Check out our New Publishers' Select for Free Articles

Analytical and Bioanalytical Chemistry (v.404, #1)

Teaching bioanalytical methods in a BSc analytical chemistry course by G. Horvai (pp. 1-3).

is Professor of Analytical Chemistry and Head of the Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics. His research interests include electrochemical sensors, molecularly imprinted polymers, and computer modeling of liquid surfaces. He is also Chairperson of the Bioanalytics Study Group of the Division of Analytical Chemistry of the European Association for Chemical and Molecular Sciences.

European Analytical Column No. 40 by Jiri Barek; Reiner Salzer; Paul J. Worsfold; Jens E. T. Andersen (pp. 5-7).

Europt(r)ode XI—11th European Conference on Optical Chemical Sensors and Biosensors in Barcelona, Spain by A. Katrin Krieg; Melanie Ewald; Barbara Schwarz (pp. 9-10).

Perry G. Wang and Weixuan He (Eds.): Hydrophilic interaction liquid chromatography (HILIC) and advanced applications by Pavel Jandera (pp. 11-12).

Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses by Tomoya Kinumi; Mari Goto; Sakae Eyama; Megumi Kato; Takeshi Kasama; Akiko Takatsu (pp. 13-21).

A certified reference material (CRM) is a higher-order calibration material used to enable a traceable analysis. This paper describes the development of a C-peptide CRM (NMIJ CRM 6901-a) by the National Metrology Institute of Japan using two independent methods for amino acid analysis based on isotope-dilution mass spectrometry. C-peptide is a 31-mer peptide that is utilized for the evaluation of β-cell function in the pancreas in clinical testing. This CRM is a lyophilized synthetic peptide having the human C-peptide sequence, and contains deamidated and pyroglutamylated forms of C-peptide. By adding water (1.00 ± 0.01) g into the vial containing the CRM, the C-peptide solution in 10 mM phosphate buffer saline (pH 6.6) is reconstituted. We assigned two certified values that represent the concentrations of total C-peptide (mixture of C-peptide, deamidated C-peptide, and pyroglutamylated C-peptide) and C-peptide. The certified concentration of total C-peptide was determined by two amino acid analyses using pre-column derivatization liquid chromatography–mass spectrometry and hydrophilic chromatography–mass spectrometry following acid hydrolysis. The certified concentration of C-peptide was determined by multiplying the concentration of total C-peptide by the ratio of the relative area of C-peptide to that of the total C-peptide measured by liquid chromatography. The certified value of C-peptide (80.7 ± 5.0) mg/L represents the concentration of the specific entity of C-peptide; on the other hand, the certified value of total C-peptide, (81.7 ± 5.1) mg/L can be used for analyses that does not differentiate deamidated and pyroglutamylated C-peptide from C-peptide itself, such as amino acid analyses and immunochemical assays.

Keywords: C-peptide; Certified reference material; Isotope-dilution mass spectrometry; Amino acid analysis

Development of a biosensor for human blood: new routes to body fluid identification by Nunzianda Frascione; Vania Pinto; Barbara Daniel (pp. 23-28).

The identification of human blood at a crime scene can provide crucial information to an investigation whilst also providing a source of nuclear material which can be targeted for DNA profiling. Here, we report on the development of an immunofluorescent biosensor for the identification of human blood which has the potential to overcome the drawbacks of the current body fluid identification techniques. An antibody (Ab) raised against human erythrocytes was conjugated to fluorescent semiconductor quantum dots (QDs) by sulfhydryl chemistry. The conjugation was verified by agarose gel electrophoresis and immunohistochemistry. Incubation of liquid blood samples with the conjugated nanocrystals was shown to quench the fluorescence emission spectra in a concentration-dependent manner. A different effect was observed with unconjugated QDs incubated in blood. Full profiles were obtained from blood samples previously treated with the Ab–QDs, demonstrating that the method does not interfere with DNA profiling. To our knowledge, this is the first example of a hybrid Ab–QD sensor that has the potential to be employed for the identification of human blood. The results of this study are expected to open up a new research direction in the field of body fluid detection.

Keywords: Anti-glycophorin A; Biosensor; Erythrocytes; Forensic; Immunofluorescence; Quantum dots

Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms by M. Caprioara-Buda; W. Meyer; B. Jeynov; P. Corbisier; S. Trapmann; H. Emons (pp. 29-42).

The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

Keywords: Genetically modified organism; Certified reference material; DNA calibrant; Plasmid; Real-time PCR; DNA copy number ratio

Validation of quantitative polymerase chain reaction methodology for monitoring DNA as a surrogate marker for species material contamination in porcine heparin by C. Auguste; S. Dereux; M. Rousset; P. Anger (pp. 43-50).

Heparin is a widely used intravenous anticoagulant comprising of a very complex mixture of glycosaminoglycan chains, mainly derived from porcine intestinal mucosa. The species of origin and the absence of contaminants from other species are important determinants of the different physicochemical characteristics of heparin. They also determine the potential for introducing infectious and adventitious agents into heparin batches destined for medicinal use. We perform routine quantitative polymerase chain reaction (Q-PCR) release tests to confirm the quality of all crude heparin batches, including those used for the manufacture of enoxaparin sodium. Here we further demonstrate that the assessment of the DNA content in crude heparin is a good surrogate marker of contamination at the mucosa level. After spiking porcine mucosa with ovine mucosa and processing this material to form crude heparin, we were able to observe similar ratios of species-specific DNA in both the starting and end products. Experiments performed with 3,000 and 1,500 ppm contamination found these concentrations to be well above the detection limit for our assay of heparin batches. Additionally this Q-PCR method can be used to detect contamination in mucosa, thus providing a tool capable of monitoring for contaminants throughout the crude heparin manufacturing process. Q-PCR analysis of industrial crude heparin samples has confirmed over time the value of this method to assess the pure porcine origin of heparin.

Keywords: Polymerase chain reaction; Quality; Heparin; Contamination

Liposomal glyco-microarray for studying glycolipid–protein interactions by Yong Ma; Irena Sobkiv; Valentinas Gruzdys; Hailong Zhang; Xue-Long Sun (pp. 51-58).

A microarray enables high-throughput interaction screening of numerous biomolecules; however, fabrication of a microarray composed of cellular membrane components has proven difficult. We report fabrication of a liposomal glyco-microarray by using an azide-reactive liposome that carries synthetic and natural glycolipids via chemically selective and biocompatible liposome immobilization chemistry. Briefly, liposomes carrying anchor lipid dipalmitoylphosphatidylethanolamine (DPPE)–PEG₂₀₀₀–triphenylphosphine and ganglioside (GM1 or GM3) were prepared first and were then printed onto an azide-modified glass slide so as to afford a liposomal glyco-microarray via Staudinger ligation. Fluorescent dye release kinetics and fluorescence imaging confirmed successful liposome immobilization and specific protein binding to the intact arrayed glycoliposomes. The liposomal glyco-microarray with different gangliosides showed their specific lectin and toxin binding with different binding affinity. The azide-reactive liposome provides a facile strategy for fabrication of either a natural or a synthetic glycolipid-based membrane-mimetic glycoarray. This liposomal glyco-microarray is simple and broadly applicable and thus will find important biomedical applications, such as studying glycolipid–protein interactions and toxin screening applications. Figure Liposome immobilization format provides a facile strategy for glycolipid-based membrane-mimetic glycoarray fabrication

Keywords: Glycoarray; Liposome; Glycolipid; Lectin; Toxin

Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe by Dmitry Y. Ryazantsev; Dmitry A. Tsybulsky; Igor A. Prokhorenko; Maksim V. Kvach; Yury V. Martynenko; Pavel M. Philipchenko; Vadim V. Shmanai; Vladimir A. Korshun; Sergey K. Zavriev (pp. 59-68).

A typical TaqMan™ real-time PCR probe contains a 5′-fluorescent dye and a 3′-quencher. In the course of the amplification, the probe is degraded starting from the 5′-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye–dye and dye–quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM–JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and “click chemistry” post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed. Figure Fluorogenic TaqMan probes with various modifications for real-time PCR

Keywords: Real-time PCR efficiency; TaqMan probes; Fluorescence quenching; Fluorescein; JOE

Application of ¹H-NMR-based metabolomics for detecting injury induced by long-term microwave exposure in Wistar rats’ urine by Li-Feng Wang; Xiang-Jun Hu; Rui-Yun Peng; Shui-Ming Wang; Ya-Bing Gao; Ji Dong; Li Zhao; Xiang Li; Hong-Yan Zuo; Chang-Zhen Wang; Rong-Lian Gao; Zhen-Tao Su; Xin-Xing Feng (pp. 69-78).

There has been growing public concern regarding exposure to microwave fields as a potential human health hazard. This study aimed to identify sensitive biochemical indexes for the detection of injury induced by microwave exposure. Male Wistar rats were exposed to microwaves for 6 min per day, 5 days per week over a period of 1 month at an average power density of 5 mW/cm² (specific absorption rate of 2.1 W/kg). Urine specimens were collected over 24 h in metabolic cages at 7 days, 21 days, 2 months, and 6 months after exposure. ¹H NMR spectroscopy data were analyzed using multivariate statistical techniques. Urine metabolic profiles of rats after long-term microwave exposure were significantly differentiated from those of sham-treated controls using principal component analysis or partial least squares discriminant analysis. Significant differences in low molecular weight metabolites (acetate, succinate, citrate, ketoglutarate, glucose, taurine, phenylalanine, tyrosine, and hippurate) were identified in the 5 mW/cm² microwave exposure group compared with the sham-treated controls at 7 days, 21 days, and 2 months. Metabolites returned to normal levels by 6 months after exposure. These data indicated that these metabolites were related to the perturbations of energy metabolism particularly in the tricarboxylic acid cycle, and the metabolism of amino acids, monoamines, and choline in urine represent potential indexes for the detection of injury induced by long-term microwave exposure.

Keywords: Microwave; ¹H-NMR; Metabolomics; Rat urine; Energy metabolism; Neurotransmitters

Flow injection chemiluminescence sensor using core-shell molecularly imprinted polymers as recognition element for determination of dapsone by Fuguang Lu; Jinlong Yang; Min Sun; Lulu Fan; Huamin Qiu; Xiangjun Li; Chuannan Luo (pp. 79-88).

This paper reports the preparation of dapsone (DDS) imprinted polymer layer-coated silica submicron particles (SiO₂) combined with chemiluminescence (CL) toward analysis of tracing DDS in practical samples. To induce the selective occurrence of surface polymerization, the amino groups were first grafted at the surface of SiO₂ by the (3-aminopropyl)triethoxysilane (APTES). The molecularly imprinted polymers (MIP) were coated at the surface of modified SiO₂ by the graft copolymerization. After the removal of templates, recognition sites of DDS were exposed in the polymer layers. The DDS-imprinted products were characterized by FT-IR, SEM, TEM, dynamic adsorption, and static adsorption tests. The proximity between the thickness of MIP layer and the spatial size of DDS indicated that the imprinted sites almost situated at the surface of MIP, leading to rapid adsorption saturation within 90 min. The apparent maximum binding amount of MIP toward DDS was evaluated as 14.98 mg·g⁻¹, which was much higher than that of non-molecularly imprinted polymers. The CL sensor provided a wide linear range for DDS within 1.0 × 10⁻⁶ to 1.0 × 10⁻⁴ mol·L⁻¹ with a detection limit of 5.27 × 10^-7 mol·L⁻¹ and the relative standard deviation of 1.8 % (n = 11) by determinations of 5.0 × 10⁻⁶ mol·L⁻¹ DDS. This method was applied to determine DDS in urine samples and satisfactory results were obtained. Figure Determination of dapsone in flow injection chemiluminescence sensor

Keywords: Dapsone; Molecularly imprinting; Core-shell; Chemiluminescence; Sensor

Multi-elemental bio-imaging of rat tissue from a study investigating the bioavailability of bismuth from shotgun pellets by Dagmar S. Urgast; Dag G. Ellingsen; Balázs Berlinger; Einar Eilertsen; Grete Friisk; Vidar Skaug; Yngvar Thomassen; John H. Beattie; In-Sook Kwun; Jörg Feldmann (pp. 89-99).

In recent years, bismuth has been promoted as a “green element” and is used as a substitute for the toxic lead in ammunition and other applications. However, the bioavailability and toxicity of bismuth is still not very well described. Following a hunting accident with bismuth-containing shots, a bioavailability study of bismuth from metal pellets inoculated into rat limb muscles was carried out. Bismuth could be found in urine and blood of the animals. Bio-imaging using laser ablation ICP-MS of thin sections of the tissue around the metal implant was carried out to find out more about the distribution of the metal diffusing into the tissue. Two laser ablation systems with different ablation cell designs were compared regarding their analytical performance. Low concentrations of bismuth showing a non-symmetrical pattern were detected in the tissue surrounding the metal implant. This was partly an artefact from cutting the thin sections but also bio-mobilisation of the metals of the implant could be seen. An accumulation of zinc around the implant was interpreted as a marker of inflammation. Challenges regarding sample preparation for laser ablation and bio-imaging of samples of diverse composition became apparent during the analysis. Figure

Keywords: Bismuth; Bio-imaging; LA-ICP-MS; Tissue thin section; Rat

Detection and characterization of cholesteryl ester hydroperoxides in oxidized LDL and oxidized HDL by use of an Orbitrap mass spectrometer by Shu-Ping Hui; Toshihiro Sakurai; Futaba Ohkawa; Hiroaki Furumaki; Shigeki Jin; Hirotoshi Fuda; Seiji Takeda; Takao Kurosawa; Hitoshi Chiba (pp. 101-112).

Oxidation of cholesteryl esters in lipoproteins by reactive oxygen species yields cholesteryl ester hydroperoxides (CEOOH). In this study, we developed a novel method for identification and characterization of CEOOH molecules in human lipoproteins by use of reversed-phase liquid chromatography with an hybrid linear ion trap–Orbitrap mass spectrometer (LC–LTQ Orbitrap). Electrospray ionization tandem mass spectrometric analysis was performed in both positive-ion and negative-ion modes. Identification of CEOOH molecules was completed by use of high-mass-accuracy (MA) mass spectrometric data obtained by using the spectrometer in Fourier-transform (FT) mode. Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy donor were oxidized by CuSO₄, furnishing oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No CEOOH molecules were detected in the nLDL and the nHDL, whereas six CEOOH molecules were detected in the oxLDL and the oxHDL. In positive-ion mode, CEOOH was detected as [M + NH₄]⁺ and [M + Na]⁺ ions. In negative-ion mode, CEOOH was detected as [M + CH₃COO]⁻ ions. CEOOH were more easily ionized in positive-ion mode than in negative-ion mode. The LC–LTQ Orbitrap method was applied to human plasma and six species of CEOOH were detected. The limit of detection was 0.1 pmol (S/N = 5:1) for synthesized CEOOH. Figure Detection and characterization of cholesteryl ester hydroperoxides in oxidized LDL and oxidized HDL by Orbitrap mass spectrometer

Keywords: Cholesteryl ester hydroperoxide; Orbitrap; Molecular species; Liquid chromatography–electrospray ionization mass spectrometry; LC–MS

MALDI-mass spectrometry imaging of desalted rat brain sections reveals ischemia-mediated changes of lipids by Hay-Yan J. Wang; Hsuan-Wen Wu; Ping-Ju Tsai; Cheng Bin Liu (pp. 113-124).

Ischemia-mediated lipidomic changes in rat brains were explored by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) profiling and imaging after in situ desalting which drastically simplified the spectral presentation of tissue lipids. Removal of interference from the massively changed cations in response to tissue damage permitted the revelation of subtle yet important lipidomic changes. The identities of the detected lipids were confirmed by MALDI tandem mass spectrometry (MALDI-MS/MS). The MALDI-MS imaging (MALDI-MSI) result of lysophosphatidylcholine 16:0 (LPC 16:0) in the desalted brain section appeared essentially identical to that of sodiated LPC 16:0 in the adjacent undesalted section and verified the suitability of the desalting method for the MALDI-MSI studies of lipids in tissue. Other than the consistently decreased phosphatidylcholine (PC) 16:0/18:1, images of PCs containing all saturated, or combined saturated and monounsaturated fatty acyl (MUFA) residues revealed their parenchymal increase by ischemia. Images of PCs containing polyunsaturated fatty acyl (PUFA) residues in normal cortex showed laminated patterns similar to cortical lamina. Ischemia reduced the abundance of PC 16:0/20:4 and PC 16:0/22:6 and disrupted the laminated distribution of the former. However, ischemia increased the subcortical abundance of PUFA-PCs containing stearoyl residue and confined their cortical increase within limited areas. Image of parenchymal sphingomyelin 18:0 (SM 18:0) showed its consistent decrease by ischemia that paralleled the increase of ceramide 18:0-H₂O in region of moderate to high SM abundance. The above results presented the lipidomic changes largely different from previous MALDI-MSI results and suggested a window of intervention that may benefit the management of cerebrovascular accident and other brain injuries.

Keywords: MALDI-mass spectrometry imaging; In situ desalting; Cerebral ischemia; Lipidomics

C-Terminal sequencing of protein by MALDI mass spectrometry through the specific derivatization of the α-carboxyl group with 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide by Chihiro Nakajima; Hiroki Kuyama; Takashi Nakazawa; Osamu Nishimura (pp. 125-132).

We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal α-carboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)-phosphonium bromide (TMPP-propylamine) through oxazolone chemistry, (2) enzymatic proteolysis of the TMPP-derivatized protein, and (3) MALDI-MS/MS analysis of the peptide mixture, in which the C-terminal peptide incorporating the TMPP group is preferably detected. In this protocol, it is possible to choose any endoproteinase such as trypsin, GluC, and AspN for digestion so that a C-terminal peptide with length appropriate for mass spectrometric sequencing could be generated. The peptide labeled with TMPP-propylamine at the C terminus tends to exhibit y-type ions in MS/MS spectra, allowing manual sequence interpretation with the simplified fragmentation pattern. The efficacy of the method was verified with five proteins, which demonstrated that the C-terminal peptides were readily distinguishable by their peak intensity and characteristic mass signature peak in MALDI-PSD analysis.

Keywords: Bioanalytical methods; Mass spectrometry/ICP-MS; Spectroscopy/instrumentation

Highly sensitive and selective measurement of underivatized methylmalonic acid in serum and plasma by liquid chromatography-tandem mass spectrometry by Chao Yuan; Jessica Gabler; Joe M. El-Khoury; Regina Spatholt; Sihe Wang (pp. 133-140).

Methylmalonic acid (MMA) is a functional biomarker of vitamin B12 deficiency. Measurement of plasma MMA is challenging due to its small molecular weight and hydrophilic nature. Several liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed for measuring plasma MMA. However, these methods involve lengthy sample preparation, long chromatographic run time, inadequate sensitivity, or interference from succinic acid (SA). Here we report a novel LC-MS/MS method for quantitation of underivatized MMA in serum or heparinized plasma with high sensitivity and selectivity. Sample preparation involved only strong anion exchange solid phase extraction. The extract was purified by online turbulent flow and analyzed on an Organic Acids column. MS/MS analysis was performed in negative electrospray mode, and the analytical time was 6 min. The use of ion ratio confirmation in combination with chromatographic resolution from SA greatly enhanced the selectivity. No interference was observed. This method was linear from 26.2 to 26,010.0 nM with an accuracy of 98–111 %. Total coefficient of variation was less than 4.6 % for three concentration levels tested. Comparison with a reference laboratory LC-MS/MS method using leftover patient serum specimens (n = 48) showed a mean bias of −2.3 nM (−0.61 %) with a Deming regression slope of 1.016, intercept of −6.6 nM, standard error of estimate of 25.3 nM, and a correlation coefficient of 0.9945. In conclusion, this LC-MS/MS method offers highly sensitive and selective quantitation of MMA in serum and plasma with simple sample preparation.

Keywords: Plasma; Serum; Methylmalonic acid; Liquid chromatography; Mass spectrometry; LC-MS/MS

Identification of volatile lung cancer markers by gas chromatography–mass spectrometry: comparison with discrimination by canines by Bogusław Buszewski; Tomasz Ligor; Tadeusz Jezierski; Anna Wenda-Piesik; Marta Walczak; Joanna Rudnicka (pp. 141-146).

In this work, a chromatographic method for identification of volatile organic compounds was compared with canine recognition. Gas chromatography and mass spectrometry (GC–TOF MS) were used for determination of concentrations of trace gases present in human breath. The technique enables rapid determination of compounds in human breath, at the parts per billion level. Linear correlations were from 0.83–234.05 ppb, the limit of detection was the range 0.31–0.75 ppb, and precision, expressed as relative standard deviation (RSD), was less than 10.00 %. Moreover, trained dogs are able to discriminate breath samples of patients with diagnosed cancer. We found a positive correlation between dog indications and the ethyl acetate and 2-pentanone content of breath (r = 0.85 and r = 0.97, respectively). The methods presented for detection of lung cancer markers in exhaled air could be used as a potential non-invasive tool for screening. In addition, the canine method is relatively simple and inexpensive in comparison with chromatography.

Keywords: Lung cancer; Canine olfaction; GC–MS

A new method for quantifying prenatal exposure to ethanol by microwave-assisted extraction (MAE) of meconium followed by gas chromatography–mass spectrometry (GC–MS) by Pamela Cabarcos; María Jesús Tabernero; Iván Álvarez; Martha Miguez; Purificación Fernández; Ana María Bermejo (pp. 147-155).

Ethanol is a legal and widely available substance. There are health and social consequences associated with its abuse. One of the most important problems is related to alcohol consumption during pregnancy. In fact, prenatal ethanol exposure can be associated with fetal alcohol spectrum disorder (FASD), a term used to describe a wide range of potentially lifelong effects that include physical, mental, behavioral, and learning disabilities. Fatty acid ethyl esters (FAEEs), which are non-oxidative metabolites of ethanol, are currently used as biomarkers of direct ethanol consumption in different matrices, including hair, blood, skin surface, and meconium. Analysis of these compounds in meconium reveals exposure to alcohol during the second and third trimesters of pregnancy. An important finding for evaluation of gestational ethanol exposure is the fact that FAEEs do not cross the placenta. Because they accumulate in the fetal gut from approximately the 20th week of gestation until birth, this provides a wide window of detection of chronic exposure to alcohol. The sum of the concentrations of all the FAEEs, with a cutoff of 2 nmol g⁻¹ or 600 ng g⁻¹ meconium, has been recommended as evidence of maternal alcohol use. We introduce a novel technique to quantify ethyl myristate, ethyl palmitate, ethyl stearate, and their deuterated analogues (as internal standards, IS) in meconium using microwave-assisted extraction (MAE) coupled with gas chromatography–mass spectrometry (GC–MS). Limits of detection and quantification were 50 and 100 ng g⁻¹ for all analytes except ethyl stearate (LOD 100 ng g⁻¹ and LOQ 500 ng g⁻¹). Calibration curves were linear from the LOQ to 5000 ng g⁻¹. The validated method was applied to the analysis of 81 meconium samples. Figure

Keywords: Meconium; Fatty acid ethyl esters; Fetal alcohol spectrum disorder; Microwave-assisted extraction; Gas chromatography; Mass spectrometry

Analysis of synthetic cannabinoids in “spice-like” herbal highs: snapshot of the German market in summer 2011 by Karoline Simolka; Rainer Lindigkeit; Hans-Martin Schiebel; Uli Papke; Ludger Ernst; Till Beuerle (pp. 157-171).

In this study, seven commercial “spice-like” products available on the German market were analyzed. They all contained significant amounts of synthetic cannabinoids and had distinctly different compositions of these adulterants. All synthetic cannabinoids were extracted and purified by different chromatographic techniques from the respective product. The structures of all compounds were elucidated by nuclear magnetic resonance spectroscopy and further characterized by mass spectrometry (MS) and ultraviolet and infrared spectroscopy to generate a full data set of each compound. Altogether, eight compounds were identified, and one deuterium-labeled cannabinoid was used as internal standard. Four products contained only one individual compound, while three products contained mixtures of two compounds. Among the eight isolated compounds, six were already known from recent publications (JWH-081, JWH-210, JWH-122, AM2201, RCS-4, and JWH-203), but the published data were not always complete. In addition, two unknown compounds (AM2201-pMe, RCS-4-(N-Me)) were isolated. Overall, compounds from three distinct classes of synthetic cannabinoids could be identified, characterized, and compared. The MS data of the different subclasses allowed the postulation of some general key fragmentations to distinguish between these subclasses. In addition, we established a general method using an isotopically labeled internal standard (JWH-018-D₃) to quantify synthetic cannabinoids in herbal mixtures. The total content of the synthetic cannabinoids ranged from 77.5 to 202 mg/g, while individual compounds were detected from 19.3 to 202 mg/g in these products. The spectroscopic data for all compounds mentioned here were collected and added en bloc as Electronic supplementary material to this manuscript. Figure Analysis of synthetic cannabinoids in herbal smoking blends

Keywords: Spice; Synthetic cannabinoids; Mass spectrometry; NMR

Pesticides in seaweed: optimization of pressurized liquid extraction and in-cell clean-up and analysis by liquid chromatography–mass spectrometry by R. A. Lorenzo; S. Pais; I. Racamonde; D. García-Rodríguez; A. M. Carro (pp. 173-181).

Chemical residues, such as insecticides and anthelmintics, are frequently redistributed from the aquatic environment to marine species. This work reports on a fast validated protocol for the analysis of azamethiphos, three avermectins, two carbamates and two benzoylurea pesticides and chemotherapeutic agents in seaweeds based on pressurized liquid extraction and separation of analytes by liquid chromatography coupled with tandem mass spectrometry. The variables affecting the efficiency of pressurized liquid extraction, including temperature, number of extraction cycles, static extraction time and percent acetonitrile flush volume, were studied using a Doehlert design. The optimum parameters were 100 °C and one cycle of 3 min with 70 % acetonitrile. Adequate in-cell clean-up of the seaweeds was achieved using 0.8 g of Florisil over 0.1 g of graphitized carbon black on the bottom of the cell. The optimized method was validated using an analyte-free seaweed sample fortified at different concentrations. The limits of quantification ranged from 3.6 μg kg⁻¹ (azamethiphos) to 31.5 μg kg⁻¹ (abamectin). The recovery was from 87 to 120 % in most cases at different spiking levels. Finally, the reproducibility of the method expressed as the relative standard deviation and evaluated at concentrations of 10 and 50 μg kg⁻¹ was in the range 9–14.3 % and 6.1–12.3 %, respectively. The applicability of the method was evaluated with five commercial and 12 wild edible seaweeds, and four target compounds were detected in two wild seaweeds at a concentration below the quantification limit.

Keywords: Seaweed; Pesticides; Pressurized liquid extraction; In-cell clean-up; Liquid chromatography coupled with tandem mass spectrometry; Experimental design

Pesticide residue analysis in cereal-based baby foods using multi-walled carbon nanotubes dispersive solid-phase extraction by Miguel Ángel González-Curbelo; María Asensio-Ramos; Antonio V. Herrera-Herrera; Javier Hernández-Borges (pp. 183-196).

In the present study, a new analytical method has been developed for the simultaneous quantification of 15 organophosphorus pesticides, including some of their metabolites, (disulfoton-sulfoxide, ethoprophos, cadusafos, dimethoate, terbufos, disulfoton, chlorpyrifos-methyl, malaoxon, fenitrothion, pirimiphos-methyl, malathion, chlorpyrifos, terbufos-sulfone, disulfoton-sulfone and fensulfothion) in three different types of commercial cereal-based baby foods. Dispersive solid-phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs) was used together with gas chromatography with nitrogen phosphorus detection. Most favorable conditions involved a previous ultrasound-assisted extraction of the sample with acetonitrile containing formic acid. After evaporation of the extract and redissolution in water, a dSPE procedure was carried out with MWCNTs. The whole method was validated in terms of repeatability, linearity, precision and accuracy and matrix effect was also evaluated. Absolute recoveries were in the range 64–105 % with relative standard deviation values below 7.6 %. Limits of quantification achieved ranged from 0.31 to 5.50 μg/kg, which were lower than the European Union maximum residue limits for pesticide residues in cereal-based baby foods.

Keywords: Pesticides; Multi-walled carbon nanotubes; Dispersive solid-phase extraction; Cereal-based baby foods; Gas chromatography–nitrogen phosphorus detection

Selective determination of mono- and dihydroxylated analogs of polybrominated diphenyl ethers in marine sponges by liquid-chromatography tandem mass spectrometry by Yoshihisa Kato; Syohei Okada; Kazutaka Atobe; Tetsuya Endo; Koichi Haraguchi (pp. 197-206).

A number of bioactive brominated secondary metabolites, including hydroxylated polybrominated diphenyl ethers, have been isolated from algae, sponges, and bacteria. In the present study, a screening method using liquid-chromatography tandem mass spectrometry was developed for the identification and selective determination of dihydroxy (diOH), hydroxy-methoxy (OH-MeO), and dimethoxy (diMeO) analogs of tetra- to hexa-BDEs in marine biota. In negative atmospheric pressure chemical ionization (APCI) mode, diOH and OH-MeO analogs provided intense [M−H]⁻ ions, whereas diMeO analogs provided characteristic [M−Br+O]⁻ and [M−CH₃]⁻ ions. This enabled the diOH-, OH-MeO-, and diMeO-PBDEs to be distinguished using selected reaction monitoring transitions in the APCI source. Recoveries of 2′-OH-6-MeO-2,3′,4,5′-tetra-BDE in spiked marine samples were 84 ± 5 %, with a limit of quantification at 9.1 ng mL⁻¹ (signal-to-noise ratio = 10). The developed method was used to analyze two sponge species collected from Palau, Micronesia; the concentration ratio of diOH-tetra-BDE:OH-MeO-tetra-BDE was 10:1 for the Lamellodysidea sp., whereas it was 1:30 for the Callyspongia sp. Figure Hydroxy-methoxy PBDEs in marine sponge (Lamellodysidea sp.)

Keywords: LC/MS/MS; APCI; Dihydroxy-PBDE; Hydroxy-methoxy-PBDE; Dimethoxy-PBDE; Marine sponge

Quantitative analysis of aberrant fatty acid composition of zebrafish hepatic lipids induced by organochlorine pesticide using stable isotope-coded transmethylation and gas chromatography-mass spectrometry by Hongying Zhong; Linjie Dong; Qingjian Dong; Changshu Ke; Jieying Fu; Xiaoli Wang; Cong Liu; Ling Dai (pp. 207-216).

Organochlorine pesticides have been extensively used worldwide for agricultural purposes. Due to their resistance to metabolism, a major public health concern has been raised. Aberrant hepatic lipid composition has been a hallmark of many liver diseases associated with exposure to various toxins and chemicals. And thus lots of efforts have been focused on the development of analytical techniques that can rapidly and quantitatively determine the changes in fatty acid composition of hepatic lipids. In this work, changes in fatty acid composition of hepatic lipids in response to DDT (dichlorodiphenyltrichloroethane) exposure were quantitatively analyzed by a gas chromatography-mass spectrometric approach based on stable isotope-coded transmethylation. It has been quantitatively demonstrated that polyunsaturated fatty acids including C20:3n3, C20:4n6, and C22:6n3 decrease in response to DDT exposure. However, saturated long chain fatty acids including C16:0, C18:0, as well as monounsaturated long chain fatty acid C18:1n9 consistently increase in a DDT-concentration-dependent manner. In particular, much higher changes in the level of hepatic C16:0 and C18:0 for male fish were observed than that for female fish. These experimental results are in accordance with qualitative histopathological analysis that revealed liver morphological alterations. The stable isotope-coded mass spectrometric approach provides a reliable means for investigating hepatotoxicity associated with fatty acid synthesis, desaturation, mitochondrial beta-oxidation, and lipid mobilization. It should be useful in elucidation of hepatotoxic mechanisms and safety assessment of environmental toxins. Figure Detection of Changes in Hepatic Lipids by Mass Spectrometry

Keywords: Gas chromatography-mass spectrometry; Stable isotope-coded fatty acid transmethylation; Fatty acids; Hepatic lipids; DDT exposure

Simultaneous determination of triazole antifungal drugs in human plasma by sweeping-micellar electrokinetic chromatography by Shu-Chiao Lin; Hsiang-Yin Liu; Shu-Wen Lin; Ming Yao; Un-In Wu; Hsiu-Po Kuo; Ching-Hua Kuo (pp. 217-228).

The number of cases of invasive fungal infections (IFIs) has risen significantly in recent years; therefore, this study developed a sensitive and effective sweeping-micellar electrokinetic chromatography (MEKC) method for the simultaneous determination of the three most frequently used triazole antifungal drugs for the treatment of IFIs, which included voriconazole, itraconazole, and posaconazole. Due to the diverse lipophilicity of the tested drugs, the analytical conditions that resulted in good resolution between itraconazole and posaconazole caused the peak for voriconazole to split. The splitting phenomenon was resolved by incorporating a high-salt stacking mechanism into the sweeping-MEKC method. The optimum background electrolyte was composed of 25 mM phosphoric acid solution (pH 2.2), 100 mM sodium dodecyl sulfate, 13 % acetonitrile, and 13 % tetrahydrofuran. The best peak shape of voriconazole was obtained when the conductivity ratio between the sample matrix and background electrolyte was 2.3. Compared to the conventional MEKC mode, the enhancement factor of the sweeping-MEKC method was 66 for itraconazole, 55 for posaconazole, and 43 for voriconazole. The sweeping-MEKC method was validated in terms of precision, accuracy, linearity, specificity, selectivity, and sensitivity. The linearity ranges of the method covered the commonly used therapeutic ranges of the three drugs. The developed sweeping-MEKC method was successfully applied to the analysis of clinical samples, thus demonstrating its applicability for clinical use. Fig

Keywords: Itraconazole; Voriconazole; Posaconazole; Sweeping-MEKC; High salt stacking; Therapeutic drug monitoring

Selectivity enhancement for the separation of tocopherols and steroids by integration of highly ordered weak interaction sites along the polymer main chain by Abul K. Mallik; Hongdeng Qiu; Makoto Takafuji; Hirotaka Ihara (pp. 229-238).

A novel alternating copolymer-based organic phase was synthesized using a new N-substituted maleimide monomer for the development of alternating copolymer-grafted silica for high-performance liquid chromatographic applications. This new monomer (DGMI) was copolymerized with octadecyl acrylate (ODA) from 3-mercaptopropyltrimethoxysilane-grafted silica to produce Sil-poly(ODA-alt-DGMI). The organic phase was characterized by the elemental analysis and the diffuse reflectance infrared Fourier transform spectroscopy. Tocopherol isomers and steroids were used as analytes for the evaluation of the chromatographic selectivity profiles of this novel stationary phase. The selectivity of this column was then compared with a polymeric ODS column and previously developed another alternating copolymer-grafted silica (without the glutamide-derived moiety) column, Sil-poly(ODA-alt-N-octadecylmaleimide). The complete baseline separation of tocopherol isomers in an isocratic mode has been achieved within 25 min with the Sil-poly(ODA-alt-DGMI). The separation of eight kinds of estrogenic steroids and corticoids has also been achieved in an isocratic mode with this column. Significant differences in separation selectivity between Sil-poly(ODA-alt-DGMI) and polymeric ODS columns were observed towards the steroids, and compared with the reference columns, a better separation profile for these analytes was obtained with the Sil-poly(ODA-alt-DGMI). The results of this investigation indicated that the enhancement of selectivity of Sil-poly(ODA-alt-DGMI) towards the test analytes arose from the multiple interaction mechanism such as hydrophobic effect, carbonyl-π and hydrogen-bonding interactions, and such integrated interactions originated from the addition of two amide groups in the N-substituted maleimide monomer.

Keywords: Alternating copolymer; Separation; Tocopherols; Steroids; Carbonyl–π interactions

Chromatographic retention behaviour, modelling and separation optimisation of the quaternary ammonium salt isometamidium chloride and related compounds on a range of reversed-phase liquid chromatographic stationary phases by Gesa J. Schad; Melvin R. Euerby; Graham G. Skellern; Justice N. A. Tettey (pp. 239-255).

This paper describes the reversed-phase liquid chromatographic behaviour of the trypanocidal quaternary ammonium salt isometamidium chloride and its related compounds on a range of liquid chromatographic phases possessing alkyl and phenyl ligands on the same inert silica. In a parallel study with various extended polar selectivity phases which possessed different hydrophobic/silanophilic (hydrogen bonding) activity ratios, the chromatographic retention/selectivities of the quaternary ammonium salts was shown to be due to a co-operative mechanism between hydrophobic and silanophilic interactions. The highly aromatic and planar isometamidium compounds were found to be substantially retained on stationary phases containing aromatic functionality via strong π–π interactions. The chemometric approach of principal component analysis was used to characterise the chromatographic behaviour of the isometamidium compounds on the differing phases and to help identify the dominant retention mechanism(s). Two-dimensional (temperature/gradient) retention modelling was employed to develop and optimise a rapid liquid chromatography method for the separation of the six quaternary ammonium salts within 2.5 min which would be suitable for bioanalysis using liquid chromatography–mass spectrometry. This is the first reported systematic study of the relationship between stationary phase chemistries and retention/selectivity for a group of quaternary ammonium salts. Fig. 1 Structure of isometamidium and related quaternary ammonium salts and their LC separation on a diverse range of RP columns.

Keywords: Quaternary ammonium salts; Isometamidium chloride (ISM); HPLC; Retention behaviour modelling and optimisation; Principal component analysis

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil in culture medium: application to fungal biotransformation studies by Thiago Barth; Raphael Conti; Mônica Tallarico Pupo; Laura Tiemi Okano; Pierina Sueli Bonato (pp. 257-266).

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid–liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100–10,000 ng mL⁻¹ for each enantiomer of DPZ (r ≥ 0.9985) and of 100–5,000 ng mL⁻¹ for each enantiomer of 5-ODD (r ≥ 0.9977) and 6-ODD (r ≥ 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

Keywords: Beauveria bassiana ; Cunninghamella elegans ; Donepezil; Enantioseparation; 5-O-Desmethyl donepezil; 6-O-Desmethyl donepezil

HPLC method for rapidly following biodiesel fuel transesterification reaction progress using a core-shell column by Samuel J. Allen; Lisa S. Ott (pp. 267-272).

There are a wide and growing variety of feedstocks for biodiesel fuel. Most commonly, these feedstocks contain triglycerides which are transesterified into the fatty acid alkyl esters (FAAEs) which comprise biodiesel fuel. While the tranesterification reaction itself is simple, monitoring the reaction progress and reaction products is not. Gas chromatography–mass spectrometry is useful for assessing the FAAE products, but does not directly address either the tri-, di-, or monoglycerides present from incomplete transesterification or the free fatty acids which may also be present. Analysis of the biodiesel reaction mixture is complicated by the solubility and physical property differences among the components of the tranesterification reaction mixture. In this contribution, we present a simple, rapid HPLC method which allows for monitoring all of the main components in a biodiesel fuel transesterification reaction, with specific emphasis on the ability to monitor the reaction as a function of time. The utilization of a relatively new, core-shell stationary phase for the HPLC column allows for efficient separation of peaks with short elution times, saving both time and solvent. Figure Separatory funnel containing a biodiesel fuel preparation (top layer, biodiesel; bottom layer, glycerol)

Keywords: Biodiesel fuel; HPLC; Analysis of reaction intermediates; Core-shell column

Real-time analysis of aromatics in combustion engine exhaust by resonance-enhanced multiphoton ionisation time-of-flight mass spectrometry (REMPI-TOF-MS): a robust tool for chassis dynamometer testing by T. W. Adam; M. Clairotte; T. Streibel; M. Elsasser; A. Pommeres; U. Manfredi; M. Carriero; G. Martini; M. Sklorz; A. Krasenbrink; C. Astorga; R. Zimmermann (pp. 273-276).

Resonance-enhanced multiphoton ionisation time-of-flight mass spectrometry (REMPI-TOF-MS) is a robust method for real-time analysis of monocyclic and polycyclic aromatic hydrocarbons in complex emissions. A mobile system has been developed which enables direct analysis on site. In this paper, we utilize a multicomponent calibration scheme based on the analytes’ photo-ionisation cross-sections relative to a calibrated species. This allows semi-quantification of a great number of components by only calibrating one compound of choice, here toluene. The cross-sections were determined by injecting nebulised solutions of aromatic compounds into the TOF-MS ion source with the help of a HPLC pump. Then, REMPI-TOF-MS was implemented at various chassis dynamometers and test cells and the exhaust of the following vehicles and engines investigated: a compression ignition light-duty (LD) passenger car, a compression ignition LD van, two spark ignition LD passenger cars, 2 two-stroke mopeds, and a two-stroke engine of a string gas trimmer. The quantitative time profiles of benzene are shown. The results indicate that two-stroke engines are a significant source for toxic and cancerogenic compounds. Air pollution and health effects caused by gardening equipment might still be underestimated.

Keywords: Fuels; Mass spectrometry/ICP-MS; Organic compounds/trace organic compounds

In-air broad beam ionoluminescence microscopy as a tool for rocks and stone artworks characterisation by Alessandro Lo Giudice; Alessandro Re; Debora Angelici; Silvia Calusi; Nicla Gelli; Lorenzo Giuntini; Mirko Massi; Giovanni Pratesi (pp. 277-281).

Broad beam ionoluminescence (IL) microscopy is a promising technique for the non-destructive characterisation of rocks and stone objects. Luminescence imaging by means of broad ion beams has been sporadically used by other authors but, to our knowledge, its potential has not yet been fully investigated, neither in geological science nor in other fields. The in-air broad beam IL microscope was developed and installed at the INFN-LABEC external microbeam in Florence. Similar to the cathodoluminescence (CL) microscope, the apparatus exploits a CCD colour camera collecting images (few square millimetres wide, with ∼10-μm spatial resolution) of the luminescence emitted by the sample hit by a defocused megaelectron volt (MeV) proton beam. The main differences with the well-established and widespread CL are the possibility of working in air (no sampling or conductive coatings required) and the possibility of combining the analysis with microbeam analysis, such as, for example, μ-IL and μ-PIXE (particle-induced X-ray emission). To show the potential of the technique, IL images of thin sections of lapis lazuli are compared with those obtained by means of an in-vacuum cold CL. An application to the study of stone artworks is also reported. This technique and apparatus will provide a valuable help for interdisciplinary applications, e.g. in geological sciences and in the cultural heritage field. Figure Experimental setup of the broad beam IL microscopy apparatus on the external microbeam line of the INFN-LABEC in Firenze during the analysis of a lapis lazuli rock

Keywords: Ion microprobe; Ionoluminescence microscopy; Cathodoluminescence; PIXE; Lapis lazuli; Archaeometry

Erratum to: Rapid determination of anilines in water samples by dispersive liquid–liquid microextraction based on solidification of floating organic drop prior to gas chromatography–mass spectrometry by Chun-Peng Diao; Chao-Hai Wei (pp. 283-284).

Erratum to: Connecting simulated, bioanalytical, and molecular docking data on the stereoselective binding of (±)-catechin to human serum albumin by Myalowenkosy I. Sabela; Njabulo J. Gumede; Laura Escuder-Gilabert; Yolanda Martín-Biosca; Khirsna Bisetty; María-Jose Medina-Hernández; Salvador Sagrado (pp. 285-285).

Sections

Personal tools

Analytical and Bioanalytical Chemistry (v.404, #1)

Sections

Personal tools

Local resources

Analytical and Bioanalytical Chemistry (v.404, #1)