Check out our New Publishers' Select for Free Articles

Analytical and Bioanalytical Chemistry (v.402, #3)

Luca Prodi, Marco Montalti and Nelsi Zaccheroni (Eds.): Luminescence applied in sensor science by Dario M. Bassani (pp. 997-998).

Wolf D. Lehmann: Protein phosphorylation analysis by electrospray mass spectrometry. A guide to concepts and practice by Jürgen H. Gross (pp. 999-1000).

Gold nanoparticle fluorescent molecular beacon for low-resolution DQ2 gene HLA typing by Valerio Beni; Taye Zewdu; Hamdi Joda; Ioanis Katakis; Ciara K. O’Sullivan (pp. 1001-1009).

Coeliac disease is an inflammation of the small intestine triggered by gluten ingestion. We present a fluorescent genosensor, exploiting molecular-beacon-functionalized gold nanoparticles, for the identification of human leukocyte antigen (HLA) DQ2 gene, a key genetic factor in coeliac disease. Optimization of sensor performance was achieved by tuning the composition of the oligonucleotide monolayer immobilized on the gold nanoparticle and the molecular beacon design. Co-immobilization of the molecular beacon with a spacing oligonucleotide (thiolated ten-thymine oligonucleotide) in the presence of ten-adenine oligonucleotides resulted in a significant increase of the sensor response owing to improved spacing of the molecular beacons and extension of the distance from the nanoparticle surface, which renders them more available for recognition. Further increase in the response (approximately 40%) was shown to be achievable when the recognition sequence of the molecular beacon was incorporated in the stem. Improvement of the specificity of the molecular beacons was also achieved by the incorporation within their recognition sequence of a one-base mismatch. Finally, gold nanoparticles functionalized with two molecular beacons targeting the DQA1*05* and DQB1*02* alleles allowed the low-resolution typing of the DQ2 gene at the nanomolar level.

Keywords: Gold nanoparticles; HLA typing; Molecular beacons; Fluorescent genosensor; Coeliac disease

Low-temperature direct bonding of glass nanofluidic chips using a two-step plasma surface activation process by Yan Xu; Chenxi Wang; Yiyang Dong; Lixiao Li; Kihoon Jang; Kazuma Mawatari; Tadatomo Suga; Takehiko Kitamori (pp. 1011-1018).

Owing to the well-established nanochannel fabrication technology in 2D nanoscales with high resolution, reproducibility, and flexibility, glass is the leading, ideal, and unsubstitutable material for the fabrication of nanofluidic chips. However, high temperature (~1,000 °C) and a vacuum condition are usually required in the conventional fusion bonding process, unfortunately impeding the nanofluidic applications and even the development of the whole field of nanofluidics. We present a direct bonding of fused silica glass nanofluidic chips at low temperature, around 200 °C in ambient air, through a two-step plasma surface activation process which consists of an O₂ reactive ion etching plasma treatment followed by a nitrogen microwave radical activation. The low-temperature bonded glass nanofluidic chips not only had high bonding strength but also could work continuously without leakage during liquid introduction driven by air pressure even at 450 kPa, a very high pressure which can meet the requirements of most nanofluidic operations. Owing to the mild conditions required in the bonding process, the method has the potential to allow the integration of a range of functional elements into nanofluidic chips during manufacture, which is nearly impossible in the conventional high-temperature fusion bonding process. Therefore, we believe that the developed low-temperature bonding would be very useful and contribute to the field of nanofluidics. Figure Direct bonding of fused silica glass nanofluidic chips at low temperature, around 200 °C in ambient air, through a two-step plasma surface activation process which consists of an O₂ RIE plasma treatment followed by a nitrogen MW radical activation

Keywords: Glass; Bonding; Low temperature; Plasma; Nanofluidics; Nanochannel

Gold nanoparticle-based fluorescence immunoassay for malaria antigen detection by Bassem S. S. Guirgis; Cláudia Sá e Cunha; Inês Gomes; Miguel Cavadas; Isabel Silva; Gonçalo Doria; Gregory L. Blatch; Pedro V. Baptista; Eulália Pereira; Hassan M. E. Azzazy; Maria M. Mota; Miguel Prudêncio; Ricardo Franco (pp. 1019-1027).

The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL⁻¹. The estimated LOD for the assay is 2.4 μg.mL⁻¹ and the LOQ is 7.3 μg.mL⁻¹. The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL⁻¹. Figure A simple and sensitive competitive immunoassay based on fluorescence-quenching by gold nanoparticles, successfully detected malaria antigens in blood cultures infected with 1,000 parasites.μL^-1.

Keywords: Malaria diagnosis; Gold nanoparticles; Nanodiagnosis; Heat shock protein; Fluorescence detection; Immunoassay; Plasmodium

Effect of carbon nanotube functionalization in micro-solid-phase extraction (μ-SPE) integrated into the needle of a syringe by Madhuleena Bhadra; Ornthida Sae-Khow; Somenath Mitra (pp. 1029-1039).

In this paper, we report the implementation of polar and nonpolar functionalized multiwalled carbon nanotubes (MWCNTs) as sorbent in μ-SPE integrated into the needle of a syringe. Excellent preconcentration of diverse pharmaceutical analytes was possible without the need for specific pH adjustments using just 300 μg of functionalized nanotubes. Enrichment factors were as high as 6.4, extraction efficiencies were as high as 25.6%, and detection limits as low as 0.08 ng/ml were obtained. The sorption on nanotubes followed Freundlich isotherms, and it was seen that polar analytes adsorbed more strongly on carboxylated MWCNTs, while amphoteric, relatively less polar and basic analytes had greater affinity for MWCNT and those with octadecylamine functionalization.

Keywords: Carbon nanotubes; Functionalization; Sorbent; Micro-SPE; SPE

Zirconium arsenate-modified silica nanoparticles for specific capture of phosphopeptides and direct analysis by matrix-assisted laser desorption/ionization mass spectrometry by Pei-Xuan Zhao; Xiao-Feng Guo; Hong Wang; Chu-Bo Qi; He-Shun Xia; Hua-Shan Zhang (pp. 1041-1056).

In this paper, we report, as far as we are aware, the first use of zirconium arsenate-modified silica nanoparticles (ZrAs-SNPs) for specific capture of phosphopeptides, followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI MS) analysis. Under the optimized enrichment conditions, the efficiency and specificity of ZrAs-SNPs were evaluated with tryptic digests of four standard proteins (α-casein, β-casein, ovalbumin, and bovine serum albumin) and compared with those of titanium arsenate-modified silica nanoparticles (TiAs-SNPs). The results showed that more selective enrichment of multiply phosphorylated peptides was observed with ZrAs-SNPs than with TiAs-SNPs whereas TiAs-SNPs resulted in slightly better recovery of singly phosphorylated peptides. ZrAs-SNPs were chosen for direct capture of phosphopeptides from diluted human serum of healthy and adenocarcinoma individuals. Our experimental profiling of serum phosphopeptides revealed that the level of phosphorylated fibrinogen peptide A was up-regulated in the serum of adenocarcinoma patients in comparison with healthy adults. This suggests the possibility of using ZrAs-SNPs for discovery of biomarkers of the pathogenesis process of tumors. Figure ZrAs-SNPs with trapped phosphopeptides

Keywords: Zirconium arsenate-modified silica nanoparticles; Phosphopeptide enrichment; Singly/multiply phosphorylated peptides; Human serum; MALDI MS

Metal-enhanced fluorescent probes based on silver nanoparticles and its application in IgE detection by Xia Wei; Hui Li; Zhonghui Li; Maika Vuki; Yu Fan; Wenying Zhong; Danke Xu (pp. 1057-1063).

In this paper, a novel metal plasmon coupled with an aptamer–nucleotide hybridized probe was fabricated and applied for protein detection. The specific aptamer and single-strand oligonucleotide were chemically bound to silver nanoparticles (AgNPs), and Cy5-labeled, complementary single-strand oligonucleotides were hybridized with the particle-bound oligonucleotides. The hybridized DNA duplexes were regarded as rigid rods that separated the fluorophore Cy5 and the surface of AgNPs to reduce the competitive quenching. Using a model system comprising human immunoglobulin E (IgE) as the analyte and goat antihuman IgE as immobilized capture antibody on glass slides, we demonstrate that the detection performance of the synthetic probe was superior to the aptamer-based fluorescent probes. The results showed a good linear correlation for human IgE in the range from 10 ng/ml to 6.25 μg/ml. The detection limit obtained was 1 ng/ml, which was 50 times lower than that using Cy5 oligonucleotide/aptamer hybrid duplex (Probe2) due to the metal-enhanced fluorescence effect. This new strategy opens the possibility for the preparation of high-sensitivity detection probes based on metal nanoparticles.

Keywords: Metal-enhanced fluorescence; Silver nanoparticles; Aptamer; IgE

Trapping cells on a stretchable microwell array for single-cell analysis by Yuli Wang; Pavak Shah; Colleen Phillips; Christopher E. Sims; Nancy L. Allbritton (pp. 1065-1072).

There is a need for a technology that can be incorporated into routine laboratory procedures to obtain a continuous, quantitative, fluorescence-based measurement of the dynamic behaviors of numerous individual living cells in parallel, while allowing other manipulations, such as staining, rinsing, and even retrieval of targeted cells. Here, we report a simple, low-cost microarray platform that can trap cells for dynamic single-cell analysis of mammalian cells. The elasticity of polydimethylsiloxane (PDMS) was utilized to trap tens of thousands of cells on an array. The PDMS microwell array was stretched by a tube through which cells were loaded on the array. Cells were trapped on the array by removal of the tube and relaxation of the PDMS. Once that was accomplished, the cells remained trapped on the array without continuous application of an external force and permitted subsequent manipulations, such as staining, rinsing, imaging, and even isolation of targeted cells. We demonstrate the utility of this platform by multicolor analysis of trapped cells and monitoring in individual cells real-time calcium flux after exposure to the calcium ionophore ionomycin. Additionally, a proof of concept for target cell isolation was demonstrated by using a microneedle to locally deform the PDMS membrane in order to retrieve a particular cell from the array.

Keywords: Bioanalytical methods; Cell systems/single-cell analysis; Biochips/high-throughput screening

Trapping cells on a stretchable microwell array for single-cell analysis by Yuli Wang; Pavak Shah; Colleen Phillips; Christopher E. Sims; Nancy L. Allbritton (pp. 1065-1072).

Keywords: Bioanalytical methods; Cell systems/single-cell analysis; Biochips/high-throughput screening

Discrimination of primitive endoderm in embryoid bodies by Raman microspectroscopy by Maha A. El-Hagrasy; Eiichi Shimizu; Masato Saito; Yoshinori Yamaguchi; Eiichi Tamiya (pp. 1073-1081).

Embryoid bodies (EBs), derived from aggregated embryonic stem (ES) cells, are capable of differentiating into all three germ layers, including the endoderm, mesoderm, and ectoderm. The initial stage of EB differentiation is the formation of a primitive endoderm (PE) layer located at the periphery of the aggregate. Raman microspectroscopy was employed to segregate PE cells from undifferentiated ES cells. The Raman spectra of the PE cells of the periphery of EBs, formed upon the withdrawal of leukemia inhibitory factor (LIF), were compared with those of the undifferentiated ES cells of the core of cell aggregates, formed in the presence of LIF. It was noticed that the PE cells have high contents of proteins and low contents of nucleic acids, lipids, and carbohydrates compared with ES cells. Also, we established the presence of another population of PE cells located in the core of the EBs. In addition, we identified some specific Raman markers to distinguish PE cells from ES cells (e.g., I ₁₀₀₃/I ₉₃₇). This is the first study to investigate the PE cells of live EBs and define some Raman markers to distinguish them from undifferentiated ES cells. Figure Comparison between the Raman signature of primitive endoderm cells (PE) and undifferentiated embryonic stem cells (ES)

Keywords: Embryoid bodies; Differentiation; Primitive endoderm; Raman microspectroscopy; Glycogen

Polystyrene-coated micropallets for culture and separation of primary muscle cells by David A. Detwiler; Nicholas C. Dobes; Christopher E. Sims; Joe N. Kornegay; Nancy L. Allbritton (pp. 1083-1091).

Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures’ surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-μm thick, polystyrene copolymer was applied to the microstructures by contact printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells.

Keywords: Bioanalytical methods; Biomaterials; Cell systems; Single cell analysis

Distinguishing breast cancer cells using surface-enhanced Raman scattering by Jing Yang; Zhuyuan Wang; Shenfei Zong; Chunyuan Song; Ruohu Zhang; Yiping Cui (pp. 1093-1100).

The detection and identification of epidermal growth factor receptor 2 (HER2)-positive breast cancer cells is crucial for the clinic therapy of breast cancer. For the aim of the detection, a novel surface-enhanced Raman scattering (SERS) probe for distinguishing breast cancers at different HER2 statuses is reported in this paper. In such a probe, anti-HER2 antibody-conjugated silver nanoparticles have been synthesized for specific targeting of HER2-positive breast cancer cells. More importantly, different from the previously reported SERS probe for targeting cancer cells, p-mercaptobenzoic acid is utilized as both the Raman reporter and the conjugation agent for attaching antibody molecules, which leads to a much simplified structure. For investigating the ability of such a probe to distinguish breast cancer cells, SKBR3 and MCF7 cells were chosen as two model systems, which are HER2-positive- and HER2-negative-expressing cells, respectively. The experimental results reveal that SKBR3 cells exhibit much stronger SERS signals than MCF7 cells, indicating that the probe could be utilized to distinguish breast cancer cells at different HER2 statuses. This kind of SERS probe holds a potential for a direct detection of living breast cancer cells with the advantages of easy fabrication, high SERS sensitivity, and biocompatibility. Figure SERS spectra of the probe in SKBR3 cells and in MCF7 cells

Keywords: Surface-enhanced Raman scattering; HER2; Cancer targeting; SKBR3; MCF7

Growth hormone secretagogues: out of competition by Armand Pinyot; Zoran Nikolovski; Jaume Bosch; Gerard Such-Sanmartín; Shinji Kageyama; Jordi Segura; Ricardo Gutiérrez-Gallego (pp. 1101-1108).

Growth hormone secretagogues (GHS) constitute a new GH deficiency treatment increasing exponentially in number and improved potency and bioavailability over the last decade. The growth hormone releasing activity makes these compounds attractive for the artificial improvement of the human sports skills, now that recombinant human growth hormone (rhGH) administration is effectively detected. The GHS family is extremely diverse both in number and chemical heterogeneity and keeps growing continuously. In this paper, a general screening test is proposed. To develop a universal method, the single common property of growth hormone secretagogues has been targeted: their capacity to bind to the GHS receptor 1a (GHS-R1a). Pretreated urine samples have been tested in a competition assay where eventually the GHS presence detached a radiolabelled ligand from the receptor in a dose-dependent manner. Blank urine samples were processed to determine potential age, gender and exercise effects, and to define a threshold beyond which a specimen is considered positive. Samples from a growth hormone releasing peptide 2 (GHRP-2) excretion study corroborated the screening assay applicability with a detection window of approximately 4.5 h, and results were confirmed by comparison with a dedicated LC–MS quantification of the intact compound.

Keywords: Receptor binding; Growth hormone secretagogues; Anti-doping

Cord blood metabolomic profiling in intrauterine growth restriction by Donata Favretto; Erich Cosmi; Eugenio Ragazzi; Silvia Visentin; Marianna Tucci; Paolo Fais; Giovanni Cecchetto; Vincenzo Zanardo; Guido Viel; Santo Davide Ferrara (pp. 1109-1121).

A number of metabolic abnormalities have been observed in pregnancies complicated by intrauterine growth restriction (IUGR). Metabolic fingerprinting and clinical metabolomics have recently been proposed as tools to investigate individual phenotypes beyond genomes and proteomes and to advance hypotheses on the genesis of diseases. Non-targeted metabolomic profiling was employed to study fetal and/or placental metabolism alterations in IUGR fetuses by liquid chromatography high-resolution mass spectrometry (LC-HRMS) analysis of cord blood collected soon after birth. Samples were collected from 22 IUGR and 21 appropriate for gestational age (AGA) fetuses. Birth weight differed significantly between IUGR and AGA fetuses (p < 0.001). Serum samples were immediately obtained and deproteinized by mixing with methanol at room temperature and centrifugation; supernatants were lyophilized and reconstituted in water for analysis. LC-HRMS analyses were performed on an Orbitrap mass spectrometer linked to a Surveyor Plus LC. Samples were injected into a 1.0 × 150-mm Luna C18 column. Spectra were collected in full-scan mode at a resolution of approximately 30,000. Data were acquired over the m/z range of 50–1,000, with measurements performed in duplicate. To observe metabolic variations between the two sets of samples, LC-HRMS data were analyzed by a principal component analysis model. Many features (e.g., ionic species with specific retention times) differed between the two classes of samples: among these, the essential amino acids phenylalanine, tryptophan, and methionine were identified by comparison with available databases. Logistic regression coupled to a receiver-operating characteristic curve identified a cut-off value for phenylalanine and tryptophan, which gave excellent discrimination between IUGR and AGA fetuses. Non-targeted LC-HRMS analysis of cord blood collected at birth allowed the identification of significant differences in relative abundances of essential amino acids between IUGR and AGA fetuses, emerging as a promising tool for studying metabolic alterations.

Keywords: IUGR; Metabolomics; HRMS; Essential amino acids; Tryptophan; Phenylalanine

Phytoestrogen biomonitoring: an extractionless LC-MS/MS method for measuring urinary isoflavones and lignans by use of atmospheric pressure photoionization (APPI) by Daniel L. Parker; Michael E. Rybak; Christine M. Pfeiffer (pp. 1123-1136).

We present here a high-performance liquid chromatography−tandem mass spectrometry (LC-MS/MS) method for quantifying phytoestrogenic isoflavones (daidzein, equol, genistein, and O-desmethylangolensin) and lignans (enterodiol and enterolactone) in urine without the use of extraction or the preconcentration techniques inherent in existing methods. The development of this concept was made possible by use of atmospheric pressure photoionization (APPI); an ionization technique that we found to improve analyte sensitivity relative to electrospray ionization and atmospheric pressure chemical ionization for this particular group of compounds. The analytical performance of this method was equal to or exceeded that of comparable methods. Between-run coefficients of variation (CVs) across three quality control (QC) pool levels analyzed in duplicate over 20 days were 3.1–5.8% CV; within-run CVs were 2.3–6.0%. Accuracy, as determined by average spike recovery in QC pools, was generally within ±10% of being quantitative (100%). Relative limits of detection were 0.04–0.4 ng/mL urine, with absolute detection limits as low as 0.1 pg. This method was applied to the analysis of >2,500 urine specimens for the 2005–2006 Centers for Disease Control and Prevention’s National Health and Nutrition Examination Survey (NHANES). The method was capable of quantifying these compounds in 95–100% of study samples. This work is the first ever report of using APPI for the LC-MS/MS determination of these compounds in urine. It is also the first method of its kind to do so without any need for analyte extraction or preconcentration prior to analysis.

Keywords: Isoflavones; Lignans; Phytoestrogens; Urine; Atmospheric pressure photoionization; Electrospray; Mass spectrometry; ESI; APCI; APPI

Comprehensive study of proteins that interact with microcystin-LR by Tomoko Mori; Takuya Kubo; Kunimitsu Kaya; Ken Hosoya (pp. 1137-1147).

We carried out a comprehensive study of proteins that exhibit specific interactions with a naturally occurring toxin, microcystin (MC)-LR, in order to gain insight into the unknown underlying mechanism of MC virulence. This audacious study employed a simple affinity test that used MC-LR immobilized on an original ethylene oxide based monolithic solid phase (Moli-gel), and swine liver lysate. Some of the proteins that interacted with MC-LR on this original affinity resin were separated by SDS-PAGE, measured by nano-LC/MS/MS after trypsin digestion, and identified using a Mascot database search. Protein sequence analyses revealed that glutathione S-transferase (GST) was one of the candidate target proteins for MC-LR. This protein was confirmed as a target protein for MC-LR based on the results of for the inhibition of an enzymatic reaction by Dhb-MC-LR. Moreover, L-3-hydroxyacyl coenzyme A dehydrogenase (HDHA) was shown to be one of the proteins that specifically interacts with MC-LR. Our results demonstrated that our analytical systems based on an original affinity resin and nano-LC/MS/MS were effective for target protein research.

Keywords: Microcystin-LR; Target proteins; Affinity resin; Nano-LC/MS/MS

Photochemical properties and sensor applications of modified yellow fluorescent protein (YFP) covalently attached to the surfaces of etched optical fibers (EOFs) by Alexey A. Veselov; Bobin George Abraham; Helge Lemmetyinen; Matti T. Karp; Nikolai V. Tkachenko (pp. 1149-1158).

Fluorescent proteins have the inherent ability to act as sensing components which function both in vitro and inside living cells. We describe here a novel study on a covalent site-specific bonding of fluorescent proteins to form self-assembled monolayers (SAMs) on the surface of etched optical fibers (EOFs). Deposition of fluorescent proteins on EOFs gives the opportunity to increase the interaction of guided light with deposited molecules relative to plane glass surfaces. The EOF modification is carried out by surface activation using 3-aminopropylthrimethoxysilane (APTMS) and bifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) which exposes sulfhydryl-reactive maleimide groups followed by covalent site-specific coupling of modified yellow fluorescent protein (YFP). Steady-state and fluorescence lifetime measurements confirm the formation of SAM. The sensor applications of YPF SAMs on EOF are demonstrated by the gradual increase of emission intensity upon addition of Ca²⁺ ions in the concentration range from a few tens of micromolars up to a few tens of millimolars. The studies on the effect of pH, divalent cations, denaturing agents, and proteases reveal the stability of YFP on EOFs at normal physiological conditions. However, treatments with 0.5% SDS at pH 8.5 and protease trypsin are found to denaturate or cleave the YFP from fiber surfaces.

Keywords: Photochemistry; Biosensor; Self-assembled monolayer; Etched optical fiber; Time-resolved fluorescence

Improved sample preparation for MALDI–MSI of endogenous compounds in skin tissue sections and mapping of exogenous active compounds subsequent to ex-vivo skin penetration by Bernd Enthaler; Julia K. Pruns; Sonja Wessel; Claudius Rapp; Markus Fischer; Klaus-Peter Wittern (pp. 1159-1167).

Localization of endogenous and exogenous compounds directly in tissue sections is a challenging task in skin research. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful label-free technique that enables determination of the distribution of a large range of biomolecules directly in tissue sections. Nevertheless, its application in this field is limited in large part by the low adhesion of skin tissue sections to indium–tin oxide-coated (ITO) glass slides. For the first time corona discharge (CD) treatment was used to modify the glass slide surface for improved adhesion. Localization of endogenous cholesterol sulfate was performed directly in human skin tissue sections. A spatial resolution of approximately 30 μm was sufficient for assignment of mass signals to skin structure morphology. Furthermore, imaging of an exogenous model compound, Nile red, was performed directly in skin tissue sections after ex-vivo penetration into porcine skin, enabling determination of the pathway and depth of penetration. Finally, the ion density map of Nile red was compared with its high resolution fluorescence micrograph. This work provides new insights into the application of MALDI–MSI in skin research. Figure MALDI-MSI results for mapping of endogenous cholesterol sulfate (m/z 465.3) directly in human skin tissue sections with spatial resolutions of 100 μm (b) and 30 μm (d). Images are overlayed with the corresponding hematoxylin and eosin staining images, which are presented in a and c, respectively

Keywords: Mass spectrometry imaging; MALDI–MSI; Skin; Penetration

Identification of phospholipid species affected by miltefosine action in Leishmania donovani cultures using LC-ELSD, LC-ESI/MS, and multivariate data analysis by L. Imbert; R. G. Ramos; D. Libong; S. Abreu; P. M. Loiseau; P. Chaminade (pp. 1169-1182).

Leishmaniasis is a widespread parasitic disease principally treated by intravenous drugs. Hexadecylphosphocholine (miltefosine) has recently proved its efficacy by oral route. Although its mechanism of action has been investigated, and principally relies on perturbations of the metabolism of lipids and especially phospholipids, further studies need to be conducted to detect precisely which metabolic pathways are impacted. For this purpose, the present work proposes a complete lipidomic study focused on membrane phospholipids of clones of Leishmania donovani non-treated (NT), treated (T) and resistant (R) to miltefosine. Firstly, a separation of phospholipids in normal phase high-performance liquid chromatography (NP-HPLC) was coupled to a mass spectrometer (MS) equipped with an electrospray (ESI) ion source, and response was compared to evaporative light scattering detection (ELSD). Secondly, a quantification of phospholipid classes was performed using NP-HPLC/ESI/MS on NT, T and R clones of L. donovani. Thirdly, full-scan acquisitions of analyzed samples were compared using orthogonal signal correction-partial least square-discriminant analysis (OSC-PLS-DA) to highlight phospholipid molecular species of interest between the three types of clones. Structural determination of the most relevant species has finally been performed using tandem mass spectrometry. A first hypothesis on the effect of miltefosine on lipid metabolic pathways is then proposed.

Keywords: Phospholipids; Mass spectrometry; Electrospray; Lipidomic; Liquid chromatography; Leishmaniasis

Monitoring metabolites consumption and secretion in cultured cells using ultra-performance liquid chromatography quadrupole–time of flight mass spectrometry (UPLC–Q–ToF-MS) by Giuseppe Paglia; Sigrún Hrafnsdóttir; Manuela Magnúsdóttir; Ronan M. T. Fleming; Steinunn Thorlacius; Bernhard Ø. Palsson; Ines Thiele (pp. 1183-1198).

Here we present an ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method for extracellular measurements of known and unexpected metabolites in parallel. The method was developed by testing 86 metabolites, including amino acids, organic acids, sugars, purines, pyrimidines, vitamins, and nucleosides, that can be resolved by combining chromatographic and m/z dimensions. Subsequently, a targeted quantitative method was developed for 80 metabolites. The presented method combines a UPLC approach using hydrophilic interaction liquid chromatography (HILIC) and MS detection achieved by a hybrid quadrupole–time of flight (Q–ToF) mass spectrometer. The optimal setup was achieved by evaluating reproducibility and repeatability of the analytical platforms using pooled quality control samples to minimize the drift in instrumental performance over time. Then, the method was validated by analyzing extracellular metabolites from acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) treated with direct (A-769662) and indirect (AICAR) AMP activated kinase (AMPK) activators, monitoring uptake and secretion of the targeted compound over time. This analysis pointed towards a perturbed purine and pyrimidine catabolism upon AICAR treatment. Our data suggest that the method presented can be used for qualitative and quantitative analysis of extracellular metabolites and it is suitable for routine applications such as in vitro drug screening. Figure UPLC-MS analysis of extracellular metabolites from acute lymphoblastic leukemia cell lines treated with AMP activated kinase (AMPK) activators points out that purine catabolism is affected upon AICAR treatment.

Keywords: Metabolomics; Mass spectrometry; UPLC; HILIC; Exo-metabolome

Simultaneous quantification of methylated purines in DNA by isotope dilution LC-MS/MS coupled with automated solid-phase extraction by Chiung-Wen Hu; Chih-Ming Chen; Hsin Hui Ho; Mu-Rong Chao (pp. 1199-1208).

Since methylation at the N-7 and O⁶ positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N ⁷-methylguanine (N ⁷-MeG), O ⁶-methylguanine (O ⁶-MeG), and N ³-methyladenine (N ³-MeA) have been suggested as good biomarkers for assessing exposure to methylating agents. Here, we report the development of a sensitive and selective assay based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) to simultaneously measure N ⁷-MeG, O ⁶-MeG, and N ³-MeA in DNA hydrolysates. With the use of isotope internal standards (¹⁵N₅-N ⁷-MeG, d ₃-O ⁶-MeG, and d ₃-N ³-MeA) and online solid-phase extraction, DNA hydrolysates can be directly analyzed within 12 min without prior sample purification. The limits of detection were 0.02, 0.002, and 0.01 ng/mL on-column (6.1, 0.6, and 3.4 fmol) for N ⁷-MeG, O ⁶-MeG, and N ³-MeA, respectively. Inter- and intraday imprecision (CV) were 3.6–9.6% and 2.7–13.6%, respectively. Mean recoveries were 96–109%. This method was then applied to quantitate the amounts of methylated purines in calf thymus DNA treated with methyl methanesulfonate (MMS). The levels of N ⁷-MeG, O ⁶-MeG, and N ³-MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N ⁷-MeG, O ⁶-MeG, and N ³-MeA in MMS-treated DNA was found to be 1.00:0.0032:0.119, respectively. This LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of methylated lesions in DNA induced by methylating agents.

Keywords: Online solid-phase extraction; LC-MS/MS; Methylated DNA; Methyl methanesulfonate

Liquid chromatography tandem mass spectrometry determination of total budesonide levels in dog plasma after inhalation exposure by Seija Berg; Marika Melamies; Minna Rajamäki; Outi Vainio; Kimmo Peltonen (pp. 1209-1215).

A sensitive and selective method to quantify budesonide in dog plasma samples was developed and fully validated. Liquid–liquid extraction was followed by solid-phase extraction and liquid chromatography–tandem mass spectrometry with electrospray ionization. After reconstitution of the analytes in the mobile phase, samples were analysed by reversed-phase liquid chromatography with isocratic elution. d8-Budesonide was used as an internal standard, and characteristic transitions of d8-budesonide and budesonide were used for quantification. The method was validated with respect to selectivity, specificity, linearity, recovery, repeatability, reproducibility and limits of detection and quantification. The validated method was successfully applied to monitor the plasma levels of budesonide in dogs exposed to clinical doses of inhaled and intravenous drug.

Keywords: Budesonide; Sample preparation; LC-MS/MS; Plasma; Inhalation

A simple assay for the simultaneous determination of rosuvastatin acid, rosuvastatin-5S-lactone, and N-desmethyl rosuvastatin in human plasma using liquid chromatography–tandem mass spectrometry (LC-MS/MS) by Joyce S. Macwan; Ileana A. Ionita; Fatemeh Akhlaghi (pp. 1217-1227).

A simple and sensitive assay was developed and validated for the simultaneous quantification of rosuvastatin acid (RST), rosuvastatin-5S-lactone (RST-LAC), and N-desmethyl rosuvastatin (DM-RST), in buffered human plasma using liquid chromatography–tandem mass spectrometry (LC-MS/MS). All the three analytes and the corresponding deuterium-labeled (d6) internal standards were extracted from 50 μL of buffered human plasma by protein precipitation. The analytes were chromatographically separated using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 μm). The mobile phase comprised of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). The analytes were separated at baseline within 6.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode. The calibration curves for all three analytes were linear (R ≥ 0.9964, n = 3) over the concentration range of 0.1–100 ng/mL for RST and RST-LAC, and 0.5–100 ng/mL for DM-RST. Mean extraction recoveries ranged within 88.0–106%. Intra- and inter-run mean percent accuracy were within 91.8–111% and percent imprecision was ≤15%. Stability studies revealed that all the analytes were stable in matrix during bench-top (6 h on ice–water slurry), at the end of three successive freeze and thaw cycles and at −80°C for 1 month. The method was successfully applied in a clinical study to determine the concentrations of RST and the lactone metabolite over 12-h post-dose in patients who received a single dose of rosuvastatin. Figure A typical chromatogram depicting the peaks of rosuvastatin acid (RST), rosuvastatin-5S-lactone (RST-LAC), and N-desmethyl rosuvastatin (DM-RST) and their respective internal standards

Keywords: Assay; Lactone; LC-MS/MS; N-Desmethyl; Pharmacokinetics; Protein precipitation; Rosuvastatin

Attribution of the discrepancy between ELISA and LC-MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies by Shujie J. Wang; Steven T. Wu; Jochem Gokemeijer; Aberra Fura; Murli Krishna; Paul Morin; Guodong Chen; Karen Price; David Wang-Iverson; Timothy Olah; Russell Weiner; Adrienne Tymiak; Mohammed Jemal (pp. 1229-1239).

High-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) methods were developed for the quantification of a PEGylated scaffold protein drug in monkey plasma samples. The LC-MS/MS method was based on the extraction of the therapeutic protein with a water-miscible organic solvent and the subsequent trypsin digestion of the extract followed by the detection of a surrogate peptide. The assay was linear over a range of 10–3,000 ng/mL. The ELISA method utilized a therapeutic target-binding format in which the recombinant target antigen was used to capture the drug in the sample, followed by detection with an anti-PEG monoclonal antibody. The assay range was 30–2,000 ng/mL. A correlation study between the two methods was performed by measuring the drug concentrations in plasma samples from a single-dose pharmacokinetic (PK) study in cynomolgus monkeys following a 5-mg/kg subcutaneous administration (n = 4). In the early time points of the PK profile, the drug concentrations obtained by the LC-MS/MS method agreed very well with those obtained by the ELISA method. However, at later time points, the drug concentrations measured by the LC-MS/MS method were consistently higher than those measured by the ELISA method. The PK parameters calculated based on the concentration data showed that the two methods gave equivalent peak exposure (C _max) at 24–48 h. However, the LC-MS/MS results exhibited about 1.53-fold higher total exposure (AUC_tot) than the ELISA results. The discrepancy between the LC-MS/MS and ELISA results was investigated by conducting immunogenicity testing, anti-drug antibody (ADA) epitope mapping, and Western blot analysis of the drug concentrations coupled with Protein G separation. The results demonstrated the presence of ADA specific to the engineered antigen-binding region of the scaffold protein drug that interfered with the ability of the drug to bind to the target antigen used in the ELISA method. In the presence of the ADAs, the ELISA method measured only the active circulating drug (target-binding), while the LC-MS/MS method measured the total circulating drug. The work presented here indicates that the bioanalysis of protein drugs may be complicated owing to the presence of drug-binding endogenous components or ADAs in the post-dose (incurred) samples. The clear understanding of the behavior of different bioanalytical techniques vis-à-vis the potentially interfering components found in incurred samples is critical in selecting bioanalytical strategies for measuring protein drugs.

Keywords: ELISA; LC-MS/MS; Free and total drug; Immunogenicity; Pharmacokinetics; Therapeutic proteins

Ultrasound-enhanced surfactant-assisted dispersive liquid–liquid microextraction and high-performance liquid chromatography for determination of ketoconazole and econazole nitrate in human blood by Yating Xia; Xiaoran Zhi; Xiuhong Wang; Mi Chen; Jing Cheng (pp. 1241-1247).

A simple and efficient method, based on ultrasound-enhanced surfactant-assisted dispersive liquid–liquid microextraction (UESA-DLLME) followed by high-performance liquid chromatography (HPLC) has been developed for extraction and determination of ketoconazole and econazole nitrate in human blood samples. In this method, a common cationic surfactant, cetyltrimethylammonium bromide (CTAB), was used as dispersant. Chloroform (40 μL) as extraction solvent was added rapidly to 5 mL blood containing 0.068 mg mL⁻¹ CTAB. The mixture was then sonicated for 2 min to disperse the organic chloroform phase. After the extraction procedure, the mixture was centrifuged to sediment the organic chloroform phase, which was collected for HPLC analysis. Several conditions, including type and volume of extraction solvent, type and concentration of the surfactant, ultrasound time, extraction temperature, pH, and ionic strength were studied and optimized. Under the optimum conditions, linear calibration curves were obtained in the ranges 4–5000 μg L⁻¹ for ketoconazole and 8–5000 μg L⁻¹ for econazole nitrate, with linear correlation coefficients for both >0.99. The limits of detection (LODs, S/N = 3) and enrichment factors (EFs) were 1.1 and 2.3 μg L⁻¹, and 129 and 140 for ketoconazole and econazole nitrate, respectively. Reproducibility and recovery were good. The method was successfully applied to the determination of ketoconazole and econazole nitrate in human blood samples.

Keywords: Ultrasound-enhanced surfactant-assisted dispersive liquid–liquid microextraction; Ketoconazole; Econazole nitrate; High-performance liquid chromatography; Human blood sample

Qualitative studies on the metabolism and the toxicological detection of the fentanyl-derived designer drugs 3-methylfentanyl and isofentanyl in rats using liquid chromatography–linear ion trap–mass spectrometry (LC-MSⁿ) by Markus R. Meyer; Julia Dinger; Andrea E. Schwaninger; Dirk K. Wissenbach; Josef Zapp; Giselher Fritschi; Hans H. Maurer (pp. 1249-1255).

The opioid 3-methylfentanyl, a designer drug of the fentanyl type, was scheduled by the Controlled Substance Act due to its high potency and abuse potential. To overcome this regulation, isofentanyl, another designer fentanyl, was synthesized in a clandestine laboratory and seized by the German police. The aims of the presented study were to identify the phase I and phase II metabolites of 3-methylfentanyl and isofentanyl in rat urine, to identify the cytochrome P450 (CYP) isoenzymes involved in their initial metabolic steps, and, finally, to test their detectability in urine. Using liquid chromatography (LC)–linear ion trap–mass spectrometry (MSⁿ), nine phase I and five phase II metabolites of 3-methylfentanyl and 11 phase I and four phase II metabolites of isofentanyl could be identified. The following metabolic steps could be postulated for both drugs: N-dealkylation followed by hydroxylation of the alkyl and aryl moiety, hydroxylation of the propanamide side chain followed by oxidation to the corresponding carboxylic acid, and, finally, hydroxylation of the benzyl moiety followed by methylation. In addition, N-oxidation of isofentanyl could also be observed. All hydroxy metabolites were partly excreted as glucuronides. Using recombinant human isoenzymes, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were found to be involved in the initial metabolic steps. Our LC-MSⁿ screening approach allowed the detection of 0.01 mg/L of 3-methylfentanyl and isofentanyl in spiked urine. However, in urine of rats after the administration of suspected recreational doses, the parent drugs could not be detected, but their common nor metabolite, which should therefore be the target for urine screening.

Keywords: 3-Methylfentanyl; Isofentanyl; Metabolism; LC-MS; Rat urine; CYP isoenzymes

Rapid, simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry by Kenji Kuwayama; Kenji Tsujikawa; Hajime Miyaguchi; Tatsuyuki Kanamori; Yuko T. Iwata; Hiroyuki Inoue (pp. 1257-1267).

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05 − 5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations. Figure Detection of MDMA and its metabolites in rat liver homogenate by TLC/MALDI/ imaging MS

Keywords: Thin-layer chromatography; Matrix-assisted laser desorption/ionization; Imaging mass spectrometry; Drug analysis; Toxicology

Characterization of sodium hyaluronate blends using frit inlet asymmetrical flow field-flow fractionation and multiangle light scattering by Muhammad Ali; Euijin Hwang; Il-Hwan Cho; Myeong Hee Moon (pp. 1269-1276).

We characterized ultrahigh molecular weight sodium hyaluronate (NaHA) and blended pharmaceutical products containing NaHA using flow field-flow fractionation and multiangle light scattering–differential refractive index (FlFFF-MALS-DRI). NaHA is a water-soluble polysaccharide with a range of molecular weights (MW; 10⁵~10⁸ Da) that is found in body fluids and tissues. NaHA is also used commercially in pharmaceutical and cosmetic applications. We used a frit inlet asymmetrical FlFFF channel to separate aqueous polymers according to their hydrodynamic size, and we used on-line measurements of light scattering to obtain the MW distribution (MWD) as well as structural information about NaHA in aqueous solution. In this study, we investigated NaHA and anti-adhesive blend mixtures of NaHA (a commercial NaHA blend mixture containing sodium carboxymethyl cellulose and a new blend with hydroxyethyl starch (HES)) to determine the molecular weight distribution MWD of NaHA and the blend mixtures and to obtain structural information about these compounds in aqueous solution. We also examined the characteristics of NaHA–HES–polylactic-co-glycolic acid film products exposed to gamma radiation for sterilization purposes.

Keywords: Flow field-flow fractionation; Multiangle light scattering; Sodium hyaluronate (NaHA); NaHA blend mixtures

A hydrolysis procedure for the analysis of total cocaine residues in wastewater by Kevin J. Bisceglia; A. Lynn Roberts; Katrice A. Lippa (pp. 1277-1287).

We report a sample pretreatment approach for the analysis of total cocaine residues in wastewater that eliminates the need for two key assumptions often made in estimating cocaine utilization from measurement of its benzoylecgonine metabolite: that benzoylecgonine is neither degraded nor generated during transport in a sewer system, and that it is excreted as a constant fraction of cocaine ingested. By adding NaOH and incubating samples at 55 °C, cocaine and its principal metabolites are efficiently hydrolyzed into ecgonine, anhydroecgonine, and norecgonine. Ecgonine, estimated to represent between 37% and 90% (on a molar basis) of cocaine residues, can be directly determined (without preconcentration via solid-phase extraction (SPE)) by reversed-phase (RP) or hydrophilic interaction liquid chromatography–tandem mass spectrometry (LC/MS/MS). If samples are subjected to SPE, anhydroecgonine can also be determined; this metabolite (and its precursors) represents ≈7% of urinary cocaine residues (based on spot collections from living individuals). Although a reference standard for norecgonine is not commercially available, such nortropanes are also a minor fraction (up to 2%) of urinary cocaine residues. The stability of two human markers (cotinine and creatinine) to the hydrolysis procedure was also investigated. Results obtained by applying the hydrolysis approach for the analysis of total cocaine in an untreated municipal wastewater sample (obtained from Baltimore, MD) were generally in excellent agreement with those obtained from split samples analyzed using a more comprehensive solid-phase extraction RPLC/MS/MS method as described in our previous work. In particular, total tropane-based cocaine residues were found to be hydrolyzed to ecgonine with 98–99% efficiency.

Keywords: HILIC; Cocaine; Ecgonine; Anhydroecgonine; Hydrolysis; Transformation

FI on-line chemiluminescence reaction for determination of MCPA in water samples by S. Torres-Cartas; C. Gómez-Benito; S. Meseguer-Lloret (pp. 1289-1296).

This paper reports an economic, simple, and rapid FI-CL method for the determination of MCPA. This method requires simple instrumentation and it is fast enough to be used in routine analyses. A chemiluminescence signal is generated by reaction between photodegraded MCPA and ferricyanide solution in alkaline medium. All physical and chemical parameters in the flow injection chemiluminescence system were optimized in the experimental setting. To eliminate interference, a solid-phase extraction stage with SDB-1 cartridges and ethanol elution is applied. The signal–MCPA concentration relation is linear in concentration intervals between 0.0015 and 0.6 μg·mL⁻¹. The calibration lines are statistically similar in different working conditions: standards with ethanol without extraction and standards with ethanol and extraction, allowing standards to be excluded from the extraction step, which simplifies the process. The detection limit (DL) is 0.5 ng·mL⁻¹, which is the same order as the maximum limit established in legislation regarding pesticide limits in water destined for human consumption. A DL of 0.13 ng·mL⁻¹ can be reached if a sample of 100 mL is preconcentrated. The interday variance coefficient is 3% and the sample throughput is 90 h⁻¹. The water analysis method is efficient with relative error percentages lower than 5% with respect to the added concentration. Figure Flow injection manifold for chemiluminescence determination of MCPA

Keywords: MCPA; Chemiluminescence; Flow injection; Solid-phase extraction; SDB-1; Water samples; SPE

Comparative proteomic analysis of grain development in two spring wheat varieties under drought stress by P. Ge; C. Ma; S. Wang; L. Gao; X. Li; G. Guo; W. Ma; Y. Yan (pp. 1297-1313).

Two spring wheat varieties Ningchun 4 and Chinese Spring with good and poor resistance to abiotic stress, respectively, were used to investigate proteomic changes in the developing grains under drought stress by a comparative proteomics approach. A total of 152 protein spots showed at least twofold differences in abundance on two-dimensional electrophoresis (2-DE) maps, of which 28 and 68 protein spots were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry, respectively. Of the 96 identified protein spots, six different expression patterns were found and they were involved in stress/defense/detoxification, carbohydrate metabolism, photosynthesis, nitrogen metabolism, storage proteins and some other important functions. Comparative proteomic analysis revealed that under the drought conditions the decreased degree of ascorbate peroxidases was more significant in Chinese Spring than in Ningchun 4 during grain development whereas translationally controlled tumor protein, which was significantly upregulated at 14 DAF, was present in Ningchun 4 and absent in Chinese Spring. The Rubisco large subunit displayed an upregulated expression pattern in Ningchun 4. In addition, two drought-tolerant proteins, triosephosphate isomerase and oxygen-evolving complex showed B and F type expression patterns in Chinese Spring, but D and B types in Ningchun 4, respectively. These differentially expressed proteins might be responsible for the stronger drought resistance of Ningchun 4 compared to Chinese Spring.

Keywords: Spring wheat; Drought resistance; Protein expression; Proteome

Determination of perfluorinated chemicals in food and drinking water using high-flow solid-phase extraction and ultra-high performance liquid chromatography/tandem mass spectrometry by Ying-Chia Chang; Wen-Ling Chen; Fang-Yu Bai; Pau-Chung Chen; Gen-Shuh Wang; Chia-Yang Chen (pp. 1315-1325).

For this study, we developed methods of determining ten perfluorinated chemicals in drinking water, milk, fish, beef, and pig liver using high-flow automated solid-phase extraction (SPE) and ultra-high performance liquid chromatography/tandem mass spectrometry. The analytes were separated on a core-shell Kinetex C18 column. The mobile phase was composed of methanol and 10-mM N-methylmorpholine. Milk was digested with 0.5 N potassium hydroxide in Milli-Q water, and was extracted with an Atlantic HLB disk to perform automated SPE at a flow rate ranged from 70 to 86 mL/min. Drinking water was directly extracted by the SPE. Solid food samples were digested in alkaline methanol and their supernatants were diluted and also processed by SPE. The disks were washed with 40% methanol/60% water and then eluted with 0.1% ammonium hydroxide in methanol. Suppression of signal intensity of most analytes by matrixes was lower than 50%; it was generally lower in fish and drinking water but higher in liver. Most quantitative biases and relative standard deviations were lower than 15%. The limits of detection for most analytes were sub-nanograms per liter for drinking water and sub-nanograms per gram for solid food samples. This method greatly shortened the time and labor needed for digestion, SPE, and liquid chromatography. This method has been applied to analyze 14 types of food samples. Perfluorooctanoic acid was found to be the highest among the analytes (median at 3.2–64 ng/g wet weight), followed by perfluorodecanoic acid (0.7–25 ng/g) and perfluorododecanoic acid (0.6–15 ng/g). Figure Automated disk-type solid-phase extractors.

Keywords: Perfluorinated chemicals; Alkaline digestion; UHPLC-MS/MS; Disk SPE; Isotope-dilution techniques; Core shell

New identification of proanthocyanidins in cinnamon (Cinnamomum zeylanicum L.) using MALDI-TOF/TOF mass spectrometry by María Luisa Mateos-Martín; Elisabet Fuguet; Carmen Quero; Jara Pérez-Jiménez; Josep Lluís Torres (pp. 1327-1336).

The inner bark of Ceylon cinnamon (Cinnamomum zeylanicum L.) is commonly used as a spice and has also been widely employed in the treatment and prevention of disease. The positive health effects associated with the consumption of cinnamon could in part be due to its phenolic composition; proanthocyanidins (PA) are the major polyphenolic component in commercial cinnamon. We present a thorough study of the PA profile of cinnamon obtained using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition to the advantages of MALDI-TOF as a sensitive technique for the analysis of high-molecular-weight compounds, the tandem arrangement allows the identification of the compounds through their fragmentation patterns from MS/MS experiments. This is the first time that this technique has been used to analyze polymeric PA. The results show that cinnamon PA are more complex than was previously thought. We show here for the first time that they contain (epi)gallocatechin and (epi)catechingallate units. As gallates (galloyl moieties) and the pyrogallol group in gallocatechins have been related to the biological activity of grape and tea polyphenols, the presence of these substructures may explain some of the properties of cinnamon extracts. MALDI-TOF/TOF reveals that cinnamon bark PA include combinations of (epi)catechin, (epi)catechingallate, (epi)gallocatechin, and (epi)afzelechin, which results in a highly heterogeneous mixture of procyanidins, prodelphinidins, and propelargonidins.

Keywords: MALDI-TOF/TOF; Cinnamon; Polyphenols; Proanthocyanidins; Mass spectrometry

Molecularly imprinted polymers for selective extraction of synephrine from Aurantii Fructus Immaturus by Jie-Ping Fan; Lu Zhang; Xue-Hong Zhang; Jun-zhong Huang; Sheng Tong; Tao Kong; Zhe-You Tian; Jian-Hang Zhu (pp. 1337-1346).

In this work, molecularly imprinted solid-phase extraction (MISPE) has been used to selectively enrich, purify, or remove synephrine from Aurantii Fructus Immaturus. To this end, a molecularly imprinted polymer (MIP) was prepared by self-assembly from the template synephrine, the functional monomer methacrylic acid, and the crosslinker ethylene glycol dimethacrylate in 1:4:20 molar ratio. Subsequent molecular interrogation of the MIP binding sites revealed preferential structural selectivity for synephrine relative to other structurally related naturally occurring compounds (i.e. octopamine and tyramine ). This selectivity was subsequently exploited to achieve substantial sample clean-up of extracts of crude Aurantii Fructus Immaturus and Aurantii Fructus Immaturus stir-baked with bran. The purity of synephrine in the extracts after MISPE represented approximately 24.21-fold enrichment of the synephrine in the untreated extracts of Aurantii Fructus Immaturus stir-baked with bran. High recoveries (85–90%) from the samples proved that the method was valid for selective enrichment, purification, or removal of synephrine from Aurantii Fructus Immaturus. Figure The Molecularly imprinted polymers for the selective extraction of synephrine

Keywords: Molecularly imprinted polymers; Solid-phase extraction; Synephrine; Aurantii Fructus Immaturus

A luminescent bacterium assay of fusaric acid produced by Fusarium proliferatum from banana by Jing Li; Guoxiang Jiang; Bao Yang; Xinhong Dong; Linyan Feng; Sen Lin; Feng Chen; Muhammad Ashraf; Yueming Jiang (pp. 1347-1354).

Fusarium proliferatum was isolated as a major pathogen causing the Fusarium disease in harvested banana fruit. One of its major compounds, fusaric acid, was identified by high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI–MS). Because the light intensity of the luminescent bacterium Vibrio qinghaiensis sp. Nov. Q67 can be inhibited by fusaric acid, the fusaric acid content of F. proliferatum was assessed and compared by both the HPLC and luminescent bacterium methods. Although both methods afforded almost similar values of fusaric acid, the latter indicated slightly lower content than the former. Czapek medium was more suitable for the growth of F. proliferatum and fusaric acid production than modified Richard medium, with an optimum pH of approximately 7.0. However, no significant (P < 0.05) correlation was obtained between the fusaric acid production and growth of mycelia of F. proliferatum. The study suggests that the bioevaluation by use of the luminescent bacterium was effective in monitoring fusaric acid production by F. proliferatum without expensive equipment.

Keywords: Bio-evaluation; Fusaric acid; Fusarium proliferatum ; Luminescence analysis

Development of a bar adsorptive micro-extraction–large-volume injection–gas chromatography–mass spectrometric method for pharmaceuticals and personal care products in environmental water matrices by N. R. Neng; J. M. F. Nogueira (pp. 1355-1364).

The combination of bar adsorptive micro-extraction using activated carbon (AC) and polystyrene–divinylbenzene copolymer (PS-DVB) sorbent phases, followed by liquid desorption and large-volume injection gas chromatography coupled to mass spectrometry, under selected ion monitoring mode acquisition, was developed for the first time to monitor pharmaceutical and personal care products (PPCPs) in environmental water matrices. Assays performed on 25 mL water samples spiked (100 ng L⁻¹) with caffeine, gemfibrozil, triclosan, propranolol, carbamazepine and diazepam, selected as model compounds, yielded recoveries ranging from 74% to 99% under optimised experimental conditions (equilibrium time, 16 h (1,000 rpm); matrix characteristics: pH 5, 5% NaCl for AC phase; LD: methanol/acetonitrile (1:1), 45 min). The analytical performance showed good precision (RSD < 18%), convenient detection limits (5–20 ng L⁻¹) and excellent linear dynamic range (20–800 ng L⁻¹) with remarkable determination coefficients (r ² > 0.99), where the PS-DVB sorbent phase showed a much better efficiency. By using the standard addition methodology, the application of the present analytical approach on tap, ground, sea, estuary and wastewater samples allowed very good performance at the trace level. The proposed method proved to be a suitable sorption-based micro-extraction alternative for the analysis of priority pollutants with medium-polar to polar characteristics, showing to be easy to implement, reliable, sensitive and requiring a low sample volume to monitor PPCPs in water matrices.

Keywords: Sorption-based methods; Bar adsorptive micro-extraction (BAμE); PPCPs; LVI-GC-MS(SIM); Environmental water matrices; Trace analysis

Determination of selected pesticides by GC with simultaneous detection by MS (NCI) and μ-ECD in fruit and vegetable matrices by M. A. Martínez-Uroz; M. Mezcua; N. Belmonte Valles; A. R. Fernández-Alba (pp. 1365-1372).

A gas chromatography–mass spectrometry method in negative chemical ionization mode has been developed incorporating simultaneous detection using a micro-electron capture detector (μ-ECD) for the determination of pesticides in fruits and vegetables. This instrument configuration uses a three-way splitter device which divides the effluent from the analytical column between the two detectors with the split ratio 1:0.1 (MSD/μ-ECD) in each run. The μ-ECD was used for confirmation purposes. Validation of the method was performed on three matrices: tomato, apple, and orange. The ethyl acetate method was assayed; recovery studies were performed at 10 and 100 μg/kg. Recoveries between 70% and 120% were achieved and relative standard deviations lower than 20% (n = 5) were obtained for all pesticides and matrices studied. Limits of quantification lower than 10 μg/kg were obtained for 100% of pesticides in all of the matrices. Limits of quantification lower than 2.5 μg/kg were achieved for 77.8% of pesticides in the tomato and apple matrices, and for 72.2% of pesticides in the orange matrix. The method showed linear response in the concentration range tested (2.5–500 μg/kg) with correlation coefficients >0.99. Good repeatability and reproducibility results were obtained in all cases, with relative standard deviations lower than 16.7% and 20%, respectively. Finally, 20 incurred samples were analyzed using the proposed method. The simultaneous use of the two detectors was satisfactory for the analysis of these real samples. The total number of pesticides identified was 25. The number of samples which contained at least one pesticide was 15—this represented 75% of the total number of samples studied.

Keywords: Negative chemical ionization; GC-Q-MS; Micro- electron capture detector; Pesticides and food analysis

Evaluation of an analytical method for determining phthalate esters in wine samples by solid-phase extraction and gas chromatography coupled with ion-trap mass spectrometer detector by Mario Vincenzo Russo; Ivan Notardonato; Giuseppe Cinelli; Pasquale Avino (pp. 1373-1381).

A solid-phase extraction (SPE) method was developed for extraction and analysis of six phthalate esters in wine samples using Carbograph 1 sorbent. The SPE procedure allowed efficient recovery of the investigated phthalates ranging between 78% and 105% with a relative standard deviation (RSD) ≤6.5 for an ethanolic phthalic acid ester (PAE) standard solution and between 73–71% and 96–99% with a RSD ≤8.4 for red wine samples spiked with 20 and 50 ng mL⁻¹ of PAE, respectively. The adsorption isotherms and breakthrough curves for Carbograph 1/water solution were reported. Gas chromatography coupled with an ion-trap mass spectrometer detector (GC/IT-MS) was used for analysis. The instrumental analytical protocol was found to yield a linear calibration in the range 0.01-10.0 μg mL⁻¹ with R ² values ≥0.9992. The limits of detection in GC/IT-MS (SIM mode) vary between 0.2 and 14 ng mL⁻¹ (RSD ≤5.6) whereas the limits of quantification range between 0.5 and 25 ng mL⁻¹ (RSD ≤5.9); the intra- and inter-day repeatabilities calculated as RSD for wine samples, were between 0.9–7.8 and 1.0–10.5, respectively. The analytical method developed was applied to several commercial wine samples. Furthermore, the investigated methods are simple, reliable, reproducible, and not expensive.

Keywords: Carbograph 1; SPE study; Wine contaminants; Phthalate esters; GC-MS

Completely automated in-syringe dispersive liquid–liquid microextraction using solvents lighter than water by Fernando Maya; José Manuel Estela; Víctor Cerdà (pp. 1383-1388).

This paper describes the development of a new multisyringe flow injection analysis set-up that enables the complete automation of the dispersive liquid–liquid microextraction (DLLME) technique using solvents lighter than water. Its hyphenation with a liquid chromatographic separation is implemented using a single multisyringe pump obtaining a compact, simple, easy to operate, and fast instrument. DLLME is carried out with a throughput of 42 h⁻¹ and DLLME for the extraction of benzo(a)pyrene and its subsequent chromatographic determination can be carried out with an analysis throughput of 7 h⁻¹. Figure The extraction of benzo(a)pyrene has been accomplished developed a new completely automated in-syringe dispersive liquid-liquid microextraction by means of the Multisyringe flow injection analysis technique.

Keywords: Dispersive liquid–liquid microextraction; Multisyringe flow injection analysis; Sample treatment automation; Monolithic columns; Long pathlength spectrophotometry

Interference of flavonoids with enzymatic assays for the determination of free fatty acid and triglyceride levels by Elise F. Hoek-van den Hil; Karsten Beekmann; Jaap Keijer; Peter C. H. Hollman; Ivonne M. C. M. Rietjens; Evert M. van Schothorst (pp. 1389-1392).

Flavonoids are bioactive food compounds with potential lipid-lowering effects. Commercially available enzymatic assays are widely used to determine free fatty acid (FFA) and triglyceride (TG) levels both in vivo in plasma or serum and in vitro in cell culture medium or cell lysate. However, we have observed that various flavonoids interfere with peroxidases used in these enzymatic assays, resulting in incorrect lower FFA and TG levels than actually present. Furthermore, addition of isorhamnetin or the major metabolite of the flavonoid quercetin in human and rat plasma, quercetin-3-O-glucuronide, to murine serum also resulted in a significant reduction of the detected TG levels, while a trend was seen for FFA levels. It is concluded that when applying these assays, vigilance is needed and alternative analytical methods, directly assessing FFA or TG levels, should be used for studying the biological effects of flavonoids on FFA and TG levels. Figure Interference of different flavonoids spiked into cell culture medium on the FFA (A) and TG (B) levels, and interference of quercetin (Q) and its major metabolite, quercetin-3-O-glucuronide (Q3G), and isorhamnetin (I) spiked into murine serum on the FFA (C) and TG (D) levels, as measured by enzymatic assays. Flavonoids analysed are quercetin (Q), (+)-catechin (C), luteolin (L), genistein (G), naringenin (N), and kaempferol (K). Asterisk indicates a significant difference (p < 0.05) from the control (0 μM)

Keywords: Free fatty acids; Triglycerides; Enzymatic assays; Flavonoids; Peroxidase

Catecholamine analysis with strong cation exchange column liquid chromatography–peroxyoxalate chemiluminescence reaction detection by Makoto Tsunoda; Takashi Funatsu (pp. 1393-1397).

A liquid chromatography–chemiluminescence detection method was developed and validated for the determination of catecholamines (norepinephrine, epinephrine, and dopamine) in mouse brains. Chromatography was performed on a strong cation exchange column (150 × 2.0-mm id) using an isocratic mobile phase of 65 mM potassium acetate/75 mM potassium phosphate (95:5, pH 3.5) at a flow rate of 0.2 mL/min following post-column fluorescence derivatization of catecholamines with ethylenediamine and peroxyoxalate chemiluminescence reaction detection. The recovery of catecholamines added to mouse brain samples was more than 95.0%, while intra- and inter-day precision of the assay were <4.8%. The validated method was used to determine norepinephrine and dopamine concentrations in mouse brains without prior sample purification.

Keywords: Brain; Dopamine; Ion exchange; Mouse

Sections

Personal tools

Analytical and Bioanalytical Chemistry (v.402, #3)

Sections

Personal tools

Local resources

Analytical and Bioanalytical Chemistry (v.402, #3)