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Analytical and Bioanalytical Chemistry (v.378, #4)

Liquid chromatography–mass spectrometry: reaching maturity rapidly by Uwe Karst (pp. 841-841).

Triple-quadrupole LC–MS–MS for quantitative determination of nitrofuran metabolites in complex food matrixes by S. Effkemann; F. Feldhusen (pp. 842-844).

Recent trends in mass spectrometer development by James W. Hager (pp. 845-850).

Trends in mass analyzer development are reviewed here with an emphasis on tandem mass spectrometers. The move toward “hybridization” of conventional mass analyzers to allow additional instrument functionality in tandem mass spectrometry is discussed.

Keywords: Mass spectrometer; Ion trap; Quadrupole; Time-of-flight MS

LC-MS analysis in the aquatic environment and in water treatment – a critical review by Christian Zwiener; Fritz H. Frimmel (pp. 851-861).

LC-MS has become an invaluable technique for trace analysis of polar compounds in aqueous samples of the environment and in water treatment. LC-MS is of particular importance due to the impetus it has provided for research into the occurrence and fate of polar contaminants, and of their even more polar transformation products. Mass spectrometric detection and identification is most widely used in combination with sample preconcentration, chromatographic separation and atmospheric pressure ionization (API). The focus of the first part of this review is directed particularly toward instruments and method development with respect to their applications for detecting emerging contaminants, microorganisms and humic substances (HS). The current status and future perspectives of 1) mass analyzers, 2) ionization techniques to interface liquid chromatography (LC) with mass spectrometry (MS), 3) methods for preconcentration and separation with respect to their application for water analysis are discussed and examples of applications are given. Quadrupole and ion trap mass analyzers with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are already applied in routine analysis. Time-of-flight (TOF) mass spectrometers are of particular interest for accurate mass measurements for identification of unknowns. For non-polar compounds, different ionization approaches have been described, such as atmospheric pressure photoionization (APPI), electrochemistry with ESI, or electron capture ionization with APCI. In sample preconcentration and separation, solid phase extraction (SPE) with different non-selective sorbent materials and HPLC on reversed-phase materials (RP-HPLC) play the dominant role. In addition, various on-line and miniaturized approaches for sample extraction and sample introduction into the MS have been used. Ion chromatography (IC), size-exclusion chromatography (SEC), and capillary electrophoresis (CE) are alternative separation techniques. Furthermore, the issues of compound identification, matrix effects on quantitation, development of mass spectral libraries and the topic of connecting analysis and toxicity bioassays are addressed.

Keywords: Mass spectrometry; Instrumentation; Contaminant; Water; Sample preparation

LC-MS analysis in the aquatic environment and in water treatment technology – a critical review by Christian Zwiener; Fritz H. Frimmel (pp. 862-874).

Environmental contaminants of recent concern are pharmaceuticals, estrogens and other endocrine disrupting chemicals (EDC) such as degradation products of surfactants, algal and cyanobacterial toxins, disinfection by-products (DBPs) and metalloids. In addition, pesticides (especially their transformation products), microorganisms, and humic substances (HS), in their function as vehicles for contaminants and as precursors for by-products in water treatment, traditionally play an important role. The present status of the application of LC-MS techniques for these water constituents are discussed and examples of application are given. Solid-phase extraction with various non-selective materials in combination with liquid chromatography (LC) on reversed-phase columns have been the most widely used methods for sample preconcentration and separation for different compound classes like pesticides, pharmaceuticals or estrogens. Electrospray ionization (ESI) and atmospheric pressure ionization (APCI) are the most frequently used ionization techniques for polar and ionic compounds, as well as for less polar non-ionic ones. The facilities of LC-MS have been successfully demonstrated for different compound classes. Polar compounds from pharmaceuticals used as betablockers, iodinated X-ray contrast media, or estrogens have been determined without derivatization down to ultratrace concentrations. LC-MS can be viewed as a prerequisite for the determination of algal and cyanobacterial toxins and the homologues and oligomers of alkylphenol ethoxylates and their metabolites. Tandem mass spectrometric techniques and the use of diagnostic ions reveal their usefulness for compound-class specific screening and unknown identification, and are also valid for the analysis of pesticides and especially for their transformation products. Structural information has been gained by the application of LC-MS methods to organometallic species. New insights into the structural variety of humic substances have been made possible by FT-ICR-MS due to its ultrahigh mass resolution. Finally, exciting possibilities for rapid detection and identification of microorganisms have been made possible by MALDI and LC-MS methods.

Keywords: Mass spectrometry; Emerging contaminant; Water; Toxin; Endocrine

Derivatization of neutral steroids to enhance their detection characteristics in liquid chromatography–mass spectrometry by Tatsuya Higashi; Kazutake Shimada (pp. 875-882).

This review article underlines the detection-oriented derivatization of neutral steroids in liquid chromatography–mass spectrometry (LC–MS). Steroids have strong biological activity at very low concentrations in target tissues and, therefore, the analysis of steroids in body fluids or tissues is necessary to elucidate the nature of the many endocrine disease processes and thus be useful for diagnosis and treatment. LC–MS has recently been used for steroid analysis because of its specificity and versatility, but the ionization efficiencies of most steroids are relatively low for the different ionization methods. Derivatization enhances the ionization efficiencies of steroids, leading to high sensitivity and specific detection. For electrospray ionization MS the introduction of permanently charged moieties or easily ionizable moieties effectively increases the sensitivity of detection of steroids. The introduction of moieties with proton affinity or electron affinity enhances the analyte signals in positive and negative atmospheric pressure chemical ionization MS, respectively.

Keywords: Liquid chromatography–mass spectrometry; Detection-oriented derivatization; Neutral steroid

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography–mass spectrometry by Harald John; Michael Walden; Sandra Schäfer; Sandra Genz; Wolf-Georg Forssmann (pp. 883-897).

Peptide quantification by liquid chromatography–mass spectrometry (LC–MS) combines the high resolving power of reversed-phase (RP) chromatography with the excellent selectivity and sensitivity of mass spectrometric detection. On the basis of comprehensive practical experience in the analysis of small molecules, pharmaceutical research is developing technologies for analysis of a growing number of peptidic drug candidates. This article is a detailed review of procedures based on LC–MS techniques for quantitative determination of peptides. With the focus on pharmaceutical applications several technologies for sample preparation, various aspects of peptide chromatography, important characteristics of ESI–MS, selectivity of MS-detection modes, the large variability of internal standards, and modern instrumentation are discussed. The demand for reliable, robust, sensitive, and accurate methods is discussed using numerous examples from the literature, complemented by experiments and results from our laboratory.

Keywords: Liquid chromatography–mass spectrometry; Electrospray; Peptide quantification; Precipitation; Solid-phase extraction; Internal standard

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites by Martin Zeller; Simone König (pp. 898-909).

Protein phosphorylation analysis is an enormous challenge. This review summarises the currently used techniques, which are based on radiolabelling and mass spectrometry as well as electrophoretic and chromatographic separation. Many methods exist, but there is still no single procedure applicable to all phosphoproteins. MS is able to deliver information about the location of phosphorylation sites, but phosphospecific properties with respect to ionisation present obstacles. Therefore, multidimensional approaches involving several analytical methods are often necessary to conquer phosphorylation site identification.

Keywords: Protein phosphorylation; Mass spectrometry; HPLC; IMAC; Radiolabelling

Evaluation of three calibration methods to compensate matrix effects in environmental analysis with LC-ESI-MS by Markus Stüber; Thorsten Reemtsma (pp. 910-916).

In quantitative analysis of environmental samples using high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC-ESI-MS) one of the major problems is the suppression or, less frequently, the enhancement of the analyte signals in the presence of matrix components. Standard addition is the most suitable method for compensating matrix effects, but it is time-consuming and laborious. In this study we compare the potential of three calibration approaches to compensate matrix effects that occurred when seven analytes (naphthalene sulfonates) were quantified in time series samples of waters with different matrices (untreated and treated industrial wastewater). The data obtained by external calibration, internal calibration with one standard, and external sample calibration (corresponding to matrix-matched calibration) were compared with those obtained by standard addition. None of the three approaches were suitable for a sample series of highly loaded, untreated wastewater with highly variable matrix. For less heavily loaded and less variable samples (treated wastewater effluents), the external sample calibration provided reasonable results for most analytes with deviations mostly below 25% as compared to standard addition. External sample calibration can be suitable to compensate matrix effects from moderately loaded samples with more uniform matrices, but it is recommended to verify this for each sample series against the standard addition approach.

Keywords: Environmental analysis; Mass spectrometry; Electrospray ionization; Matrix effects; Compensation methods; Standard addition

Liquid chromatography–electrochemistry–mass spectrometry of polycyclic aromatic hydrocarbons by Suze M. van Leeuwen; Heiko Hayen; Uwe Karst (pp. 917-925).

An efficient method for fast elucidation of the electrochemical reactions of polycyclic aromatic hydrocarbons (PAH) has been set up by applying post-column electrochemistry in liquid chromatography–mass spectrometry (LC–MS). With this set-up strong improvement of sensitivity in the LC–MS analysis of PAH is observed. Due to their low redox potentials, the non-polar PAH are converted into the respective radical cations, which may further react with constituents of the mobile phase and in additional electrochemical oxidation steps. Among other products, mono-, di-, and trioxygenated species are observed in aqueous solutions, alkoxylated compounds in alcohols, and solvent adducts in the presence of acetonitrile. While more different products are observed by using atmospheric pressure chemical ionization in the positive-ion mode (APCI(+)), the deprotonation of hydroxylated species results in very clear spectra in the negative-ion mode (APCI(−)). Deuterated PAH and deuterated solvents were used to gain additional information on the formation of the reaction products.

Keywords: Carcinogens; PAH; Electrochemistry; LC–MS; On-line LC–electrochemistry–MS

HPLC-MS investigations of acidic contaminants in ammunition wastes using volatile ion-pairing reagents (VIP-LC-MS) by Torsten C. Schmidt; Ulf Buetehorn; Klaus Steinbach (pp. 926-931).

In order to hyphenate ion pairing chromatography and MS detection we used several types of formates as volatile ion pairing reagents (IPRs) instead of common tetraalkylammonium salts, as these salts tend to precipitate in the ion source. The formates were prepared by mixing formic acid with the corresponding amine. Both tributyl- and trihexylammonium formate proved to be valuable IPRs for the separation of acidic compounds like nitrobenzoic acids, nitrobenzenesulfonic acids and nitrated phenols. Due to the weaker retention of the ion-pairs with trialkylammonium formates compared with tetraalkylammonium compounds, either less organic modifier or a higher concentration of the IPR had to be used. With negative atmospheric pressure chemical ionization mass spectrometry and electrospray ionization mass spectrometry it was possible to unambiguously identify several acidic oxidation products of 2,4,6-trinitrotoluene (TNT) in ammunition wastewater and soil extracts. 2-amino-4,6-dinitrobenzoic acid was often found to be the main metabolite of TNT in such water samples.

Keywords: Liquid chromatography-mass spectrometry; Ion-pairing chromatography; Ammunition waste; 2,4,6-Trinitrotoluene ; Nitrobenzoic acids

Development of a LC-MS/MS method for the analysis of volatile primary and secondary amines as NIT (naphthylisothiocyanate) derivatives by Anna-Sara Claeson; Anders Östin; Anna-Lena Sunesson (pp. 932-939).

High-performance liquid chromatography with mass spectrometric detection was used for the structure elucidation of eighteen primary and secondary amines and ammonia derivatised with naphthylisothiocyanate (NIT). A fragmentation scheme was established using reference compounds and the scheme was applied to real air samples from a tyre repair shop and from the air above a bacterial culture. The sampling was performed using a solid sorbent, XAD-2, impregnated with NIT, and the derivatives were extracted with acetonitrile and analysed with LC-MS/MS. A three-step process was developed for screening and identifying of volatile amines. The first step, selected reaction monitoring; SRM was applied in order to screen the samples for NIT derivatives. In the second step, a precursor ion scan gave the [M+H]⁺ ion, and in the third step a product ion scan gave the fragments needed for identification. The detection limits varied between 0.12 and 0.25 ng μL⁻¹ when screening for unknown derivatised amines. It was possible to separate and identify all the amines with the structural information obtained and the method proved to be general, sensitive and well suited for sampling and analysis of complex environmental samples.

Keywords: LC-MS; Primary amines; Secondary amines; Naphthylisothiocyanate (NIT); Air sampling; Bacteria

Multianalyte determination of different classes of pesticides (acidic, triazines, phenyl ureas, anilines, organophosphates, molinate and propanil) by liquid chromatography-electrospray-tandem mass spectrometry by Ana C. Borba da Cunha; Maria J. López de Alda; Damià Barceló; Tania M. Pizzolato; Joao Henrique Z. dos Santos (pp. 940-954).

This work describes the optimization of a liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS-MS) method for the multianalyte determination of twenty pesticides, selected based on current regulations and extent of use. Chromatographic separation was carried out on a Purospher STAR RP-18e column using gradient acetonitrile-water as mobile phase. Triazines, phenylureas, organophosphates, anilines, and molinate were determined in the positive ionization mode, and acidic pesticides and propanil in the negative ion mode. Two different precursor ion-product ion transitions were selected for each analyte and monitored under time scheduled multiple reaction monitoring (MRM) conditions. The optimized method was shown to be linear in the range 1 to 1000 ng/mL with correlation coefficients higher than 0.99 for all but one (diazinon) of the analytes, very sensitive (with limits of detection between 0.010 and 4.528 ng/mL), and repeatable (with relative standard deviations, calculated from the replicate analysis of standard mixtures, lower than 14%). The present work was also devoted to the elucidation of the structures of the principal fragment ions obtained after collision-induced dissociation of the pesticides investigated, an aspect often overlooked in the literature.

Keywords: Pesticides; Fragmentation; Electrospray; Tandem mass spectrometry

Liquid chromatography with triple-quadrupole or quadrupole-time of flight mass spectrometry for screening and confirmation of residues of pharmaceuticals in water by Alida A. M. Stolker; Willem Niesing; Elbert A. Hogendoorn; Johanna F. M. Versteegh; Regine Fuchs; Udo A. Th. Brinkman (pp. 955-963).

LC–MS–MS has been performed with triple-quadrupole (QqQ) and quadrupole-time of flight (Q-ToF) instruments and has been used for screening and confirmation of pharmaceuticals in surface, drinking, and ground water. Screening was based on monitoring of one specific MS–MS ion of the target compounds. Confirmation of the identity of the pharmaceuticals was based either on the monitoring of two specific MS–MS ions and calculation of the ratio of their intensities, or on the exact masses of MS–MS product ions obtained for a molecular ion by use of LC–Q-ToF MS. The set of pharmaceuticals included four analgesics (acetylsalicylic acid, diclofenac, ibuprofen, and paracetamol), three antibiotics (sulfamethoxazole, erythromycin, and chloramphenicol), five blood-lipid regulators and beta-blockers (fenofibrate, bezafibrate, clofibric acid, bisoprolol, and metoprolol), and the anti-epileptic drug carbamazepine. Limits of quantification ranged from 5 to 25 ng L⁻¹. Fifty-six samples were analysed and residues of the pharmaceuticals were detected in almost all surface and groundwater and in some drinking water samples. The identity of the compounds could be confirmed by use of both QqQ- and Q-ToF-based LC–MS–MS. However, the latter technique has the distinct advantage that a large number of pharmaceuticals can be screened and confirmed at low concentrations (1–100 ng L⁻¹) in one run.

Keywords: LC-QqQ MS; LC–Q-ToF MS; identification; EU criteria

A simple device for the extraction of TLC spots: direct coupling with an electrospray mass spectrometer by Heinrich Luftmann (pp. 964-968).

A new device is described which allows the recovery of compounds from thin layer chromatograms in short times, within a small volume, and without contamination. This apparatus can be coupled online to an electrospray mass spectrometer, but can also be used with other detectors or for micropreparations.

Keywords: TLC; Extraction ; Electrospray mass spectrometry; ES

Structural study of spirolide marine toxins by mass spectrometry by Lekha Sleno; Anthony J Windust; Dietrich A Volmer (pp. 969-976).

A novel group of toxins, the spirolides, has been investigated by several mass spectrometric (MS) methods to enable structure elucidation and metabolite identification. These macrocyclic compounds, produced by the dinoflagellate Alexandrium ostenfeldii, are a new class of marine phycotoxin with characteristic spiro-linked tricyclic ether and imine moieties. A crude phytoplankton extract has been shown to contain known spirolides and several unknown compounds, present at low yet significant levels. This study has focused on mass spectrometric characterization of the main component of this extract, 13-desmethyl spirolide C. Collision-induced dissociation (CID) spectra were collected on triple-quadrupole and quadrupole linear ion-trap instruments. High-resolution Fourier-transform ion cyclotron resonance MS data revealed the accurate masses of the protonated molecule and the product ions formed by infrared multiphoton dissociation. A fragmentation scheme for this toxin has been proposed to explain the formation of the collision-induced fragments. Charge-remote fragmentations dominate the CID spectra, because there is only one predominantly basic site in this molecule, and prove to be structurally informative. Extensive MS characterization of 13-desmethyl spirolide C will undoubtedly be useful in the characterization of known and unknown spirolides and other related compounds.

Keywords: Triple-quadrupole mass spectrometry; Quadrupole-linear ion-trap mass spectrometry; Collision-induced dissociation mass spectrometry; Infrared multiphoton dissociation mass spectrometry; Fourier-transform ion cyclotron resonance mass spectrometry; MS fragmentation pathways; Charge-remote fragmentation; Phytoplankton; Alexandrium ostenfeldii ; Spirolide marine toxins; 13-Desmethyl spirolide C

Structural study of spirolide marine toxins by mass spectrometry by Lekha Sleno; Michael J. Chalmers; Dietrich A. Volmer (pp. 977-986).

The spirolides are a family of marine biotoxins derived from the dinoflagellate Alexandrium ostenfeldii, recently isolated from contaminated shellfish and characterized. A crude phytoplankton extract has been extensively studied for mass spectrometric determination and characterization of several known spirolides and previously unreported compounds. The complex sample was initially analyzed by full-scan mass spectrometry in an ion-trap instrument, enabling identification of several components. Subsequent analysis by selected-ion monitoring in a triple-quadrupole instrument resulted in the confirmation of the identities of the compounds detected in the ion trap. Purification of the crude extract was performed using an automated mass-based fractionation system, yielding several fractions with different relative contributions of the spirolide components. Collision-induced dissociation (CID) in the triple-quadrupole instrument produced significant fragment ions for all identified species. Selective enrichment of some minor compounds in certain fractions enabled excellent CID spectra to be generated; this had previously been impossible, because of interferences from the major toxins present. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry was then performed for accurate determination of the masses of MH⁺ ions of all the species present in the sample. Additionally, infrared multiphoton dissociation in the FTICR instrument generated elemental formulae for product ions, including those formed in the previous collisional activation experiments. Collection of these results and the fragmentation scheme proposed for the main component of the extract, 13-desmethyl spirolide C, from part I of this study, enabled elucidation of the structures of some uncharacterized spirolides and some biogenetically related compounds present at previously unreported masses.

Keywords: Ion-trap mass spectrometry; Triple-quadrupole mass spectrometry; Fourier-transform ion cyclotron resonance mass spectrometry; Full-scan mass spectrometry; Selected-ion monitoring; Collision-induced dissociation; Infrared multiphoton dissociation; Automated mass-based fractionation; Phytoplankton; Spirolide marine toxins; Alexandrium ostenfeldii

Development of a simplified method for the simultaneous determination of retinol, α-tocopherol, and β-carotene in serum by liquid chromatography–tandem mass spectrometry with atmospheric pressure chemical ionization by Roberta Andreoli; Paola Manini; Diana Poli; Enrico Bergamaschi; Antonio Mutti; Wilfried M. A. Niessen (pp. 987-994).

A new and simple method for the determination of fat-soluble vitamins (retinol, α-tocopherol, and β-carotene) in human serum was developed and validated by using liquid chromatography–tandem mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS-MS). Different solvent mixtures were tested to obtain deproteinization and extraction of the analytes from the matrix. As a result, a volume of 240 μL of a 1:1 (v/v) ethanol/ethyl acetate mixture added to 60 μL of serum was found to be suitable for both protein precipitation and antioxidants solubilization, giving the best recovery for all three analytes. Deproteinized samples (20 μL) were injected after dilution, without the need for concentration or evaporation to dryness and reconstruction of the sample. Vitamins were separated on a C-8 column using a 95:5 (v/v) methanol/dichloromethane mixture and ionized in the positive-ion mode; detection was performed in the selected-reaction monitoring mode. Linearity of the LC-APCI-MS-MS method was established over 5 orders of magnitude for retinol and α-tocopherol, whereas in the case of β-carotene it was limited to 4 orders. Lower limits of quantitation were 1.7, 2.3, and 4.1 nM for retinol, α-tocopherol, and β-carotene, respectively. Serum concentrations of retinol, α-tocopherol, and α+β-carotene determined in a group of healthy volunteers were 2.48, 38.07, and 0.50 μM, respectively, in samples collected in winter (n=122) and 2.69, 45.88, and 0.90 μM during summer (n=66).

Keywords: Retinol; α-Tocopherol; β-Carotene; Serum; LC-APCI-MS-MS; Ozone

Flavonoids in Leguminosae: Analysis of extracts of T. pratense L., T. dubium L., T. repens L., and L. corniculatus L. leaves using liquid chromatography with UV, mass spectrometric and fluorescence detection by Eva de Rijke; Herman Zappey; Freek Ariese; Cees Gooijer; Udo A. T. Brinkman (pp. 995-1006).

Reversed-phase LC on C-18 bonded silica with a methanol–ammonium formate gradient was used to determine the main flavonoids in leaves of four species of the Leguminosae family. The detection modes were diode-array UV absorbance, fluorescence, and (tandem) mass spectrometry. LC–UV was used for a general screening, sub-classification, and the calculation of total flavonoid contents. LC–FLU was included to identify isoflavones on the basis of their native fluorescence. Most structural information regarding aglycons, sugar moieties, and acidic groups was derived from LC–MS in both the full-scan and extracted-ion mode, using negative-ion atmospheric pressure chemical ionization. MS/MS did not provide much additional information, because the same fragments were observed as in full-scan MS.In T. pratense and T. repens, the main constituents were flavonoid glucoside–(di)malonates, while T. dubium and L. corniculatus mainly contained flavonoid (di)glycosides. Satellite sets comprising an aglycon, the glucoside and glucoside–malonates or –acetates, were abundantly present only in T. pratense. Generally speaking, the main aglycons and sugars in the four plant species are surprisingly different. In addition, while the results for T. pratense are similar to those reported in the literature, there is little agreement in the case of the other species. Finally, total flavonoid contents ranged from 50–65 mg/g for L. corniculatus and T. dubium, to 15 mg/g for T. pratense and only 1 mg/g for T. repens.

Keywords: Mass spectrometry; Fluorescence; Flavonoids; Glycosides; Leguminosae

Liquid chromatography coordination ion-spray mass spectrometry (LC–CIS–MS) of docosahexaenoate ester hydroperoxides by Jennifer R. Seal; Ned A. Porter (pp. 1007-1013).

Coordination ion-spray mass spectrometry (CIS–MS) is a useful tool in the detection and identification of complex mixtures of cholesterol ester and phospholipid hydroperoxides. The methyl ester, cholesterol ester, and phospholipid hydroperoxides of docosahexaenoic acid were analyzed by LC–CIS–MS and their elution orders were identified. Their corresponding alcohols were also identified. The methyl hydroperoxydocosahexaenoate (HPDHE) elution order is 14, 17, 16, 13, 20, 11, 10, 4, 7, 8 while the methyl hydroxydocosahexaenoate (HDHE) elution order is 14 ≥ 17, 16, 13, 11, 10, 20, 7, 8, 4. The cholesteryl HPDHE elution order is 14, 17, 16, 13, 20, 11 ≥ 10, 4, 7, 8 and the cholesteryl HDHE elution order is 17, 14, 16, 13, 11, 20, 10, 7, 8, 4. The elution order of the 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphatidylcholine (PDPC) hydroperoxides and alcohols is 20, 16, 17, 13, 14, 10, 11, 7, 8, 4.

Keywords: Docosahexaenoic acid; Coordination ion-spray-mass spectrometry (CIS–MS); Lipid peroxidation; Silver coordination

A simple and robust set-up for on-column sample preconcentration – nano-liquid chromatography – electrospray ionization mass spectrometry for the analysis of N-acylhomoserine lactones by Moritz Frommberger; Philippe Schmitt-Kopplin; Guichen Ping; Heinz Frisch; Michael Schmid; Yukui Zhang; Anton Hartmann; Antonius Kettrup (pp. 1014-1020).

A simple method for the simultaneous, rapid and sensitive determination of N-acylhomoserine lactone signaling molecules in bacterial isolates, without prior sample preconcentration and with minimal sample cleanup, is presented. The analysis relies on the combination of analyte preconcentration and separation on a single device: a relatively large sample volume (1–5 μL) is directly loaded onto a laboratory-made, miniaturized (75 μm i. d.) reverse phase nano-liquid chromatography column, connected on-line to a microelectrospray-ionization ion trap mass spectrometer. In a first step the analyte is adsorbed (and so concentrated) at the beginning of the column, and is eluted and selectively separated in a second step by the organic mobile phase. Sample preconcentration follows the mechanisms of solid phase extraction on a nano-scale, while separation takes place according to classical liquid chromatography separation principles. The columns can be manufactured easily, are simply connected, and used with minimal solvent amounts; this makes this method extremely robust and cost-effective. The analytical setup was found to be routinely quantitative down to a concentration of 10 ng/mL (corresponding to a total analyte amount of 10 pg or ca. 50 fmol). The limit of detection was reached at 1 ng/mL (1 pg, ca. 5 fmol). Compared to the classical AHL analysis of bacterial cultures with biosensors, where selectivity and sensitivity is often limited, this rapid analytical technique is a substantial qualitative and quantitative improvement. Two unsubstituted N-acylhomoserine lactones could be identified and quantified from a Burkholderia cepacia culture supernatant in a chloroform extract.

Keywords: Quorum sensing; AHL; Nano-HPLC; Sample preconcentration; Mass spectrometry

Trace determination of peptides in water samples using packed capillary liquid chromatography with UV and MS detection and characterization of peptide oxidation products by MS by Thomas Bjellaas; Anders Holm; Paal Molander; John Aasulf Tørnes; Tyge Greibrokk; Elsa Lundanes (pp. 1021-1030).

A capillary liquid chromatographic column switching method has been developed for fast and sensitive determination of peptides in water samples. Sample volumes of 1 mL were loaded onto a (320 μm I.D. ×30 mm) 10 μm Kromasil C₁₈ pre-column, providing on-line analyte enrichment, prior to back-flushed elution onto a (320 μm I.D. ×150 mm) 3.5 μm Kromasil C₁₈ analytical column. Loading flow rates of 250 μL/min and a mobile phase composition of acetonitrile/water/trifluoroacetic acid (22/77.9/0.1, v/v) provided a total analysis time of less than 25 minutes for the test peptides angiotensin II, bombesin, bradykinin, corazonin, neurotensin and substance P, using temperature programmed elution. In addition, solvent gradient elution and combined solvent gradient elution and temperature programming were explored. Using on-capillary UV detection at 210 nm resulted in a concentration limit of detection (cLOD) of about 1 ng/mL. The method was validated over the concentration range 1–100 ng/mL, yielding a coefficient of correlation of 0.997 or better. The within-assay (n=6) and between-assay (n=6) precisions of peak areas were on average 6% RSD and 5% RSD, respectively.When the method was applied to spiked chlorinated tap water samples, it was found that peptides containing methionine, tryptophan and cystine were oxidized. Identification of the oxidation products of the peptides in hypochlorite-treated water was done with positive electrospray ionization time-of-flight mass spectrometric detection.

Keywords: Capillary liquid chromatography; Mass spectrometry; Peptides; Hypochlorite-treated water; Oxidation

Characterization of peptide–protein interactions using photoaffinity labeling and LC/MS by Olaf Jahn; Klaus Eckart; Hossein Tezval; Joachim Spiess (pp. 1031-1036).

The combination of photoaffinity labeling (PAL) with modern mass spectrometric techniques is a powerful approach for the characterization of peptide–protein interactions. Depending on the analytical strategy applied, a PAL experiment can provide different levels of information ranging from the identification of interaction partners to the structural characterization of ligand-binding sites. On the basis of LC/MS data generated in the framework of the identification of the binding site of the neuropeptide corticotropin-releasing factor (CRF) on its binding protein (CRFBP), the key role of LC/MS in the characterization of photoadducts on different structural levels was demonstrated. Covalent photoadducts of rat CRFBP (rCRFBP) were obtained by PAL with different mono- and bifunctional benzophenone photoprobes designed on the basis of the sequence of the synthetic CRF fragment human/rat CRF^6–33 which binds to CRFBP with high affinity. In view of the stoichiometry, LC/MS analysis revealed that the photoadducts consisted of one molecule of photoprobe and one molecule of rCRFBP. For a further characterization of the photoadducts on the oligopeptide level, enzymatic digests of unlabeled rCRFBP and of the respective photoadduct were compared by peptide mapping monitored with LC/MS. Thereby, it was found that the photoprobe that contained the photophore at its N-terminus labeled the amino acid sequence rCRFBP(34–38), whereas the photoprobe that contained the photophore at its C-terminus labeled rCRFBP(12–26). On the basis of the characterization of the photoadduct formed by rCRFBP and the bifunctional photoprobe that contained photophores on both termini, semiquantitative comparison of different enzymatic digests was accomplished by application of the mass-selective multiple ion chromatogram strategy.

Keywords: Photoaffinity labeling; Benzophenone photoprobe; LC/MS; Multiple ion chromatogram (MIC); Corticotropin-releasing factor (CRF)

Nanoscale proteomics by Y. Shen; N. Tolić; C. Masselon; L. Paša-Tolić; D. G. Camp II; M. S. Lipton; G. A. Anderson; R. D. Smith (pp. 1037-1045).

Efforts to develop a liquid chromatography (LC)/mass spectrometry (MS) technology for ultra-sensitive proteomics studies (i.e., nanoscale proteomics) are described. The approach combines high-efficiency nanoscale LC (separation peak capacity of ≈10³; 15-μm-i.d. packed capillaries with flow rates of 20 nL min⁻¹, the optimal separation linear velocity) with advanced MS, including high-sensitivity and high-resolution Fourier transform ion cyclotron resonance MS, to perform both single-stage MS and tandem MS (MS/MS) proteomic analyses. The technology enables broad protein identification from nanogram-size proteomics samples and allows the characterization of more abundant proteins from sub-picogram-size samples. Protein identification in such studies using MS is demonstrated from <75 zeptomole of a protein. The average proteome measurement throughput is ~50 proteins h⁻¹ using MS/MS during separations, presently requiring approximately 3 h sample⁻¹. Greater throughput (~300 proteins h⁻¹) and improved detection limits providing more comprehensive proteome coverage can be obtained by using the “accurate mass and time” tag approach developed in our laboratory. This approach provides a dynamic range of at least 10⁶ for protein relative abundances and an improved basis for quantitation. These capabilities lay the foundation for studies from single or limited numbers of cells.

Keywords: LC; NanoLC; NanoESI; LC/MS; LC/MS/MS; Proteomics

Biomolecular and element analytical spectroscopy: new methods and application perspectives for two associated disciplines by Detlef Günther; Michael Przybylski; Gerhard Schlemmer (pp. 1046-1047).

A mini-review of mass spectrometry using high-performance FTICR-MS methods by R. M. A. Heeren; A. J. Kleinnijenhuis; L. A. McDonnell; T. H. Mize (pp. 1048-1058).

Structural characterization of macromolecules is currently delivering new insights into the behavior of individual molecules or molecular ensembles. Technological advances have made it possible to examine smaller and smaller amounts (down to single molecules) of larger and larger molecular systems. Mass spectrometry in particular is capable of the detailed study of extremely small quantities (down to a single molecule) of very large (biological) molecules. The advent of new ionization techniques such as electrospray and matrix-assisted laser desorption are mainly responsible for these advances. As a result, mass spectrometry has evolved into an enabling discipline that plays an increasingly important role in combinatorial chemistry, polymer science, biochemistry, medicine, environmental and marine science, and archaeology and conservation science. This paper will review a selection of methodological developments in the field of high-performance Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of these macromolecules.

Keywords: FTICR-MS; Proteomics; High-resolution; Peptides; ESI; MALDI

ICP–MS trace-element analysis as a forensic tool by Andrea Ulrich; Christoph Moor; Heinz Vonmont; Hans-Rudolf Jordi; Martin Lory (pp. 1059-1068).

Careful tracing of evidence at the site of a crime must be performed before suspects can be convicted of a crime or homicide. Fingerprints and ballistic control samples are important evidence. A common method used to examine lead bullets is comparison of physical properties such as weight, dimensions, shape, and distinctive markings. However ballistic investigations, for example comparison of characteristic scratches and marks left on fired bullets, do not always give sufficient information. Ballistic abrasion patterns can change for a variety of reasons, e.g. deformation or mechanical strain. Sometimes only particles remain in a victim’s body. In such cases trace-element composition and lead-isotope ratios can be compared with those of controls. Elemental composition of particles and deformed bullets have been compared with the elemental fingerprints and isotope ratios of potential bullet types found on suspects. The applicability of the method was studied for two different cases. Data interpretation and several limitations of the technique are also discussed in this paper.

Keywords: Forensic; Bullet alloys; Inductively coupled plasma mass spectrometry (ICP–MS); Trace elements; Lead-isotope ratio

Size-related vaporisation and ionisation of laser-induced glass particles in the inductively coupled plasma by Hans-Rudolf Kuhn; Marcel Guillong; Detlef Günther (pp. 1069-1074).

Ongoing discussions about the origin of elemental fractionation occurring during LA-ICP-MS analysis show that this problem is still far from being well understood. It is becoming accepted that all three possible sources (ablation, transport, excitation) contribute to elemental fractionation. However, experimental data about the vaporisation size limit of different particles in the ICP, as produced in laser ablation, have not been available until now. This information should allow one to determine the signal contributing mass within the ICP and would further clarify demands on suitable laser ablation systems and gas atmospheres in terms of their particle size distribution.The results presented here show a vaporisation size limit of laser induced particles, which was found at particle sizes between 90 nm and 150 nm using an Elan 6000 ICP-MS. Due to the fact that the ICP-MS response was used as evaluation parameter, vaporisation and ionisation limits are not distinguishable.The upper limit was determined by successively removing the larger particles from the aerosol, which was created by ablation of a NIST 610 glass standard at a wavelength of 266 nm, using a recently developed particle separation device. Various particle fractions were separated from the aerosol entering the ICP. The decrease in signal intensity is not proportional to the decrease in volume, indicating that particles above 150 nm in diameter are not completely ionised in the ICP. Due to the limited removal range of the particle separation device, which cannot remove particles smaller than 150 nm, single hole ablations were used to determine the lower vaporisation limit. This is based on measurements showing that larger particles occur dominantly during the first 100 laser pulses only. After this period, the ratio of ICP-MS counts and total particle volume was found to be constant while most of the particles are smaller than 90 nm, indicating complete vaporisation and ionisation of these particles.To describe the influence of different plasma forward powers on the vaporisation limit, the range 1000–1600 W was studied. Results indicate that optimum vaporisation and ionisation occurs at 1300 W. However, an increase of the particle ionisation limit towards larger particles was not observed within the accuracy of this study using the full range of parameters available for optimisation on commonly used ICP-MS instruments.

Keywords: Laser-ablation-ICP-MS; Ionisation; Plasma; Particles; Aerosol

Ultratrace determination of mercury in water following EN and EPA standards using atomic fluorescence spectrometry by Thomas Labatzke; Gerhard Schlemmer (pp. 1075-1082).

Chemical vapour generation has been used in combination with atomic fluorescence spectrometry to determine mercury at ultratrace concentrations down to 0.1 ng L⁻¹. A time-based injection of 1 mL of solution for measurement was sufficient to generate a steady-state detector response in the direct mode of measurement. The detection limit calculated from a ten-point calibration curve according to DIN 32645 was 0.26 ng L⁻¹. Instrument noise is limited by reflected radiation from the light source rather than by the dark current of the photomultiplier. The detection limit is directly influenced by the reagent blank which was 2 ng L⁻¹ in the experiments described. Focusing by amalgamation and subsequent thermal desorption generates a detector response which is about eight times higher in peak intensity and about twice as large in integrated intensity. The detection limit under these conditions is 0.09 ng L⁻¹ which can be further improved by preconcentration of larger volumes of solution for measurement. The cycle time for one individual reading is about 40 s without amalgamation and 125 s with amalgamation. The linear dynamic range of the system is five orders of magnitude with a single photomultiplier gain setting. The carry-over is less than 0.3% in direct measurement mode. Reference water samples and a surface water containing approximately 5 ng L⁻¹ were used to prove the validity of the method for real samples. Good accuracy and recoveries of 103% were calculated using the fast direct determination technique.

Keywords: Atomic fluorescence spectrometry; Chemical vapour generation; Water; Mercury

Application of the reference-element technique for fast sequential flame atomic-absorption spectrometry by Hans-Dieter Projahn; Ute Steeg; John Sanders; Eric Vanclay (pp. 1083-1087).

Fast sequential flame atomic-absorption spectrometry (FS-FAAS) is able to measure a sequence of analyte wavelengths in one monochromator scan and so achieves or exceeds the analysis speed of sequential ICP–OES. The requirements and implementation of an FS-FAAS instrument are presented in detail. FS-FAAS is a sequential multi element technique which retains the advantages of conventional FAAS, for example ease of use. While the FS-FAAS technique is an ideal tool for routine determination of elements in the mg L⁻¹ concentration range, it is still subject to common sources of error such as transport problems and long-term drift. The reference-element technique can be used to correct for these types of common interference; other advantages include correction of some errors which are induced during sample preparation, and improved analytical accuracy and precision. The reference-element technique when utilizing the FS- FAAS technique is described in detail and benefits of the technique will be demonstrated by comparing performance for selected applications, for example a mineral sample and a scrap wood digest.

Keywords: Flame atomic-absorption spectrometry; Fast sequential FAAS; Reference-element technique; Interferences; Accuracy; Precision

Determination of trace elements in planktonic microcrustaceans using total reflection X-ray fluorescence (TXRF): First results from two Chilean lakes by Stefan Woelfl; Margarete Mages; Susana Mercado; Lorena Villalobos; Mihály Óvári; Francisco Encina (pp. 1088-1094).

First results are described from the application of a recently developed dry method for determination of elements in single specimens of freshwater microcrustaceans, using total reflection X-ray fluorescence spectrometry (TXRF). This method is a powerful, non-destructive technique for quantifying the trace element content of minute biological samples with a dry weight of 3–50 μg. Three different freshwater microcrustaceans were sampled, from the natural, uncontaminated Lake Laja and from the artificial Rapel reservoir which is slightly contaminated by drainage water from a copper mine. Single specimens of Daphnia pulex, Bosmina chilensis, and Ceriodaphnia dubia were prepared using a modification of the dry method and measured by TXRF. The results showed that both As, Mn, Fe, Ni, Zn, and Cu content and the bioaccumulation of these metals were usually significantly different between the microcrustaceans from the two lakes. The largest difference was found for Cu which was eight times more concentrated in the two microcrustaceans from Rapel reservoir than it was in D. pulex from Lake Laja.

Keywords: Trace elements; Zooplankton; Total reflection X-ray fluorescence; Bioaccumulation; Metals

Biofilms as bio-indicator for polluted waters? by Margarete Mages; Mihály Óvári; Wolf v. Tümpling Jr; Krisztina Kröpfl (pp. 1095-1101).

The aim of this work was to investigate the heavy metal accumulation by natural biofilms living in the catchment area of the Tisza river in Hungary, as well as in biofilms cultivated in vitro. Laboratory tests have demonstrated that metals can be adsorbed on biofilms, depending on their concentration and on the availability of free sorptive places. Biofilms were cultivated in vitro in natural freshwater from the Saale river, Germany. After reaching the plateau phase, Cu was added to reach a concentration of 100 µg/L. An increase of its mass fraction in the biofilm was observed, which caused the decrease of the concentration in the water phase. Unfortunately, the reactor wall was also found to act as adsorbent for Cu. More detailed results of our in vitro experiments will be published in a forthcoming paper. Naturally grown biofilm samples from exposed as well as background places at the Hungarian rivers Szamos and Tisza were collected in 2000 and 2002 after the cyanide spill, and analysed using total reflection X-ray fluorescence analysis (TXRF). Metal mass fraction differences as high as two orders of magnitude were found between polluted and unpolluted (background) sampling points. Extremely high concentration values, e.g. 5600 µg/g Zn in biofilm, were found at highly polluted sampling points. This means an enrichment factor of ca. 10,000 compared to the water phase.

Keywords: Total reflection X-ray fluorescence; Trace element accumulation; Biofilms; Tisza river; Trace element determination; Rotating annular reactor

"Affinity-proteomics": direct protein identification from biological material using mass spectrometric epitope mapping by Marcus Macht; Andreas Marquardt; Sören-Oliver Deininger; Eugen Damoc; Markus Kohlmann; Michael Przybylski (pp. 1102-1111).

We describe here a new approach for the identification of affinity-bound proteins by proteolytic generation and mass spectrometric analysis of their antibody bound epitope peptides (epitope excision). The cardiac muscle protein troponin T was chosen as a protein antigen because of its diagnostic importance in myocardial infarct, and its previously characterised epitope structure. Two monoclonal antibodies (IgG1-1B10 and IgG1-11.7) raised against intact human troponin T were found to be completely cross reactive with bovine heart troponin T. A combination of immuno-affinity isolation, partial proteolytic degradation (epitope excision), mass spectrometric peptide mapping, and database analysis was used for the direct identification of Tn T from bovine heart cell lysate. Selective binding of the protein was achieved by addition of bovine heart cell lysate to the Sepharose-immobilised monoclonal antibodies, followed by removal of supernatant material containing unbound protein. While still bound to the affinity matrix the protein was partially degraded thereby generating a set of affinity-bound, overlapping peptide fragments comprising the epitope. Following dissociation from the antibody the epitope peptides were analysed by matrix assisted laser desorption-ionisation (MALDI) and electrospray-ionisation (ESI) mass spectrometry. The peptide masses identified by mass spectrometry were used to perform an automated database search, combined with a search for a common "epitope motif". This procedure resulted in the unequivocal identification of the protein from biological material with only a minimum number of peptide masses, and requiring only limited mass-determination accuracy. The dramatic increase of selectivity for identification of the protein by combining the antigen–antibody specificity with the redundancy of peptide sequences renders this "affinity-proteomics" approach a powerful tool for mass spectrometric identification of proteins from biological material.

Keywords: Affinity-proteomics; Protein identification; Biological material; Mass spectrometric epitope mapping; Immuno-affinity isolation; Partial proteolytic degradation; Matrix assisted laser desorption-ionisation (MALDI) mass spectrometry; Electrospray-ionisation (ESI) mass spectrometry; Database analysis

Interpreting conformational effects in protein nano-ESI-MS spectra by Maria Šamalikova; Irena Matečko; Norbert Müller; Rita Grandori (pp. 1112-1123).

Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is employed here to describe equilibrium protein conformational transitions and to analyze the influence of instrumental settings, pH, and solvent surface tension on the charge-state distributions (CSD). A first set of experiments shows that high flow rates of N₂ as curtain gas can induce unfolding of cytochrome c (cyt c) and myoglobin (Mb), under conditions in which the stability of the native protein structure has already been reduced by acidification. However, it is possible to identify conditions under which the instrumental settings are not limiting factors for the conformational stability of the protein inside ESI droplets. Under such conditions, equilibrium unfolding transitions described by ESI-MS are comparable with those obtained by other established biophysical methods. Experiments with the very stable proteins ubiquitin (Ubq) and lysozyme (Lyz) enable testing of the influence of extreme pH changes on the ESI process, uncoupled from acid-induced unfolding. When HCl is used for acidification, Ubq and Lyz mass spectra do not change between pH~7 and pH 2.2, indicating that the CSD is highly characteristic of a given protein conformation and not directly affected by even large pH changes. Use of formic or acetic acid for acidification of Ubq solutions results in major spectral changes that can be interpreted in terms of protein unfolding as a result of the increased hydrophobicity of the solvent. On the other hand, Lyz, cyt c, and Mb enable direct comparison of protein CSD (corresponding to either the folded or the unfolded protein) in HCl or acetic acid solutions at low pH. The values of surface tension for these solutions differ significantly. Confirming indications already present in the literature, we observe very similar CSD under these solvent conditions for several proteins in either compact or disordered conformations. The same is true for comparison between water and water–acetic acid for folded cyt c and Lyz. Thus, protein CSD from water–acetic solutions do not seem to be limited by the low surface tension of acetic acid as previously suggested. This result could reflect a general lack of dependence of protein CSD on the surface tension of the solvent. However, it is also possible that the effect of acetic acid on the precursor ESI droplets is smaller than generally assumed.

Keywords: Charge-state distributions; Protein folding; Rayleigh limit; Surface tension; Nano-electrospray-ionization mass spectrometry; Electrostatic effects

Reaction monitoring of enzyme-catalyzed ester cleavage by time-resolved fluorescence and electrospray mass spectrometry: method development and comparison by Tanja Steinkamp; André Liesener; Uwe Karst (pp. 1124-1128).

Two complementary methods for reaction monitoring of the esterase-catalyzed cleavage of bis(2-pyridylmethyl)(2-acetoxyphenyl)amine are developed and compared. While enzyme-amplified lanthanide luminescence (EALL) allows for the time-resolved fluorescence determination of the intrinsically non-fluorescent product, both substrate and product of the enzymatic reaction may be determined simultaneously by electrospray mass spectrometry (ESI-MS). Excitation wavelength for the Tb(III) complex of the reaction product is 297 nm and emission was detected at 545 nm, which is the characteristic emission wavelength of the terbium(III) ion. In contrast to other EALL techniques, the presented method allows for the direct monitoring of an enzymatic conversion without any further sample preparation (e.g., rebuffering). For the mass spectrometric measurements the mass traces were set to m/z=306, 328, 348, and 370 for the protonated ester, the resulting phenol and their sodium adducts, respectively.

Identification of N-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry by Claus Albach; Eugen Damoc; Thomas Denzinger; Melitta Schachner; Michael Przybylski; Brigitte Schmitz (pp. 1129-1135).

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform–ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.

Keywords: N-Glycosylation sites; NCAM; MALDI-TOF-MS; MALDI-FTICR-MS

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Analytical and Bioanalytical Chemistry (v.378, #4)