%) hair-care products may elevate fatty acid ethyl ester (FAEE) concentrations in hair. In this case series, nine individuals were identified by FAEE analysis to be chronic alcohol abusers in the context of child-welfare substance abuse monitoring. Based on patient claims of moderate or no alcohol consumption, the presence of ethanol in the patients’ hair-care regimens was investigated. Samples were additionally tested for the presence of ethyl glucuronide (EtG). From a total of nine patients, 12 hair samples were submitted for analysis. Patient histories were obtained as well as Material Safety Data Sheets (MSDS) listing hair-care product ethanol content. Hair samples were pre-washed to remove external contamination and analyzed for FAEE and EtG by GC-MS. According to the Society of Hair Testing consensus guidelines, FAEE levels exceeding 0.50 ng/mg and/or EtG levels exceeding 30 pg/mg indicate chronic excessive alcohol consumption. Upon initial analysis, the nine samples exhibited positive FAEE findings ranging from 0.496 to 4.984 ng/mg. MSDS review revealed the presence of ethanol from 10% to 95% by volume in at least one hair-care product used by each individual. Results of the EtG analysis ranged from 1.9 to 23.5 pg/mg. These findings indicate that regular use of products with ethanol content as low as 10% can impact FAEE results. EtG analysis should be used to confirm FAEE findings and appears to be unaffected by hair-care products, likely due to alternative mechanisms of incorporation.

Keywords: Fatty acid ethyl ester (FAEE); Ethyl glucuronide (EtG); Alcohol; Ethanol; Hair

Validated method for the determination of ethylglucuronide and ethylsulfate in human urine by Jochen Beyer; Tu N. Vo; Dimitri Gerostamoulos; Olaf H. Drummer (pp. 189-196).

Detection of the alcohol metabolites ethylglucuronide (EtG) and ethylsulfate (EtS) has become routine in many forensic laboratories over the last few years. Most previously published methods using liquid chromatography coupled with electrospray tandem mass spectrometry require a post-chromatographic addition of solvent and/or extensive sample preparation prior to analysis. The aim of the study was to develop a simplified method. To 20 μL urine, internal standard containing EtG-d5 and EtS-d 5 was added and the mixture was treated with elution buffer internal standard. EtG and EtS were separated using a Shimadzu Prominence high performance liquid chromatography (HPLC) system with a C18 separation column (Restek Ultra Aqueous C18, 4.6 × 150 mm, 5 μm), using isocratic elution with a mobile phase consisting of 10 mM ammonium acetate buffer pH 7 (total run time, 6 min). The compounds were detected using an Applied Biosystems API 5000 liquid chromatography tandem mass spectrometry system (atmospheric pressure chemical ionization, multiple-reaction monitoring mode). The method was fully validated according to international guidelines. The assay was found to be selective for the compounds of interest. It was linear from 0.1 to 10 mg/L for all analytes (R ² > 0.99). Matrix effects studies showed the presence of a slight but consistent ion enhancement (n = 10 different urine samples) at low concentrations and no effects at higher concentrations. Accuracy data were between 0.75% and 8.1% bias for EtG and between −5.0% and −11.3% bias for EtS. Precision data were between 4.3% and 6.9% relative standard deviations (RSD) for EtG and between 6.0% and 7.5% RSD for EtS. No instability was observed after repeated freezing and thawing. This fast, reliable, and accurate method enables the detection and quantification of alcohol metabolites in urine. The method is easier to use and more sensitive than previously published methods.

Keywords: Ethylglucuronide; Ethylsulfate; Urine; Liquid chromatography; Mass spectrometry

Sensing mispaired thymines in DNA heteroduplexes using an electroactive osmium marker: towards electrochemical SNP probing by Pavel Kostečka; Luděk Havran; Miroslava Bittová; Hana Pivoňková; Miroslav Fojta (pp. 197-204).

A complex OsO₄, 2,2′-bipyridine (Os,bipy), has been used for electroactive labeling of biopolymers as well as for probing of nucleic acids and protein structure and interactions. In DNA, Os,bipy forms electrochemically active adducts with pyrimidine nucleobases, exhibiting highly selective modification of thymine residues in single-stranded DNA. Here, we show that modification of rare thymine residues (one thymine among several tens of unreactive purine bases) can easily be detected by means of a simple ex situ voltammetric analysis using carbon electrodes. Based on this remarkable sensitivity of detection, Os,bipy has been used as an electroactive probe for unpaired and/or mismatched thymine residues within DNA heteroduplexes. Site-specific chemical modification of the DNA with the Os,bipy has allowed a clear distinction between perfectly base-paired DNA homoduplexes and mismatched heteroduplexes, as well as discrimination among heteroduplexes containing one or two mispaired thymines, a single thymine insertion, or combination of a mispair and an insertion. Figure Sensing of single base mismatches using osmium tetroxide, 2,2′-bipyridine (Os,bipy). Os,bipy binds selectively to mispaired thymines, giving electroactive adducts easily detectable at a pyrolytic graphite electrode

Keywords: Single-base mismatch; Nucleobase insertion; Osmium tetroxide; Carbon electrodes; Voltammetry; Chemical probe; Electroactive label

Micro-solid phase equilibrium extraction with highly ordered TiO₂ nanotube arrays: a new approach for the enrichment and measurement of organochlorine pesticides at trace level in environmental water samples by Qingxiang Zhou; Yunrui Huang; Junping Xiao; Guohong Xie (pp. 205-212).

Ordered TiO₂ nanotube arrays have been widely used in many fields such as photocatalysis, self-cleaning, solar cells, gas sensing, and catalysis. This present study exploited a new functional application of the ordered TiO₂ nanotube arrays. A micro-solid phase equilibrium extraction using ordered TiO₂ nanotube arrays was developed for the enrichment and measurement of organochlorine pesticides prior to gas chromatography-electron capture detection. Ordered TiO₂ nanotube arrays exhibited excellent merits on the pre-concentration of organochlorine pesticides and lower detection limits of 0.10, 0.10, 0.10, 0.098, 0.0076, 0.0097, 0.016, and 0.023 μg L⁻¹ for α-HCH, β-HCH, γ-HCH, δ-HCH, p,p’-DDE, p,p’-DDD, o,p’-DDT, and p,p’-DDT, respectively, were achieved. Four real water samples were used for validation, and the spiked recoveries were in the range of 78–102.8%. These results demonstrated that the developed micro-solid phase equilibrium extraction using ordered TiO₂ nanotube arrays would be very constructive and have a great beginning with a brand new prospect in the analysis of environmental pollutants. Figure

Keywords: Ordered TiO₂ nanotube array; Organochlorine pesticides; Micro-solid phase equilibrium extraction; Gas chromatography-electron capture detection

Semi-automated image analysis: detecting carbonylation in subcellular regions of skeletal muscle by Vratislav Kostal; Kiara Levar; Mark Swift; Erik Skillrud; Mark Chapman; LaDora V. Thompson; Edgar A. Arriaga (pp. 213-222).

The level of carbonylation in skeletal muscle is a marker of oxidative damage associated with disease and aging. While immunofluorescence microscopy is an elegant method to identify carbonylation sites in muscle cross-sections, imaging analysis is manual, tedious, and time consuming, especially when the goal is to characterize carbonyl contents in subcellular regions. In this paper, we present a semi-automated method for the analysis of carbonylation in subcellular regions of skeletal muscle cross-sections visualized with dual fluorescent immunohistochemistry. Carbonyls were visualized by their reaction with 2,4-dinitrophenylhydrazine (DNPH) followed by immunolabeling with an Alexa488-tagged anti-DNP antibody. Mitochondria were probed with an anti-COXI primary antibody followed by the labeling with an Alexa568-tagged secondary antibody. After imaging, muscle fibers were individually analyzed using a custom-designed, lab-written, computer-aided procedure to measure carbonylation levels in subsarcolemmal and interfibrillar mitochondrial regions, and in the cytoplasmic and extracellular regions. Using this procedure, we were able to decrease the time necessary for the analysis of a single muscle fiber from 45 min to about 1 min. The procedure was tested by four independent analysts and found to be independent on inter-person and intra-person variations. This procedure will help increase highly needed throughput in muscle studies related to ageing, disease, physical performance, and inactivity that use carbonyl levels as markers of oxidative damage.

Keywords: Carbonylation; Fluorescence microscopy; Image analysis; Mitochondria; Aging; Muscle fiber; ImageJ; MatLab

Design and probing of efflux functions of EGFP fused ABC membrane transporters in live cells using fluorescence spectroscopy by Feng Ding; Kerry J. Lee; Ardeschir Vahedi-Faridi; Tao Huang; Xiao-Hong Nancy Xu (pp. 223-235).

We have designed and constructed fusion genes of C-terminal (Ct) or N-terminal (Nt) bmrA with EGFP vectors and successfully expressed them in ΔBmrA (BmrA deletion strain of Bacillus subtilis), generating two new strains of B. subtilis (Ct-BmrA-EGFP and Nt-BmrA-EGFP). The fusion genes were characterized using gel electrophoresis and DNA sequencing. Their expression in live cells was determined by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy and spectroscopy. The efflux function of the new strains was studied by measuring their accumulation kinetics of intracellular Hoechst dye molecules (a pump substrate) using fluorescence spectroscopy, which were compared with wild-type (WT-BmrA) and ΔBmrA strains. Both new strains show lower accumulation rates than ΔBmrA, and their efflux kinetics are inhibited by a pump inhibitor (orthovanadate). The results suggest that both strains extrude the dye molecules and the fusion proteins retain the efflux function of BmrA (ATP-binding cassette, ABC, transporter). Notably, Nt-BmrA-EGFP strain shows lower accumulation rates (higher efflux rates) than Ct-BmrA-EGFP. Modeled structures of the fusion proteins illustrate a highly flexible linker region connecting EGFP with BmrA, suggesting a minimal obstruction of EGFP to the BmrA. A closer distance of two C termini (∼14 Å) than two N termini (47.9 Å) of the “closed” BmrA dimer depicts the larger steric effect of C-terminal fusion. This study also shows that glucose affects the fluorescence study of efflux function of BmrA, suggesting that efflux kinetics of ABC membrane transporters in live cells must be characterized in the absence of glucose. Figure Design and characterization of efflux functions of ABC-EGFP transporters

Keywords: ABC (BmrA) membrane transporter; ABC-EGFP fusion transporters; Single cell imaging; Multidrug resistance; Bacillus subtilis ; Fluorescence spectroscopy

Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine by John H. Miller IV; Philip A. Poston; H. Thomas Karnes (pp. 237-244).

A in-line desorption device was developed, which allows for direct analysis of dried blood spots eliminating the need for punching disks from the filter paper cards. Using this device, we have validated a method to quantify biomarkers related to maple syrup urine disease (MSUD), a metabolism disorder that often requires a second-tier test for confirmation. Direct analysis of newborn screening cards is conducted in-line with a high-resolution chromatographic separation with mass spectrometry using electrospray ionization and multiple-reaction monitoring. Quantification of leucine and isoleucine using an isotopically labeled internal standard encompasses a range suitable for MSUD assessment. Precision and accuracy of the technique was acceptable with relative standard deviations within 10% at three fortified concentrations and an unfortified level. A post-column infusion test shows minimum matrix suppression was observed using this direct sampling technique.

Keywords: Direct analysis; Maple syrup urine disease; Newborn screening; Human whole blood; LC/MS/MS

Culturing and investigation of stress-induced lipid accumulation in microalgae using a microfluidic device by Ryan E. Holcomb; Lucas J. Mason; Kenneth F. Reardon; Donald M. Cropek; Charles S. Henry (pp. 245-253).

There is increasing interest in using microalgae as a lipid feedstock for the production of biofuels. Lipids used for these purposes are triacylglycerols that can be converted to fatty acid methyl esters (biodiesel) or decarboxylated to “green diesel.” Lipid accumulation in most microalgal species is dependent on environmental stress and culturing conditions, and these conditions are currently optimized using slow, labor-intensive screening processes. Increasing the screening throughput would help reduce the development cost and time to commercial production. Here, we demonstrated an initial step towards this goal in the development of a glass/poly(dimethylsiloxane) (PDMS) microfluidic device capable of screening microalgal culturing and stress conditions. The device contained power-free valves to isolate microalgae in a microfluidic growth chamber for culturing and stress experiments. Initial experiments involved determining the biocompatibility and culturing capability of the device using the microalga Tetraselmis chuii. With this device, T. chuii could be successfully cultured for up to 3 weeks on-chip. Following these experiments, the device was used to investigate lipid accumulation in the microalga Neochloris oleabundans. It was shown that this microalga could be stressed to accumulate cytosolic lipids in a microfluidic environment, as evidenced with fluorescence lipid staining. This work represents the first example of microalgal culturing in a microfluidic device and signifies an important expansion of microfluidics into the biofuels research arena.

Keywords: Microalgae; Microfluidics; Valves; Lipids; Biofuels; Cell culturing

Urinary profile of methylprednisolone acetate metabolites in patients following intra-articular and intramuscular administration by Alessia Panusa; Luca Regazzoni; Giancarlo Aldini; Marica Orioli; Arrigo Giombini; Paola Minghetti; Carlo Tranquilli; Marina Carini (pp. 255-267).

A study on urinary metabolites of methylprednisolone acetate (MPA) has been performed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) in precursor ion scanning (PIS) and neutral loss (NL) modes. Patients suffering from joint inflammation have been treated with Depo-Medrol® (MPA marketed suspension, 40 mg) intra-articularly (IA) and after a wash-out period, intramuscularly (IM) at the same dose. Urine samples have been collected after both the administration routes. Metabolites were identified in PIS mode by setting the fragment ion at m/z 161 which is specific for MPA, methylprednisolone (MP), methylprednisolone hemisuccinate, and in NL mode by selecting the losses of 54, 72, 176 and 194 Da. The MP-related structure of each target ion detected in both the MS modes was then confirmed by MS/MS acquisitions, and by accurate mass experiments. By using this approach, 13 MPA metabolites (M1–M13) have been identified, nine already reported in the literature and four unknown and for which the chemical structures have been proposed. No differences in the metabolic pattern of MPA when administered IM or IA were observed. The relative abundances of metabolites compared with the internal standard (MP-D2) were monitored by multiple reaction monitoring analysis for 19 days after both the administration routes. Figure Urine of patients before (dotted lines) and after (1st day, thick lines) the IM administration of Depo-Medrol®. Extracted ion chromatograms of glucuronic metabolites of MP with [M+H]⁺ at m/z 551. PIS m/z 161 and NL 194 Da traces (25 and 20 eV collision energies).

Keywords: Methylprednisolone acetate; Metabolites; Intra-articular; Intramuscular; LC–ESI–MS/MS

Fast capillary electrophoresis–time-of-flight mass spectrometry using capillaries with inner diameters ranging from 75 to 5 μm by Marco Grundmann; Frank-Michael Matysik (pp. 269-278).

Fast electrophoretic separations in fused silica capillaries (CE) coupled to time-of-flight mass spectrometry (TOF-MS) are presented. CE separations of the model analytes (epinephrine, norepinephrine, dopamine, histidine, and isoproterenol) under conditions of high electric field strengths of up to 1.25 kV cm⁻¹ are completed in 20 s. Coupling of CE with TOF-MS is accomplished using a coaxial sheath liquid electrospray ionization interface. The influence of parameters inherent to the interface and their effects, including suction pressure and dilution, are discussed. In addition to standard capillaries of 75 and 50 μm inner diameter (ID), separations in capillaries with IDs of 25, 15, and 5 μm have been successfully applied to this setup. The analytical performance is compared over this range of capillary dimensions, and both advantages and disadvantages are discussed.

Keywords: Capillary electrophoresis; Time-of-flight mass spectrometry; Electrospray ionization; Fused silica capillaries; High electric field strengths; Catecholamines

In situ FTIR and generalized 2D IR correlation spectroscopic studies on the crystallization behavior of solution-cast PHB film by He Huang; Wenjuan Guo; Hong Chen (pp. 279-288).

The crystallization behavior of biosynthesized poly(3-hydroxybutyrate) film cast from 1,1,2,2-tetrachloromethane was studied by in situ FTIR spectroscopy and two-dimensional (2D) correlation analysis. Time-dependent in situ FTIR spectral variations in the C=O stretching region (1,780–1,700 cm^-1) were monitored and analyzed by a series of data processing methods, including calculation of difference spectrum and second derivative spectrum, Fourier self-deconvolution, curve-fitting and 2D correlation analysis. Four bands have been resolved from the 2D correlation analysis, and the following overall sequential order among the intensity changes of the four bands has been obtained at 1,750 > 1,739 > 1,722 > 1,715 cm^-1. Combining with the other data processing methods, a curve-fitting approach has been employed to reveal that there are probably five component bands under the C=O band profile, centered at 1,746; 1,737; 1,728; 1,722; and 1,712 cm^-1. Detailed analysis on the in situ component band intensity variations in the C=O stretching region indicates that the crystalline and amorphous band intensities change simultaneously during the crystallization process, with no local sequential order. Further analysis on the relative area percentage changes of the five component bands suggests that the crystalline component only changes in a fully cooperative manner with part of the amorphous component at the initial crystallization period. Band area change of each state with crystallization time

Keywords: Poly(3-hydroxybutyrate) (PHB); Crystallization; Curve-fitting; 2D correlation analysis; Sequential order

Determination of cocaine on banknotes through an aptamer-based electrochemiluminescence biosensor by Qihong Cai; Lifen Chen; Fang Luo; Bin Qiu; Zhenyu Lin; Guonan Chen (pp. 289-294).

A novel electrochemiluminescence (ECL) “sandwich” biosensor has been developed to detect cocaine. The sandwich biosensor was fabricated on the basis of the fact that a single aptamer could be split into two fragments and the two dissociated parts could form a folded, associated complex in the presence of targets. One of these (capture probe), which had hexane–thiol at its 5′-terminus, was immobilized on a gold electrode via thiol–gold binding. The other one (detection probe) was labeled with the ECL reagent tris(2,2′-bipyridyl)ruthenium(II)-doped silica nanoparticles (RuSiNPs) at its 3′-terminus. Owing to the weak interaction between the two fragments, the sensor exhibited a low ECL signal in the absence of cocaine. After the target cocaine had been added to the solution, it induced association of the two fragments and stabilized the associated complexes, leading to immobilization of RuSiNPs on the electrode surface, and the ECL detected on the electrode surface was enhanced. The enhanced ECL intensity was directly proportional to the logarithm of the cocaine concentration in the range from 1.0 × 10⁻⁹ to1.0 × 10⁻¹¹ mol/L, with a detection limit of 3.7 × 10⁻¹² mol/L. The biosensor was applied to detect trace amounts of cocaine on banknotes with satisfactory results. Figure Scheme of the ECL aptasensor for cocaine detection in a sandwich manner. Note: each part is not according to the proportion

Keywords: Electrochemiluminescence; Biosensor; Cocaine; Aptamer; Banknote

Capillary electrophoresis-mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals by R. Haselberg; V. Brinks; A. Hawe; G. J. de Jong; G. W. Somsen (pp. 295-303).

In this work, the usefulness of capillary electrophoresis–electrospray ionization time-of-flight–mass spectrometry for the analysis of biopharmaceuticals was studied. Noncovalently bound capillary coatings consisting of Polybrene-poly(vinyl sulfonic acid) or Polybrene-dextran sulfate-Polybrene were used to minimize protein and peptide adsorption, and achieve good separation efficiencies. The potential of the capillary electrophoresis-mass spectrometry (CE-MS) system to characterize degradation products was investigated by analyzing samples of the drugs, recombinant human growth hormone (rhGH) and oxytocin, which had been subjected to prolonged storage, heat exposure, and/or different pH values. Modifications could be assigned based on accurate masses as obtained with time-of-flight–mass spectrometry (TOF-MS) and migration times with respect to the parent compound. For heat-exposed rhGH, oxidations, sulfonate formation, and deamidations were observed. Oxytocin showed strong deamidation (up to 40%) upon heat exposure at low pH, whereas at medium and high pH, mainly dimer (>10%) and trisulfide formation (6–7%) occurred. Recombinant human interferon-β-1a (rhIFN-β) was used to evaluate the capability of the CE-MS method to assess glycan heterogeneity of pharmaceutical proteins. Analysis of this N-glycosylated protein revealed a cluster of resolved peaks which appeared to be caused by at least ten glycoforms differing merely in sialic acid and hexose N-acetylhexosamine composition. Based on the relative peak area (assuming an equimolar response per glycoform), a quantitative profile could be derived with the disialytated biantennary glycoform as most abundant (52%). Such a profile may be useful for in-process and quality control of rhIFN-β batches. It is concluded that the separation power provided by combined capillary electrophoresis and TOF-MS allows discrimination of highly related protein species.

Keywords: Biopharmaceuticals; Capillary electrophoresis; Electrospray ionization; Mass spectrometry; Noncovalent coatings

Proficiency testing has improved the quality of data of total vitamin B₂ analysis in liquid dietary supplement by Mark Sykes; Joanne Croucher; Rosemary Ann Smith (pp. 305-310).

A previously reported proficiency test for the analysis of vitamin B₂ in liquid dietary supplement demonstrated bimodality. The same trend has now been observed in four subsequent tests of this type. The trend would not so easily have been observed without applying a fit-for-purpose standard deviation that is more generous than that predicted by the Horwitz equation. Since originally reporting the bimodal problem and hypothesising its cause by incomplete enzymic digestion of riboflavin-5-phosphate, there has been a general improvement in the reporting of the higher mode. This is thought to correspond to free riboflavin following complete digestion of the sample. Several individual participants appear to have learned from the experience and have changed their reporting of the lower mode to the higher mode. Figure Analysis of dietary supplements is subject to proficiency testing

Keywords: Proficiency testing; Vitamin analysis; Enzymic digestion

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Analytical and Bioanalytical Chemistry (v.400, #1)