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Analytical and Bioanalytical Chemistry (v.399, #8)

Advances in analytical mass spectrometry by Maria Careri (pp. 2583-2584).

has been Full Professor of Analytical Chemistry at the Faculty of Sciences of Parma University since 2001. Since 2002 she has been Director of the university master course “Chemistry Laboratory Quality Systems.” She is also Director of the National School of “Analytical Methodologies Based on Mass Spectrometry” of the Italian Chemical Society, which has been held in Parma since 2005. She is Past-President of the Division of Analytical Chemistry of the Italian Chemical Society. She is a member of the editorial boards of Analytical and Bioanalytical Chemistry, Current Analytical Chemistry, and Journal of Chromatography A. For the last 10 years her research activities have centered on the development of novel materials for solvent-free extraction techniques for food safety and environmental monitoring and the development of innovative methods for structural and functional proteomics using mass-spectrometry-based techniques. Her current research interests include the development of novel materials for desorption electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry with special focus on the characterization of the surfaces.

Trends in analytical atomic and molecular mass spectrometry in biology and the life sciences by Maria Careri; Alessandro Mangia (pp. 2585-2595).

Trends in analytical atomic and molecular mass spectrometry (MS) are reviewed here with an emphasis on problems related to biology and the life sciences. The move towards novel desorption/ionization techniques including matrix-free laser desorption-ionization MS and recent technological developments in MS imaging of elements, small molecules, and proteins are discussed. Recent developments in the interfacing of microfluidics with MS are also reviewed. Metabolic profiling, evaluation of biomarkers, pharmaceutical metabolite identification, and many related topics are addressed.

Keywords: Mass spectrometry imaging; Ion mobility mass spectrometry; Laser ablation; ICP–MS; Matrix-free laser desorption/ionization MS; Microfluidics; Miniature mass spectrometers; Multiplexed analysis; Biosciences

Recent advances in SALDI-MS techniques and their chemical and bioanalytical applications by K. P. Law; James R. Larkin (pp. 2597-2622).

Although laser desorption mass spectrometry was introduced in the 1960s, the potential of laser mass spectrometry was not realised until the introduction of matrix-assisted laser desorption/ionisation (MALDI) in the 1980s. The technique relies on light-absorbing compounds called matrices that are co-crystallised with the analyte to achieve high ionisation and desorption efficiencies. MALDI offers a lot of advantages and is an indispensable tool in macromolecule analysis. However, the presence of the matrix also produces a high chemical background in the region below m/z 700 in the mass spectrum. Surface-assisted laser desorption/ionisation (SALDI) substitutes the chemical matrix of MALDI for an active surface, which means that matrix interference can be eliminated. SALDI mass spectrometry has evolved in recent years into a technique with great potential to provide insight into many of the challenges faced in modern research, including the growing interest in “omics” and the demands of pharmaceutical science. A great variety of materials have been reported to work in SALDI. Examples include a number of nanomaterials and surfaces. The unique properties of nanomaterials greatly facilitate analyte desorption and ionisation. This article reviews recent advances made in relation to carbon- and semiconductor-based SALDI strategies. Examples of their environmental, chemical and biomedical applications are discussed with the aim of highlighting progression in the field and the robustness of the technique, as well as to evaluate the strengths and weaknesses of individual approaches. In addition, this article describes the physical and chemical processes involved in SALDI and explains how the unique physical and electronic properties of nanostructured surfaces allow them to substitute for the matrix in energy transfer processes.

Keywords: SALDI; DIOS; NALDI; Nanomaterial; Laser mass spectrometry

Size, weight and position: ion mobility spectrometry and imaging MS combined by András Kiss; Ron M. A. Heeren (pp. 2623-2634).

Size, weight and position are three of the most important parameters that describe a molecule in a biological system. Ion mobility spectrometry is capable of separating molecules on the basis of their size or shape, whereas imaging mass spectrometry is an effective tool to measure the molecular weight and spatial distribution of molecules. Recent developments in both fields enabled the combination of the two technologies. As a result, ion-mobility-based imaging mass spectrometry is gaining more and more popularity as a (bio-)analytical tool enabling the determination of the size, weight and position of several molecules simultaneously on biological surfaces. This paper reviews the evolution of ion-mobility-based imaging mass spectrometry and provides examples of its application in analytical studies of biological surfaces.

Keywords: Mass spectrometry; Ion chromatography; Imaging; Genomics/proteomics

Fast liquid chromatography combined with mass spectrometry for the analysis of metabolites and proteins in human body fluids by Linda Kortz; Christin Helmschrodt; Uta Ceglarek (pp. 2635-2644).

In the last decade various analytical strategies have been established to enhance separation speed and efficiency in high performance liquid chromatography applications. Chromatographic supports based on monolithic material, small porous particles, and porous layer beads have been developed and commercialized to improve throughput and separation efficiency. This paper provides an overview of current developments in fast chromatography combined with mass spectrometry for the analysis of metabolites and proteins in clinical applications. Advances and limitations of fast chromatography for the combination with mass spectrometry are discussed. Practical aspects of, recent developments in, and the present status of high-throughput analysis of human body fluids for therapeutic drug monitoring, toxicology, clinical metabolomics, and proteomics are presented.

Keywords: Body fluids; Liquid chromatography; Mass spectrometry; Rapid chromatography; Ultra-high-performance liquid chromatography; Monolithic columns

Advances in mass spectrometry applied to pharmaceutical metabolomics by Dieter M. Drexler; Michael D. Reily; Petia A. Shipkova (pp. 2645-2653).

Metabolomics, also referred to in the literature as metabonomics, is a relatively new systems biology tool for drug discovery and development and is increasingly being used to obtain a detailed picture of a drug’s effect on the body. Metabolomics is the qualitative assessment and relative or absolute quantitative measurement of the endogenous metabolome, defined as the complement of all native small molecules (metabolites less than 1,500 Da). A metabolomics study frequently involves the comparative analysis of sample sets from a normal state and a perturbed state, where the perturbation can be of any nature, such as genetic knockout, administration of a drug, or change in diet or lifestyle. Advances in mass spectrometry (MS) technologies including direct introduction or in-line chromatographic separation modes, ionization techniques, mass analyzers, and detection methods have provided powerful tools to assess the molecular changes in the metabolome. This review focuses on advances in MS pertaining to the analytical data generation for the main metabolomics methods, namely, fingerprinting, nontargeted, and targeted approaches, as they are applied to pharmaceutical drug discovery and development. Figure Three-dimensional image of a liquid chromatography-mass spectrometry (LC-MS) profile.

Keywords: Metabolomics; Metabonomics; Mass spectrometry

Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures by Jeroen Kool; Martin Giera; Hubertus Irth; Wilfried M. A. Niessen (pp. 2655-2668).

In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets—pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches. Schematic view of an on-line bioaffinity analysis or HRS setup with MS based bioassay detection

Keywords: Mass spectrometry; On-line based bioassays; Post-column bioaffinity screening; Bioactive mixture analysis

Recent developments in protein–ligand affinity mass spectrometry by Niels Jonker; Jeroen Kool; Hubertus Irth; Wilfried M. A. Niessen (pp. 2669-2681).

This review provides an overview of direct and indirect technologies to screen protein–ligand interactions with mass spectrometry. These technologies have as a key feature the selection or affinity purification of ligands in mixtures prior to detection. Specific fields of interest for these technologies are metabolic profiling of bioactive metabolites, natural extract screening, and the screening of libraries for bioactives, such as parallel synthesis libraries and small combichem libraries. The review addresses the principles of each of the methods discussed, with a focus on developments in recent years, and the applicability of the methods to lead generation and development in drug discovery. Figure Schematic view of the principle of filtration based 96-well affinity selection MS binding assays

Keywords: Mass spectrometry; Protein–ligand interactions; Affinity; Bioassay; Pre- and on-column mixture screening

Electron ionization in LC-MS: recent developments and applications of the direct-EI LC-MS interface by Pierangela Palma; Giorgio Famiglini; Helga Trufelli; Elisabetta Pierini; Veronica Termopoli; Achille Cappiello (pp. 2683-2693).

The purpose of this article is to underline the possibility of efficiently using electron ionization (EI) in liquid chromatography (LC) and mass spectrometry (MS). From a historical perspective, EI accompanied the first attempts in LC-MS but, owing to several technical shortcomings, it was soon outshined by soft, atmospheric pressure ionization (API) techniques. Nowadays, two modern approaches, supersonic molecular beam LC-MS and direct-EI LC-MS, offer a valid alterative to API, and preserve the advantages of EI also in LC-MS applications. These advantages can be summarized in three crucial aspects: automated library identification; identification of unknown compounds, owing to EI extensive fragment information; inertness to coeluted matrix interferences owing to very unlikely ion–ion and ion–molecule interactions in the EI gas-phase environment. The direct-EI LC-MS interface is a simple and efficient solution able to produce high-quality, interpretable EI spectra from a wide range of low molecular weight molecules of different polarity. Because of the low operative flow rates, this interface relies on a nano-LC technology that helps in reducing the impact of the mobile phase on the gas-phase environment of EI. This review provides an extensive discussion on the role of EI in LC-MS interfacing, and presents in detail several performance aspects of the direct-EI LC-MS interface, especially in terms of response, mass-spectral quality, and matrix effects. In addition, several key applications are also reported. Figure Range of HPLC amenable compound classes by LC-EIMS

Keywords: Nano liquid chromatography; Mass spectrometry; Electron ionization

Mass spectrometry as test bench for medicinal chemistry studies by Roberta Seraglia; Pietro Traldi (pp. 2695-2710).

This review describes how mass spectrometry can be used as a powerful test bench to obtain information on the biological activity of target compounds. Considering that mass spectrometry is based on the chemical reactivity of the analytes, it is possible to investigate the stability of the active compounds, to predict their behaviour in the environment of interest, and to obtain structure–reactivity relationships for new molecules of pharmacological interest. Electron ionization and metastable ion studies give evidence of the correlation between the mutagenic properties of a series of aryl and heteroaryl triazenes and mass spectrometric data. A linear relationship between the energetics of C(O)–O bond cleavage of some carbamic acid O-aryl esters and their FAAH inhibition activity has been proved by electrospray-ionization ion-trap mass spectrometry. An inverse correlation between the stability and cytotoxic activity of some copper complexes has been clearly established by electrospray-ionization mass spectrometry. Moreover, because of the sensitivity and specificity of mass spectrometry, it has been possible to determine and characterize impurities that in some cases can be the real bioactive compound.

Keywords: Structure-activity relationships; Electron ionization; Electrospray ionization; Collisionally induced decomposition

High-resolution, imaging TOF-SIMS: novel applications in medical research by Per Malmberg; Eva Jennische; Daniel Nilsson; Håkan Nygren (pp. 2711-2718).

The use of high-resolution, imaging TOF-SIMS is described and examples are made to demonstrate the application of the method in medical research. Cytochemistry by TOF-SIMS is shown by localization of diacylglycerol (DG) in cryostat sections of hyaline cartilage and by localization of corticosterone in cryostat sections of the adrenal gland cortex. Quantitative measurements and comparison of groups is shown by comparing the lipid content of adipose tissue from two mouse strains, transgenic mouse expressing the FOXC2 gene and wild-type controls. Finally, biopsies made for histopathological diagnosis of infantile reversible cytochrome c oxidase deficiency myopathy were analyzed in order to define the chemical content of areas showing a pathological structure in the light microscope. The use of high-resolution, imaging TOF-SIMS in medical research allows analysis of intact tissue and probe-free localization of specific target molecules in cells and tissues. The TOF-SIMS analysis is not dependent on penetration of reagents into the sample and also independent of probe reactivity such as cross-reactivity or background staining. The TOF-SIMS method can be made quantitative and allows for analysis of specific target molecules in defined tissue compartments.

Keywords: TOF-SIMS; Cytochemistry; Histopathological diagnosis

Innovative methodology to transfer conventional GC-MS heroin profiling to UHPLC-MS/MS by B. Debrus; J. Broséus; D. Guillarme; P. Lebrun; P. Hubert; J.-L. Veuthey; P. Esseiva; S. Rudaz (pp. 2719-2730).

Nowadays, in forensic laboratories, heroin profiling is frequently carried out by gas chromatography coupled with mass spectrometry (GC-MS). This analytical technique is well established, provides good sensitivity and reproducibility, and allows the use of large databases. Despite those benefits, recently introduced analytical techniques, such as ultra-high-pressure liquid chromatography (UHPLC), could offer better chromatographic performance, which needs to be considered to increase the analysis throughput for heroin profiling. With the latter, chromatographic conditions were optimized through commercial modeling software and two atmospheric pressure ionization sources were evaluated. Data obtained from UHPLC–MS/MS were thus transferred, thanks to mathematical models to mimic GC-MS data. A calibration and a validation set of representative heroin samples were selected among the database to establish a transfer methodology and assess the models’ abilities to transfer using principal component analysis and hierarchical classification analysis. These abilities were evaluated by computing the frequency of successful classification of UHPLC–MS/MS data among GC-MS database. Seven mathematical models were tested to adjust UHPLC–MS/MS data to GC-MS data. A simplified mathematical model was finally selected and offered a frequency of successful transfer equal to 95%. Figure

Keywords: Heroin profiling; Principal component analysis; Hierarchical classification analysis; UHPLC–MS/MS; Multiple linear regression; Transfer

The use of cation exchange matrix separation coupled with ICP-MS to directly determine platinum group element (PGE) and other trace element emissions from passenger cars equipped with diesel particulate filters (DPF) by Warren R. L. Cairns; Antonella De Boni; Giulio Cozzi; Massimo Asti; Edoardo Merlone Borla; Flavio Parussa; Ezio Moretto; Paolo Cescon; Claude Boutron; Jacopo Gabrieli; Carlo Barbante (pp. 2731-2740).

Inductively coupled plasma-mass spectrometry coupled with cation exchange matrix separation has been optimised for the direct determination of platinum group element (PGE) and trace element emissions from a diesel engine car. After matrix separation method detection limits of 1.6 ng g⁻¹ for Pd, 0.4 ng g⁻¹ for Rh and 4.3 ng g⁻¹ for Pt were achieved, the method was validated against the certified reference material BCR 723, urban road dust. The test vehicle was fitted with new and aged catalytic converters with and without diesel particulate filters (DPF). Samples were collected after three consecutive New European Driving Cycle (NEDC) of the particulate and “soluble” phases using a home-made sampler optimised for trace element analysis. Emission factors for the PGEs ranged from 0.021 ng km⁻¹ for Rh to 70.5 ng km⁻¹ for Pt; when a DPF was fitted, the emission factors for the PGEs actually used in the catalysts dropped by up to 97% (for Pt). Trace element emission factors were found to drop by a maximum of 92% for Ni to a minimum of 18% for Y when a DPF was fitted; a new DPF was also found to cause a reduction of up to 86% in the emission of particulate matter.

Keywords: ICP-MS; Cation exchange; Emission factors; PGEs; Trace elements; DPF

Planar solid-phase microextraction-ion mobility spectrometry: a diethoxydiphenylsilane-based coating for the detection of explosives and explosive taggants by M. Mattarozzi; F. Bianchi; F. Bisceglie; M. Careri; A. Mangia; G. Mori; A. Gregori (pp. 2741-2746).

A novel diethoxydiphenylsilane-based coating for planar solid-phase microextraction was developed using sol–gel technology and used for ion mobility spectrometric detection of the explosives 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and of the explosive taggant ethylene glycol dinitrate. The trap was characterized in terms of coating thickness, morphology, inter-batch repeatability, and extraction efficiency. An average thickness of 143 ± 13 μm with a uniform distribution of the coating was obtained. Good performances of the developed procedure in terms of both intra-batch and inter-batch repeatability with relative standard deviations <7% were obtained. Experimental design and desirability function were used to find the optimal conditions for simultaneous headspace extraction of the investigated compounds: the optimal values were found in correspondence of a time and a temperature of extraction of 45 min and 40 °C, respectively. Detection and quantitation limits in low nanogram levels were achieved proving the superior extraction capability of the developed coating, obtaining ion mobility spectrometric responses at least two times higher than those achieved using commercial teflon and paper traps.

Keywords: Planar solid-phase microextraction; Ion mobility spectrometry; Coating; Experimental design; Explosives

Metabolism studies of the Kratom alkaloid speciociliatine, a diastereomer of the main alkaloid mitragynine, in rat and human urine using liquid chromatography-linear ion trap mass spectrometry by Anika A. Philipp; Dirk K. Wissenbach; Armin A. Weber; Josef Zapp; Hans H. Maurer (pp. 2747-2753).

Mitragyna speciosa (Kratom) is currently used as a drug of abuse. When monitoring its abuse in urine, several alkaloids and their metabolites must be considered. In former studies, mitragynine (MG), its diastereomer speciogynine (SG), and paynantheine and their metabolites could be identified in rat and human urine using LC-MSⁿ. In Kratom users' urines, besides MG and SG, further isomeric compounds were detected. To elucidate whether the MG and SG diastereomer speciociliatine (SC) and its metabolites represent further compounds, the phase I and II metabolites of SC were identified first in rat urine after the administration of the pure alkaloid. Then, the identified rat metabolites were screened for in the urine of Kratom users using the above-mentioned LC-MSⁿ procedure. Considering the mass spectra and retention times, it could be confirmed that SC and its metabolites are so far the unidentified isomers in human urine. In conclusion, SC and its metabolites can be used as further markers for Kratom use, especially by consumption of raw material or products that contain a high amount of fruits of the Malaysian plant M. speciosa.

Keywords: Kratom ; Speciociliatine; Metabolism; LC-MS; Urine

Quantum dot-based array for sensitive detection of Escherichia coli by Nuria Sanvicens; Núria Pascual; María Teresa Fernández-Argüelles; Javier Adrián; José Manuel Costa-Fernández; Francisco Sánchez-Baeza; Alfredo Sanz-Medel; M.-Pilar Marco (pp. 2755-2762).

A fluorescent quantum dot-based antibody array, used in sandwich format, has been developed to detect Escherichia coli O157:H7. Numerous parameters such as solid support, optimal concentration of immunoreagents, blocking reagents, and assay time were optimized for array construction. Quantum dot-conjugated anti-IgG was used as the detecting system. The array allows the detection of E. coli O157:H7 at concentrations below 10 CFU mL⁻¹ without sample enrichment, exhibiting an increase of three orders of magnitude in the limit of detection compared to ELISA. The interference caused by Gram (+) and Gram (−) bacteria was negligible at low concentrations of bacteria. Figure

Keywords: Bacteria; E. coli ; Antibody array; Quantum dot

Label-free detection of nucleic acids by turn-on and turn-off G-quadruplex-mediated fluorescence by Jiangtao Ren; Haixia Qin; Jiahai Wang; Nathan W. Luedtke; Erkang Wang; Jin Wang (pp. 2763-2770).

In this study we have used two fluorescent probes, tetrakis(diisopropylguanidino)-zinc-phthalocyanine (Zn-DIGP) and N-methylmesoporphyrin IX (NMM), to monitor the reassembly of “split” G-quadruplex probes on hybridization with an arbitrary “target” DNA. According to this approach, each split probe is designed to contain half of a G-quadruplex-forming sequence fused to a variable sequence that is complementary to the target DNA. Upon mixing the individual components, both base-pairing interactions and G-quadruplex fragment reassembly result in a duplex–quadruplex three-way junction that can bind to fluorescent dyes in a G-quadruplex-specific way. The overall fluorescence intensities of the resulting complexes were dependent on the formation of proper base-pairing interactions in the duplex regions, and on the exact identity of the fluorescent probe. Compared with samples lacking any “target” DNA, the fluorescence intensities of Zn-DIGP-containing samples were lower, and the fluorescence intensities of NMM-containing samples were higher on addition of the target DNA. The resulting biosensors based on Zn-DIGP are therefore termed “turn-off” whereas the biosensors containing NMM are defined as “turn-on”. Both of these biosensors can detect target DNAs with a limit of detection in the nanomolar range, and can discriminate mismatched from perfectly matched target DNAs. In contrast with previous biosensors based on the peroxidase activity of heme-bound split G-quadruplex probes, the use of fluorescent dyes eliminates the need for unstable sensing components (H₂O₂, hemin, and ABTS). Our approach is direct, easy to conduct, and fully compatible with the detection of specific DNA sequences in biological fluids. Having two different types of probe was highly valuable in the context of applied studies, because Zn-DIGP was found to be compatible with samples containing both serum and urine whereas NMM was compatible with urine, but not with serum-containing samples.

Keywords: G-quadruplex; Conformational constraint; Split probe; Fluorescent probe

New marker of tumor cell death revealed by ATR-FTIR spectroscopy by Lucia Di Giambattista; Deleana Pozzi; Paola Grimaldi; Silvia Gaudenzi; Stefania Morrone; Agostina Congiu Castellano (pp. 2771-2778).

Fourier transform infrared spectroscopy in attenuated total reflection can be used to discriminate the necrotic from the apoptotic cell death in a tumoral T cell line irradiated by a UV source able to induce both apoptosis and necrosis. Using Jurkat cells as the model system, significant spectral differences in the irradiated cells vs. time were observed in the lipid–proteins ratio absorbance band at 1,397 cm⁻¹ and in lactic acid IR band at 1,122 cm⁻¹; these spectral features are inversely correlated with the percentage of apoptotic cells assessed by flow cytometry. From the analysis of second derivatives in the IR spectral region between 1,800 and 900 cm⁻¹, we have detected two significant spectral changes: the first centered at 1,621 cm⁻¹ by analyzing the components of the amide I band and the second centered at 1,069 cm⁻¹ due to C–O stretching vibration of the DNA backbone sensitive to the dehydrated state of DNA; these identified differences in the intracellular biomolecules have been allowed to monitor the necrotic process. The variations in the spectral data set have been identified by the Kruskal–Wallis test and confirmed by the hierarchical cluster analysis.

Keywords: FTIR-ATR spectroscopy; UVB radiation; Apoptosis; Necrosis

Influence of amino acid relative position on the oxidative modification of histidine and glycine peptides by Ana Reis; Conceição Fonseca; Elisabete Maciel; Pedro Domingues; M. Rosário M. Domingues (pp. 2779-2794).

The radical oxidation of isomeric peptides containing one reactive amino acid [histidine (H)] and another less reactive amino acid [glycine (G)] in the form of dipeptides (HG and GH) and tripeptides (HGG, GHG, and GGH) was studied by mass spectrometry coupled to liquid chromatography (LC-MS) for detection and LC-MSⁿ for structural characterization. The oxidation products identified were keto, hydroxy, keto-hydroxy, and hydroperoxide derivatives for both di- and tripeptides. Among these, it was found that insertion of oxygen atoms occurred at histidine for HG and HGG, and both histidine and glycine for GH, GHG, and GGH. In addition, oxidation products formed by alkoxyl rearrangement reactions with cleavage of the peptide chain were also identified for GH, GHG, and GGH, corroborating hydrogen abstraction step in G residues. These findings were supported through the identification of radical intermediate species formed and trapped with 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) spin trap. The observation of DMPO adducts bearing two spin trap molecules reinforced the abstraction of two hydrogen atoms from the same molecule.Overall, modification sites identified showed that the outcome of oxidative behavior of peptides is influenced not only by the reactivity of the amino acid in the peptide sequence but also by its relative position within the sequence.

Keywords: Hydroxyl radical; DMPO; Mass spectrometry; Oxidation; Peptides

Use of polyethylenimine-modified magnetic nanoparticles for highly specific enrichment of phosphopeptides for mass spectrometric analysis by Cheng-Tung Chen; Lee-Yih Wang; Yen-Peng Ho (pp. 2795-2806).

Phosphopeptides have been isolated and concentrated by use of polyethyleneimine (PEI)-modified magnetic nanoparticles as an extremely specific affinity probe. The particles specifically captured phosphopeptides from a tryptic digest of a protein mixture that contained 0.07% (mole/mole) phosphoproteins, which is the highest specificity obtained to date. The time required for enrichment of the phosphopeptides was 1 min only. PEI-modified magnetic nanoparticles carry positive charges over a wide range of pH—between 3 and 11. This feature means the particles are effectively dispersed in solution during phosphopeptide capture. Mass spectrometric analysis revealed the very high efficiency of enrichment of phosphopeptides that contain both single and multiply-phosphorylated sites. The detection limit in the analysis of phosphopeptides obtained from both bovine α-casein and β-casein by matrix-assisted laser desorption/ionization mass spectrometry was 5 fmol. This approach was also used to enrich the phosphopeptides in a protein digest obtained from non-fat milk.

Keywords: Phosphopeptides; Mass spectrometry; Polyethyleneimine; Nanoparticles; Enrichment

Predicting the partitioning of biological compounds between room-temperature ionic liquids and water by means of the solvation-parameter model by Juan M. Padró; Agustín Ponzinibbio; Leidy B. Agudelo Mesa; Mario Reta (pp. 2807-2820).

The partition coefficients, P _IL/w, for different probe molecules as well as for compounds of biological interest between the room-temperature ionic liquids (RTILs) 1-butyl-3-methylimidazolium hexafluorophosphate, [BMIM][PF₆], 1-hexyl-3-methylimidazolium hexafluorophosphate, [HMIM][PF₆], 1-octyl-3-methylimidazolium tetrafluoroborate, [OMIM][BF₄] and water were accurately measured. [BMIM][PF₆] and [OMIM][BF₄] were synthesized by adapting a procedure from the literature to a simpler, single-vessel and faster methodology, with a much lesser consumption of organic solvent. We employed the solvation-parameter model to elucidate the general chemical interactions involved in RTIL/water partitioning. With this purpose, we have selected different solute descriptor parameters that measure polarity, polarizability, hydrogen-bond–donor and hydrogen-bond–acceptor interactions, and cavity formation for a set of specifically selected probe molecules (the training set). The obtained multiparametric equations were used to predict the partition coefficients for compounds not present in the training set (the test set), most being of biological interest. Partial solubility of the ionic liquid in water (and water into the ionic liquid) was taken into account to explain the obtained results. This fact has not been deeply considered up to date. Solute descriptors were obtained from the literature, when available, or else calculated through commercial software. An excellent agreement between calculated and experimental log P _IL/w values was obtained, which demonstrated that the resulting multiparametric equations are robust and allow predicting partitioning for any organic molecule in the biphasic systems studied. Figure

Keywords: Ionic liquids; Partition coefficients; Liquid–liquid extraction; Solvation-parameter model; RTIL synthesis

Improvement of reproducibility and sensitivity of CE analysis by using the capillary coated dynamically with carboxymethyl chitosan by Yi Liu; Xiaofang Fu; Yu Bai; Maolin Zhai; Yiping Liao; Jie Liao; Huwei Liu (pp. 2821-2829).

Analysis reproducibility and detection sensitivity of capillary electrophoresis (CE) are often questioned by applied scientists, which has hindered its application as a routine method. To address these issues, a simple, precise, and reproducible dynamic coating method was developed by applying carboxymethyl chitosan (CMC) dynamic coating on fused silica capillary. The proposed coating was accomplished by simply rinsing the capillary with CMC solution for 1 min in between runs, with no regeneration procedure or buffer additives needed. Electroosmotic flow could be well controlled by adjusting the pH of background electrolyte, and the adsorption of analytes onto the capillary inner wall was effectively eliminated. The main parameters of the coating condition were optimized, and extensive applications of these CMC-dynamically coated capillaries in CE separations were then firmly confirmed. By using proteins, aristolochic acids, and inorganic anions as model analytes, the coating showed a good stability, high reproducibility, as well as improved sensitivity. Baseline separations could be obtained with high efficiency. The reduced adsorption was impressively effective for basic proteins, with an average plate number of 90,000/m for each protein, apart from the good resolution on the chromatogram. A high sensitive detection of α-lactalbumin was achieved with a limit of detection (S/N = 3) of 3.5 nM, and the number of theoretical plates was as high as 1,200,000/m. In addition, the combination of the CMC coating with nonaqueous CE and CE-mass spectrometry proved to be practical. All results showed that the CMC-dynamically coated capillary has special properties and obvious superiority over the uncoated ones for CE analysis. Figure

Keywords: CE-MS; Carboxymethyl chitosan; Dynamic coating; Reproducibility; Sensitivity

Capillary electrophoretic separation of nanoparticles by Sławomir Oszwałdowski; Katarzyna Zawistowska-Gibuła; Kenneth P. Roberts (pp. 2831-2842).

In the present work, CdSe nanocrystals (NCs) synthesized with a trioctylphosphine surface passivation layer were modified using amphiphilic molecules to form a surface bilayer capable of providing stable NCs aqueous solutions. Such modified nanocrystals were used as a test solute in order to analyze new electrophoretic phenomena, by applying a micellar plug as a separation tool for discriminating nanocrystals between micellar and micelle-free zones during electrophoresis. The distribution of NCs between both zones depended on the affinity of nanocrystals towards the micellar zone, and this relies on the kind of surface ligands attached to the NCs, as well as electrophoretic conditions applied. In this case, the NCs that migrated within a micellar zone can be focused using a preconcentration mechanism. By modifying electrophoretic conditions, NCs were forced to migrate outside the micellar zone in the form of a typical CZE peak. In this situation, a two-order difference in separation efficiencies, in terms of theoretical plates, was observed between focused NCs (N ~ 10⁷) and a typical CZE peak for NCs (N ~ 10⁵). By applying the amino-functionalized NCs the preconcentration of NCs, using a micellar plug, was examined, with the conclusion that preconcentration efficiency, in terms of the enhancement factor for peak height (SEF_height) can be, at least 20. The distribution effect was applied to separate CdSe/ZnS NCs encapsulated in silica, as well as surface-modified with DNA, which allows the estimation of the yield of conjugation of biologically active molecules to a particle surface. Figure Micellar plug as a tool to separate nanoparticles: nanocrystals coated with an ionic surfactant form a pseudomicellar system with a non-ionic surfactant. Appling this system in the form of a plug, in capillary electrophoresis, nanocrystals preconcentration within a plug or release from a plug, depending on applied conditions, were observed. This was used to discriminate particles vs. particles bioconjugated with aptamer

Keywords: Capillary electrophoresis; Micelles; Nanocrystals; Focusing; Oligomer

Inhibitor screening of pharmacological chaperones for lysosomal β-glucocerebrosidase by capillary electrophoresis by Meera Shanmuganathan; Philip Britz-McKibbin (pp. 2843-2853).

Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, β-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-β-d-glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC 50) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease. Figure Inhibitor screening of previously approved drugs that function as pharmacological chaperones for glucocerebrosidase by capillary electrophoresis.

Keywords: Enzyme kinetics; Inhibitor screening; Glucocerebrosidase; Pharmacological chaperone; Capillary electrophoresis; Gaucher disease

Field-amplified sample injection combined with pressure-assisted capillary electrophoresis UV detection for the simultaneous analysis of allantoin, uric acid, and malondialdehyde in human plasma by Angelo Zinellu; Salvatore Sotgia; Luca Deiana; Ciriaco Carru (pp. 2855-2861).

The allantoin/uric acid (All/UA) ratio and malondialdehyde (MDA) plasma levels have been proposed as important markers for monitoring oxidation triggered by the action of free radicals (FR). Here, we describe an easy field amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of All, UA, and free MDA in human plasma. The plasma samples were simply filtered through centrifugation membrane tubes for protein elimination and directly injected on a capillary without complex cleanup and/or sample derivatization procedures. The use of a run buffer composed of 300 mmol/L sodium borate at pH 10 with 50 mmol/L of N-methyl-d-glucamine and an overimposed pressure/voltage of 0.1 psi during the electrophoretic run allows basline resolution of the analytes within 17 min. The electrokinetic injection allows a detection limit of 15 nmol/L for All, 20 nmol/L for UA and 10 nmol/L for MDA in a plasma sample, thus significantly improving the LOD of previous described methods based on capillary electrophoresis. Precision tests indicate a good repeatability of our method both for migration times (CV = 1.85%) and areas (CV = 2.87%). Moreover, a good reproducibility of intra- and inter-assay tests was obtained (CV = 4.63% and CV = 6.59% respectively). The suitability of the method was tested by measuring analyte levels in 40 healthy volunteers.

Keywords: Capillary electrophoresis; FASI; Allantoin; Uric acid; Malondialdehyde; Oxidative stress

Comparison of the efficiency of different extraction methods for the simultaneous determination of mycotoxins and pesticides in milk samples by ultra high-performance liquid chromatography-tandem mass spectrometry by M. M. Aguilera-Luiz; P. Plaza-Bolaños; R. Romero-González; J. L. Martínez Vidal; A. Garrido Frenich (pp. 2863-2875).

A rapid multi-analyte method has been developed for the simultaneous determination of pesticides and mycotoxins in milk by ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC–QqQ–MS/MS). A variety of methodologies has been evaluated, including solid-phase extraction (SPE), “dilute-and-shoot” (liquid–liquid extraction-based procedures), and QuEChERS (quick, easy, cheap, effective, rugged, and safe)-based methods. The optimization and development process was carried out considering that the maximum residue level for aflatoxin M1 (AFM1) in milk in the European Union (EU) is set at 0.05 μg kg⁻¹, which is the lowest tolerance in the target compounds. The selected method consisted of an extraction by SPE using C18 as sorbent and methanol as elution solvent. The final determination was performed by UHPLC–QqQ–MS/MS. Matrix-matched standard calibration was used for quantification, obtaining recoveries in the range 60–120% with relative standard deviations <25%, at three spiking levels: 0.5, 10, and 50 μg kg⁻¹ (ten times lower for AFM1). Limits of quantification ranged from 0.20 to 0.67 μg kg⁻¹, which were always below or equal to the established tolerance levels by the EU. Finally, the selected method was applied to different types of milk.

Keywords: Mycotoxins; Pesticides; Milk; Analysis; SPE; UHPLC–QqQ–MS/MS

Development of a liquid chromatography–mass spectrometry method for the determination of ursolic acid in rat plasma and tissue: Application to the pharmacokinetic and tissue distribution study by Qinhua Chen; Shangwen Luo; Yulin Zhang; Zilin Chen (pp. 2877-2884).

A fast and sensitive liquid chromatography–mass spectrometry method was developed for the determination of ursolic acid (UA) in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was performed on a 3.5 μm Zorbax SB-C18 column (30 mm × 2.1 mm) with a mobile phase consisting of methanol and aqueous 10 mM ammonium acetate using gradient elution. Quantification was performed by selected ion monitoring with (m/z)⁻ 455 for UA and (m/z)⁻ 469 for the IS. The method was validated in the concentration range of 2.5 − 1470 ng mL⁻¹ for plasma samples and 20 − 11760 ng g⁻¹ for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 1.6% to 7.1% and 3.7% to 9.0%, respectively, and the intra- and inter-day assay accuracy was 84.2 − 106.9% and 82.1 − 108.1%, respectively. Recoveries in plasma and tissues ranged from 83.2% to 106.2%. The limits of detections were 0.5 ng mL⁻¹ or 4.0 ng g⁻¹. The recoveries for all samples were >90%, except for liver, which indicated that ursolic acid may metabolize in liver. The main pharmacokinetic parameters obtained were T _max = 0.42 ± 0.11 h, C _max = 1.10 ± 0.31 μg mL⁻¹, AUC = 1.45 ± 0.21 μg h mL⁻¹ and K _a = 5.64 ± 1.89 h⁻¹. The concentrations of UA in rat lung, spleen, liver, heart, and cerebellum were studied for the first time. This method is validated and could be applicable to the investigation of the pharmacokinetics and tissue distribution of UA in rats. Figure The mean plasma concentration–time curve and tissue distributions of UA in rats after oral administration of UA

Keywords: Ursolic acid; HPLC-MS; Pharmacokinetics; Tissue distribution

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Analytical and Bioanalytical Chemistry (v.399, #8)

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Analytical and Bioanalytical Chemistry (v.399, #8)