Analytica Chimica Acta (v.954, #C)

Microalgae as embedded environmental monitors by Zachary L. Ogburn; Frank Vogt (1-13).
In marine ecosystems, microalgae are an important component as they transform large quantities of inorganic compounds into biomass and thereby impact environmental chemistry. Of particular relevance is phytoplankton's sequestration of atmospheric CO2, a greenhouse gas, and nitrate, one cause of harmful algae blooms. On the other hand, microalgae sensitively respond to changes in their chemical environment, which initiates an adaptation of their chemical composition. Analytical methodologies were developed in this study that utilize microalgae's adaptation as a novel approach for in-situ environmental monitoring. Longterm applications of these novel methods are investigations of environmental impacts on phytoplankton's sequestration performance and their nutritional value to higher organisms feeding on them.In order to analyze the chemical composition of live microalgae cells (Nannochloropsis oculata), FTIR-ATR spectroscopy has been employed. From time series of IR spectra, the formation of bio-sediment can be monitored and it has been shown that the nutrient availability has a small but observable impact. Since this bio-sediment formation is governed by several biological parameters of the cells such as growth rate, size, buoyancy, number of cells, etc., this enables studies of chemical environment's impact on biomass formation and the cells' physical parameters.Moreover, the spectroscopic signature of these microalgae has been determined from cultures grown under 25 different CO2 and NO3 mixtures (200 ppm-600 ppm CO2, 0.35 mM-0.75 mM NO3 ). A novel, nonlinear modeling methodology coined ‘Predictor Surfaces’ is being presented by means of which the nonlinear responses of the cells to their chemical environment could reliably be described. This approach has been utilized to measure the CO2 concentration in the atmosphere over the phytoplankton culture as well as the nitrate concentration dissolved in their growing environment. The achieved precision of concentration predictions were a few percent of the measurement range. Moreover, the Predictor Surface itself allows for a chemical interpretation of the cells' response to a shift in their chemical environment. This will open new approaches to study the link between concentration levels in an ecosystem and the biological consequences for this ecosystem.Novel Predictor Surfaces describe how the IR signature of e.g. the marine microalgae Nannochloropsis oculata changes as a function of their chemical environment. By means of these nonlinear calibration models, for instance atmospheric CO2 can be quantified accurately.Display Omitted
Keywords: Environmental monitoring; FTIR-ATR spectroscopy; Nonlinear chemometrics; Microalgae;

The support vector machine (SVM) is a powerful classifier that has recently been implemented in a classification tree (SVMTreeG). This classifier partitioned the data by finding gaps in the data space. For large and complex datasets, there may be no gaps in the data space confounding this type of classifier. A novel algorithm was devised that uses fuzzy entropy to find optimal partitions for situations when clusters of data are overlapped in the data space. Also, a kernel version of the fuzzy entropy algorithm was devised. A fast support vector machine implementation is used that has no cost C or slack variables to optimize. Statistical comparisons using bootstrapped Latin partitions among the tree classifiers were made using a synthetic XOR data set and validated with ten prediction sets comprised of 50,000 objects and a data set of NMR spectra obtained from 12 tea sample extracts.Display Omitted
Keywords: Chemometrics; SVM classification tree; Fuzzy entropy; SVMTreeG; SVMTreeH; Tea; Authentication; Kernel entropy of classification;

Convolutional neural networks for vibrational spectroscopic data analysis by Jacopo Acquarelli; Twan van Laarhoven; Jan Gerretzen; Thanh N. Tran; Lutgarde M.C. Buydens; Elena Marchiori (22-31).
In this work we show that convolutional neural networks (CNNs) can be efficiently used to classify vibrational spectroscopic data and identify important spectral regions. CNNs are the current state-of-the-art in image classification and speech recognition and can learn interpretable representations of the data. These characteristics make CNNs a good candidate for reducing the need for preprocessing and for highlighting important spectral regions, both of which are crucial steps in the analysis of vibrational spectroscopic data.Chemometric analysis of vibrational spectroscopic data often relies on preprocessing methods involving baseline correction, scatter correction and noise removal, which are applied to the spectra prior to model building. Preprocessing is a critical step because even in simple problems using ‘reasonable’ preprocessing methods may decrease the performance of the final model.We develop a new CNN based method and provide an accompanying publicly available software. It is based on a simple CNN architecture with a single convolutional layer (a so-called shallow CNN). Our method outperforms standard classification algorithms used in chemometrics (e.g. PLS) in terms of accuracy when applied to non-preprocessed test data (86% average accuracy compared to the 62% achieved by PLS), and it achieves better performance even on preprocessed test data (96% average accuracy compared to the 89% achieved by PLS). For interpretability purposes, our method includes a procedure for finding important spectral regions, thereby facilitating qualitative interpretation of results.Display Omitted
Keywords: Vibrational spectroscopy; Convolutional neural networks; Preprocessing;

Standardization from a benchtop to a handheld NIR spectrometer using mathematically mixed NIR spectra to determine fuel quality parameters by Neirivaldo Cavalcante da Silva; Cláudia Jéssica Cavalcanti; Fernanda Araújo Honorato; Jose Manuel Amigo; Maria Fernanda Pimentel (32-42).
The interest in performing in field measures using portable instruments is growing increasingly. Calibration transfer techniques can be used to enable models, predicted values or spectra obtained in a benchtop instrument be used in portable instrument, saving money and time required for a complete recalibration. Most of the calibration transfer methods require a set of transfer samples which spectra have to be acquired in both spectrometers. The present work evaluates the use of virtual standards as transfer samples in the reverse standardization (RS) method in order to standardize very dissimilar spectral responses of fuel samples (gasoline and biodiesel blends) from a high-resolution benchtop Frontier FT-NIR (PerkinElmer) spectrometer and a handheld MicroNIR™1700 (JDSU). These virtual standards can be created by mathematically mixing spectra from the pure solvents present in gasoline or diesel/biodiesel (D/B) blends, to avoid volatilization and changes in the composition of the compounds during storage and/or transportation of the real transfer fuel samples. Virtual standards were created using ten and five pure solvents for gasoline and D/B blends, respectively. Partial least squares regression (PLS) models were built for five quality parameters of gasoline (distillation temperatures at 10%, 50%, 90% and final boiling point (FBP) volume recovered and density) and one of D/B blends (biodiesel content). The RMSEP values obtained after the standardization approaches were equivalent to the reproducibility of the reference methods, except for density and biodiesel content parameters obtained for the virtual samples standardization approach. RS procedure provided promising results showing that it is possible to transfer gasoline or D/B blend spectra acquired with a high-resolution benchtop instrument to the handheld MicroNIR using virtual standards as transfer samples.Display Omitted
Keywords: Reverse standardization; Virtual standards; Near infrared; Handheld spectrometer; Fuel;

Integration of noble metal nanomaterials on graphene nanosheets potentially paves one way to improve their electronic, chemical and electrochemical properties. The study reported synthesis of palladium@gold nanoalloys/nitrogen and sulphur-functionalized multiple graphene aerogel composite (Pd@Au/N,S-MGA). The as-prepared composite offers a well-defined three-dimensional architecture with rich of mesopores. The Pd@Au nanoalloys were dispersed on the graphene framework networks and their active sites were fully exposed. The unique structure achieves to ultra high electron/ion conductivity, electrocatalytic activity and structural stability. The sensor based on the Pd@Au/N,S-MGA creates ultrasensitive electrochemical response towards dopamine due to significantly electrochemical synergy between Pd, Au and N,S-MGA. Its differential pulse voltammetric signal linearly increases with the increase of dopamine concentration in the range from 1.0 × 10−9 M to 4.0 × 10−5 M with the detection limit of 3.6 × 10−10 M (S/N = 3). The analytical method provides the advantage of sensitivity, reproducibility, rapidity and long-term stability. It has been successfully applied in the detection of trace dopamine in biological samples. The study also opens a window on the electronic properties of graphene aerogel and metal nanomaterials as well their nanohybrids to meet needs of further applications as nanoelectronics in diagnosis, bioanalysis and catalysis.We reported a new palladium@gold nanoalloys/nitrogen and sulphur-functionalized multiple graphene aerogel. The sensor based on the nanohybrid exhibits ultrahigh sensitivity, reproducibility and stability to electrochemical detection of dopamine.Display Omitted
Keywords: Palladium@gold nanoalloys; Nitrogen and sulphur-functionalized multiple graphene aerogel; Dopamine; Electrochemical sensor; Detection;

Surface-coated wooden-tip electrospray ionization mass spectrometry for determination of trace fluoroquinolone and macrolide antibiotics in water by Jiewei Deng; Tiantian Yu; Yao Yao; Qi Peng; Lijuan Luo; Baowei Chen; Xiaowei Wang; Yunyun Yang; Tiangang Luan (52-59).
In this study, a surface-coated wooden-tip electrospray ionization mass spectrometry (SCWT-ESI-MS) method was developed for rapid and sensitive analysis of trace fluoroquinolone and macrolide antibiotics in water. First, a SCWT solid-phase microextration (SPME) probe was prepared, via silanization and sulfonation for modification of a layer of adsorbent containing both C8-chain and sulfo group on the surface of wooden tips. Then, the SCWT-SPME probe was applied for extraction of trace fluoroquinolone and macrolide antibiotics in water. The specially designed adsorbent gave the probe desirable enrichment capacity towards fluoroquinolone and macrolide antibiotics, with enrichment factors of approximately 100–500 folds for six target analytes. After extraction, the loaded SCWT-SPME probe was directly applied for ambient MS analysis. With the application of a high voltage and some spray solvent on the SCWT-SPME probe, analytes enriched on the probe was desorbed and ionized for mass spectrometric analysis under ambient and open-air conditions. The method was sensitive, with limits of detection and quantification of 1.8–4.5 ng/L and 5.9–15.1 ng/L, respectively. The method also showed good linearity, with correlation coefficient values (r 2) of no less than 0.9940 for six target analytes. The developed method was successfully applied for the analysis of six fluoroquinolone and macrolide antibiotics in tap and river water samples, and no antibiotic was found in tap water but some antibiotics were detected in river water with concentrations at dozens to hundreds nanogram-per-liter level. Standard addition experiments were also performed, and the obtained recoveries were 89–102% for tap waters and 82–92% for river waters, respectively. All the experimental results demonstrated that our proposed SCWT-ESI-MS method was rapid, sensitive, and reliable for analyzing trace antibiotics in water.Display Omitted
Keywords: Surface-coated wooden-tip; Mass spectrometry; Ambient ionization; Solid-phase microextraction; Antibiotics;

β-carboline alkaloids of the harmala group (HAlks)—a family of compounds with pharmacologic effects—can be found at trace levels (<25 μg kg−1 algae) in the edible invasive algae Undaria pinnatifida, known commonly as wakame. In this study, we present a simple and sensitive method to detect and quantify at low parts-per-trillion levels the six HAlks more frequently found in those plants. The method is based on on-line solid phase extraction capillary electrophoresis mass spectrometry using a C18 sorbent. First, the methodology was optimized and validated with standard solutions through the use of ultraviolet (UV) and mass spectrometry (MS) detection. Second, the optimized method for MS detection was applied to an analysis of the HAlks in U. pinnatifida extracts. The method achieved limits of detection between 2 and 77 pg mL−1 for standards, producing an analyte preconcentration of about 1000-times in comparison to CE-MS. Some matrix effects were observed for the complex wakame extracts, especially for the most polar HAlks (harmol and harmalol), which bear aromatic hydroxyl groups. Harmine, harmaline, and norharmane were not detected in the algal extracts, whereas harmane was found at 70 pg mL−1 (70 ng kg−1 dry algae). The results underscored that C18-SPE-CE-MS may be considered as a powerful method to detect trace levels of alkaloids and other bioactive small molecules in complex plant extracts.Display Omitted
Keywords: Algae; Capillary electrophoresis; Harmala alkaloids; In-line or on-line preconcentration; Mass spectrometry;

Silicon isotope ratio measurements by inductively coupled plasma tandem mass spectrometry for alteration studies of nuclear waste glasses by Alkiviadis Gourgiotis; Thomas Ducasse; Evelyne Barker; Patrick Jollivet; Stéphane Gin; Sylvain Bassot; Charlotte Cazala (68-76).
High-level, long-lived nuclear waste arising from spent fuel reprocessing is vitrified in silicate glasses for final disposal in deep geologic formations. In order to better understand the mechanisms driving glass dissolution, glass alteration studies, based on silicon isotope ratio monitoring of 29Si-doped aqueous solutions, were carried out in laboratories.This work explores the capabilities of the new type of quadrupole-based ICP-MS, the Agilent 8800 tandem quadrupole ICP-MS/MS, for accurate silicon isotope ratio determination for alteration studies of nuclear waste glasses. In order to avoid silicon polyatomic interferences, a new analytical method was developed using O2 as the reaction gas in the Octopole Reaction System (ORS), and silicon isotopes were measured in mass-shift mode. A careful analysis of the potential polyatomic interferences on SiO+ and SiO2 + ion species was performed, and we found that SiO+ ion species suffer from important polyatomic interferences coming from the matrix of sample and standard solutions (0.5M HNO3). For SiO2 +, no interferences were detected, and thus, these ion species were chosen for silicon isotope ratio determination. A number of key settings for accurate isotope ratio analysis like, detector dead time, integration time, number of sweeps, wait time offset, memory blank and instrumental mass fractionation, were considered and optimized. Particular attention was paid to the optimization of abundance sensitivity of the quadrupole mass filter before the ORS. We showed that poor abundance sensitivity leads to a significant shift of the data away from the Exponential Mass Fractionation Law (EMFL) due to the spectral overlaps of silicon isotopes combined with different oxygen isotopes (i.e. 28Si16O18O+, 30Si16O16O+). The developed method was validated by measuring a series of reference solutions with different 29Si enrichment. Isotope ratio trueness, uncertainty and repeatability were found to be <0.2%, <0.5% and <0.6%, respectively. These performances meet the requirements of the studies of nuclear glasses alteration and open up possibilities to use this method for precise determination of silicon content in natural samples by Isotope Dilution.Display Omitted
Keywords: Silicon isotope ratios; Triple quadrupole; ICP-QQQ; ICP-MS/MS; Nuclear glasses; Tandem mass spectrometry;

Highly sensitive quantification for human plasma-targeted metabolomics using an amine derivatization reagent by Naoko Arashida; Rumi Nishimoto; Masashi Harada; Kazutaka Shimbo; Naoyuki Yamada (77-87).
Amino acids and their related metabolites play important roles in various physiological processes and have consequently become biomarkers for diseases. However, accurate quantification methods have only been established for major compounds, such as amino acids and a limited number of target metabolites. We previously reported a highly sensitive high-throughput method for the simultaneous quantification of amines using 3-aminopyridyl-N-succinimidyl carbamate as a derivatization reagent combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS). Herein, we report the successful development of a practical and accurate LC-MS/MS method to analyze low concentrations of 40 physiological amines in 19 min. Thirty-five of these amines showed good linearity, limits of quantification, accuracy, precision, and recovery characteristics in plasma, with scheduled selected reaction monitoring acquisitions. Plasma samples from 10 healthy volunteers were evaluated using our newly developed method. The results revealed that 27 amines were detected in one of the samples, and that 24 of these compounds could be quantified. Notably, this new method successfully quantified metabolites with high accuracy across three orders of magnitude, with lowest and highest averaged concentrations of 31.7 nM (for spermine) and 18.3 μM (for α-aminobutyric acid), respectively.Display Omitted
Keywords: High performance liquid chromatography; Tandem mass spectrometry; Derivatization; Plasma; Targeted metabolomics;

This paper describes a simple method for the fabrication of screen-printed based electrodes for amperometric detection on microchip electrophoresis (ME) devices. The procedure developed is quite simple and does not require expensive instrumentation or sophisticated protocols commonly employed on the production of amperometric sensors, such as photolithography or sputtering steps. The electrodes were fabricated through manual deposition of home-made conductive carbon ink over patterned acrylic substrate. Morphological structure and electrochemical behavior of the carbon electrodes were investigated by scanning electron microscopy and cyclic voltammetry. The produced amperometric sensors were coupled to polydimethylsiloxane (PDMS) microchips at end-channel configuration in order to evaluate their analytical performance. For this purpose, electrophoretic experiments were carried out using nitrite and ascorbic acid as model analytes. Separation of these substances was successfully performed within 50s with good resolution (R = 1.2) and sensitivities (713.5 pA/μM for nitrite and 255.4 pA/μM for ascorbate). The reproducibility of the fabrication method was evaluated and revealed good values concerning the peak currents obtained (8.7% for nitrite and 9.3% for ascorbate). The electrodes obtained through this method exhibited satisfactory lifetime (ca. 400 runs) over low fabrication cost (less than $1 per piece). The feasibility of the proposed device for real analysis was demonstrated through the determination of nitrite concentration levels in drinking water samples. Based on the results achieved, the approach proposed here shows itself as an interesting alternative for simple fabrication of carbon-based electrodes. Furthermore, the devices indicate great promise for other kind of analytical applications involving ME devices.Display Omitted
Keywords: Electrochemical detection; Microfabrication; Microfluidic devices; PDMS microchips; Working electrode;

A benzothiazole-based fluorescent probe for distinguishing and bioimaging of Hg2+ and Cu2+ by Biao Gu; Liyan Huang; Wei Su; Xiaoli Duan; Haitao Li; Shouzhuo Yao (97-104).
A new benzothiazole-based fluorescent probe 2-(benzo[d]thiazol-2-yl)-4-(1,3- dithian-2-yl)phenol (BT) with two different reaction sites, a thioacetal group (site 1 for Hg2+), and O and N atoms of the benzothiazole dye (site 2 for Cu2+), was designed and synthesized. The probe BT showed ratiometric fluorescent response to Hg2+ and fluorescence quenching behavior to Cu2+, which induces naked-eye fluorescent color changes from green to blue and colorless, respectively. Moreover, it displayed highly sensitivity and selectivity toward Hg2+ and Cu2+ without interference from other metal ions. The sensing mechanisms were also confirmed by 1H NMR titration, mass spectrum and Job's plot analyses. Finally, probe BT was successfully used for fluorescent imaging of Hg2+ and Cu2+ in living cells, demonstrating its potential applications in biological science.Display Omitted
Keywords: Fluorescent probe; Hg2+ and Cu2+; Selectivity; Fluorescent imaging;

Robust l-malate bienzymatic biosensor to enable the on-site monitoring of malolactic fermentation of red wines by Pablo Giménez-Gómez; Manuel Gutiérrez-Capitán; Fina Capdevila; Anna Puig-Pujol; Cesar Fernández-Sánchez; Cecilia Jiménez-Jorquera (105-113).
Monitoring the malolactic fermentation process is strictly required to guarantee the sensorial quality and freshness of red wines. This could be achieved by in-field and real-time continuous measurements of l-malate concentration in the fermentation tanks. The potential of a miniaturized amperometric bienzymatic biosensor as an analytical tool to be applied in such scenario is described in this paper. The biosensor comprises a thin-film gold electrode as transducer, malate dehydrogenase (MDH) and diaphorase (DP) enzymes together with nicotinamide adenine dinucleotide (NAD+) cofactor as the selective receptor and an adequate redox mediator to record the corresponding amperometric signal. Three different biosensor architectures are studied, whose main differences lie in the immobilization of the different chemical components onto the electrode surface. In all cases a fast-electrosynthethized polypyrrole (PPy) membrane is generated for this purpose. The experimental conditions are optimized and the best architecture shows a sensitivity of 1365 ± 110 mA M−1 cm−2 and a detection limit of 6.3 × 10−8 M in a concentration range of 1 × 10−7 M – 1 × 10−6 M. The biosensor presents an excellent working stability as it retains above 90% of its sensitivity after 37 days, thus enabling the monitoring of the malolactic fermentation of three red wines. The obtained results show excellent agreement with the standard colorimetric method.Display Omitted
Keywords: Amperometric bienzymatic biosensor; Electrosynthesized polypyrrole membrane; Immobilization of redox mediator; l-malic acid analysis; Malolactic fermentation;

High-sensitive surface plasmon resonance microRNA biosensor based on streptavidin functionalized gold nanorods-assisted signal amplification by Kaihong Hao; Yu He; Huiting Lu; Shaotao Pu; Yingnan Zhang; Haifeng Dong; Xueji Zhang (114-120).
Herein, a facile and sensitive microRNA (miRNA) biosensor was designed by using interfacial biotinylated thiolated DNA molecular beacon (MB) as probe and streptavidin functionalized gold nanorods (Stre-GNRs) as tag for the enhanced surface plasmon resonance (SPR) signal. The MB probe with two terminals labeled with biotin and thiol groups, respectively, was modified on the gold film via thiol-gold interaction. Upon hybridization with the target, the biotinylated group became accessible to the Stre-GNRs. The introduction of the Stre-GNRs tag to the gold film produced strong SPR signal for detection. Our work has illustrated that the plasmonic field extension generated from the gold film to GNRs and the mass increase due to the GNRs have led to drastic sensitivity enhancement. Under optimal conditions, this proposed approach allowed detection of miRNA with the limit of detection (LOD) down to 0.045 pM. The results have shown that the MB probe functionalized sensing film, together with streptavidin-conjugated GNRs, was readily served as a plasmonic coupling partner that can be used as a powerful ultrasensitive sandwich assay for miRNA detection, and GNRs were readily served as promising amplification labels in SPR sensing technology.Display Omitted
Keywords: MicroRNA detection; Streptavidinylated gold nanorods; Surface plasmon resonance (SPR); Signal amplification;

Single nucleotide polymorphism discrimination with and without an ethidium bromide intercalator by Renzo A. Fenati; Ashley R. Connolly; Amanda V. Ellis (121-128).
Single nucleotide polymorphism (SNP) genotyping is an important aspect in understanding genetic variations. Here, we discriminate SNPs using toe-hold mediated displacement reactions. The biological target is an 80 nucleotide long double-stranded–DNA from the mtDNA HV1 region, associated with maternal ancestry. This target has been specially designed with a pendant toehold and a cationic fluorophore, ATTO 647N, as a reporter, produced in a polymerase chain reaction. Rates of reaction for the toehold-polymerase chain reaction products (TPPs) with their corresponding complementary displacing sequences, labelled with a Black Hole Quencher 1, followed the order TPP–Cytosine > TPP–Thymine > TPP–Adenine ≥ TPP–Guanine. Non-complementary rates were the slowest with mismatches involving cytosine. These reactions, operating in a static/or contact mode, gave averaged readouts between SNPs within 15 min (with 80–90% quenching), compared to 25–30 min in previous studies involving fluorescence resonance energy transfer. Addition of an intercalating agent, ethidium bromide, retarded the rate of reaction in which cytosine was involved, presumably through stabilization of the base pairing, which resulted in markedly improved discrimination of cytosine containing SNPs.Display Omitted
Keywords: Nucleotide polymorphism; Strand displacement; Fluorescence quenching; Genotyping; Kinetics;

A weak cation exchange liquid chromatography stationary phase (nylon-COOH) was prepared by grafting polyacrylic acid on to native nylon 6 capillary-channeled polymer (C-CP) fibers via a microwave-assisted radical polymerization. To the best of our knowledge, this is the first study of applying microwave-assisted grafting polymerization to affect nylon material for protein separation. The C-CP fiber surfaces were characterized by attenuated total reflection (ATR) infrared spectroscopy and scanning electron microscope (SEM). The anticipated carbonyl peak at 1722.9 cm−1 was found on the nylon-COOH fibers, but was not found on the native fiber, indicating the presence of the polyacrylic acid on nylon fibers after grafting. The nylon-COOH phase showed a ∼12× increase in lysozyme dynamic binding capacity (∼12 mg mL−1) when compared to the native fiber phase (∼1 mg mL−1). The loading capacity of the nylon-COOH phase is nearly independent of the lysozyme loading concentration (0.05–1 mg mL−1) and the mobile phase linear velocity (7.3–73 mm s−1). The reproducibility of the lysozyme recovery from the nylon-COOH (RSD = 0.3%, n = 10) and the batch-to-batch variability in the functionalization (RSD = 3%, n = 5) were also investigated, revealing very high levels of consistency. Fast baseline separations of myoglobin, α-chymotrypsinogen A, cytochrome c and lysozyme were achieved using the nylon-COOH column. It was found that a 5× increase in the mobile phase linear velocity (7.3-to-36.5 mm s−1) had little effect on the separation resolution. The microwave-assisted grafting polymerization has great potential as a generalized surface modification methodology across the applications of C-CP fibers.Display Omitted
Keywords: Capillary-channeled polymer; Fiber; Surface modification; Microwave-assisted polymerization; Ion exchange; Chromatography;

The confident identification and in-depth profiling of molecular lipid species remain to be a challenge in lipidomics analysis. In this work, an off-line two-dimensional mixed-mode and reversed-phase liquid chromatography (RPLC) method combined with high-field quadrupole orbitrap mass spectrometer (Q Exactive HF) was developed to profile lipids from complex biological samples. In the first dimension, 22 different lipid classes were separated on a monolithic silica column with elution order from neutral to polar lipids. A total of 13 fractions were collected and run on a RPLC C30 column in the second dimension for further separation of the lipid molecular species based on their hydrophobicity, with the elution order being determined by both the length and degree of unsaturation in the fatty-acyl chain. The method was applied to analyze lipids extracted from rat plasma and rat liver. Fatty acid methyl ester analysis by gas chromatography-mass spectrometry was used to identify the fatty acyls from total lipid extracts, which provided a more confident identification of the lipid species present in these samples. More than 800 lipids were identified in each sample and their molecular structures were confidentially confirmed using tandem mass spectrometry (MS/MS). The number of lipid molecular species identified in both rat plasma and rat liver by this off-line two-dimensional method is approximately twice of that by one-dimensional RPLC-MS/MS employing a C30 column. This off-line two-dimensional mixed-mode LC-RPLC-MS/MS method is a promising technique for comprehensive lipid profiling in complex biological matrices.Display Omitted
Keywords: Rat plasma lipidome; Rat liver lipidome; Off-line two-dimensional liquid chromatography; Untargeted lipidomics; Monolithic column; Accucore C30 column;

Novel assay of antibacterial components in manuka honey using lucigenin-chemiluminescence-HPLC by Koji Karasawa; Shiomi Haraya; Sachie Okubo; Hidetoshi Arakawa (151-158).
Five components (hydrogen peroxide, methylglyoxal, dihydroxyacetone, fructose and glucose) of New Zealand manuka honey (Leptospermum scoparium) were analyzed using lucigenin chemiluminescence high-performance liquid chromatography (lucigenin-CL-HPLC). We focused on active oxygen species produced from the components in order to easily detect these five components contained in manuka honey. H2O2 and O2 generated from these components were identified by lucigenin-CL and electron spin resonance (ESR), and the bactericidal effect of ROS was confirmed using E. coli. The previously reported assays for Manuka honey components have low specificities and require complicated preprocessing methods. As our results, the detection and identification of these components were possible within 30 min in lucigenin-CL-HPLC system, without any special treatment. It is considered that lucigenin-CL-HPLC is useful for the quality control and the analysis of various honey.Display Omitted
Keywords: Manuka honey; Lucigenin; HPLC; Chemiluminescence; Active oxygen;