Analytical and Bioanalytical Chemistry (v.400, #7)

In recent years, interest has increased regarding the identification of volatile organic compounds (VOCs) for metabolic profiling, human scent identification of the living and deceased, and diagnostic potentials for certain diseases that are known for its association with distinct odor. In this study, a method has been developed that is capable of sampling, identifying, and differentiating the VOCs present in various biological specimens of forensic importance (blood, breath, buccal cells, and urine) taken from the same individuals. The developed method requires a pretreatment step to remove targeted VOCs from the sampling apparatus prior to sampling of the individual specimens. The VOCs collected from the biological specimens were characterized by solid-phase microextraction and gas chromatography/mass spectrometry with ratios of the most abundant and frequent VOCs compared using qualitative and semiquantitative methods. Blood, breath, and buccal cells required extraction procedures ranging from 18 to 21 h in order to optimize the limit of detection, which averaged 5–15 ng across these specimens. The optimal method for measuring urine VOCs was complete in less than an hour; however, the limit of detection was higher with a range of 10–40 ng quantifiable. The demonstrated sensitivity and reproducibility of the methods developed allow for population studies of human scent VOCs from various biological specimen collection kits used in the forensic and clinical fields.
Keywords: Forensic science; SPME-GC/MS; Biological specimens; Volatile organic compounds; Human scent

Biomedical mass spectrometry by Mitsutoshi Setou; Hisao Oka (1827-1827).
has been full professor of Anatomy and Cell Biology at the Hamamatsu University School of Medicine since 2008. His research interests include development and application of imaging mass spectrometry and systems biology with omics technologies. Dr Setou is the author of over 100 peer-reviewed papers, 150 scientific papers, 10 book chapters, and editor of a book on imaging mass spectrometry. has been full professor of analytical chemistry at the Kinjo Gakuin University School of Pharmacy since 2005. His research interests include identification and determination of bioactive compounds by liquid chromatography–mass spectrometry. Dr Oka is the author of over 150 peer-reviewed papers and 30 book chapters, and editor of a book on food safety and applications of liquid chromatography–mass spectrometry.

Secreted phospholipase A2, lipoprotein hydrolysis, and atherosclerosis: integration with lipidomics by Kei Yamamoto; Yuki Isogai; Hiroyasu Sato; Yoshitaka Taketomi; Makoto Murakami (1829-1842).
Phospholipase A2 (PLA2) is a group of enzymes that hydrolyze the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. Of many PLA2s or related enzymes identified to date, secreted PLA2s (sPLA2s) comprise the largest family that contains 10 catalytically active isozymes. Besides arachidonic acid released from cellular membranes for eicosanoid synthesis, several if not all sPLA2s have recently been implicated in hydrolysis of phospholipids in lipoprotein particles. The sPLA2-processed low-density lipoprotein (LDL) particles contain a large amount of lysophospholipids and exhibit the property of “small-dense” or “modified” LDL, which facilitates foam cell formation from macrophages. Transgenic overexpression of these sPLA2s leads to development of atherosclerosis in mice. More importantly, genetic deletion or pharmacological inhibition of particular sPLA2s significantly attenuates atherosclerosis and aneurysm. In this article, we will give an overview of current understanding of the role of sPLA2s in atherosclerosis, with recent lipidomics data showing the action of a subset of sPLA2s on lipoprotein phospholipids.
Keywords: Lipids; Enzymes; Mass spectrometry

Urinary 2-hydroxy-5-oxoproline, the lactam form of α-ketoglutaramate, is markedly increased in urea cycle disorders by Tomiko Kuhara; Yoshito Inoue; Morimasa Ohse; Boris F. Krasnikov; Arthur J. L. Cooper (1843-1851).
α-Ketoglutaramate (KGM) is the α-keto acid analogue of glutamine, which exists mostly in equilibrium with a lactam form (2-hydroxy-5-oxoproline) under physiological conditions. KGM was identified in human urine and its concentration quantified by gas chromatography/mass spectrometry (GC/MS). The keto acid was shown to be markedly elevated in urine obtained from patients with primary hyperammonemia due to an inherited metabolic defect in any one of the five enzymes of the urea cycle. Increased urinary KGM was also noted in other patients with primary hyperammonemia, including three patients with a defect resulting in lysinuric protein intolerance and one of two patients with a defect in the ornithine transporter I. These findings indicate disturbances in nitrogen metabolism, most probably at the level of glutamine metabolism in primary hyperammonemia diseases. Urinary KGM levels, however, were not well correlated with secondary hyperammonemia in patients with propionic acidemia or methylmalonic acidemia, possibly as a result, in part, of decreased glutamine levels. In conclusion, the GC/MS procedure has the required lower limit of quantification for analysis of urinary KGM, which is markedly increased in urea cycle disorders and other primary hyperammonemic diseases. Figure The glutaminase II pathway.
Keywords: α-Ketoglutaramate; 2-hydroxy-5-oxoproline; GC/MS analysis of urine; Primary hyperammonemia; Secondary hyperammonemia; Urea cycle disorders

Triacylglycerol/phospholipid molecular species profiling of fatty livers and regenerated non-fatty livers in cystathionine beta-synthase-deficient mice, an animal model for homocysteinemia/homocystinuria by Kazutaka Ikeda; Akiko Kubo; Noriyuki Akahoshi; Hidenori Yamada; Naoya Miura; Takako Hishiki; Yoshiko Nagahata; Tomomi Matsuura; Makoto Suematsu; Ryo Taguchi; Isao Ishii (1853-1863).
Fatty liver is one of the typical manifestations in homocysteinemia/homocystinuria patients and their genetic animal model, mice lacking cystathionine β-synthase (Cbs −/−). The vast majority of Cbs −/− die within 4 weeks after birth via yet unknown mechanisms, whereas a small portion survive to adulthood, escaping fatty degeneration of the liver during lactation periods, through regeneration. To investigate the molecular basis of such fatty changes, we analyzed lipid components in fatty livers of 2-week-old Cbs −/− and regenerated non-fatty livers of 8-week-old Cbs −/− survivors using a chip-based nanoESI (electrospray ionization)-MS system, which allows quantitative detection of triacylglycerol/phospholipid molecular species. Hepatic levels of all major triacylglycerol species were much higher in Cbs −/− than in wild-type mice at 2 weeks, although not at 8 weeks. Levels of some phospholipid species were either up- or downregulated in 2-week-old Cbs −/−; e.g. saturated (16:0 and 18:0) or mono-unsaturated (16:1 and 18:1) fatty acids-containing phosphatidylcholine/phosphatidylethanolamine species were upregulated, while poly-unsaturated fatty acids-containing phosphatidylcholine (18:2–18:2 and 18:2–20:5), phosphatidylethanolamine (18:1–20:4), and phosphatidylinositol (18:0–20:4) were downregulated. Capillary electrophoresis-MS analysis identified high-level accumulation of S-adenosylmethionine and S-adenosylhomocysteine in fatty livers of 2-week-old Cbs −/− but much less in non-fatty livers of 8-week-old Cbs −/−. Although hepatic S-adenosylmethionine/S-adenosylhomocysteine ratios were comparable between 2-week-old Cbs −/− and wild-type, global protein arginine methylation was disturbed in fatty livers of Cbs −/−. Our results suggest that cellular signaling mediated by altered phospholipid contents might be involved in pathogenesis of fatty liver in Cbs −/–.
Keywords: Capillary electrophoresis; Hepatic steatosis; Methylation; NanoESI-MS; Neutral loss scanning; Precursor ion scanning

Authenticity assessment of beef origin by principal component analysis of matrix-assisted laser desorption/ionization mass spectrometric data by Nobuhiro Zaima; Naoko Goto-Inoue; Takahiro Hayasaka; Hirofumi Enomoto; Mitsutoshi Setou (1865-1871).
It has become necessary to assess the authenticity of beef origin because of concerns regarding human health hazards. In this study, we used a metabolomic approach involving matrix-assisted laser desorption/ionization imaging mass spectrometry to assess the authenticity of beef origin. Highly accurate data were obtained for samples of extracted lipids from beef of different origin; the samples were grouped according to their origin. The analysis of extracted lipids in this study ended within 10 min, suggesting this approach can be used as a simple authenticity assessment before a definitive identification by isotope analysis.
Keywords: Beef; Imaging mass spectrometry; Principal component analysis; Lipids

Distribution of phospholipid molecular species in autogenous access grafts for hemodialysis analyzed using imaging mass spectrometry by Hiroki Tanaka; Nobuhiro Zaima; Naoto Yamamoto; Minoru Suzuki; Yuuki Mano; Hiroyuki Konno; Naoki Unno; Mitsutoshi Setou (1873-1880).
Arteriovenous fistulae (AVF) using vein grafts are frequently used for vascular access in hemodialysis. When superficial veins are used as autogenous access grafts for hemodialysis, atherosclerotic-like tissue degeneration often causes stenosis and obstruction. Although the differences between the pathology of degeneration in AVF and atherosclerosis (i.e., peripheral artery occlusive disease (PAD)) are known, their underlying molecular mechanisms are not. We determined the characteristic abnormal lipid metabolism of AVF. Oil red O staining clearly showed the accumulation of lipid molecules in AVF and PAD tissues. We found that the staining pattern was different between AVF and PAD tissues. The media and adventitia of AVF and the intima and media of PAD were intensely stained. Quantitative lipid analysis revealed that the amount of PL was significantly increased in AVF and PAD. Next, we performed matrix-assisted laser desorption/ionization imaging mass spectroscopy and determined the characteristic distribution of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) in AVF. The distribution patterns of LPC (1-acyl 16:0) and PC (diacyl 16:0/20:4) were consistent with the Oil red O staining images, suggesting that metabolisms related to LPC (1-acyl 16:0) and PC (diacyl 16:0/20:4) are altered in AVF.
Keywords: Arteriovenous fistula; Imaging mass spectrometry; Lysophosphatidylcholine; Arachidonic acid

A GC/MS-based metabolomic approach for diagnosing citrin deficiency by Tomiko Kuhara; Morimasa Ohse; Yoshito Inoue; Arthur J. L. Cooper (1881-1894).
Citrin is the hepatic mitochondrial aspartate–glutamate carrier that is encoded by the gene SLC25A13. Citrin deficiency often leads to hyperammonemia, for which the current treatment concept is different from that for primary hyperammonemias. Metabolite level diagnosis, often referred to as chemical diagnosis, is not always successful in identifying citrin deficiency immediately or in a timely fashion. We previously made the chemical diagnosis of citrin deficiency in ten patients from nine families. In order to devise a more rapid and more accurate chemical diagnosis of this disorder than is currently available, we reinvestigated the gas chromatography/mass spectrometry-based urine metabolome in these patients. In patients aged 2 to 5 months, prominent biomarkers detected included one or more of the following metabolites: tyrosine, p-hydroxyphenyllactate, p-hydroxyphenylpyruvate, and N-acetyltyrosine, galactose, galactitol and galactonate, glucose, glucitol, and cystathionine. These biomarkers are less prominent in older patients, but are not increased in argininosuccinate synthetase deficiency or other hyperammonemias. α-Ketoglutaramate (KGM), a recently recognized urinary biomarker of primary hyperammonemias associated with defects of the urea cycle, was increased in most patients with citrin deficiency studied here in spite of normal urinary levels of glutamine (the immediate precursor of KGM), 5-oxoproline, glutamate, aspartate, and asparagine. Other important urinary biomarkers that should be measured for differential diagnosis of hyperammonemias, including orotate, uracil, and β-ureidopropionate, were not increased. The presence of citrulline and citrulline-derived metabolites was noted in all cases. The present study shows that noninvasive urine metabolomics, together with an analysis of selected metabolites or groups of metabolites, provides a more reliable and rapid chemical diagnosis of citrin deficiency than was previously available and more readily differentiates this disorder from other hyperammonemic syndromes.
Keywords: Citrin deficiency; Orotate; Uracil; α-Ketoglutaramate; Gas chromatography/mass spectrometry; Metabolome

Semi-quantitative analyses of metabolic systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG mice by Akiko Kubo; Mitsuyo Ohmura; Masatoshi Wakui; Takahiro Harada; Shigeki Kajihara; Kiyoshi Ogawa; Hiroshi Suemizu; Masato Nakamura; Mitsutoshi Setou; Makoto Suematsu (1895-1904).
Analyses of energy metabolism in human cancer have been difficult because of rapid turnover of the metabolites and difficulties in reducing time for collecting clinical samples under surgical procedures. Utilization of xenograft transplantation of human-derived colon cancer HCT116 cells in spleens of superimmunodeficient NOD/SCID/IL-2Rγnull (NOG) mice led us to establish an experimental model of hepatic micrometastasis of the solid tumor, whereby analyses of the tissue sections collected by snap-frozen procedures through newly developed microscopic imaging mass spectrometry (MIMS) revealed distinct spatial distribution of a variety of metabolites. To perform intergroup comparison of the signal intensities of metabolites among different tissue sections collected from mice in fed states, we combined matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI–TOF-IMS) and capillary electrophoresis–mass spectrometry (CE–MS), to determine the apparent contents of individual metabolites in serial tissue sections. The results indicated significant elevation of ATP and energy charge in both metastases and the parenchyma of the tumor-bearing livers. To note were significant increases in UDP-N-acetyl hexosamines, and reduced and oxidized forms of glutathione in the metastatic foci versus the liver parenchyma. These findings thus provided a potentially important method for characterizing the properties of metabolic systems of human-derived cancer and the host tissues in vivo.
Keywords: Imaging mass spectrometry (IMS); Matrix-assisted laser desorption/ionization (MALDI); Capillary electrophoresis–mass spectrometry (CE–MS); Tumor-bearing liver; Metabolome; Glycoprotein biosynthesis

Use of the MALDI BioTyper system with MALDI–TOF mass spectrometry for rapid identification of microorganisms by Kazuyuki Sogawa; Masaharu Watanabe; Kenichi Sato; Syunsuke Segawa; Chisato Ishii; Akiko Miyabe; Syota Murata; Tomoko Saito; Fumio Nomura (1905-1911).
In a clinical diagnosis microbiology laboratory, the current method of identifying bacterial isolates is based mainly on phenotypic characteristics, for example growth pattern on different media, colony morphology, Gram stain, and various biochemical reactions. These techniques collectively enable great accuracy in identifying most bacterial isolates, but are costly and time-consuming. In our clinical microbiology laboratory, we prospectively assessed the ability of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) to identify bacterial strains that were routinely isolated from clinical samples. Bacterial colonies obtained from a total of 468 strains of 92 bacterial species isolated at the Department of Clinical Laboratory at Chiba University were directly placed on target MALDI plates followed by addition of CHCA matrix solution. The plates were then subjected to MALDI–TOF MS measurement and the microorganisms were identified by pattern matching with the libraries in the BioTyper 2.0 software. Identification success at the species and genus levels was 91.7% (429/468) and 97.0% (454/468), respectively. MALDI–TOF MS is a rapid, simple, and high-throughput proteomic technique for identification of a variety of bacterial species. Because colony-to-colony differences and effects of culture duration on the results are minimal, it can be implemented in a conventional laboratory setting. Although for some pathogens, preanalytical processes should be refined, and the current database should be improved to obtain more accurate results, the MALDI–TOF MS based method performs, in general, as well as conventional methods and is a promising technology in clinical laboratories.
Keywords: Rapid microorganism identification; Clinical sample; MALDI–TOF MS; MALDI BioTyper

The mammalian tongue is one of the most important organs during food uptake because it is helpful for mastication and swallowing. In addition, taste receptors are present on the surface of the tongue. Lipids are the second most abundant biomolecules after water in the tongue. Lipids such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) are considered to play fundamental roles in the mediation of cell signaling. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of lipids across sections of dissected tissue. In this study, we identified and visualized the PC, LPC, and SM species in a mouse tongue body section with matrix-assisted laser desorption/ionization (MALDI)-IMS. The ion image constructed from the peaks revealed that docosahexaenoic acid (DHA)-containing PC, LPC, linoleic acid-containing PC and SM (d18:1/16:0), and oleic acid-containing PC were mainly distributed in muscle, connective tissue, stratified epithelium, and the peripheral nerve, respectively. Furthermore, the distribution of SM (d18:1/16:0) corresponded to the distribution of nerve tissue relating to taste in the stratified epithelium. This study represents the first visualization of PC, LPC and SM localization in the mouse tongue body.
Keywords: Imaging mass spectrometry; Mouse tongue body; Phosphatidylcholine; Lysophosphatidylcholine; Sphingomyelin; Matrix-assisted laser desorption/ionization

Liquid chromatography–mass spectrometric determination of plasmalogens in human plasma by Shu-Ping Hui; Hitoshi Chiba; Takao Kurosawa (1923-1931).
A new liquid chromatography–mass spectrometry (LC/MS) method has been developed for the quantitative analysis of plasmalogens in human plasma using a nonendogenous plasmalogen (1-O-1′-(Z)-tricosenyl-2-oleoyl-rac-glycero-3-phosphocholine, PLS 23:0/18:1) as an internal standard. 1-O-1′-(Z)-Tricosenyl glyceryl ether was prepared by reacting lithioalkoxyallyl with 1-iodoeicosane as the key intermediate in the formation of PLS 23:0/18:1. In LC/MS analyses, PLS 23:0/18:1 generated significant fragment ions in positive and negative modes. In positive ion mode, the [M+H]+ of PLS 23:0/18:1 yielded unique fragments with cleavages at the sn-1 and sn-2 positions of the glycerol backbone. In negative ion mode, the [M+CH3COO] of PLS 23:0/18:1 resulted in characteristic fragmentation at the sn-2 and sn-3 positions. 1-O-1′-(Z)-Hexadecenyl-2-linoleoyl-rac-glycero-3-phosphocholine (PLS 16:0/18:2) and 2-arachidonoyl-O-1′-(Z)-hexadecenyl-rac-glycero-3-phosphocholine (PLS 16:0/20:4) were chemically synthesized as PLS 23:0/18:1. The calibration curves obtained for PLS 16:0/18:2 and PLS 16:0/20:4 were linear throughout the calibration range (0.04–1.60 pmol). The LOD (S/N = 5:1) was 0.008 pmol and the LOQ (S/N = 6:1) was 0.01 pmol for both PLS 16:0/18:2 and PLS 16:0/20:4. Plasma concentrations of PLS 16:0/18:2 and PLS 16:0/20:4 were 4.0 ± 1.3 μM and 3.5 ± 1.2 μM (mean ± SD), respectively, in five healthy volunteers. Figure SRM chromatograms were obtained by the quantitative LC/MS system in positive mode: (A) synthetic PLS 16:0/18:2, (B) synthetic PLS 16:0/20:4, (C) IS, (A') plasma extract (precursor ion: m/z 742.6), (B') plasma extract (precursorion: m/z 766.6), (C') plasma extract after addition of the IS.
Keywords: Liquid chromatography/electrospray ionization mass spectrometry; LC/MS; Plasmalogen; Analysis; Synthesis

Abnormal phospholipids distribution in the prefrontal cortex from a patient with schizophrenia revealed by matrix-assisted laser desorption/ionization imaging mass spectrometry by Junya Matsumoto; Yuki Sugiura; Dai Yuki; Takahiro Hayasaka; Naoko Goto-Inoue; Nobuhiro Zaima; Yasuto Kunii; Akira Wada; Qiaohui Yang; Keisuke Nishiura; Hiroyasu Akatsu; Akira Hori; Yoshio Hashizume; Takayuki Yamamoto; Keiko Ikemoto; Mitsutoshi Setou; Shin-ichi Niwa (1933-1943).
Schizophrenia is one of the major psychiatric disorders, and lipids have focused on the important roles in this disorder. In fact, lipids related to various functions in the brain. Previous studies have indicated that phospholipids, particularly ones containing polyunsaturated fatty acyl residues, are deficient in postmortem brains from patients with schizophrenia. However, due to the difficulties in handling human postmortem brains, particularly the large size and complex structures of the human brain, there is little agreement regarding the qualitative and quantitative abnormalities of phospholipids in brains from patients with schizophrenia, particularly if corresponding brain regions are not used. In this study, to overcome these problems, we employed matrix-assisted laser desorption/ionization imaging mass spectrometry (IMS), enabling direct microregion analysis of phospholipids in the postmortem brain of a patient with schizophrenia via brain sections prepared on glass slides. With integration of traditional histochemical examination, we could analyze regions of interest in the brain at the micrometric level. We found abnormal phospholipid distributions within internal brain structures, namely, the frontal cortex and occipital cortex. IMS revealed abnormal distributions of phosphatidylcholine molecular species particularly in the cortical layer of frontal cortex region. In addition, the combined use of liquid chromatography/electrospray ionization tandem mass spectrometry strengthened the capability for identification of numerous lipid molecular species. Our results are expected to further elucidate various metabolic processes in the neural system.
Keywords: Schizophrenia; Postmortem brain; Imaging mass spectrometry (IMS); Matrix-assisted laser desorption/ionization (MALDI); Phospholipids

Determination of cyanide in blood by electrospray ionization tandem mass spectrometry after direct injection of dicyanogold by Kayoko Minakata; Hideki Nozawa; Kunio Gonmori; Itaru Yamagishi; Masako Suzuki; Koutaro Hasegawa; Kanako Watanabe; Osamu Suzuki (1945-1951).
An electrospray ionization tandem mass spectrometric (ESI-MS-MS) method has been developed for the determination of cyanide (CN) in blood. Five microliters of blood was hemolyzed with 50 μL of water, then 5 μL of 1 M tetramethylammonium hydroxide solution was added to raise the pH of the hemolysate and to liberate CN from methemoglobin. CN was then reacted with NaAuCl4 to produce dicyanogold, Au(CN) 2 , that was extracted with 75 μL of methyl isobutyl ketone. Ten microliters of the extract was injected directly into an ESI-MS-MS instrument and quantification of CN was performed by selected reaction monitoring of the product ion CN at m/z 26, derived from the precursor ion Au(CN) 2 at m/z 249. CN could be measured in the quantification range of 2.60 to 260 μg/L with the limit of detection at 0.56 μg/L in blood. This method was applied to the analysis of clinical samples and the concentrations of CN in the blood were as follows: 7.13 ± 2.41 μg/L for six healthy non-smokers, 3.08 ± 1.12 μg/L for six CO gas victims, 730 ± 867 μg for 21 house fire victims, and 3,030 ± 97 μg/L for a victim who ingested NaCN. The increase of CN in the blood of a victim who ingested NaN3 was confirmed using MS-MS for the first time, and the concentrations of CN in the blood, gastric content and urine were 78.5 ± 5.5, 11.8 ± 0.5, and 11.4 ± 0.8 μg/L, respectively.
Keywords: Cyanide; Blood; Gold; Electrospray ionization; Tandem mass spectrometry

Methylmalonic aciduria is a common inherited metabolic disorder. Methylmalonic acid (MMA), a key indicator of methylmalonic aciduria, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the MMA in amniotic fluid can be measured by stable-isotope dilution gas chromatography-mass spectrometry. Here, we quantified the MMA in cell-free amniotic fluid samples that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. Our results indicated that the filter paper method was reproducible and accurate enough to be applied to clinical analysis. We also used the filter paper method to screen at-risk fetuses and obtained a clear diagnosis in each case. We conclude that our method enables the prenatal diagnosis of methylmalonic aciduria using practical procedures and a simplified method for transporting the samples.
Keywords: Dried amniotic fluid on filter paper; Methylmalonic aciduria; Prenatal diagnosis; Methylmalonic acid; GC-MS

High-throughput and simultaneous analysis of eight central-acting muscle relaxants in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry in the positive and negative ionization modes by Tadashi Ogawa; Hideki Hattori; Rina Kaneko; Kenjiro Ito; Masae Iwai; Yoko Mizutani; Tetsuya Arinobu; Akira Ishii; Hiroshi Seno (1959-1965).
In this report, a high-throughput and sensitive method for analysis of eight central-acting muscle relaxants in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) in the positive and negative ionization modes using tolbutamide as internal standard is presented. After pretreatment of a plasma sample by solid-phase extraction with an Oasis HLB cartridge, muscle relaxants were analyzed by UPLC with Acquity UPLC BEH C18 column and Acquity TQD tandem quadrupole mass spectrometer equipped with an electrospray ionization interface. The calibration curves for muscle relaxants spiked into human plasma equally showed good linearities in the nanogram per milliliter order range. The detection limits (signal-to-noise ratio = 3) was as low as 0.1–2 ng/mL. The method gave satisfactory recovery rates, accuracy, and precision for quality control samples spiked with muscle relaxants. To further validate the present method, 250 mg of chlorphenesin carbamate was orally administered to a healthy male volunteer, and the concentrations of chlorphenesin carbamate in plasma were measured 0.5, 1, 2, 4, 6, and 8 h after dosing; their concentrations in human plasma were between 0.62 and 2.44 μg/mL. To our knowledge, this is the first report describing simultaneous analysis of over more than two central-acting muscle relaxants by liquid chromatography–tandem mass spectrometry. This has been realized by the capability of our instrument for simultaneous multiple reaction monitoring of the target compounds in both positive and negative ionization modes. Therefore, the present method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.
Keywords: UPLC; LC-MS/MS; High-throughput; Central-acting muscle relaxant; Dantrolene

Next-generation sequencing provides technologies which sequence whole prokaryotic and eukaryotic genomes in days, perform genome-wide association studies, chromatin immunoprecipitation followed by sequencing and RNA sequencing for transcriptome studies. An exponentially growing volume of sequence data can be anticipated, yet functional interpretation does not keep pace with the amount of data produced. In principle, these data contain all the secrets of living systems, the genotype–phenotype relationship. Firstly, it is possible to derive the structure and connectivity of the metabolic network from the genotype of an organism in the form of the stoichiometric matrix N. This is, however, static information. Strategies for genome-scale measurement, modelling and predicting of dynamic metabolic networks need to be applied. Consequently, metabolomics science—the quantitative measurement of metabolism in conjunction with metabolic modelling—is a key discipline for the functional interpretation of whole genomes and especially for testing the numerical predictions of metabolism based on genome-scale metabolic network models. In this context, a systematic equation is derived based on metabolomics covariance data and the genome-scale stoichiometric matrix which describes the genotype–phenotype relationship.
Keywords: Genotype–phenotype relationship; Systems biology; Metabolomics; Proteomics; Genome annotation; Gene models; Metabolic modelling; Flux balance analysis; Dynamic modelling; Stochastic differential equations; Covariance; Principal components analysis; Phenotypic plasticity

β-Secretase 1 (BACE1) is the enzyme involved in the abnormal production of the amyloidogenic peptide Aβ42, one of the major causes of histological hallmarks of Alzheimer's disease. Thus, BACE1 represents a key target protein in the development of new potential drugs for the non-symptomatic treatment of Alzheimer’s disease. Since the discovery of BACE1 one decade ago, both in the pharmaceutical industry and in academia there has been an intense search for novel inhibitors to be developed as new effective drugs. There is a great deal of interest in the discovery of selective non-peptide BACE1 inhibitors with a new chemical skeleton, suited for central nervous system penetration and endowed with more appropriate pharmacokinetic properties. Therefore, the selection of appropriate methods for screening and characterization of BACE1 inhibitors is crucial. This review focuses on the description of the in vitro methods to test BACE1 activity and inhibition, with particular emphasis on fluorescence resonance energy transfer (FRET) methods, aiming at critically highlighting advantages and drawbacks. An overview of BACE1 inhibitors is given, underlying the variability of the FRET methods reported in the literature, and the structure evolution of inhibitors active in cellular cultures and in vivo, from peptide to small synthetic and natural structures.
Keywords: β-Secretase 1; Fluorescence resonance energy transfer methods; In vitro assay; Alzheimer’s disease; β-Secretase 1 inhibitors

Dopamine-loaded chitosan nanoparticles: formulation and analytical characterization by Elvira De Giglio; Adriana Trapani; Damiana Cafagna; Luigia Sabbatini; Stefania Cometa (1997-2002).
The formulation and characterization of dopamine (DA)-loaded chitosan nanoparticles (CSNPs) are described as preliminary steps for the development of potential DA carrier systems intended for Parkinson’s disease treatment. For this purpose, CSNPs were firstly produced and, afterwards, they were incubated in a DA aqueous solution to promote neurotransmitter loading. The characterization of the resulting nanoparticles started with Fourier transform infrared spectroscopy analysis to ascertain the presence of DA in the nanocarrier, whereas X-ray photoelectron spectroscopy analysis provided evidence of the localization of DA on the nanoparticle surface. A quartz crystal microbalance with dissipation monitoring (QCM-D) was then exploited to investigate both swelling of CSNPs and interaction of DA with CSNPs. In particular, the QCM-D revealed that this interaction is fast and so this allows a stable nanostructured system to be obtained. Fig. 1 QCM-D investigation on dopamine/chitosan nanoparticles interaction
Keywords: Chitosan; Nanoparticles; Dopamine; QCM-D; FT-IR; XPS

Real-time cell-impedance sensing assay as an alternative to clonogenic assay in evaluating cancer radiotherapy by Wilson Roa; Xiaoyan Yang; Linghong Guo; Biao Huang; Shima Khatibisepehr; Stephan Gabos; Jie Chen; James Xing (2003-2011).
Intrinsic radiosensitivity of normal and tumour tissues has been shown to be an independent prognostic factor for patients’ response to radiotherapy. This study compares the real-time cell-impedance sensing (RT-CES) assay with the conventional clonogenic assay in terms of in-vitro radiosensitivity. One objective in this study was to predict in-vivo response to gold nanoparticle (GNP) treatment on the basis of in-vitro RT-CES testing results. Four adenocarcinoma cancer cell lines were tested using both the RT-CES and the clonogenic assays. Cell-survival curves were plotted, and the mean SF2 values obtained by these two different assay methods were compared using ANOVA. Radiation sensitivities obtained in-vitro were then compared with the in-vivo results. On the basis of the measurement of cell colonies, the RT-CES assay has similar radiosensitivity to the clonogenic assay, but significantly shortens the testing time from 14–21 days to only 72 h. Intrinsic GNP enhanced radiation sensitivity using tumour volume (mm3) in vivo is comparable with that using RT-CES cell survival assay in vitro. Furthermore, the RT-CES system provides real-time information regarding the state of cell radiosensitivity that may give useful information towards personalizing radiotherapy. The RT-CES assay enables more reliable and time-efficient results in the evaluation of radiosensitivity. Figure Real-time cell-impedance sensing assay in evaluating cancer radiotherapy
Keywords: Cancer radiotherapy; Radiosensitivity; Clonogenic assay; RT-CES cell-proliferation assay

Collection, storage, and electrophoretic analysis of nanoliter microdialysis samples collected from awake animals in vivo by Meng Wang; Neil D. Hershey; Omar S. Mabrouk; Robert T. Kennedy (2013-2023).
Microdialysis sampling is an important tool for chemical monitoring in living systems. Temporal resolution is an important figure of merit that is determined by sampling frequency, assay sensitivity, and dispersion of chemical zones during transport from sampling device to fraction collector or analytical system. Temporal resolution has recently been improved by segmenting flow into plugs, so that nanoliter fractions are collected at intervals of 0.1–2 s, thus eliminating temporal distortion associated with dispersion in continuous flow. Such systems, however, have yet to be used with behaving subjects. Furthermore, long-term storage of nanoliter samples created by segmented flow has not been reported. In this work, we have addressed these challenges. A microdialysis probe was integrated to a plug generator that could be stably mounted onto behaving animals. Long-term storage of dialysate plugs was achieved by collecting plugs into high-purity perfluoroalkoxy tubes, placing the tube into hexane and then freezing at −80°C. Slow warming with even temperatures prevented plug coalescence during sample thawing. As a demonstration of the system, plugs were collected from the striatum of behaving rats using a 0.5-mm-long microdialysis probe. Resulting plugs were analyzed 1–4 days later by chip-based electrophoresis. To improve throughput of plug analysis over previous work, the speed of electrophoretic separation was increased by using forced air cooling and 1-butyl-2,3-dimethylimidazolium tetrafluoroborate as a separation buffer additive, allowing resolution of six neuroactive amino acids in 30 s. Concentration changes induced by K+ microinjections were monitored with 10 s temporal resolution. The improvements reported in this work make it possible to apply segmented flow microdialysis to the study of behaving animals and enable experiments where the analytical system cannot be placed close to the animal.
Keywords: Behaving; Electrophoresis; Microdialysis; Offline analysis; Plug storage; Segmented flow; Spatial resolution; Temporal resolution

Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies by Lauren M. Ramsay; Nathan Cermak; Oluwatosin O. Dada; Norman J. Dovichi (2025-2030).
Capillary isoelectric focusing tends to suffer from poor reproducibility, particularly for the analysis of complex protein samples from cellular or tissue homogenates. This poor reproducibility appears to be associated with erratic variations in electroosmotic flow. One cause of electroosmotic flow variation is degradation of the capillary coating caused by the extremely basic solution commonly used during mobilization and focusing; this degradation of the capillary coating can be reduced by employing a CAPS mobilization buffer at pH 9. Another cause of variation is protein adsorption to the capillary wall, which causes an increase in electroosmotic flow. The effects of protein adsorption can be reduced by use of surfactants in the buffer and by employing an extremely low sample loading. We report the use of CAPS mobilization buffer in combination with an ultrasensitive laser-induced fluorescence detector for the reproducible analysis of ∼2 ng of protein from a Barrett's esophagus biopsy.
Keywords: Capillary isoelectric focusing; Laser-induced fluorescence; Capillary coatings; Electrophoretic reproducibility

Hydrophobin can prevent secondary protein adsorption on hydrophobic substrates without exchange by Bernhard von Vacano; Rui Xu; Sabine Hirth; Ines Herzenstiel; Markus Rückel; Thomas Subkowski; Ulf Baus (2031-2040).
By combining several surface analytical tools, we show that an adsorbed layer of the protein H*Protein B prevents the adsorption of secondary proteins bovine serum albumin, casein, or collagen at low-salinity conditions and at pH 8. H*Protein B is an industrially producible fusion protein of the hydrophobin family, known for its high interfacial activity. While applications of hydrophobin have been reported to facilitate adhesion of proteins under different pH conditions, careful analysis by quartz-crystal microbalance and ellipsometry prove that no additional adsorption can be found on top of the H*Protein B layer in this study. Surface analysis by X-ray photoelectron spectroscopy and secondary ion mass spectrometry proves that the hydrophobin layer stays intact even after hours of exposure to solutions of the secondary proteins and that no exchange of proteins can be detected. Fig. 1 The combination of surface analytical methods shows that an adsorbed layer of H*Protein B, a technically available representative of the hydrophobin family, prevents adsorption of secondary proteins at low-salinity conditions and pH 8. Due to the multi-method approach it can be proven that no exchange of the adsorbed hydrophobin protein is taking place
Keywords: Hydrophobin; Protein adsorption; Secondary ion mass spectrometry; Quartz-crystal microbalance; X-ray photoelectron spectroscopy

In this study, a novel and enzyme-free electrochemical immunoassay was developed for the detection of alpha-fetoprotein (AFP; as a model biomarker) by using a novel redox nanocatalyst, patterned gold-iridium oxide nanoparticle assemblies (Au-IrOx), on a Prussian blue (PB) functionalized glassy carbon electrode. The as-prepared Au-IrOx nanospheres were not only used for the labeling of secondary anti-AFP antibody but also executed for water oxidation. With a sandwich immunoassay format, the electrochemical measurement was carried out in 0.1 M KNO3-HCl, pH 3.2, containing 1.0 mM p-aminophenol (AP). Initially, the added AP was oxidized to p-quinone imine (QI) with the help of the immobilized PB on the electrode, and then the generated QI was reduced back to AP by hydrogenation reaction from water oxidation. The self-produced QI reactants were catalytically recycled and thus amplified the signal of electrochemical response. Under optimal conditions, the electrochemical immunosensor displayed a wide working range of 0.005 to 200 ng/mL with a low detection limit of 0.5 pg/mL AFP (at 3sB). Intra- and inter-assay coefficients of variation were below 10%. In addition, the methodology was validated with real serum samples, receiving a good agreement with the results obtained from commercially available electrochemiluminescence automated analyzer. Figure A novel and enzyme-free electrochemical immunoassay was designed for the detection of alpha-fetoprotein buy using a novel redox nanocatalyst, patterned goldiridium oxide nanopaticle assemblies, on the Prussian blue functionalized glassy carbon electrode with catalytic recycling of self-produced reactants.
Keywords: Electrochemical immunoassay; Alpha-fetoprotein; Iridium oxide nanospheres; Catalytic recycling

Multi-step temperature control in a polymerase chain reaction (PCR) is a limiting factor in device miniaturization and portability. In this study, we propose the fabrication of a three-dimensional (3D) microdevice employing a single heater to minimize temperature control required for an on-chip continuous-flow PCR as well as the overall footprint by stacking the device in multi-layers. Two poly(dimethylsiloxane) (PDMS) layers with differing thicknesses are vertically stacked with their microchannel-engraved sides facing down. Through-holes are made in the thicker PDMS layer, which is sandwiched between a glass substrate at the bottom and the thinner PDMS layer at the top. In this way, a fluidic conduit is realized in a 3D configuration. The assembled 3D microdevice is then placed onto a heater glass-side down. The interface of the two PDMS layers displays a relatively lower temperature than that of the PDMS and glass layers due to the low thermal conductivity of the PDMS and its physical distance from the heater. The denaturation temperature can be controlled by adjusting the temperature of the heater, while the annealing/extension temperature can be controlled automatically by molding the thicker bottom PDMS layer into the appropriate thickness calculated using a numerical derivation proposed in this study. In this way, a cumbersome temperature measurement step is eliminated. DNA amplification was successfully carried out using the proposed 3D fluidic microdevice, and the intensity of the resulting amplicon was comparable to that obtained using a thermal cycler. This novel concept of adopting a single heating source greatly simplifies the temperature control issue present in an on-chip continuous-flow PCR. It also allows the use of a commercialized hot plate as a potential heat source, paving the way for device miniaturization and portability in a highly cost-effective manner. In this study, a simple and facile technique to make arrays of through-holes for the fluidic interconnection inside a 3D channel configuration is also addressed. Figure PCR inside a 3D fluidic conduit and a resulting target amplicon (~230 bp)
Keywords: On-chip polymerase chain reaction (PCR); Multi-layer microdevice; 3D fluidic conduit; A single heater; DNA amplification

Previous work from this laboratory has reported the chemical synthesis of N-acetylcysteine (NAC) conjugates of natural bile acids (BAs) and shown that such novel conjugates can be formed in vivo in rats to which NAC has been administered. The subsequent fate of such novel conjugates is not known. One possible biotransformation is sulfation, a major pathway for BAs N-acylamidates in patients with cholestatic liver disease. Here, we report the chemical synthesis of the 3-sulfates of the S-acyl NAC conjugates of five natural BAs (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic). We also measured the sulfation of N-acetylcysteine–natural bile acid (BA-NAC) conjugates when they were incubated with a rat liver cytosolic fraction. The chemical structures of the BA-NAC 3-sulfates were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation of singly and doubly charged deprotonated molecules, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. When BA-NACs were incubated with a rat liver cytosolic fraction to which 3’-phosphoadenosine 5’-phosphosulfate was added, sulfation occurred, but the dominant reaction was hydrolysis of the S-acyl linkage to form the unconjugated BAs. Subsequent sulfation occurred at C-3 on the unconjugated BAs that had been formed from the BA-NACs. Such sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus, NAC conjugates of BAs as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes.
Keywords: Bile acid mercapturates; Bile acid sulfation

Quantitative analysis of erythropoietin in human plasma by tandem mass spectrometry by Fabien Xuereb; Stéphane Chaignepain; Dominique Breilh; Frédéric Godde; Marie-Claude Saux; Christof Lenz; Matthias Glueckmann; Jean-Marie Schmitter (2073-2084).
The extended use of protein drugs in therapeutics has created the need for their quantification in human plasma. A methodology using the therapeutic protein itself as internal standard for quantitative analysis by multiple reaction monitoring (MRM) has been designed and applied to epoetin beta, a recombinant human erythropoietin (rhEPO). After depletion of major proteins, plasma samples were desalted and enriched in rhEPO by reversed phase liquid chromatography prior to tryptic cleavage. Differential isotopic labeling of peptides was performed by derivatization with 2-methoxy-4,5-dehydro-imidazole. A light version (four hydrogen atoms) of this reagent was used for plasma peptides. Tryptic peptides obtained from pure rhEPO were derivatized with a heavy version (four deuterium atoms) of the same reagent and used as internal standards. Two rhEPO tryptic peptides with three MRM transitions per peptide were selected for quantification. This strategy provided a quantification limit close to 50 amol of epoetin beta per microliter of plasma (equivalent to 1.7 ng/mL), i.e., well below the expected therapeutic concentrations in plasma (around 100–500 amol/μL).
Keywords: Protein drug; Erythropoietin; Mass spectrometry; Multiple reaction monitoring; Isotopic labeling; Quantification

The gold-nanoparticle-based surface plasmon resonance light scattering and visual DNA aptasensor for lysozyme by Xinyi Wang; Yao Xu; Yang Chen; Limei Li; Feng Liu; Na Li (2085-2091).
We developed a new simple and sensitive assay for lysozyme based on gold nanoparticle plasmon resonance light scattering (PRLS) measurement and naked-eye detection using for the first time the lysozyme DNA aptamer as the recognition element. Lysozyme DNA aptamer could stabilize gold nanoparticles (AuNPs) at high ionic strength. Introducing lysozyme to the system easily triggered the aggregation of AuNPs, producing a red-to-blue color change of the solution, red-shifted plasmon absorption, and enhanced plasmon resonance light scattering. The linear range was found to be 0.2∼4 nM for 0.7 nM AuNPs, 0.3∼6 nM for 1.4 nM AuNPs and 0.6∼8 nM for 2.1 nM AuNPs. About 0.1 nM lysozyme can produce an observable enhancement of PRLS signal. For visual detection, 1 nM lysozyme can produce a very distinctive color change. Satisfactory recoveries were obtained for simulated saliva and diluted urine samples, indicating that the method has potential for analyses of clinical samples. The simplicity and high sensitivity that are consistent with the resources and needs of many laboratories makes this method a good choice for routine analysis. Figure Schematic description and demonstration of aggregation of DNA aptamer stabalized AuNPs for colorimetric and PRLS sensing of lysozyme.
Keywords: Gold nanoparticles; Aggregation; Lysozyme; DNA aptamer; Plasmon resonance light scattering; Visual detection

Multi-analyte procedures are of great interest in clinical and forensic toxicology making the analytical process much simpler, faster, and cheaper and allow monitoring of analytes of different drug classes in one single body sample. The aim of the present study was to validate an ultra high performance liquid chromatographic-tandem mass spectrometric approach for fast target screening and quantification of 34 antidepressants in plasma after simple liquid–liquid extraction as part of a multi-analyte procedure for over 130 drugs. The validation process including recovery, matrix effects, process efficiency, ion suppression/enhancement of co-eluting analytes (already published), selectivity, cross talk, accuracy and precision, stabilities, and limits of quantification and detection showed that the approach was selective, sensitive, accurate, and precise for 28 of the 34 tested drugs. The applicability was successfully tested by analyzing authentic plasma samples and external quality control samples. Furthermore, it could be shown that time- and cost-saving one-point calibration was applicable for 21 drugs for daily routine and especially in emergency cases.
Keywords: UHPLC-MS/MS; Antidepressants; Plasma; Validation

Application of molecularly imprinted polymer solid-phase extraction for salivary cotinine by Ricardo Vilela Vitor; Matheus Coutinho Gonçalves Martins; Eduardo Costa Figueiredo; Isarita Martins (2109-2117).
A method constituted by molecularly imprinted solid-phase extraction (MISPE) with high-performance liquid chromatography coupled to diode array detector (HPLC-DAD) was developed for cotinine analysis in saliva samples. For this purpose, the separation was carried out with a C18 reversed-phase column at 20 °C. The mobile phase which was composed of a mixture of 09:91 (v/v) acetonitrile/phosphate buffer, pH 6.3, was delivered with isocratic flow rate at 1.4 mL min−1. Employing MISPE, the best conditions were achieved with 1.5 mL of saliva plus 1.5 mL of 0.1 mol L−1 of acetate buffer, pH 5.5, which were then passed through a cartridge previously conditioned with 2 mL acetonitrile, 2 mL methanol, and 2 mL of 0.1 mol L−1 sodium acetate buffer, pH 5.5. The washing was carried out with 1 mL deionized water, 1 mL of 0.1 mol L−1 sodium hydroxide, and 1 mL hexane; finally; the cotinine elution was carried out with 3 mL methanol/water (97.5: 2.5, v/v). Linearity ranged from 30 to 500 ng mL−1 with r > 0.99. Intra-assay, interassay precision, and accuracy ranged from 3.1% to 10.1%, 5.2% to 15.9%, and 99.22% to 111.17%, respectively. The detection and quantification limits were 10 and 30 ng mL−1, respectively. This investigation has provided a reliable method for routine cotinine determination in saliva, and it is an important tool for monitoring cigarette smoke exposure in smokers. The method was applied in five smokers’ samples who consumed around five to 20 cigarettes per day and the values of cotinine in saliva were from 66.7 to 316.16 ng mL−1.
Keywords: Molecularly imprinted polymer; Solid-phase extraction; Cotinine; Saliva; Liquid chromatography

Polysorbates can be separated according to their functionality and architecture by liquid chromatography on a hydrophilic interaction chromatography (HILIC) column in acetone–water mobile phases containing 90–97% acetone. The different polymer homologous series are separated according to the number of terminal hydroxy groups and elute as narrow peaks. The hydrophilic part (ethoxylates of sorbitan, isosorbide, and poly(ethyleneglycol)) and amphiphilic funtionalites (ethoxylated mono-, di-, tri-, and tetraesters) were separated by HILIC mode of high-performance liquid chromatography (HPLC). All these separated functionaities are identified and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). This combination of HPLC and MALDI-TOF MS has been proven to be an excellent tool for the characterization of heterogenous complex samples.
Keywords: Polysorbate; Tween; Liquid chromatography under critical conditions; HILIC; MALDI-TOF MS

Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive by Nan-Sook Hong; Lian Hua Shi; Ji Seon Jeong; Inchul Yang; Sook-Kyung Kim; Sang-Ryoul Park (2131-2140).
A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2′-deoxyribonucleoside 5′-monophosphates (dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities of the negatively charged dNMPs. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision, and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA.
Keywords: Capillary electrophoresis; dNMPs; DNA; Reversal of EOF; MEKC

Determination of phenylenediamine isomers in hair dyes by coal cinders micro-column extraction and MEKC by Yiwei Wu; Feng Jiang; Lin Chen; Jing Zheng; Zhenli Deng; Qing Tao; Jing Zhang; Lijuan Han; Xiaoshu Wei; Aimin Yu; Haili Zhang (2141-2147).
A new micellar electrokinetic chromatography (MEKC) method using beta-cyclodextrins (β-CDs) and 1-butyl-3-methylimidazolium hexafluorophosphates (ionic liquids) as additives was successfully developed for determination of para-, meta-, and ortho-phenylenediamines isomers (p-P, m-P, and o-P) in hair dyes. To improve the sensitivity of the MEKC-UV, a simple and cheap flow injection (FI) technique using a micro-column packed with coal cinders (the by-products from combustion in a boiler) as solid-phase extractant was also investigated. In the presence of 20 mmol L−1 phosphates at pH 5.5, addition of 12 mmol L−1 ionic liquids and 8 mmol L−1 β-CDs greatly improved the separation efficiency. The three analytes could be quantitatively adsorbed by coal cinders, and desorbed readily with 0.15 mL of 0.01 mol L−1 NaOH. Under the optimum conditions, an enrichment factor (EF) of 33.3 was obtained, and determination limits of p-P, m-P, and o-P were 1.97 × 10−7, 0.99 × 10−7, and 0.61 × 10−7 mol L−1, respectively. The adsorption capacities of the coal cinders micro-column for p-P, m-P, and o-P were all 1.20 mg g−1. The presented procedure was successfully applied to the determination of p-P, m-P, and o-P in hair dyes with satisfactory results. Figure Electropherograms of 0.1 mmol L−1 p-P, m-P, and o-P obtained by a direct MEKC and b SPE-MEKC; SPE conditions: sample solution flow rate 0.30 mL min−1, eluent flow rate 0.45 mL min−1, desorption by 0.15 mL of 0.01 mol L−1 NaOH; MEKC conditions: 20 mmol L−1 phosphates containing 12 mmol L−1 ionic liquid and 8 mmol L−1 β-CD at pH 5.5; injection time 50 s
Keywords: Coal cinders micro-column extraction; Hair dye; MEKC; Phenylenediamine isomers

Dispersive liquid–liquid microextraction (DLLME) and hollow fiber liquid–liquid–liquid microextraction (HF-LLLME) combined with HPLC–DAD have been applied for the determination of three narcotic drugs (alfentanil, fentanyl, and sufentanil) in biological samples (human plasma and urine). Different DLLME parameters influencing the extraction efficiency such as type and volume of the extraction solvent and the disperser solvent, concentration of NaOH, and salt addition were investigated. In the HF-LLLME, the effects of important parameters including organic solvent type, concentration of NaOH as donor solution, concentration of H2SO4 as acceptor phase, salt addition, stirring rate, temperature, and extraction time were investigated and optimized. The results showed that both extraction methods exhibited good linearity, precision, enrichment factor, and detection limit. Under optimal condition, the limits of detection ranged from 0.4 to 1.9 μg/L and from 1.1 to 2.3 μg/L for DLLME and HF-LLLME, respectively. For DLLME, the intra- and inter-day precisions were 1.7–6.4% and 14.2–15.9%, respectively; and for HF-LLLME were 0.7–5.2% and 3.3–10.1%, respectively. The enrichment factors were from 275 to 325 and 190 to 237 for DLLME and HF-LLLME, respectively. The applicability of the proposed methods was investigated by analyzing biological samples. For analysis of human plasma and urine samples, HF-LLLME showed higher precision, more effective sample clean-up, higher extraction efficiency, lower organic solvent consumption than DLLME. Figure Schematic diagram of dispersive liquid-liquid microextraction and hollow fiber liquid-liquid-liquid microextraction combined with liquid chromatography-diode array detection for the determination of fentanyl, alfentanil and sufentanil.
Keywords: Dispersive liquid–liquid microextraction; Hollow fiber liquid–liquid–liquid microextraction; Liquid chromatography; Human plasma and urine; Narcotic drugs

An analytical procedure involving solid-phase extraction (SPE) and high-performance liquid chromatography-mass spectrometry has been developed for the determination of nine high-intensity sweeteners authorised in the EU; acesulfame-K (ACS-K), aspartame (ASP), alitame (ALI), cyclamate (CYC), dulcin (DUL), neohesperidin dihydrochalcone (NHDC), neotame (NEO), saccharin (SAC) and sucralose (SCL) in a variety of food samples (i.e. beverages, dairy and fish products). After extraction with a buffer composed of formic acid and N,N-diisopropylethylamine at pH 4.5 in ultrasonic bath, extracts were cleaned up using Strata-X 33 μm Polymeric SPE column. The analytes were separated in gradient elution mode on C18 column and detected by mass spectrometer working with an electrospray source in negative ion mode. To confirm that analytical method is suitable for its intended use, several validation parameters, such as linearity, limits of detection and quantification, trueness and repeatibilty were evaluated. Calibration curves were linear within a studied range of concentrations (r 2  ≥ 0.999) for six investigated sweeteners (CYC, ASP, ALI, DUL, NHDC, NEO). Three compounds (ACS-K, SAC, SCL) gave non-linear response in the investigated concentration range. The method detection limits (corresponding to signal-to-noise (S/N) ratio of 3) were below 0.25 μg mL−1 (μg g−1), whereas the method quantitation limits (corresponding to S/N ratio of 10) were below 2.5 μg mL−1 (μg g−1). The recoveries at the tested concentrations (50%, 100% and 125% of maximum usable dose) for all sweeteners were in the range of 84.2 ÷ 106.7%, with relative standard deviations <10% regardless of the type of sample matrix (i.e. beverage, yoghurt, fish product) and the spiking level. The proposed method has been successfully applied to the determination of the nine sweeteners in drinks, yoghurts and fish products. The procedure described here is simple, accurate and precise and is suitable for routine quality control analysis of foodstuffs.
Keywords: High-intensity sweeteners; Solid-phase extraction; Liquid chromatography; Mass spectrometry; Food analysis

We report on investigations of plasmas produced by laser ablation of fresh potatoes using infrared nanosecond laser radiation. A twin laser system consisting of two Nd:YAG oscillators was used to generate single or double pulses of adjustable interpulse delay. The potatoes were irradiated under ambient air with moderate pulse energies of about 10 mJ. The expansion dynamics of the ablation plume was characterized using fast imaging with a gated camera. In addition, time-resolved optical emission spectroscopy was applied to study the spectral line emission of the various plasma species. The electron density was deduced from Stark broadening, and the plasma temperature was inferred from the relative emission intensities of spectral lines. The relative concentrations of metals were estimated from the comparison of the measured emission spectra to the spectral radiance computed for a plasma in local thermal equilibrium. It is shown that the plasma produced by double pulses has a larger volume and a lower density. These properties lead to an increase of the signal-to-noise ratio by a factor of 2 and thus to an improved measurement sensitivity. Figure Laser-induced breakdown spectroscopy for food analysis
Keywords: Laser plasma; Plasma modeling; Calibration-free LIBS; Quality control of food

Multiresidue method to quantify pesticides in fish muscle by QuEChERS-based extraction and LC-MS/MS by Angélique Lazartigues; Laure Wiest; Robert Baudot; Marielle Thomas; Cyril Feidt; Cécile Cren-Olivé (2185-2193).
Pesticide residues in fish muscle are an environmental and a health safety concern which requires analytical methods presenting high sensitivity and low limits of quantification. In this study, adapted QuEChERS method, coupled to liquid chromatography tandem mass spectrometry (Scheduled MRM-5500 QTRAP), was developed to quantify 13 pesticides (azoxystrobin, clomazone, diflufenican, dimethachlor, carbendazim, iprodion, isoproturon, mesosulfuron-methyl, metazachlor, napropamid, quizalofop and thifensulfuron-methyl) in muscle of fish. Quantification limits were below 1 ng g−1 except for clomazone (1.8 ng g−1) and quizalofop (7.4 ng g−1). Best recoveries were observed for perch (>80%) and roach (>68%), except for thifensulfuron-methyl. Lower recoveries had been observed for carp (6% to 86%). Relative standard deviation was lower than 28% for intra-day and 29% for inter-day analysis, respectively. This method was successfully tested on three fish species, naturally or orally exposed: roach (Rutilus rutilus), perch (Perca fluviatilis) and carp (Cyprinus carpio). Few levels were observed in fish naturally exposed, but carp and perch orally contaminated showed measurable levels in their muscles.
Keywords: Multiresidue; QuEChERS; Pesticides; Fish; LC-MS/MS

This paper presents the development of a fast and sensitive analytical method for the simultaneous determination of UV filters and degradation products having quite different polarities (log Kow 2.19–6.88) in sediment, by means of pressurized liquid extraction (PLE) with in-cell purification and analysis by ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS/MS). Analytes were simultaneously concentrated and purified by placing aluminium oxide as clean-up sorbent in the extraction cell for a faster sample pre-treatment. Under optimized conditions, quantitative recoveries (only one compound below 80%) and satisfactory precision (RSD, 5–15%) were obtained. Low limits of detection were achieved of 0.5–15 ng/g dry weight (dw). The use of PLE extraction and purification and UPLC technology enabled all the compounds to be separated chromatographically in less than 9 min, and with a total chromatographic analysis time of 18 min. This method significantly decreased the overall time of analysis as compared to those of previously developed. Finally, the optimized methodology was applied to investigate the occurrence of the target UV filters in sediment samples collected along the Ebro river basin (Spain). UV filters were detected in 95% of the sediment samples analysed. Results revealed a widespread presence of octocrylene (OC), reaching concentrations up to 24 × 102 ng/g dw, the highest reported so far. Ethylhexyl dimethyl PABA (OD-PABA) and benzophenone-3 (BP3) were also frequently detected (60–65%), but at lower concentrations (4.4–27 ng/g dw). 4DHB (an estrogenic degradation product of BP3) was present in three samples at concentrations between 12 and 21 ng/g dw. These results constitute the first data on the occurrence of OD-PABA and 4DHB in sediments.
Keywords: UV filters; River sediments; UPLC-MS/MS analysis; Pressurized liquid extraction; In-cell purification

A simple, sensitive, and specific analytical method has been developed for the quantitative determination of 15 reducing carbohydrates in the soil solution of crop rhizosphere. Reducing carbohydrates were derivatized with 1-phenyl-3-methyl-5-pyrazolone, separated by reversed-phase high-performance liquid chromatography and detected by electrospray ionization tandem mass spectrometry. Lower limits of quantitation of 2 ng/mL were achieved for all carbohydrates. Quantitation was performed using peak area ratios (analyte/internal standard) and a calibration curve spiked in water with glucose-d2 as the internal standard. Calibration curves showed excellent linearity over the range 2–100 ng/mL (10–1,000 ng/mL for glucose). The method has been tested with quality control samples spiked in water and soil solution samples obtained from the rhizosphere of wheat and canola and has been found to provide accurate and precise results.
Keywords: Carbohydrates; Mass spectrometry; Quantification; Derivatization; PMP; Soil solutions

An automated dynamic leaching test integrated in a portable flow-based setup is herein proposed for reliable determination of readily bioaccessible Cr(VI) under worst-case scenarios in soils containing varying levels of contamination. The manifold is devised to accommodate bi-directional flow extraction followed by processing of extracts via either in-line clean-up/preconcentration using multi-walled carbon nanotubes or automatic dilution at will, along with Cr(VI) derivatization and flow-through spectrophotometric detection. The magnitude of readily mobilizable Cr(VI) pools was ascertained by resorting to water extraction as promulgated by current standard leaching tests. The role of carbon nanomaterials for the uptake of Cr(VI) in soil leachates and the configuration of the packed column integrated in the flow manifold were investigated in detail. The analytical performance of the proposed system for in vitro bioaccessibility tests was evaluated in chromium-enriched soils at environmentally relevant levels and in a standard reference soil material (SRM 2701) with a certified value of total hexavalent chromium. The automated method was proven to afford unbiased assessment of water-soluble Cr(VI) in soils as a result of the minimization of the chromium species transformation. By combination of the kinetic leaching profile and a first-order leaching model, the water-soluble Cr(VI) fraction in soils was determined in merely 6 h against >24 h taken in batchwise steady-state standard methods. Schematic representation of the compact flow set-up for automated dynamic extraction and determination of readily bioaccessible Cr(VI) in soils
Keywords: Hexavalent chromium; Bioaccessibility test; Soil; Automation; Miniaturization; Carbon nanoparticles

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based approach was applied for the detection of various lipid classes, such as triacylglycerols (TAGs) and phospholipids (PLs), and their oxidation by-products in extracts of small (50–100 μg) samples obtained from painted artworks. Ageing of test specimens under various conditions, including the presence of different pigments, was preliminarily investigated. During ageing, the TAGs and PLs content decreased, whereas the amount of diglycerides, short-chain oxidative products arising from TAGs and PLs, and oxidized TAGs and PLs components increased. The examination of a series of model paint samples gave a clear indication that specific ions produced by oxidative cleavage of PLs and/or TAGs may be used as markers for egg and drying oil-based binders. Their elemental composition and hypothetical structure are also tentatively proposed. Moreover, the simultaneous presence of egg and oil binders can be easily and unambiguously ascertained through the simultaneous occurrence of the relevant specific markers. The potential of the proposed approach was demonstrated for the first time by the analysis of real samples from a polyptych of Bartolomeo Vivarini (fifteenth century) and a “French school” canvas painting (seventeenth century).
Keywords: Paintings; Lipids; Binders; MALDI; Lipid oxidation

Determination of detection limits for SEM-EDS and m-FTIR analysis of artwork by C. Sessa; A. Vila; J. F. García (2241-2251).
Scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDS) and micro-Fourier transform infrared (m-FTIR) spectroscopy are two of the primary analytical techniques used for the characterisation of artwork (Casadio and Toniolo, J. Cult. Herit. 2:71–78, 2001; Bruni et al., Vibr. Spectrosc. 20:15–25, 1999; Bouchard et al., e-Preserv. Sci. 6:27–37, 2009). Despite wide application of these techniques, no studies have been performed to evaluate their detection limits for this type of analysis. The characterisation of minor components used in a piece of art is important because these components may provide key information about the process of creation and answer questions regarding conservation and restoration (Casadio and Toniolo, J. Cult. Herit. 2:71–78, 2001; Bicchieri et al., Spectrochim. Acta B At. Spectrosc. 56:915–922, 2001). This study focused on easel paintings. Several mock-ups were prepared, and the painting layers were created with binary mixtures of three different blues (Prussian blue, phthalocyanine blue and ultramarine blue). Blue pigments have been used extensively in many art pieces and several studies have described problems related to their determination in low concentrations (Bouchard et al., e-Preserv. Sci. 6:27–37, 2009; Bicchieri et al., Spectrochim. Acta B At. Spectrosc. 56:915–922, 2001; Osticiolia et al., Spectrochim. Acta A Mol. Biomol. Spectrosc. 73:525–531, 2009; Giaccai, Mater. Res. Soc. Symp. Proc. 1047:233–242, 2008; Lutzenberger and Stege, e-Preserv. Sci. 6:89–100, 2009). This study indicated that the minimum concentrations at which the presence of the minor pigment is detected in at least half of the determinations performed are between 0.9% and 1.5% for phthalocyanine blue, between 0.8% and 1.3% for Prussian blue and between 0.3% and 1% for ultramarine blue with SEM-EDS; with m-FTIR spectroscopy, the minimum concentrations are between 0.9% and 1.5% for phthalocyanine blue, 2.4% or more for Prussian blue and 19% or more for ultramarine blue. The concentrations measured for some pigments were high and may result in errors in the description of the composition of a piece of art. The use of complementary techniques may address this limitation. On the basis of these results, the application of analytical procedures that include replicate determinations and analyses of areas of 100 × 100 μm2 or greater and exclude single determinations are recommended to more accurately describe the material composition of a work of art.
Keywords: Fourier transform infrared; Scanning electron microscopy and energy-dispersive X-ray spectroscopy; Detection limit; Paintings; Heterogeneity; Ultramarine blue; Prussian blue; Phthalocyanine blue

Characterization of non-enzymatic acylation of amino or thiol groups of bionucleophiles by the acyl-adenylate or acyl-CoA thioester of cholic acid by Kuniko Mitamura; Eriko Aoyama; Toshihiro Sakai; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa (2253-2259).
Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are highly electrophilic acyl-linked metabolites which can undergo transacylation reactions with amino and thiol groups of nucleophilic groups on acceptor molecules such as amino acids, peptides, and proteins. Here, non-enzymatic acylation at pH 7.4 of glycine, taurine, glutathione (GSH), and N-acetylcysteine (NAC) by cholyl-adenylate (CA-AMP) was compared with that mediated by cholyl-CoA thioester (CA-CoA) using a 1:1 mixture of stable isotopically labeled CA-AMP and unlabeled CA-CoA. The transacylation products of these substrates were analyzed by liquid chromatography/electrospray ionization linear ion-trap mass spectrometry in negative-ion detection mode. CA-AMP was more reactive than CA-CoA with the amino group of glycine or taurine than with the thiol group of GSH or NAC. In contrast, CA-CoA was more reactive than CA-AMP with the thiol group of GSH or NAC and was far less reactive with the amino group of glycine or taurine. These differences in the reactivity of CA-AMP as compared with that of CA-CoA towards amino and thiol groups may be attributed to the electrophilicity of the carbonyl carbon of these acyl-linked cholic acid metabolites and the nucleophilicity of the amino and thiol group in the bionucleophiles that were studied.
Keywords: Glutathione; N-acetylcysteine; Glycine; Taurine; Liquid chromatography/electrospray ionization-mass spectrometry

The electrode characteristics and selectivities of PVC-based thiocyanate selective polymeric membrane electrode (PME) incorporating the newly synthesized zinc complex of 6,7:14,15-Bzo2-10,11-(4-methylbenzene)-[15]-6,8,12,14-tetraene-9,12-N2-1,5-O2 (I 1 ) and zinc complex of 6,7:14,15-Bzo2-10,11-(4-methylbenzene)-[15]-6,14-diene-9,12-dimethylacrylate-9,12-N2-1,5-O2 (I 2 ) are reported here. The best response was observed with the membrane having a composition of I2:PVC:o-NPOE:HTAB in the ratio of 6:33:59:2 (w/w; milligram). This electrode exhibited Nernstian slope for thiocyanate ions over working concentration range of 4.4 × 10−7 to 1.0 × 10−2 mol L−1 with detection limit of 2.2 × 10−7 mol L−1. The performance of this electrode was compared with coated graphite electrode (CGE), which showed better response characteristics w.r.t Nernstian slope 59.0 ± 0.2 mV decade−1 activity, wide concentration range of 8.9 × 10−8 to 1.0 × 10−2 mol L−1 and detection limit of 6.7 × 10−8 mol L−1. The response time for CGE and PME was found to be 8 and 10 s, respectively. The proposed electrode (CGE) was successfully applied to direct determination of thiocyanate in biological and environmental samples and also as indicator electrode in potentiometric titration of SCN ion.
Keywords: Macrocyclic Zn(II) complexes; Coated graphite electrode; Polymeric membrane electrode; Thiocyanate selective sensor; Potentiometry; Selectivity

Erratum to: A GC/MS-based metabolomic approach for diagnosing citrin deficiency by Tomiko Kuhara; Morimasa Ohse; Yoshito Inoue; Arthur J. L. Cooper (2271-2271).