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Analytical and Bioanalytical Chemistry (v.405, #4)

Surviving toxic work environments by Apryll M. Stalcup (pp. 1145-1147).

is currently Professor of Chemical Sciences at Dublin City University and Director of the Irish Separation Science Cluster. She has over 20 years of experience in the area of intermolecular interactions, using separations as the primary interrogative tool. Both fundamental research and practical applications have been emphasized in her work. Her research group has used a variety of separation platforms, including gas chromatography, liquid chromatography, capillary electrophoresis and preparative electrophoresis. Her initial research focused on the development of new strategies for chiral separations and includes the introduction of sulfated β-cyclodextrin and heparin as chiral selectors for capillary electrophoresis and preparative electrophoretic chiral separations. Her research group has also been engaged in the synthesis of new chromatographic stationary phases, including surface-confined ionic liquids, and their characterization using linear solvation energy relationships. She is a member of the American Chemical Society, the American Association for the Advancement of Science, the American Nuclear Society and Sigma Xi.

Surviving toxic work environments by Apryll M. Stalcup (pp. 1145-1147).

61st Annual Conference on Application of X-ray Analysis: Denver X-ray Conference by Magnus Menzel (pp. 1149-1150).

Hermann Mascher: HPLC methods for clinical pharmaceutical analysis. A user’s guide by Petr Solich (pp. 1151-1152).

Effects of the expression level of epidermal growth factor receptor on the ligand-induced restructuring of focal adhesions: a QCM-D study by Marcela P. Garcia; Ammar Shahid; Jennifer Y. Chen; Jun Xi (pp. 1153-1158).

Epidermal growth factor receptor (EGFR) plays a major role in cell migration and invasion and is considered to be the primary source of activation of various malignant tumors. To gain insight into how elevated levels of EGFR influence cellular function, particularly cell motility, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to examine restructuring of focal adhesions in MCF-10A cells induced by epidermal growth factor. Engineered cells that overexpress epidermal growth factor receptor (EGFR) exhibited a very different kinetic profile from wildtype MCF-10A cells that have a lower level of EGFR with a higher rate for the initial disassembly of focal adhesion and a much lower rate for the later reassembly of focal adhesions. It is conceivable that these effects exhibited by EGFR-overexpressing cells may promote the initiation and maintenance of a more favorable adhesion state for cell migration. This study has demonstrated the capability of the dissipation monitoring function of the QCM-D to quantitatively assess kinetic aspects of cellular processes with a high temporal resolution and sensitivity. Figure Characterization of the effects of the expression level of epidermal growth factor receptor on the kinetics of the epidermal growth factor-induced restructuring of focal adhesions with the quartz crystal microbalance with dissipation monitoring.

Keywords: Focal adhesions; Cell motility; EGFR overexpression; Cancer invasion; QCM-D; Mechanical sensor

MALDI imaging in human skin tissue sections: focus on various matrices and enzymes by Bernd Enthaler; Maria Trusch; Markus Fischer; Claudius Rapp; Julia K. Pruns; Jens-Peter Vietzke (pp. 1159-1170).

Matrix-assisted laser/desorption ionization (MALDI) mass-spectrometric imaging (MSI), also known as MALDI imaging, is a powerful technique for mapping biological molecules such as endogenous proteins and peptides in human skin tissue sections. A few groups have endeavored to apply MALDI-MSI to the field of skin research; however, a comprehensive article dealing with skin tissue sections and the application of various matrices and enzymes is not available. Our aim is to present a multiplex method, based on MALDI-MSI, to obtain the maximum information from skin tissue sections. Various matrices were applied to skin tissue sections: (1) 9-aminoacridine for imaging metabolites in negative ion mode; (2) sinapinic acid to obtain protein distributions; (3) α-cyano-4-hydroxycinnamic acid subsequent to on-tissue enzymatic digestion by trypsin, elastase, and pepsin, respectively, to localize the resulting peptides. Notably, substantial amounts of data were generated from the distributions retrieved for all matrices applied. Several primary metabolites, e.g. ATP, were localized and subsequently identified by on-tissue postsource decay measurements. Furthermore, maps of proteins and peptides derived from on-tissue digests were generated. Identification of peptides was achieved by elution with different solvents, mixing with α-cyano-4-hydroxycinnamic acid, and subsequent tandem mass spectrometry (MS/MS) measurements, thereby avoiding on-tissue MS/MS measurements. Highly abundant peptides were identified, allowing their use as internal calibrants in future MALDI-MSI analyses of human skin tissue sections. Elastin as an endogenous skin protein was identified only by use of elastase, showing the high potential of alternative enzymes. The results show the versatility of MALDI-MSI in the field of skin research. This article containing a methodological perspective depicts the basics for a comprehensive comparison of various skin states. Figure Matrix-assisted laser/desorption ionization (MALDI) mass-spectrometric imaging (MSI), also known as MALDI imaging, is a powerful technique for mapping biological molecules in human skin tissue sections. In this body of work, a multiplex method, based on MALDI-MSI, is presented to obtain maximum information from skin tissue sections. Therefore, various matrices were applied to skin tissue sections: (1) 9-aminoacridine (9-AA) for imaging small molecules in negative ion mode; (2) sinapinic acid (SA) to obtain protein distributions; (3) α-cyano-4-hydroxycinnamic acid (α-HCHA) subsequent to on-tissue enzymatic digestion by trypsin, elastase, and pepsin, respectively, to localize the resulting peptides. Of note, identification of metabolites was achieved by post-source decay (PSD) MALDI, and proteins were identified subsequent to enzymatic digestion via the resulting peptides which were eluted from the skin tissue section and afterwards analyzed with use of a tandem time-of-flight (ToF) mass spectrometer. The application of alternative enzymes, such as pepsin and elastase, is highlighted within this article

Keywords: Matrix-assisted laser/desorption ionization mass-spectrometric imaging; Matrix-assisted laser/desorption ionization imaging; Matrix-assisted laser/desorption ionization matrices; Enzymes; Skin tissue

Effect-based proteomic detection of growth promoter abuse by Terence F. McGrath; Jeroen A. van Meeuwen; Anne-Cécile Massart; Edwin de Pauw; Philippe Delahaut; Jos Buijs; Aldert A. Bergwerff; Christopher T. Elliott; Mark H. Mooney (pp. 1171-1179).

Unregulated growth promoter use in food-producing animals is an issue of concern both from food safety and animal welfare perspectives. However, the monitoring of such practices is analytically challenging due to the concerted actions of users to evade detection. Techniques based on the monitoring of biological responses to exogenous administrations have been proposed as more sensitive methods to identify treated animals. This study has, for the first time, profiled plasma proteome responses in bovine animals to treatment with nortestosterone decanoate and 17β-oestradiol benzoate, followed by dexamethasone administration. Two-dimensional fluorescence differential in-gel electrophoresis analysis revealed a series of hepatic and acute-phase proteins within plasma whose levels were up- or down-regulated within phases of the treatment regime. Surface plasmon resonance (SPR) immuno-assays were developed to quantify responses of identified protein markers during the experimental treatment study with a view to developing methods which can be used as screening tools for growth promoter abuse detection. SPR analysis demonstrated the potential for plasma proteins to be used as indicative measures of growth promoter administrations and concludes that the sensitivity and robustness of any detection approach based on plasma proteome analysis would benefit from examination of a range of proteins representative of diverse biological processes rather being reliant on specific individual markers.

Keywords: Biomarker discovery; Growth promoters; Calves; 2D-DIGE; Depleted plasma; SPR protein assays

Characterisation and quantification of liposome-type nanoparticles in a beverage matrix using hydrodynamic chromatography and MALDI–TOF mass spectrometry by Johannes P. F. G. Helsper; Ruud J. B. Peters; Lambertus Brouwer; Stefan Weigel (pp. 1181-1189).

This paper describes the characterisation of liposome-type nanoparticles (NPs) dispersed in a beverage matrix. Characterisation is based on a two-step procedure: first, liposomes are separated on the basis of size in the nanometre range by use of hydrodynamic chromatography (HDC); second, chemical characterisation is performed by use of MALDI–TOF mass spectrometry (MS). Characterisation of three types of Coatsome liposome, a commercially available type of empty liposome, was investigated. All three liposome types, Coatsome A = anionic, N = neutral, and C = cationic, gave single peaks in HDC, reflecting diameters of 153, 187, and 205 nm, respectively. Subsequent MALDI–TOF MS in positive mode furnished major signals at m/z = 734.5 ([M + H]⁺ adduct) and m/z = 756.6 ([M + Na]⁺ adduct) of l-(α)-dipalmitoylphosphatidylcholine (DPPC) monomer and dimeric adducts at m/z = 1468.1 and m/z = 1490.1, respectively. MALDI–TOF MS in negative mode gave a signal at m/z = 721.3 ([M − H]⁻ adduct) of l-(α)-dipalmitoylphosphatidylglycerol (DPPG), except for Coatsome C which lacks this phospholipid. After HDC separation of Coatsome A NPs the major DPPC and DPPG signals can be detected in the expected fractions by use of MALDI–TOF MS in positive and negative modes, respectively. Validation of the analytical strategy revealed linearity (R ² > 0.99), repeatability (relative standard deviation <10 %), and reproducibility (relative standard deviation between days <10 %) were good, recovery was 61 ± 5 %, and the limit of quantification was 1 mg mL⁻¹ in this matrix. With 4 mg Coatsome A mL⁻¹ 20 out of 20 samples furnished the 734.5 and 756.6 signals typical of DPPC in MALDI–TOF MS characterisation.

Keywords: Hydrodynamic chromatography; Liposomes; MALDI–TOF; Organic nanoparticles; Validation

Gold nanorod separation and characterization by asymmetric-flow field flow fractionation with UV–Vis detection by Julien Gigault; Tae Joon Cho; Robert I. MacCuspie; Vincent A. Hackley (pp. 1191-1202).

The application of asymmetric-flow field flow fractionation (A4F) for low aspect ratio gold nanorod (GNR) fractionation and characterization was comprehensively investigated. We report on two novel aspects of this application. The first addresses the analytical challenge involved in the fractionation of positively charged nanoparticles by A4F, due to the interaction that exists between the negatively charged native membrane and the analyte. We show that the mobile phase composition is a critical parameter for controlling fractionation and mitigating the membrane-analyte interaction. A mixture of ammonium nitrate and cetyl trimethyl ammonium bromide at different molar ratios enables separation of GNRs with high recovery. The second aspect is the demonstration of shape-based separation of GNRs in A4F normal mode elution (i.e., Brownian mode). We show that the elution of GNRs is due both to aspect ratio and a steric-entropic contribution for GNRs with the same diameter. This latter effect can be explained by their orientation vector inside the A4F channel. Our experimental results demonstrate the relevance of the theory described by Beckett and Giddings for non-spherical fractionation (Beckett and Giddings, J Colloid and Interface Sci 186(1):53–59, 1997). However, it is shown that this theory has its limit in the case of complex GNR mixtures, and that shape (i.e., aspect ratio) is the principal material parameter controlling elution of GNRs in A4F; the apparent translational diffusion coefficient of GNRs increases with aspect ratio. Finally, the performance of the methodology developed in this work is evaluated by the fractionation and characterization of individual components from a mixture of GNR aspect ratios.

Keywords: Field flow fractionation; Gold; Nanoparticle; Nanorod; Shape separation; Hyphenated technique; Elution mechanism; Positive charge

Milk and serum standard reference materials for monitoring organic contaminants in human samples by Michele M. Schantz; Gauthier Eppe; Jean-François Focant; Coreen Hamilton; N. Alan Heckert; Rebecca M. Heltsley; Dale Hoover; Jennifer M. Keller; Stefan D. Leigh; Donald G. Patterson Jr; Adam L. Pintar; Katherine E. Sharpless; Andreas Sjödin; Wayman E. Turner; Stacy S. Vander Pol; Stephen A. Wise (pp. 1203-1211).

Four new Standard Reference Materials (SRMs) have been developed to assist in the quality assurance of chemical contaminant measurements required for human biomonitoring studies, SRM 1953 Organic Contaminants in Non-Fortified Human Milk, SRM 1954 Organic Contaminants in Fortified Human Milk, SRM 1957 Organic Contaminants in Non-Fortified Human Serum, and SRM 1958 Organic Contaminants in Fortified Human Serum. These materials were developed as part of a collaboration between the National Institute of Standards and Technology (NIST) and the Centers for Disease Control and Prevention (CDC) with both agencies contributing data used in the certification of mass fraction values for a wide range of organic contaminants including polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, polybrominated diphenyl ether (PBDE) congeners, and polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners. The certified mass fractions of the organic contaminants in unfortified samples, SRM 1953 and SRM 1957, ranged from 12 ng/kg to 2200 ng/kg with the exception of 4,4′-DDE in SRM 1953 at 7400 ng/kg with expanded uncertainties generally <14 %. This agreement suggests that there were no significant biases existing among the multiple methods used for analysis. Figure Comparison of Concentrations of Selected Compounds in Human Serum and Human Milk Standard Reference Materials (SRMs)

Keywords: Human Serum; Human Milk; SRMs; PCBs; Pesticides PBDEs; PCDFs; PCDDs

Comparison of different extraction methods for simultaneous determination of B complex vitamins in nutritional yeast using LC/MS-TOF and stable isotope dilution assay by Kristel Hälvin; Toomas Paalme; Ildar Nisamedtinov (pp. 1213-1222).

The application of LC/MS-TOF method combined with stable isotope dilution assay was studied for determination of thiamine, riboflavin, nicotinamide, nicotinic acid, pantothenic acid, pyridoxal, and pyridoxine in food. Nutritional yeast powder was used as a model food matrix. Acid extraction was compared with various enzymatic treatments in ammonium formate buffer to find a suitable method for the conversion of more complex vitamers into the same forms as the used isotope-labeled internal standards. The enzyme preparations α-amylase, takadiastase, β-glucosidase, and acid phosphatase were all able to liberate thiamine and riboflavin. The diastatic enzyme preparations α-amylase and takadiastase also expressed proteolytic side activities resulting in the formation of small peptides which interfered with the mass spectra of thiamine and riboflavin. Liberation of nicotinamide and pantothenic acid from NAD⁺ and CoA, respectively, could not be achieved with any of the studied enzyme preparations. Hydrochloric acid extraction at 121 °C for 30 min was found to be destructive to pantothenic acid, but increased the liberation of pyridoxal. Figure Comparison of different extraction methods for B complex vitamins determination in nutritional yeast

Keywords: B complex vitamins; Isotope dilution mass spectrometry; Liquid chromatography; MS-TOF; Nutritional yeast

Populus nigra L. bud absolute: a case study for a strategy of analysis of natural complex substances by Patrizia Rubiolo; Cristina Casetta; Cecilia Cagliero; Hugues Brevard; Barbara Sgorbini; Carlo Bicchi (pp. 1223-1235).

The new European regulations (e.g., REACH) require that Natural Complex Substances such as essential oils, absolutes, concretes, and resinoids are registered. This need implies that the chemical composition of these complex mixtures is characterized as exhaustively as possible in view of defining their toxicological risk. This study proposes an analysis strategy to be applied to the chemical characterization of poplar absolute as an example of Natural Complex Substances of vegetable origin. In the first part, the proposed strategy is described, and the advantages and the limitations related to the combination of conventional analytical techniques such as gas chromatography (GC) without and with sample derivatization and high-performance liquid chromatography (HPLC) are critically discussed. In the second part, the qualitative data obtained with GC and HPLC analysis of poplar bud absolute confirm the sample complexity which mainly consists of phenolic components. Fourteen compounds (i.e., phenolic acids, phenylpropanoids, and flavonoids) were then chosen as markers representative of the main classes of components characterizing poplar bud absolute. The marker quantitation carried out by GC-SIM-MS and HPLC-PDA analyses gives similar results confirming the reliability of both techniques. These results demonstrate that conventional analytical techniques can positively and effectively contribute to the study of the the composition of Natural Complex Substances, i.e., matrices for which highly effective separation is necessary, consisting mainly of isomers or homologous components. The combination of GC and HPLC techniques is ever more necessary for routine quality control when conventional instrumentations are used. Figure Elucidation of Natural Complex Substances (NCS)

Keywords: Natural Complex Substances (NCS); Populus nigra L.; High and low volatility compounds; GC derivatization; Qualitative analysis; Quantitative analysis

A concept study on non-targeted screening for chemical contaminants in food using liquid chromatography–mass spectrometry in combination with a metabolomics approach by Erik Tengstrand; Johan Rosén; Karl-Erik Hellenäs; K. Magnus Åberg (pp. 1237-1243).

A generic method to screen for new or unexpected contaminants at ppm levels in food has been developed. The method comprises an acidic acetonitrile extraction, detection with ultra-high-pressure liquid chromatography coupled to electrospray ionisation time-of-flight mass spectrometry and statistical evaluation using a metabolomics approach comparing suspected contaminated food with uncontaminated foods. The method was tested for 26 model contaminants from 100 μg/g down to 0.4 μg/g in three brands of fresh orange juice. Blinded statistical evaluation revealed signals from all added contaminants detectable by liquid chromatography–electrospray ionisation using positive ionisation mode, while only two false-positive signals were reported. The method is primarily intended to be used for investigation of food samples suspected to be contaminated with unknown substances. Additionally it could be used to continuously monitor for appearance of new food contaminants as a complement to the specific targeted analysis that is today’s foundation of food safety analysis.

Keywords: Chemometrics/statistics; Foods/beverages; HPLC; Mass spectrometry/ICP-MS

Ionic liquid-salt aqueous two-phase extraction based on salting-out coupled with high-performance liquid chromatography for the determination of sulfonamides in water and food by Juan Han; Yun Wang; Yan Liu; Yanfang Li; Yang Lu; Yongsheng Yan; Liang Ni (pp. 1245-1255).

Ionic liquid-salt aqueous two-phase extraction coupled with high-performance liquid chromatography with ultraviolet detection was developed for the determination of sulfonamides in water and food samples. In the procedure, the analytes were extracted from the aqueous samples into the ionic liquid top phase in one step. Three sulfonamides, sulfamerazine, sulfamethoxazole, and sulfamethizole were selected here as model compounds for developing and evaluating the method. The effects of various experimental parameters in extraction step were studied using two optimization methods, one variable at a time and Box–Behnken design. The results showed that the amount of sulfonamides did not have effect on the extraction efficiency. Therefore, a three-level Box–Behnken experimental design with three factors, which combined the response surface modeling, was used to optimize sulfonamides extraction. Under the most favorable extraction parameters, the detection limits (S/N = 3) and quantification limits (S/N = 10) of the proposed method for the target compounds were achieved within the range of 0.15–0.3 ng/mL and 0.5–1.0 ng/mL from spiked samples, respectively, which are lower than or comparable with other reported approaches applied to the determination of the same compounds. Finally, the proposed method was successfully applied to the determination of sulfonamide compounds in different water and food samples and satisfactory recoveries of spiked target compounds in real samples were obtained.

Keywords: Ionic liquid-salt aqueous two-phase extraction; Sulfonamides; High-performance liquid chromatography; Box–Behnken design; Water samples; Foods

Simultaneous determination of 24 or more acidic and alkaline phytohormones in femtomole quantities of plant tissues by high-performance liquid chromatography–electrospray ionization–ion trap mass spectrometry by Shichang Liu; Weiqi Chen; Long Qu; Ying Gai; Xiangning Jiang (pp. 1257-1266).

Phytohormones act at relatively low concentrations as major regulatory factors of plant growth and development, and cross talk of phytohormones is currently of great interest throughout the plant science community. To meet this demand, a method that is capable of simultaneously analyzing diverse plant hormones is essential. This paper introduces a high-performance liquid chromatographic separation technique coupled with sensitive and selective ion trap mass spectrometry to simultaneously determine 24 or more acidic and alkaline phytohormones, including auxin, cis- and trans-abscisic acid, 11 cytokinins, and 10 gibberellins, in a single injection of sample. A binary solid-phase extraction using Oasis MCX cartridges for cations and Oasis MAX cartridges for anions was used to prepurify more than 24 acidic and alkaline phytohormones from a single plant extract. The method showed good linearity for all 24 phytohormones with R ² values ranging from 0.9903 to 0.9997. Limits of detection for most of the phytohormones were in the femtomole range with some extending into the sub-femtomole range. This method was applied to hundreds of plant samples comprising different tissues from various plants, including herbaceous, woody climbing, and woody plants to demonstrate feasibility and to validate the methodology.

Keywords: HPLC–ESI–MSn; Phytohormone; Solid-phase extraction; Plant sample

Detection of flagellin by interaction with human recombinant TLR5 immobilized in liposomes by Y. Olguín; P. Villalobos; L. G. Carrascosa; M. Young; E. Valdez; L. Lechuga; R. Galindo (pp. 1267-1281).

Digestive diseases caused by flagellated bacteria are a huge public health problem worldwide and rapid detection methods are needed for contaminated environments. In this study, we propose a method to detect patterns associated with pathogens based on the properties of the innate immune system. Specifically, we use Toll-like receptor 5 (TLR5), a transmembrane protein that specifically recognizes flagellin (the structural protein of bacterial flagella). TLR5, which was obtained by recombinant production in insect cells, was immobilized into liposomes to form TLR5-proteoliposomes. Through surface plasmon resonance (SPR) and competition flow cytometry assays, the sensitivity of proteoliposomes to recognize Escherichia coli and Salmonella typhimurium flagellin was evaluated. In addition, we compared the results obtained by immobilizing anti-flagellin antibodies into liposomes. The results of the flagellin-affinity tests, expressed as an SPR kinetic rate constant ratio in the equilibrium equation K _D = k _d/k _a, showed values of 13.8 × 10⁻⁹ and 7.73 × 10⁻⁹ M for the TLR5-proteoliposomes and anti-flagellin antibodies, respectively, against S. typhimurium. The anti-flagellin affinity results for E. coli showed K _D of 84.1 × 10⁻⁸ M for SPR assays and K _D of 3.5 × 10⁻⁸ M for competitive flow cytometry, which was used as a detection system without the immobilization of proteoliposomes. This research demonstrates the practical possibility of using proteoliposomes as recognition elements in the generation of systems for the rapid detection of flagellated bacteria, which could help avoid consumption of contaminated food by humans and thereby prevent intestinal infections.

Keywords: Toll-like receptor 5; Baculovirus; Proteoliposomes; Flagellin; Surface plasmon resonance; Competitive flow cytometry

Strategy for quantifying trace levels of BMAA in cyanobacteria by LC/MS/MS by Liying Jiang; Eric Johnston; K. Magnus Åberg; Ulrika Nilsson; Leopold L. Ilag (pp. 1283-1292).

The cyanobacterial neurotoxin β-N-methylamino-l-alanine (BMAA) is an amino acid that is putatively associated with the pathology of amyotrophic lateral sclerosis/Parkinsonism–dementia complex (ALS-PDC) disease. It raises serious health risk concerns since cyanobacteria are ubiquitous thus making human exposure almost inevitable. The identification and quantification of BMAA in cyanobacteria is challenging because it is present only in trace amounts and occurs alongside structurally similar compounds such as BMAA isomers. This work describes an enhanced liquid chromatography/tandem mass spectrometry platform that can distinguish BMAA from its isomers β-amino-N-methyl-alanine, N-(2-aminoethyl) glycine (AEG), and 2,4-diaminobutyric acid, thus ensuring confident identification of BMAA. The method's sensitivity was improved fourfold by a post-column addition of acetonitrile. The instrument and method limits of detection were shown to be 4.2 fmol/injection (or 0.5 pg/one column) and 0.1 μg/g dry weight of cyanobacteria, respectively. The quantification method uses synthesized deuterated BMAA as an internal standard and exhibits good linearity, accuracy, and precision. Matrix effects were also investigated, revealing an ion enhancement of around 18 %. A lab-cultured cyanobacterial sample (Leptolyngbya PCC73110) was analyzed and shown to contain about 0.73 μg/g dry weight BMAA. The isomer AEG, whose chromatographic properties closely resemble those of BMAA, was also detected. These results highlight the importance of distinguishing BMAA from its isomers for reliable identification as well as providing a sensitive and accurate quantification method for measuring trace levels of BMAA in cyanobacterial samples.

Keywords: ALS-PDC; Isomers; AEG; BMAA; Post-column addition; Matrix effect

Exploring in vivo violacein biosynthesis by application of multivariate curve resolution on fused UV–VIS absorption, fluorescence, and liquid chromatography–mass spectrometry data by Clecio Dantas; Romà Tauler; Márcia Miguel Castro Ferreira (pp. 1293-1302).

In this work, the application of multivariate curve resolution-alternating least squares (MCR-ALS) is proposed for extracting information from multitechnique fused multivariate data (UV–VIS absorption, fluorescence, and liquid chromatography–mass spectrometry) gathered during the biosynthesis of violacein pigment. Experimental data sets were pretreated and arranged in a row-wise augmented data matrix before their chemometric investigation. Five different chemical components were resolved. Kinetic and spectral information about these components were obtained and their relationship with violacein biosynthesis was established. Three new chemical compounds with molar masses of 453, 465, and 479 u, until now not reported in the literature, were identified and proposed as intermediates in the biosynthesis of other indolocarbazoles. The precursor (tryptophan), one intermediate (deoxyviolacein), and the final product (violacein) of violacein biosynthesis were identified and characterized using the proposed approach. The chemometric procedure based on the MCR-ALS method has proved to be a powerful tool to investigate violacein biosynthesis and its application can be easily extended to the study of other bioprocesses.

Keywords: In vivo violacein biosynthesis; Multivariate curve resolution-alternating least squares; Data fusion; UV–VIS; Fluorescence spectroscopy; LC–MS

Identification of PET radiometabolites by cytochrome P450, UHPLC/Q-ToF-MS and fast radio-LC: applied to the PET radioligands [¹¹C]flumazenil, [¹⁸F]FE-PE2I, and [¹¹C]PBR28 by Nahid Amini; Ryuji Nakao; Magnus Schou; Christer Halldin (pp. 1303-1310).

A general method is presented for the identification of radiometabolites in plasma of human and monkey subjects after administration of positron emission tomography (PET) radioligands. The radiometabolites are first produced in vitro, using liver microsomes, subsequently separated using fast radio-liquid chromatography (radio-LC), and individually collected and identified by ultra high-performance liquid chromatography/quadrupole-time of flight-mass spectrometry in MS and MS^E mode. Fast radio-LC provided superior resolution compared to conventional radio-LC, resulting in separation of a greater number of metabolites. The radiometabolites produced in vivo are then compared to and identified based on the in vitro results. This approach was applied to three PET radioligands, [¹¹C]flumazenil, [¹⁸F]FE-PE2I, and [¹¹C]PBR28, resulting in the identification of five, two, and one radiometabolites, respectively. This procedure can easily be adopted to identify the radiometabolites produced in vivo from a variety of PET radioligands.

Keywords: Positron emission tomography (PET) radioligands; Radiometabolites; Fast radio-liquid chromatography (radio-LC); Ultra high-performance liquid chromatography/quadrupole-time of flight-mass spectrometry (UHPLC/Q-ToF-MS); Cytochrome P450

The effect of optical substrates on micro-FTIR analysis of single mammalian cells by Katia Wehbe; Jacob Filik; Mark D. Frogley; Gianfelice Cinque (pp. 1311-1324).

The study of individual cells with infrared (IR) microspectroscopy often requires living cells to be cultured directly onto a suitable substrate. The surface effect of the specific substrates on the cell growth—viability and associated biochemistry—as well as on the IR analysis—spectral interference and optical artifacts—is all too often ignored. Using the IR beamline, MIRIAM (Diamond Light Source, UK), we show the importance of the substrate used for IR absorption spectroscopy by analyzing two different cell lines cultured on a range of seven optical substrates in both transmission and reflection modes. First, cell viability measurements are made to determine the preferable substrates for normal cell growth. Successively, synchrotron radiation IR microspectroscopy is performed on the two cell lines to determine any genuine biochemically induced changes or optical effect in the spectra due to the different substrates. Multivariate analysis of spectral data is applied on each cell line to visualize the spectral changes. The results confirm the advantage of transmission measurements over reflection due to the absence of a strong optical standing wave artifact which amplifies the absorbance spectrum in the high wavenumber regions with respect to low wavenumbers in the mid-IR range. The transmission spectra reveal interference from a more subtle but significant optical artifact related to the reflection losses of the different substrate materials. This means that, for comparative studies of cell biochemistry by IR microspectroscopy, it is crucial that all samples are measured on the same substrate type. Figure Cell separation by PCA due to the refractive index of the substrate used, revealing transmission artifact.

Keywords: Single-cell analysis; Synchrotron radiation IR microspectroscopy; IR optical substrates; Transmission; Reflection; PCA

Vibrational spectroscopies for the analysis of cutaneous permeation: experimental limiting factors identified in the case of caffeine penetration by Sana Tfaili; Cyril Gobinet; Gwendal Josse; Jean-François Angiboust; Arlette Baillet; Michel Manfait; Olivier Piot (pp. 1325-1332).

Caffeine is utilised as a reference for permeation studies in dermatology and cosmetology. The present work aimed to monitor the permeation of a caffeine solution through the skin. For this purpose, Raman and infrared studies were performed. Raman microspectroscopy permitted a dynamic follow-up of the caffeine diffusion. In complementary, infrared microimaging provided information of the caffeine localization in the skin by applying multivariate statistical processing on skin tissue sections. Herein, we prove the possibility of tracking low concentrations of caffeine through the skin and we highlight some experimental limitations of vibrational spectroscopies.

Keywords: IR spectroscopy; Raman spectroscopy; Drug screening; Chemometrics; Skin

Rapid phytochemical analysis of birch (Betula) and poplar (Populus) foliage by near-infrared reflectance spectroscopy by Kennedy F. Rubert-Nason; Liza M. Holeski; John J. Couture; Adam Gusse; Daniel J. Undersander; Richard L. Lindroth (pp. 1333-1344).

Poplar (Populus) and birch (Betula) species are widely distributed throughout the northern hemisphere, where they are foundation species in forest ecosystems and serve as important sources of pulpwood. The ecology of these species is strongly linked to their foliar chemistry, creating demand for a rapid, inexpensive method to analyze phytochemistry. Our study demonstrates the feasibility of using near-infrared reflectance spectroscopy (NIRS) as an inexpensive, high-throughput tool for determining primary (e.g., nitrogen, sugars, starch) and secondary (e.g., tannins, phenolic glycosides) foliar chemistry of Populus and Betula species, and identifies conditions necessary for obtaining reliable quantitative data. We developed calibrations with high predictive power (residual predictive deviations ≤ 7.4) by relating phytochemical concentrations determined with classical analytical methods (e.g., spectrophotometric assays, liquid chromatography) to NIR spectra, using modified partial least squares regression. We determine that NIRS, although less sensitive and precise than classical methods for some compounds, provides useful predictions in a much faster, less expensive manner than do classical methods. Graphical abstract Near-infrared reflectance spectroscopy with calibrations based on modified partial least squares regression can provide quantitative measurements of foliar nitrogen, carbohydrate, tannin, and phenolic glycoside content in poplar and birch

Keywords: Carbohydrate; NIRS; Nitrogen; Phenolic glycoside; Populus ; Tannin

Intracellular in vitro probe acylcarnitine assay for identifying deficiencies of carnitine transporter and carnitine palmitoyltransferase-1 by Jamiyan Purevsuren; Hironori Kobayashi; Yuki Hasegawa; Kenji Yamada; Tomoo Takahashi; Masaki Takayanagi; Toshiyuki Fukao; Seiji Fukuda; Seiji Yamaguchi (pp. 1345-1351).

Mitochondrial fatty acid oxidation (FAO) disorders are caused by defects in one of the FAO enzymes that regulates cellular uptake of fatty acids and free carnitine. An in vitro probe acylcarnitine (IVP) assay using cultured cells and tandem mass spectrometry is a tool to diagnose enzyme defects linked to most FAO disorders. Extracellular acylcarnitine (AC) profiling detects carnitine palmitoyltransferase-2, carnitine acylcarnitine translocase, and other FAO deficiencies. However, the diagnosis of primary carnitine deficiency (PCD) or carnitine palmitoyltransferase-1 (CPT1) deficiency using the conventional IVP assay has been hampered by the presence of a large amount of free carnitine (C0), a key molecule deregulated by these deficiencies. In the present study, we developed a novel IVP assay for the diagnosis of PCD and CPT1 deficiency by analyzing intracellular ACs. When exogenous C0 was reduced, intracellular C0 and total AC in these deficiencies showed specific profiles clearly distinguishable from other FAO disorders and control cells. Also, the ratio of intracellular to extracellular C0 levels showed a significant difference in cells with these deficiencies compared with control. Hence, intracellular AC profiling using the IVP assay under reduced C0 conditions is a useful method for diagnosing PCD or CPT1 deficiency.

Keywords: Fatty acid oxidation; Carnitine cycle disorder; Acylcarnitine profile; ESI-MS/MS

Quantitative analysis of multiple genes’ expressions based on a novel competitive RT-PCR assay by Jia Li; Li-hui Lin; Juan Wang; Xia Peng; Ya-nan Liu; Jun-hua Xiao; Yu-xun Zhou; Li Li (pp. 1353-1360).

We established a novel gene expression analysis platform, Multiplex Competitive RT-PCR Using Fluorescent Universal Primers (MCF-PCR), to study multi-gene expression patterns simultaneously. This platform combines fluorescent universal primers, multiplex competitive RT-PCR, and capillary electrophoretic separation, which ensures MCF-PCR a reliable, medium-throughput, cost-effective technology for gene expression profiling. With cloned standard DNAs, the detection limits, precision, and sensitivity of MCF-PCR were evaluated and compared with that of the assay without adding competitive templates and real-time PCR, respectively. The results showed that detection limit was 3.125 × 10³ to 3.2 × 10⁶ copies, and 10 % copy differences between two samples can be detected by MCF-PCR. To validate MCF-PCR, we analyzed expression profile of five genes in interleukin (IL)-4/IL-13 pathway in peripheral blood of 20 healthy adults and 20 allergic dermatitis patients; three genes including IL-4, IL-13, and STAT6 were found differentially expressed in the two sample groups, which maybe key players in IL-4/IL-13 immunological signaling pathway and need further function analysis. Figure Principle of MCF-PCR. cDNA was amplified using chimeric primers, each containing 18-20 nt target-complementary sequence (solid blue/black) and 18 nt universal primer-complementary sequence (solid red). Subsequent PCR amplifications using universal primers to yield fluorescent-labeled amplification products.

Keywords: Fluorescent universal primers; Multiplex competitive RT-PCR; Gene expression profiling; Real-time PCR; IL-4/IL-13 pathway

Comparative studies of thiol-sensitive fluorogenic probes for HAT assays by Tielong Gao; Chao Yang; Yujun George Zheng (pp. 1361-1371).

Histone acetyltransferases (HATs) catalyze the acetylation of specific lysine residues in histone and nonhistone proteins. Recent studies showed that acetylation is widely distributed among cellular proteins, suggestive of diverse functions of HATs in cellular pathways. Nevertheless, currently available assays for HAT activity study are still quite limited. Here, we evaluated a series of thiol-sensitive fluorogenic compounds for the detection of the enzymatic activities of different HAT proteins. Upon conjugation to the thiol group of HSCoA, these molecules gain enhanced quantum yields and strong fluorescence, permitting facile quantitation of HAT activities. We investigated and compared the assay performances of these fluorogenic compounds for their capability as HAT activity reporters, including kinetics of reaction with HSCoA, influence on HAT activity, and fluorescence amplification factors. Our data suggest that CPM and coumarin maleic acid ester are excellent HAT probes owing to their fast reaction kinetics and dramatic fluorescence enhancement during the HAT reaction. Further, the microtiter plate measurements show that this fluorescent approach is robust and well suited for adaption to high-throughput screening of small molecule inhibitors of HATs, highlighting the value of this assay strategy in new drug discovery.

Keywords: Histone acetyltransferase; HAT; Fluorescent probe; Epigenetics; Chromatin modification

Development, optimization, and use of an APCI source with temperature-controlled vaporization of solid and liquid samples by Sonja Krieger; Alexandra von Trotha; Kelvin Sze-Yin Leung; Oliver J. Schmitz (pp. 1373-1381).

A trend is observed in mass spectrometry, in which solid samples without prior dissolution and chromatographic separation are brought directly into the ion source and are ionized, e.g., by corona discharge (Atmospheric Solids Analysis Probe) or plasma (Direct Analysis in Real Time). The Direct Inlet Probe-atmospheric-pressure chemical ionization (APCI) ion source presented here, which was coupled to a high-resolution quadrupole time-of-flight–mass spectrometer, differs from most of the other ion sources in having temperature-programmed heating of the sample. The resulting possibility to reduce ion suppression and ion-molecule reactions in the ion source was shown by the separation of two fatty acid methyl esters as a result of their boiling point difference. Using caffeine as sample, certain source parameters such as the auxiliary gas flow, the drying gas flow, and the position of the probe tip in the ion source were optimized. The ability to perform quantitative analyses was shown by the linear concentration response (R ² = 0.9984) observed when analyzing different caffeine concentrations. An extract of a Chinese medicinal herb was used to examine the reproducibility (relative standard deviations of the most abundant m/z signals were ≤8.1 %). It was also possible to distinguish milled samples of Radix Angelicae sinensis and Radix Angelicae gigas from each other and to identify the coumarins they contain without sample preparation. Supplying synthetic air instead of nitrogen to the ion source makes APCI in the negative mode possible as well; this was proven by the analysis of n-nonyl-β-d-maltoside.

Keywords: Ambient ionization; Atmospheric-pressure chemical ionization; Chinese herbal medicine; DIP-APCI; Mass spectrometry

Determination of 32 cathinone derivatives and other designer drugs in serum by comprehensive LC-QQQ-MS/MS analysis by Madeleine J. Swortwood; Diane M. Boland; Anthony P. DeCaprio (pp. 1383-1397).

Recently, clandestine drug lab operators have attempted to bypass controlled substances laws and regulations with “designer” compounds chemically and pharmacologically similar to controlled substances. For example, “bath salts” have erupted onto the scene as “legal highs” containing cathinone analogs that have produced severe side effects in users worldwide. These products have sparked concern among law enforcement agencies, and emergency bans have been placed on the sale of such items. Despite the increasing number of designer drugs available, there are few comprehensive screening techniques for their detection and quantification in biological specimens. The liquid chromatography triple quadrupole tandem mass spectrometry (LC-QQQ-MS/MS) method presented here encompasses over thirty important compounds within the phenethylamine, tryptamine, and piperazine designer drug classes. Analytes were determined by LC-QQQ-MS/MS in the multiple-reaction monitoring mode after mixed-mode solid-phase extraction. The bioanalytical method was fully validated according to recommended international guidelines. The assay was selective for all analytes with acceptable accuracy and precision. Limits of quantification were in the range of 1–10 ng/mL for each compound with limits of detection near 10 pg/mL. In order to evaluate its applicability in a forensic toxicological setting, the validated method was used to analyze post-mortem specimens from two cases that were suspected of containing designer drugs. The method was able to identify and quantify seven of these compounds at concentrations as low as 11 ng/mL. The method should have wide applicability for rapid screening of important new drugs of abuse at high sensitivity in both post- and ante-mortem forensic analysis. Figure LC-MS chromatogram (intensity vs. retention time) of primary MRM transitions for 32 targeted analytes

Keywords: Cathinone derivatives; Designer drugs; Bath salts; Serum; LC-MS/MS; Method validation

Simultaneous and sensitive LC–MS/MS determination of tetrahydrocannabinol and metabolites in human plasma by N. Ferreirós; S. Labocha; C. Walter; J. Lötsch; G. Geisslinger (pp. 1399-1406).

Cannabis is not only a widely used illicit drug but also a substance which can be used in pharmacological therapy because of its analgesic, antiemetic, and antispasmodic properties. A very rapid and sensitive method for determination of ∆⁹-tetrahydrocannabinol (THC), the principal active component of cannabis, and two of its phase I metabolites in plasma has been developed and validated. After solid-phase extraction of plasma (0.2 mL), the clean extracts were analyzed by tandem mass spectrometry after a 5-min liquid chromatographic separation. The linear calibration ranges were from 0.05 to 30 ng mL⁻¹ for THC and 11-nor-∆⁹-carboxy-tetrahydrocannabinol (THC-COOH) and from 0.2 to 30 ng mL⁻¹ for ∆⁹-(11-OH)-tetrahydrocannabinol (11-OH-THC). Imprecision and inaccuracy were always below 7 and 12 % (expressed as relative standard deviation and relative error), respectively. The method has been successfully applied to determination of the three analytes in plasma obtained from healthy volunteers after oral administration of 20 mg dronabinol.

Keywords: THC; Metabolites; LC–MS/MS; Cannabinoids

Optimizing hollow-fiber-based pharmacokinetic assay via chemical stability study to account for inaccurate simulated drug clearance of rifampicin by Lee Sun New; Tze Peng Lim; Jing Wen Oh; Gavin Jia Sheng Cheah; Andrea L. Kwa; Eric Chun Yong Chan (pp. 1407-1415).

With increasing multidrug resistance coupled to a poor development pipeline, clinicians are exploring antimicrobial combinations to improve treatment outcomes. In vitro hollow-fiber infection model (HFIM) is employed to simulate human in vivo drug clearance and investigate pharmacodynamic synergism of antibiotics. Our overarching aim was to optimize the HFIM-based pharmacokinetic (PK) assay by using rifampicin and polymyxin B as probe drugs. An ultrapressure liquid chromatography tandem mass spectrometry method was validated for the quantification of rifampicin and polymyxin B components. In vitro profiling studies demonstrated that the experimental PK profiles of polymyxin B monotherapy were well correlated with the human population PK data while monotherapy with rifampicin failed to achieve the expected maximum plasma concentration. Chemical stability studies confirmed polymyxin B was stable in broth at 37 °C up to 12 h while rifampicin was unstable under the same conditions over 12 and 80 h. The calculated mean clearance of rifampicin due to chemical degradation was 0.098 ml/min accounting for 12.2 % of its clinical total clearance (CL = 0.8 ml/min) based on population PK data. Our novel finding reinforces the importance to optimize HFIM-based PK assay by performing chemical stability study so as to account for potential discrepancy between experimental and population PK profiles of antimicrobial agents. Figure Optimizing hollow-fiber-based pharmacokinetic assay

Keywords: Hollow-fiber infection model; Polymyxin B; Rifampicin; Multidrug-resistant bacteria; Pharmacokinetics; Clearance

Improvements to the compressed-sample (CS) technique for MALDI-TOF mass spectrometry by Lukas Hyzak; Susanne Giese; Hans-Willi Kling; Volker Wulf; David Melchior; Michael Köhler; Oliver J. Schmitz (pp. 1417-1424).

A recently developed solvent-free compressed-sample technique for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis allows the reproducible analysis of synthetic polymers and peptides up to 3,500 Da. In this work, we present an improvement in resolution, an increase in intensity and a decrease of the variation coefficient, as illustrated by the analysis of PEG 2000 and MALDI imaging experiments. These advantages were achieved by homogenization of the electrical field, which was disturbed by the drills in the original MALDI target. In order to homogenize the electrical field, a new target with smaller drills was developed, metal powder was added to the matrix/analyte mixture and a round laser raster was used. Furthermore, a ball mill was implemented for the sample preparation to replace the extremely user-dependent grinding in a mortar. The new conditions were successfully applied to the quantification of several peptides of higher molecular weight and gave higher precision than had previously been achieved with the compressed-sample technique.

Keywords: Mass spectrometry; MALDI; Compressed sample; Solvent-free sample preparation

Fluorescence assay for protein post-translational tyrosine sulfation by Bo-Han Chen; Chen-Chu Wang; Lu-Yi Lu; Kuo-Sheng Hung; Yuh-Shyong Yang (pp. 1425-1429).

We developed a fluorescent assay to conveniently determine the kinetics of protein sulfation, which is essential for understanding interface between protein sulfation and protein–protein interactions. Tyrosylprotein sulfotransferase (TPST) catalyzes protein sulfation using 3′-phosphate 5′-phosphosulfate (PAPS) as sulfuryl group donor. In this report, PAPS was regenerated following sulfuryl group transfer between adenosine 3′,5′-diphosphate and 4-methylumbelliferyl sulfate catalyzed by phenol sulfotransferase (PST). The TPST and PST coupled enzyme platform continuously generated fluorescent 4-methylumbelliferone (MU) that was used to real-time monitor protein sulfation. Using a recombinant N utilization substance protein A fused Drosophila melanogaster tyrosylprotein sulfotransferase, we demonstrated that the activity of TPST determined through MU fluorescence directly correlated with protein sulfation. Kinetic constants obtained with small P-selectin glycoprotein ligand-1 peptide (PSGL-1 peptide, MW 1541) or its large glutathione S-transferase fusion protein (GST-PSGL-1, MW 27833) exhibited significant variation. This assay can be further developed to a high-throughput method for the characterization of TPSTs and for the identification and screening of their protein substrates. Figure Fluorophore to report the progress of post-translational protein tyrosine sulfation: Protein sulfation was continuously monitored through a PAPS regeneration system that produced MU (fluorophore) and PAPS (sulfuryl group donor) from MUS and PAP. MU is a fluorescent reporter and PAPS is one of the substrates of TPST.

Keywords: Protein sulfation; Tyrosylprotein sulfotransferase (TPST); Phenol sulfotransferase (PST); Fluorescence enzyme assay

Erratum to: New “hyphenated” CPC-HPLC-DAD-MS strategy for simultaneous isolation, analysis and identification of phytochemicals: application to xanthones from Garcinia mangostana by Thomas Michel; Emilie Destandau; Laëtitia Fougère; Claire Elfakir (pp. 1431-1431).

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Analytical and Bioanalytical Chemistry (v.405, #4)

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Analytical and Bioanalytical Chemistry (v.405, #4)