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Analytical and Bioanalytical Chemistry (v.405, #7)

Monolithic columns in liquid phase separations by Luis A. Colon (pp. 2093-2094).

is Professor of Chemistry and Associate Dean of the Graduate School at The State University of New York at Buffalo. His current research interests are in the fields of microchemistry/nanochemistry and separation science in general, including the development of chromatographic media for liquid-phase separations and detection schemes for monitoring mass-limited samples, the use of nanotechnology in separations, and the development of new strategies to separate and analyze complex chemical and biochemical sample mixtures.

Recent advances in monolithic columns for protein and peptide separation by capillary liquid chromatography by Yu Liang; Lihua Zhang; Yukui Zhang (pp. 2095-2106).

Capillary liquid chromatography (cLC) has great potential for protein and peptide separation, with advantages of high efficiency, high resolution, low sample consumption, and high sensitivity when coupled with mass spectrometry. In recent years, monoliths have been widely used as the stationary phases for capillary columns, owing to easy preparation, high permeability, fast mass transfer, and low backpressure. This review summarizes recent advances (2007–2012) in monolithic columns for protein and peptide separation by cLC. After a brief introduction on the preparation of monolithic capillary columns, the emphasis of this review is focused on the recent application of such columns for protein and peptide separation by cLC. Furthermore, the challenges and potential hot points of monolithic capillary columns in the future are discussed.

Keywords: Monoliths; Capillary liquid chromatography; Proteins; Peptides; Separation

Monolithic columns in plant proteomics and metabolomics by Marilda Rigobello-Masini; José Carlos Pires Penteado; Jorge Cesar Masini (pp. 2107-2122).

Since “omics” techniques emerged, plant studies, from biochemistry to ecology, have become more comprehensive. Plant proteomics and metabolomics enable the construction of databases that, with the help of genomics and informatics, show the data obtained as a system. Thus, all the constituents of the system can be seen with their interactions in both space and time. For instance, perturbations in a plant ecosystem as a consequence of application of herbicides or exposure to pollutants can be predicted by using information gathered from these databases. Analytical chemistry has been involved in this scientific evolution. Proteomics and metabolomics are emerging fields that require separation, identification, and quantification of proteins, peptides, and small molecules of metabolites in complex biological samples. The success of this work relies on efficient chromatographic and electrophoretic techniques, and on mass spectrometric detection. This paper reviews recent developments in the use of monolithic columns, focusing on their applications in “top-down” and “bottom-up” approaches, including their use as supports for immobilization of proteolytic enzymes and their use in two-dimensional and multidimensional chromatography. Whereas polymeric columns have been predominantly used for separation of proteins and polypeptides, silica-based monoliths have been more extensively used for separation of small molecules of metabolites. Representative applications in proteomics and in analysis of plant metabolites are given and summarized in tables.

Keywords: Plant; Proteomics; Metabolomics; Monolithic columns; Liquid chromatography

Recent advances in the design of organic polymer monoliths for reversed-phase and hydrophilic interaction chromatography separations of small molecules by Jiri Urban; Pavel Jandera (pp. 2123-2131).

Owing to their favorable porous structure with pore size distribution shifted towards large flow-through pores, organic polymer monoliths have been mainly employed for the separation of macromolecules in gradient elution liquid chromatography. The absence of significant amounts of small pores with a stagnant mobile phase and the resulting low surface area were considered as the main reason for their poor behavior in the isocratic separation of small molecules. Several recent efforts have improved the separation power of organic polymer monoliths for small molecules offering column efficiency up to tens of thousands of plates per meter. These attempts include optimization of the composition of polymerization mixture, including the variation of functional monomer, the cross-linking monomer, and the porogen solvents mixture, adjustment of polymerization temperature, and time. Additionally, post-polymerization modifications including hypercross-linking and the use of carbon nanostructures showed significant improvement in the column properties. This review describes recent developments in the preparation of organic polymer monoliths suitable for the separation of small molecules in the isocratic mode as well as the main factors affecting the column efficiency.

Keywords: Organic polymer monoliths; Small molecules; Reversed-phase chromatography; Hydrophilic interaction chromatography; Efficiency

Affinity monolith chromatography: a review of principles and recent analytical applications by Erika L. Pfaunmiller; Marie Laura Paulemond; Courtney M. Dupper; David S. Hage (pp. 2133-2145).

Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis, or study of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments and applications of this method, with particular emphasis being given to work that has appeared in the last 5 years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths, and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns, and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal ions, and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized-metal-ion affinity chromatography, dye–ligand affinity chromatography, chiral separations, and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing, and biotechnology. Current trends and possible directions in AMC are also discussed.

Keywords: Affinity monolith chromatography; Monolithic supports; Affinity chromatography; Bioaffinity chromatography; Immunoaffinity chromatography; Immobilized-metal-ion affinity chromatography; Dye–ligand affinity chromatography; Biointeraction chromatography

Synthesis and theoretical study of molecularly imprinted monoliths for HPLC by Chao Zheng; Yan-Ping Huang; Zhao-Sheng Liu (pp. 2147-2161).

Molecularly imprinted monoliths integrate the high permeability of monolithic materials and the high selectivity and affinity of molecularly imprinted polymers (MIP). Thus, in recent years, development of this novel MIP format in HPLC has expanded quickly, particularly use of organic materials. This review focuses on the principal aspects of good practice in polymerization, theoretical studies, and recent developments in molecularly imprinted monoliths. Some thoughts on perspectives of MIP monoliths are also expressed.

Keywords: Molecularly imprinted polymer; Monolithic column; HPLC

Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins by Jens Sproß; Sebastian Brauch; Friedrich Mandel; Moritz Wagner; Stephan Buckenmaier; Bernhard Westermann; Andrea Sinz (pp. 2163-2173).

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label. Figure Schematic representations of the online 3D-nano-HPLC/nano-ESI-Q-TOF-MS/MS setup; LP loading pump, NP nano-pump

Keywords: Affinity enrichment; Avidin; Biotin; Monolithic column; Multidimensional HPLC; Protein identification

Nanoparticle-modified monolithic pipette tips for phosphopeptide enrichment by Jana Krenkova; Frantisek Foret (pp. 2175-2183).

We have developed nanoparticle-modified monoliths in pipette tips for selective and efficient enrichment of phosphopeptides. The 5 μL monolithic beds were prepared by UV-initiated polymerization in 200 μL polypropylene pipette tips and either iron oxide or hydroxyapatite nanoparticles were used for monolith modification. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of iron oxide nanoparticles on the pore surface of the monolith was obtained via multivalent electrostatic interactions of citrate ions on the surface of nanoparticles with a quaternary amine functionalized poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith. Hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The nanoparticle-modified monoliths were compared with commercially available titanium dioxide pipette tips. Performance of the developed and commercially available sorbents was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and β-casein digests followed by off-line MALDI/MS analysis.

Keywords: Nanoparticles; Monolith; Pipette tip; Enrichment; Phosphopeptides

New monolithic stir-cake-sorptive extraction for the determination of polar phenols by HPLC by Xiaojia Huang; Yulei Wang; Dongxing Yuan; Xiaojing Li; Shuyu Nong (pp. 2185-2193).

A novel porous monolith has been prepared and used as a sorbent in stir-cake-sorptive extraction (SCSE). The monolithic material was prepared by in-situ copolymerization of allyl thiourea (AT) and divinylbenzene (DB) in the presence of dimethylformamide as a porogen solvent. To optimize the polymerization conditions, different monoliths with different ratios of functional monomer to porogenic solvent were prepared, and their extraction efficiency was investigated in detail. The monolith was characterized by elemental analysis, scanning electron microscopy, mercury intrusion porosimetry, and infrared spectroscopy. Analysis of polar phenols in environmental water samples by a combination of ATDB-SCSE and HPLC with diode-array detection was selected as a model for the practical application of the new sorbent. Several extraction conditions, including extraction and desorption time, pH, and ionic strength of the sample matrix were optimized. The results showed that the new monolith had high affinity for polar phenols and could be used to extract them effectively. Under the optimum conditions, low detection (S/N = 3) and quantification (S/N = 10) limits were achieved for the phenols, within the ranges 0.18–0.90 and 0.59–2.97 μg L⁻¹, respectively. The linearity of the method was good, and the method enabled simple, practical, and low-cost extraction of these analytes. The distribution coefficients between ATDB and water (K _ATDB/W) were calculated for the phenolic compounds and compared with K _O/W. Finally, the proposed method was successfully applied to the determination of the compounds in three environmental water samples, with acceptable recovery and satisfactory repeatability. Figure HPLC chromatograms of real water sample treated with ATDB-SCSE (a) and spiked water sample treated with ATDB-SCSE (b)

Keywords: Stir-cake-sorptive extraction; Poly-(allylthiourea–divinylbenzene); Monolithic material; Phenols; Water samples

Biocatalytic reactors based on ribonuclease A immobilized on macroporous monolithic supports by E. A. Ponomareva; M. V. Volokitina; D. O. Vinokhodov; E. G. Vlakh; T. B. Tennikova (pp. 2195-2206).

Immobilized enzyme reactors (IMERs) produced by the covalent attachment of ribonuclease A to macroporous methacrylate-based monolithic supports using different experimental approaches are discussed and compared. Enzyme immobilization was carried out by direct covalent binding, as well as through attachment via a polymer spacer. The kinetic properties of an IMER operating in either recirculation mode or zonal elution mode were studied. Additionally, the effect of flow rate on the bioconversion efficiency of each IMER sample was examined. Figure Enzyme immobilization via aldehyde-bearing macromolecular spacer on the surface of epoxy-containing monoliths

Keywords: Polymer monoliths; Enzyme immobilization; Flow-through immobilized enzyme reactors; Polymer spacer

Iminodiacetic acid functionalised organopolymer monoliths: application to the separation of metal cations by capillary high-performance chelation ion chromatography by Áine Moyna; Damian Connolly; Ekaterina Nesterenko; Pavel N. Nesterenko; Brett Paull (pp. 2207-2217).

Lauryl methacrylate-co-ethylene dimethacrylate monoliths were polymerised within fused silica capillaries and subsequently photo-grafted with varying amounts of glycidyl methacrylate (GMA). The grafted monoliths were then further modified with iminodiacetic acid (IDA), resulting in a range of chelating ion-exchange monoliths of increasing capacity. The IDA functional groups were attached via ring opening of the epoxy group on the poly(GMA) structure. Increasing the amount of attached poly(GMA), via photo-grafting with increasing concentrations of GMA, from 15 to 35 %, resulted in a proportional and controlled increase in the complexation capacity of the chelating monoliths. Scanning capacitively coupled contactless conductivity detection (sC⁴D) was used to characterise and verify homogenous distribution of the chelating ligand along the length of the capillaries non-invasively. Chelation ion chromatographic separations of selected transition and heavy metals were carried out, with retention factor data proportional to the concentration of grafted poly(GMA). Average peak efficiencies of close to 5,000 N/m were achieved, with the isocratic separation of Na, Mg(II), Mn(II), Co(II), Cd(II) and Zn(II) possible on a 250-mm-long monolith. Multiple monolithic columns produced to the same recipes gave RSD data for retention factors of <15 % (averaged for several metal ions). The monolithic chelating ion-exchanger was applied to the separation of alkaline earth and transition metal ions spiked in natural and potable waters.

Keywords: Porous polymer monolith; Iminodiacetic acid; Photo-grafting; Capillary chelation ion chromatography

Comparison of the performance of Chromolith Performance RP-18e, 1.8-μm Zorbax Eclipse XDB-C18 and XTerra MS C18, based on modelling approaches by S. Pous-Torres; J. R. Torres-Lapasió; M. C. García-Álvarez-Coque (pp. 2219-2231).

Achievement of the highest separation efficiency and quick delivery of results are key requirements in liquid chromatography for enhancing productivity and reducing analysis cost, especially in the pharmaceutical industry. This work concerns two of the most popular current solutions to get fast separations: the use of a silica-based monolithic column (Chromolith Performance RP-18e) and a small-particle packed column (1.8-μm Zorbax Eclipse XDB-C18, which needs dedicated instruments allowing higher backpressures). Both columns succeeded in the full separation of phenols and β-adrenolytic drugs, which are compounds that interact with residual silanols, giving rise to wider peaks. The results were compared with those obtained with a special column designed to avoid silanol interaction, containing 5 μm particles (XTerra MS C18). Chromolith gave the shortest times at the expense of higher solvent consumption at the high flow rates needed. In contrast to other studies, comprehensive conclusions on the chromatographic performance, in terms of selectivity, peak shape, resolution, and analysis time, are derived from the inspection of the whole experimental domain using retention and peak shape modelling. In the literature, column comparison is usually carried out based on the performance for selected mobile phases (very often a single one), which offers deceiving results.

Keywords: Chromolith; UHPLC; Acetonitrile; Flow rate; Retention modelling; Chromatographic performance; Phenols; β-adrenolytic drugs

Epoxy-based monoliths for capillary liquid chromatography of small and large molecules by Mohammad Talebi; R. Dario Arrua; Andras Gaspar; Nathan A. Lacher; Qian Wang; Paul R. Haddad; Emily F. Hilder (pp. 2233-2244).

A versatile epoxy-based monolith was synthesised by polycondensation polymerisation of glycidyl ether 100 with ethylenediamine using a porogenic system consisting of polyethylene glycol, M _w = 1000, and 1-decanol. Polymerisation was performed at 80 °C for 22 h. A simple acid hydrolysis of residual epoxides resulted in a mixed diol-amino chemistry. The modified column was used successfully for hydrophilic interaction liquid chromatography (HILIC) of small molecule probes such as nucleic acid bases and nucleosides, benzoic acid derivatives, as well as for peptides released from a tryptic digest of cytochrome c. The mixed-mode chemistry allowed both hydrophilic partitioning and ion-exchange (IEX) interactions to contribute to the separation, providing flexibility in selectivity control. Residual epoxide groups were also exploited for incorporating a mixed IEX chemistry. Alternatively, the surface chemistry of the monolith pore surface rendered hydrophobic via grafting of a co-polymerised hydrophobic hydrogel. The inherent hydrophilicity of the monolith scaffold also enabled high performance separation of proteins under IEX and hydrophobic interaction modes and in the absence of non-specific interactions.

Keywords: Epoxy-based monolith; Hydrophilic interaction chromatography (HILIC); Capillary liquid chromatography; Proteins; Peptides; Liquid chromatography-mass spectrometry (LC-MS)

Preparation and characterization of an imprinted monolith by atom transfer radical polymerization assisted by crowding agents by Lu Ban; Liang Zhao; Bang-Li Deng; Yan-Ping Huang; Zhao-Sheng Liu (pp. 2245-2253).

A method based on reverse atom transfer radical polymerization (R-ATRP) and molecular crowding has been used for design and synthesis of monolithic molecularly imprinted polymers (MIPs) capable of recognizing ibuprofen (IBU). 4-Vinylpyridine (4-VP) was used as the functional monomer, and ethylene glycol dimethacrylate (EDMA) was the crosslinking monomer. Azobisisobutyronitrile (AIBN)–CuCl₂–N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) was used as the initiating system. Compared with conventional radical polymerization-based IBU-MIPs, the imprinting effects of the obtained IBU-MIPs was enhanced, suggesting the merit of combination of reverse ATRP and molecular crowding. In addition, it was found that the polymerization time of the molecularly imprinted monolithic column, the amount of template, the degree of crosslinking, and the composition of mobile phase greatly affected retention of the template and the performance of molecular recognition. Figure Schematic representation of molecular imprinting under molecular crowding conditions in the presence of R-ATRP

Keywords: Atom transfer radical polymerization; Ibuprofen; Molecularly imprinted polymer; Molecular crowding; Molecular recognition; Monolith

High-resolution monolithic columns—a new tool for effective and quick separation by Hana Sklenářová; Petr Chocholouš; Petra Koblová; Lukáš Zahálka; Dalibor Šatínský; Ludmila Matysová; Petr Solich (pp. 2255-2263).

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a “classic” monolithic column, the first generation (Onyx™ monolithic C₁₈, 100 mm × 4.6 mm, Phenomenex); a “narrow” monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm × 3 mm, Merck); and a recently introduced “high-resolution” monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm × 4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile–phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column. Figure Chromatogram of separation using different flow rates (corresponded to optimal separation conditions); 1 0.5 mL min^-1; 2 0.6 mL min^-1; 3 0.3 mL min^-1

Keywords: High-performance liquid chromatography; HighResolution; Monolithic column; Silica monoliths; Sorbent development; Van Deemter curve

Preparation of a butyl–silica hybrid monolithic column with a “one-pot” process for bioseparation by capillary liquid chromatography by Zhenbin Zhang; Fangjun Wang; Junjie Ou; Hui Lin; Jing Dong; Hanfa Zou (pp. 2265-2271).

A butyl–silica hybrid monolithic column for bioseparation by capillary liquid chromatography (cLC) was prepared with butyl methacrylate and alkoxysilanes through a “one-pot” process. The effects of polycondensation temperature, volume percentage of N,N′-dimethylformamide, and content of cetyltrimethylammonium bromide and butyl methacrylate on the morphologies of the hybrid monolithic columns prepared were investigated in detail. Baseline separations of proteins and small peptides on the hybrid monolithic column were achieved by cLC with gradient elution. In addition, the resulting hybrid column was also applied for analysis of tryptic digests of bovine serum albumin by cLC coupled with tandem mass spectrometry. The results demonstrate its potential application in separation of complex biological samples.

Keywords: One pot; Sol–gel; Butyl–silica hybrid monolith; Capillary liquid chromatography; Bioseparation

Quality assurance challenge 11 by Manfred Reichenbächer; Jürgen W. Einax (pp. 2273-2275).

Solution to the Olympic Games challenge by Reinhard Meusinger (pp. 2277-2278).

Real-time size discrimination and elemental analysis of gold nanoparticles using ES-DMA coupled to ICP-MS by Sherrie Elzey; De-Hao Tsai; Lee L. Yu; Michael R. Winchester; Michael E. Kelley; Vincent A. Hackley (pp. 2279-2288).

We report the development of a hyphenated instrument with the capacity to quantitatively characterize aqueous suspended gold nanoparticles (AuNPs) based on a combination of gas-phase size separation, particle counting, and elemental analysis. A customized electrospray-differential mobility analyzer (ES-DMA) was used to achieve real-time upstream size discrimination. A condensation particle counter and inductively coupled plasma mass spectrometer (ICP-MS) were employed as downstream detectors, providing information on number density and elemental composition, respectively, of aerosolized AuNPs versus the upstream size selected by ES-DMA. A gas-exchange device was designed and optimized to improve the conversion of air flow (from the electrospray) to argon flow required to sustain the ICP-MS plasma, the key compatibility issue for instrumental hyphenation. Our work provides the proof of concept and a working prototype for utilizing this construct to successfully measure (1) number- and mass-based distributions; (2) elemental compositions of nanoparticles classified by size, where the size classification and elemental analysis are performed within a single experiment; (3) particle concentrations in both solution (before size discrimination) and aerosol (after size discrimination) phases; and (4) the number of atoms per nanoparticle or the nanoparticle density.

Keywords: DMA; Electrospray; Gold; Nanoparticle; ICP-MS; Quantitative analysis

Putting a spin on LA-ICP-MS analysis combined to isotope dilution by Fanny Claverie; Julien Malherbe; Naomi Bier; John L. Molloy; Stephen E. Long (pp. 2289-2299).

The determination of Zn, Sr, Ba, and Pb in solid samples has been achieved by laser ablation inductively coupled plasma isotope dilution mass spectrometry using a spinning platform. The fast rotation of a sample and an isotopically enriched spike placed close together on a sample holder allowed performing the isotope dilution directly inside the ablation cell. The proportion of spike versus sample of the aerosol mixture obtained has been determined online by isotope dilution in order to correct for differences in ablation rate although both materials were placed on the axis of rotation of the motor. Homogeneous, time-stable, and reusable samples were prepared by lithium borate fusion. A unique isotopically enriched spike glass was used to analyze four Standard Reference Materials of different matrix (after a simple polishing): two sediments Standard Reference Material (SRM) 1944 and SRM 2702 and two soils SRM 2586 and SRM 2711a. The proposed method yielded mass fractions with a deviation from the certified value usually lower than 12 % and a precision of less than 9 % RSD (except for Zn in SRM 2586 and 2711a). Although direct spiking of the solid before fusion could presumably provide better isotopic mixing, the presented methodology allows the reuse of the spike glass (thus, decreasing drastically the cost of the analysis) and is relatively faster because the spike does not need to be weighted, added, and evaporated each time. These results demonstrate the potential of this newly developed method for fast analysis of solid samples using isotope dilution at a low cost.

Keywords: Isotope dilution; LA-ICP-MS; Aerosol mixing; Spinning platform

Manganese speciation in paired serum and CSF samples using SEC-DRC-ICP-MS and CE-ICP-DRC-MS by B. Michalke; M. Lucio; A. Berthele; B. Kanawati (pp. 2301-2309).

Occupational manganese (Mn) overexposure leads to accumulation in the brain and has been shown to cause progressive, permanent, neuro-degenerative damage with syndromes similar to idiopathic Parkinsonism. Mn is transported by an active mechanism across neural barriers (NB) finally into the brain; but to date, modes of Mn neurotoxic action are poorly understood. This paper investigates the relevant Mn-carrier species which are responsible for widely uncontrolled transport across NB. Mn speciation in paired serum/cerebrospinal fluid (CSF) samples was performed by size exclusion chromatography–inductively coupled plasma–dynamic reaction cell–mass spectrometry (SEC-ICP-DRC-MS) and capillary zone electrophoresis coupled to ICP-DRC-MS in a 2D approach for clear identification. For additional species verification, electrospray ionization–Fourier transform ion cyclotron resonance–mass spectrometry was used after SEC-ICP-DRC-MS (second 2D approach). The Mn species from the different sample types were interrelated and correlation coefficients were calculated. In serum protein-bound Mn species like Mn-transferrin/albumin (Mn-Tf/HSA) were dominant, which had the main influence on total Mn in serum if Mn_total was <1.5 μg/L. Above serum Mn_total concentration of 1.6 μg/L the serum Mn_total concentration was correlated with increasing Mn-citrate (Mn-Cit) concentration. In parallel Mn_total and Mn species in CSF were determined. It turned out that Mn_total from CSF was about half of Mn_total in serum; Mn-Tf/HSA was only about 10 % compared to serum. It turned out that above 1.6 μg/L Mn_total in serum Mn-Cit was not only the leading Mn species in serum but also was the main influencing factor of both Mn_total and Mn-Cit concentration in CSF. These results were further investigated using two statistical models (orthogonal partial least squares discriminant analysis, canonical discriminant analysis). Both models discriminated the samples in two groups where CSF samples were either correlated to Mn_total and Mn-Cit (samples with serum Mn_total > 1,550 ng/L) or correlated to Mn-Tf/HSA (samples with serum Mn_total < 1,550 ng/L). We conclude that elevated Mn-Cit_serum could be a valuable marker for increased total Mn in CSF (and brain), i.e., it could be a marker for elevated risk of Mn-dependent neurological disorders such as manganism in occupational health.

Keywords: Manganese; Speciation; Cerebrospinal fluid; Serum; SEC-ICP-DRC-MS; CE-ICP-DRC-MS

Non-invasive detection of drug toxicity in rats by solid-phase extraction and MALDI-TOF analysis of urine samples by Ibon Iloro; Esperanza Gonzalez; Virginia Gutierrez-de Juan; Jose M. Mato; Juan M. Falcon-Perez; Felix Elortza (pp. 2311-2320).

An access to fast and non-invasive techniques to infer or predict the drug-induced injury caused by newly developed drugs and to monitor therapeutic efficacy of established drugs during treatment are of the outmost importance in pharmaceutical industry and clinical diagnosis. Peptidome and low molecular weight proteome profiling is an emerging technique that allows the recognition of distinctive patterns and differentiation among diverse physiopathological conditions. In this article, we evaluated the utility of peptide/small protein profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with WCX magnetic bead-based solid-phase extraction as a screening tool for drug toxicity assessment in urine samples. Given that drug-induced injury is primarily reflected in liver, three different, well-described hepatotoxic drugs were chosen for this work. These were: carbon tetrachloride (CCl₄) which induces liver fibrosis, d(+)-galactosamine as a model for acute liver injury, and Escherichia coli-derived lipopolysaccharide to study the damage caused by endotoxins. The profiles obtained with a correct clustering analysis show that this methodology can be used as a non-invasive and straightforward approach to test for potential drug toxicity. Pharmaceutical research and drug development studies could benefit from this methodology as liver injury inducer compounds could be easily detected in vivo by non-invasive means, accelerating the launch of safer drugs to the market.

Keywords: MALDI-TOF profiling; Solid-phase extraction; Systems toxicology; Drug-induced injury; Urine peptidomics

Improved detection specificity for plasma proteins by targeting cysteine-containing peptides with photo-SRM by Quentin Enjalbert; Marion Girod; Romain Simon; Jérémy Jeudy; Fabien Chirot; Arnaud Salvador; Rodolphe Antoine; Philippe Dugourd; Jérôme Lemoine (pp. 2321-2331).

Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as an alternative to immunoassays for protein quantification owing to faster development time and higher multiplexing capability. However, the SRM strategy is faced with the high complexity of peptide mixtures after trypsin digestion of whole plasma or the cellular proteome that most of the time causes contamination, irremediably, by interfering compounds in the transition channels monitored. This problem becomes increasingly acute when the targeted protein is present at a low concentration. In this work, the merit of laser-induced photo-dissociation in the visible region at 473 nm implemented in an hybrid quadrupole linear ion-trap mass spectrometer (photo-SRM) was evaluated for detection specificity of cysteine-containing peptides in a group of plasma proteins after tagging with a dabcyl chromophore. Compared with conventional SRM, photo-SRM chromatograms have improved detection specificity for most of peptides monitored. Comparison of the signals obtained for the best proteotypic peptides in SRM mode and those recorded by photo-SRM of cysteine-containing peptides for the same proteins reveals either increased (up to 10-fold) or similar signal to photo-SRM detection. Finally, photo-SRM has extended response linearity across a calibration plot obtained by diluting human plasma in rat plasma, down to the lowest concentrations. Hence, photo-SRM may advantageously complement conventional SRM in assay of proteins in complex biological matrices.

Keywords: Photo-SRM; Cysteine-containing peptides quantification; Chromophore derivatization; Plasma proteins

Mass spectrometry based N- and C-terminal sequence determination of a hepatopancreas-type prophenoloxidase from the kuruma prawn, Marsupenaeus japonicus by Hiroki Kuyama; Taro Masuda; Chihiro Nakajima; Kyosuke Momoji; Tatsuya Sugawara; Osamu Nishimura; Takashi Hirata (pp. 2333-2340).

We previously identified and characterized a novel hepatopancreas-type prophenoloxidase from kuruma prawn, Marsupenaeus japonicus. In the characterization, this enzyme was indicated to have a feature of a signal peptide at its N-terminus. The putative primary structure was then proposed but its N- and C-terminal sequences remained undetermined. In the present study, the N- and C-terminal amino acid sequences of this prophenoloxidase were determined by de novo sequencing methods using matrix-assisted laser desorption ionization mass spectrometry. The sequence analyses revealed that the N-terminus of the prophenoloxidase was processed, whereas the C-terminus was not. This finding suggests that this enzyme has a signal peptide, and that it is synthesized at the endoplasmic reticulum in hepatopancreas cells and secreted to hemolymph plasma, similar to the case of hemocyanin, another member of the class III copper proteins.

Keywords: Prophenoloxidase; Crustacean; Signal peptide; De novo sequencing; Matrix-assisted laser desorption ionization mass spectrometry; TMPP

A strategy for the identification of plants in illegal pharmaceutical preparations and food supplements using chromatographic fingerprints by E. Deconinck; C. De Leersnijder; D. Custers; P. Courselle; J. O. De Beer (pp. 2341-2352).

The detection of regulated and forbidden herbs in pharmaceutical preparations and nutritional supplements is a growing problem for laboratories charged with the analysis of illegal pharmaceutical preparations and counterfeit medicines. This article presents a feasibility study of the use of chromatographic fingerprints for the detection of plants in pharmaceutical preparations. Fingerprints were developed for three non-regulated common herbal products—Rhamnus purshiana, Passiflora incarnata L. and Crataegus monogyna—and this was done by combining three different types of detection: diode-array detection, evaporative light scattering detection and mass spectrometry. It is shown that these plants could be detected in respective triturations of the dry extracts with lactose and three different herbal matrices as well as in commercial preparations purchased on the open market. Figure Detection of Passiflora incarnata in three commercial preparations using chromatographic fingerprints

Keywords: Chromatographic fingerprints; Illegal herbal products; Diode-array detection; Evaporative light scattering detection; Mass spectrometry

Non-stochastic sampling error in quantal analyses for Campylobacter species on poultry products by Peter Irwin; Sue Reed; Jeffrey Brewster; Ly Nguyen; Yiping He (pp. 2353-2369).

Using primers and fluorescent probes specific for the most common food-borne Campylobacter species (Campylobacter jejuni and Campylobacter coli), we developed a multiplex, most probable number (MPN) assay using quantitative PCR (qPCR) as the determinant for binomial detection: i.e., number of p positive pathogen growth responses out of n = 6 observations each of 4 mL (V) per dilution. Working with media washes of thrice frozen-thawed chicken pieces which had been spiked with known levels of C. jejuni and C. coli, we found that about 20 % of the experiments had a significant amount of error in the form of either greater than 25 % MPN calculation error (Δε) and/or a low apparent recovery rate (R less than 1 = MPN observed ÷ CFU spiked). Assuming such errors were exacerbated by an excessively small n, we examined computer-generated MPN enumeration data from the standpoint of stochastic sampling error (Δ) and found that such binomial-based assays behaved identically to Poisson-based methods (e.g., counting data) except that fewer technical replicates (n) appeared to be required for the same number of cells per test volume (μ). This result implies that the qPCR detection-based MPN protocol discussed herein should accurately enumerate a test population with a μ ≥ 1 using n = 6 observations per dilution. For our protocol, this equates to ≥ 8 cells per 400–500 g of sampled product. Based on this analysis, the error rate we saw in spiked experiments (where μ > > 1) implied a non-stochastic source. In other experiments we present evidence that this source was, at least in part, related to the cell concentration step (i.e., centrifugation). We also demonstrate that the error rate lessened (from ∼38 % to ∼13 %) at lower Campylobacter levels (μ ≤ 40) as would most likely exist in nature. Using this protocol, we were able to quantify 14 to 1,226 MPN per 450 g of naturally contaminated chicken for skinless pieces and 11 to 244 MPN per 450 g for wings, breasts, legs, and thighs (skin on) whereupon about 50 % of the 29 samples tested negative for both species. Four of these chicken wash samples did have substantially lower Campylobacter levels (1 to 6 MPN per 450 g) which might be better enumerated using a larger n. However, we established that the limit of quantification of this protocol diminishes for n > 6 because one is ever more diluting the sample, or lessening V, to achieve the requisite n.

Keywords: Food-borne pathogens; Campylobacter ; Real-time PCR; qPCR; MPN

Enzyme-based online monitoring and measurement of antioxidant activity using an optical oxygen sensor coupled to an HPLC system by Michela Quaranta; Endry Nugroho Prasetyo; Klaus Koren; Gibson S. Nyanhongo; Michael Murkovic; Ingo Klimant; Georg M. Guebitz (pp. 2371-2377).

It is estimated that up to 50 % of the adult population take antioxidant products on a daily basis to promote their health status. Strangely, despite the well-recognized importance of antioxidants, currently there is no international standard index for labeling owing to the lack of standardized methods for antioxidant measurement in complex products. Here, an online high-performance liquid chromatography (HPLC)-based method to detect and measure the total antioxidant capacity of antioxidant samples is presented. In this approach, complex samples containing antioxidants are separated by the HPLC system, which is further coupled to an antioxidant measuring system consisting of an optical oxygen sensor, laccase, and tetramethoxy azobismethylene quinone (TMAMQ). The antioxidants, separated via HPLC, reduce TMAMQ to syringaldazine, which is then reoxidized by laccase while simultaneously consuming O₂. The amount of consumed oxygen is directly proportional to the concentration of antioxidants and is measured by the optical oxygen sensor. The sensor is fabricated by coating a glass capillary with an oxygen-sensitive thin layer made of platinum(II) meso-tetra(4-fluorophenyl)tetrabenzoporphyrin and polystyrene, which makes real-time analysis possible (t ₉₀ = 1.1 s in solution). Four selected antioxidants (3 mM), namely, catechin, ferulic acid, naringenin (used as a control), and Trolox, representing flavonol, hydrocinnamic acid, flavanone, and vitamin E, respectively, were injected into the online antioxidant monitoring system, separated, and then mixed with the TMAMQ/laccase solution, which resulted in oxygen consumption. This study shows that, with the use of such a system, the antioxidant activity of individual antioxidant molecules in a sample and their contribution to the total antioxidant activity of the sample can be correctly assigned.

Keywords: Online measurements; Antioxidant; Tetramethoxy azobismethylene quinone/syringaldazine; Optical oxygen sensor

Fluorescent system based on bacterial expression of hybrid KcsA channels designed for Kv1.3 ligand screening and study by Kseniya S. Kudryashova; Oksana V. Nekrasova; Alexey I. Kuzmenkov; Alexander A. Vassilevski; Anastasia A. Ignatova; Yuliya V. Korolkova; Eugene V. Grishin; Mikhail P. Kirpichnikov; Alexey V. Feofanov (pp. 2379-2389).

Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K⁺-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch–clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the “mix and read” mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom. Figure The scheme of a fluorescent analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K⁺-conducting pore.

Keywords: Voltage-gated; Binding assay; Confocal; Spheroplasts

Correction of peripheral blood mononuclear cell cytosolic protein for hemoglobin contamination by Dick Pluim; Bart A. W. Jacobs; Martin D. Krähenbühl; Anna E. M. Ruijter; Jos H. Beijnen; Jan H. M. Schellens (pp. 2391-2395).

Pharmacodynamic (PD) analysis requires accurate and precise quantification of enzyme activity targeted by anticancer agents in surrogate cells like peripheral blood mononuclear cells (PBMCs). Enzyme activity is normally reported per mass unit of protein input. However, high and fluctuating hemoglobin (Hb) contamination strongly influences the protein content of PBMC cytosolic lysate. We present the development and validation of a spectrophotometrical Hb quantification method to correct for this contamination. The applicability of Hb correction was demonstrated by determination of the dihydropyrimidine dehydrogenase enzyme activity in PBMC cytosolic lysates.

Keywords: PBMC; Protein; Pharmacodynamics; Hemoglobin; RBC

Application of reversed-phase high-performance liquid chromatography with fluorimetric detection for simultaneous assessment of global DNA and total RNA methylation in Lepidium sativum: effect of plant exposure to Cd(II) and Se(IV) by Eunice Yanez Barrientos; Kazimierz Wrobel; Adolfo Lopez Torres; Felix Gutiérrez Corona; Katarzyna Wrobel (pp. 2397-2404).

In the present work, application of the previously established reversed-phase liquid chromatography procedure based on fluorescent labeling of cytosine and methylcytosine moieties with 2-bromoacetophenone (HPLC-FLD) is presented for simultaneous evaluation of global DNA and total RNA methylation at cytosine carbon 5. The need for such analysis was comprehended from the recent advances in the field of epigenetics that highlight the importance of non-coding RNAs in DNA methylation and suggest that RNA methylation might play a similar role in the modulation of genetic information, as previously demonstrated for DNA. In order to adopt HPLC-FLD procedure for DNA and RNA methylation analysis in a single biomass extract, two extraction procedures with different selectivity toward nucleic acids were examined, and a simplified calibration was designed allowing for evaluation of methylation percentage based on the ratio of chromatographic peak areas: cytidine/5-methylcytidine for RNA and 2´-deoxycytidine/5-methyl-2′-deoxycytidine for DNA. As a proof of concept, global DNA and total RNA methylation were determined in Lepidium sativum hydroponically grown in the presence of different Cd(II) or Se(IV) concentrations, expecting that plant exposure to abiotic stress might affect not only global DNA but also total RNA methylation. The results obtained showed the increase of DNA methylation in the treated plants up to concentration levels 2 mg L⁻¹ Cd and 1 mg L⁻¹ Se in the growth medium. For higher stressors’ concentration, global DNA methylation tended to decrease. Most importantly, an inverse correlation was found between DNA and RNA methylation levels (r = −0.6788, p = 0.031), calling for further studies of this particular modification of nucleic acids in epigenetic context.

Keywords: Bioanalytical methods; HPLC; 5-Methylcytosine; Nucleic acids; Epigenetics

Dibenzo-p-dioxins and dibenzofurans in human breast milk collected in the area of Taranto (Southern Italy): first case study by Giuliana Bianco; Rosalia Zianni; Giuseppe Anzillotta; Achille Palma; Vincenzo Vitacco; Laura Scrano; Tommaso R. I. Cataldi (pp. 2405-2410).

We report on the content of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in 15 breast milk samples of nursing women living in the city of Taranto (Southern, Italy) or nearby. Breast milk samples were collected over the 2008–2009 period and analyzed by gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) upon accelerated solvent extraction (ASE) using acetone/n-hexane mixture 1:1 (v/v). The method was validated demonstrating good performing features. Profiles of PCDD/PCDF congeners in breast milk samples exhibited a prevalence of PCDFs compared to PCDDs. Toxic equivalents (TEQs in picogram per gram fat) of four breast milk were far above the legal limit for human consumption of 3.0 pg/g; their estimated daily and weekly dietary intake were almost 5–20 and 10–40 times higher, respectively, than the tolerable intake values established by the World Health Organization. Figure Occurrence of the toxic micropollutants in breast milk samples of mothers living in or nearby Taranto

Keywords: Polychlorinated dibenzo-p-dioxins; Polychlorinated dibenzofurans; Breast milk; High-resolution mass spectrometry; Gas chromatography

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Analytical and Bioanalytical Chemistry (v.405, #7)