Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

Gliotoxin is produced by non-ribosomal peptide synthesis and secreted from certain fungi, including Aspergillus fumigatus. It is an epipolythiodioxopiperazine that contains an intact disulphide bridge and is the focus of intense research as a consequence of its negative immunomodulatory properties. Gliotoxin detection is generally enabled by reversed-phase–high-performance liquid chromatography (RP-HPLC), with absorbance detection (220–280 nm), or liquid chromatography-mass spectrometry, yet detection is not readily achievable by matrix-assisted laser desorption ionisation–time-of-flight mass spectrometry (MALDI-ToF MS). We have developed a single-pot derivatisation strategy which uses sodium borohydride-mediated reduction of gliotoxin followed by immediate alkylation of exposed thiols by 5′-iodoacetamidofluorescein to yield a stable product, diacetamidofluorescein-gliotoxin (GT-(AF)₂), of molecular mass 1103.931 Da ((M + H)+). This product is readily detectable by RP-HPLC and exhibits a 6.8-fold increase in molar absorptivity compared with gliotoxin, which results in a higher sensitivity of detection (40 ng; 125 pmoL). GT-(AF)₂ also fluoresces (excitation/emission, 492:518 nm). Unlike free gliotoxin, the product (>800 fmol) is detectable by MALDI-ToF MS. Sporidesmin A can also be detected by RP-HPLC and MALDI-ToF MS (>530 fmol) using this strategy. We also demonstrate that the strategy facilitates detection of gliotoxin (mean ± SD = 3.55 ± 0.07 μg 100 μL⁻¹; n = 2) produced by A. fumigatus, without the requirement for organic extraction of culture supernatants and associated solvent removal. GT-(AF)₂ is also detectable (150 ng; 460 pmol) by thin-layer chromatography.     

Sensitive determination of a glyoxal-DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry

A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal-deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water-acetonitrile-formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. x 5 mm Hypercarb (5 mum) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 mul, and was subsequently back-flushed onto a 0.30 mm I.D. x 150 mm Lichrospher diol (5 mum) analytical column. The samples were loaded with a flow rate of 40 mul min(-1) and glyoxal-deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water-acetonitrile-formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 mul min(-1). Mass spectrometric determination of glyoxal-deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H](+)m/z 326 --> m/z 210. The method was evaluated over the concentration range 0.25-50 ng ml(-1) of glyoxal-deoxyguanosine in the hydrolysate of 5 mug DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2-11% and 1.4-11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg mul(-1) DNA hydrolysate solution, corresponding to 48 adducts per 10(6) normal nucleosides. The method was applied for the determination of glyoxal-deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.     

Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry and their trace detection in complex matrices by gas chromatography-tandem mass spectrometry.

Bacterial cellular polysaccharides are composed of a variety of sugar monomers. These sugars serve as chemical markers to identify specific species or genera or to determine their physiological status. Some of these markers can also be used for trace detection of bacteria or their constituents in complex clinical or environmental matrices. Analyses are performed, in our hands, employing hydrolysis followed by the alditol acetate derivatization procedure. Substantial improvements have been made to sample preparation including simplification and computer-controlled automation. For characterization of whole cell bacterial hydrolysates, sugars are analyzed by gas chromatography-mass spectrometry (GC-MS). Simple chromatograms are generated using selected ion monitoring (SIM). Using total ion GC-MS, sugars can be readily identified. In more complex clinical and environmental samples, markers for bacteria are present at sufficiently low concentrations that more advanced instrumentation, gas chromatography-tandem mass spectrometry (GC-MS-MS), is preferred for optimal analysis. Using multiple reaction monitoring, MS-MS is used (replacing more conventional SIM) to ignore extraneous chromatographic peaks. Triple quadrupole and ion trap GC-MS-MS instruments have both been used successfully. Absolute chemical identification of sugar markers at trace levels is achieved, using MS-MS, by the product spectrum.     

Preparative separation of a multicomponent peptide mixture by mass spectrometry

We report on the first multiplex preparative separation by mass spectrometry of bio-organic molecules in the 200-350 Da mass range that is typical for synthetic drugs. A five-component mixture consisting of two di- and three tripeptides has been separated by mass using a specially designed mass spectrometer. The instrument for preparative separations consists of an electrospray ionization (ESI) source, ion transfer optics, an electrostatic sector, and an inhomogeneous-field magnetic mass analyzer that achieves linear mass dispersion of ion beams. Protonated peptides produced by electrospray were separated, nondestructively landed on a 16-channel array of dry collector plates, and reconstituted in solution. The preparation procedures and the instrumental conditions have been optimized to maximize the ion currents. The significant features of the special mass spectrometer are high ion currents and simultaneous separation and collection of mixture components.     

Evaluation of nano-liquid chromatography-tandem mass spectrometry in a column switching setup for the absolute quantification of peptides in the picomolar range

A standard nanospray-liquid chromatography-tandem mass spectrometry system in a column switching setup for the absolute quantification of leucine-enkephalin was evaluated. Analytes were loaded on a C18 trapping column and back-flushed in the 75 microm analytical column. Quantification was performed with a triple quadrupole instrument. Validation results show that it is feasible, with a conventional nano-LC system in the column switching setup, to quantify peptides as low as 500 amol on column (50 pmol/L). Weighted linear regression analysis proves a good linearity in a dynamic range of almost three orders of magnitude. Nevertheless, robustness remains a key issue in nano-LC-MS/MS.     

Coupling ultra high-pressure liquid chromatography with single quadrupole mass spectrometry for the analysis of a complex drug mixture

The coupling of ultra high-pressure liquid chromatography with a single quadrupole mass spectrometer was investigated for the analysis of several cytochromes P450 (CYP450) substrates and respective metabolites. The effect of numerous operating parameters (e.g. mobile phase pH, flow rate, gradient length, MS acquisition mode, dwell time, polarity switching, etc.) on selectivity, sensitivity and acquisition rate was studied. It was demonstrated that basic pH conditions provided the best compromise in terms of sensitivity and chromatographic selectivity with both acidic and basic compounds. The optimal mobile phase flow rate for UHPLC-MS experiments should be comprised between 300 and 600 μL/min for 2.1 mm ID columns, while a higher flow rate generated up to 3-fold loss in sensitivity. It was also demonstrated that the fast polarity switching mode represented a valuable tool to improve throughput, maintaining acceptable performance. Finally, limits of detection were included in the range [1-50 ng/mL] in positive ionization mode and [50-250 ng/mL] in negative ionization mode, for investigated compounds.     

Isolation of individual capsaicinoids from a mixture and their characterization by (13)C NMR spectrometry

Individual compounds were isolated from a laboratory mixture of capsaicinoids by a multi-stage approach. First, the capsaicinoids were fractionated into capsaicins and non-capsaicins by argentation solid phase extraction (SPE) on a silver-charged propyl sulfonate resin. Second, compounds in each fraction were isolated by semi-preparative liquid chromatography on a C₃₀ phase in aqueous methanol. Third, the individual components of the original mixture were concentrated by reversed phased (C₁₈) SPE. The structure of each purified compound was confirmed by ¹³C NMR spectrometry and spectral comparison to known standards, purchased or synthesized locally. The chemical shifts of 15 capsaicinoid standards were measured on a 600 MHz instrument, and their assignments to particular carbons were made by reference to Distortionless Enhancement by Polarization Transfer (DEPT) NMR experiments and NMR spectral prediction software.     

Nano-particle labelling of nucleic acids for enhanced detection by inductively-coupled plasma mass spectrometry (ICP-MS)

Oligonucleotides containing a biotin functionality were successfully labelled with a streptavidin nanogold conjugate and subsequently separated and analysed by high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS).     

Evaluation of false positive responses by mass spectrometry and ion mobility spectrometry for the detection of trace explosives in complex samples

Secondary electrospray ionization-ion mobility-time of flight mass spectrometry (SESI-IM-TOFMS) was used to evaluate common household products and food ingredients for any mass or mobility responses that produced false positives for explosives. These products contained ingredients which shared the same mass and mobility drift time ranges as the analyte ions for common explosives. The results of this study showed that the vast array of compounds in these products can cause either mass or mobility false positive responses. This work also found that two ingredients caused either enhanced or reduced ionization of the target analytes. Another result showed that an IMS can provide real-time separation of ion species that impede accurate mass identifications due to overlapping isotope peak patterns. The final result of this study showed that, when mass and mobility values were used to identify an ion, no false responses were found for the target explosives. The wider implication of these results is that the possibility exists for even greater occurrences of false responses from complex mixtures found in common products. Neither IMS nor MS alone can provide 100% assurance from false responses. IMS, due to its low cost, ease of operation, rugged reliability, high sensitivity and tunable selectivity, will remain the field method of choice for the near future but, when combined with MS, can also reduce the false positive rate for explosive analyses.     

Sample preparation and direct electrospray ionization on a tip column for rapid mass spectrometry analysis of complex samples

A handheld pipette tip column electrospray ionization source (PTC-ESI source) was developed for rapid mass spectrometry analysis at ambient pressure. The PTC-ESI source was made up of three main component parts including a micro DC high voltage (HV) power supply, a micropipette and a disposable micropipette tip filled with a plug of adsorbent. A DC high voltage was applied to the sharp point of the micropipette tip column to induce electrospray ionization. The PTC-ESI source was successfully used for direct analysis of basic organic compounds, organic acids and peptides in a simple matrix. In the case of complex samples, micro-extraction based on the adsorbent phase filled in the pipette tip was used to remove impurities and concentrate target analytes prior to ionization. The eluting solution was not pipetted out, but directly dispersed in the form of electrospray from the pipette tip for ionization. The effectiveness of the PTC-ESI source has been further demonstrated by fast analysis of therapeutic compounds and endogenous bioactive chemicals in complex biological samples.     

Identification of tellurium-containing compounds by means of mass spectrometry

A general mass spectrophotometric method for the identification of tellurium-containing compounds is described. The method is based on the analysis of the typical pattern of cluster peaks containing tellurium due to -Te-, -Te₂- or

(X = Cl, Br). A comparison of the computer calculated and experimental mass spectra of some of the compounds containing tellurium is given.     

Simultaneous determination of capecitabine and its metabolite 5-fluorouracil by column switching and liquid chromatographic/tandem mass spectrometry

A liquid chromatographic/tandem mass spectrometric method was developed and validated for the quantitation of capecitabine and its metabolite 5-fluorouracil in human plasma. The simultaneous determination of both analytes was achieved by a column switching method using a trapping column and two analytical columns with different stationary phases. Isocratic elution was used for the separation of capecitabine on a C18 column whereas 5-fluorouracil was separated using gradient elution on an non-polar carbon phase. The calibration curves were linear for both compounds with a correlation factor (R2) > 0.9993 for 5-fluorouracil and >0.9942 for capecitabine. The assay was validated in the concentration range 5.00-1000 ng ml(-1) for both compounds. The intra-day precision was better than 10% for 5-fluorouracil and better than 11% for capecitabine whereas the inter-day precision was better than 8% for 5-fluorouracil and better than 14% for capecitabine.     

Quantitative analysis of eight testosterone metabolites using column switching and liquid chromatography/tandem mass spectrometry

The rate at which testosterone is metabolized to different singly hydroxylated metabolites has been widely used as an in vitro marker for activity of different CYP450 enzymes. The interest in extra-hepatic metabolism, e.g. due to metabolism in the gut wall, has increased during the last decade. Measurement of extra-hepatic enzyme activity using testosterone as a substrate requires a highly sensitive analytical method. A new liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using column switching for online cleaning and desalting of samples, was developed and validated for analysis of 2alpha-, 2beta-, 6alpha-, 6beta-, 7alpha-, 16alpha-, and 16beta-hydroxytestosterone and androstenedione. The samples were injected on a SB-CN column and detection was performed using MS/MS. The limits of quantification ranged from 0.3 to 3.33 nM for the different metabolites. The validated method was used to quantify the enzyme activity in rat intestine mucosa. The formation rates of 16alpha-, 16beta-hydroxytestosterone and androstenedione were quantified, and 2beta-and 6beta-hydroxytestosterone were formed above the limits of detection.     

A new naphthimidazole derivative for saccharide labeling with enhanced sensitivity in mass spectrometry detection

A series of saccharides, including maltoheptose, blood type B antigen, pullulan and the glucan of Ganoderma lucidum, are easily converted into the naphthimidazole (NAIM) derivatives in high yields by the iodine‐promoted oxidative condensation. The NAIM‐labeled saccharides, without further purification, show enhanced signals in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS). The combined use of NAIM derivatization and MALDI‐TOFMS analysis thus provides a rapid method for identification of saccharides even in less than 1 pmol of saccharide in the sample. Characterization of the biologically active saccharides and complex polysaccharides is also achieved through the NAIM‐derivatization method. This study can be further applied to facilitate the isolation and analysis of novel saccharides. Copyright © 2009 John Wiley & Sons, Ltd.     

Selective detection of phosphopeptides in complex mixtures by electrospray liquid chromatography/mass spectrometry

A mass spectrometry-based method that does not involve the use of radiolabeling was developed for selective detection of phosphopeptides in complex mixtures. Mixtures of phosphorylated and nonphosphorylated peptides at the low picomole level are analyzed by negative ion electrospray liquid chromatography/mass spectrometry using C-18 packed fused-silica columns (≤320-μm i.d.). Peptides and phosphopeptides in the chromatographic eluant undergo collision-induced dissociation in the free-jet expansion region prior to the mass analyzing quadrupole. Using relatively high collisional excitation potentials, phospho|peptides containing phosphoserine, phosphothreonine, and phosphotyrosine fragment to yield diagnostic ions at m/z 63 and 79 corresponding to PO₂ ^?; and PO₃ ^?, respectively. Chromatographic peaks containing phosphopeptides are indicated where these diagnostic ions maximize. The highest sensitivity for phosphopeptide detection is obtained using selected-ion monitoring for m/z 63 and 79. Full-scan mass spectra that exhibit the diagnostic phosphopeptide fragment ions, together with pseudomolecular ions, may be obtained by stepping the collisional excitation potential from a high value during the portion of each scan in which the low-mass-to-charge ratio diagnostic marker ions are being detected to a lower value while the upper mass-to-charge ratio range is being scanned. Good sensitivity for phosphopeptide detection was achieved using standard trifluoroacetic acid containing mobile phases for reversed-phase high-performance liquid chromatography. Data illustrating the selectivity and sensitivity of the approach are presented for mixtures of peptides and phosphopeptides containing the three commonly phosphorylated amino acids.     

Micro-scale analysis of aminoglycoside antibiotics in human plasma by capillary liquid chromatography and nanospray tandem mass spectrometry with column switching

A simple liquid chromatographic method combined with tandem mass spectrometry (LC-MS-MS) is described for the analysis of aminoglycoside antibiotics. Clinically these antibiotics may cause both ototoxicity and nephrotoxicity; therefore, the monitoring of aminoglycoside levels in patient plasma is required for protecting human health. In this study separation of the method is based on ion-pair chromatographic technology on a short capillary reversed-phase C18 column. The method was successfully applied to analyze amikacin in human plasma. In human plasma after deproteinisation with HFBA, an aliquot of 1 microL supernatant was injected into the chromatographic system. Only a small amount of plasma sample, 10 microL, is sufficient for the monitoring of amikacin levels in clinically therapeutic range. The relative standard deviations (RSD) of the method for intra- and inter-day analyses (n=5) are less than 5.8%. Application of this method for the trace analysis of amikacin in human plasma proved simple and workable.     

The temperature behaviour of a ferroelectric smectic mixture and the detection of ferroelectric switching by NMR

The orientational order of a ferroelectric mixture has been measured by proton NMR. The alignment of the mesogenic units is reflected in the splitting of the NMR signal into a doublet. Up to a cell thickness of 200 μm, it was possible to produce well-oriented layers in the bookshelf geometry by magnetic orientation of the substances in the B₀-field. The angular dependences of the line width of the NMR signal on the tilt angle of the director have been calculated. A ferroelectric switching was detected by measuring the angular dependence of the line width in the switched state. The tilt angle and the orientational order parameter S of the bookshelf samples were estimated at various temperatures.     

Identification of trimethyllead in urine by high-performance liquid chromatography with column switching and chemical reaction detection and by liquid chromatography-mass spectrometry

Incorporated tetraalkyllead compounds are metabolized in the liver and the highly toxic trialkyllead species are excreted via the urine. The procedure for the determination of these metabolites in urine consists of solid-phase enrichment, reversed-phase pre-column high-performance liquid chromatography (HPLC) and chemical reaction detection. As urine is a very complex matrix, it must be questioned whether the retention time alone is a sufficient criterion for the identification of the analytes. For the trimethyllead ion the validity of the results was examined by selectivity checks of the chemical reaction detector, by the application of different stationary and mobile phases in single and dual pre-column HPLC systems and by the use of thermospray LC-mass spectrometry as an independent method. The results demonstrated that the recommended method is accurate for the determination of trimethyllead in urine samples.     

Analysis of thin layers by means of spark-source mass spectrometry

Summary A device has been constructed with which the surface of a specimen can be scanned with a spark drawn from a fixed counter electrode. The mean depth of penetration is of the order of microns and limits of detection of 0.1 ppm are possible. Repeated scanning allows profile analysis, too.

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Analyse dünner Schichten mit Hilfe der Funken-Massenspektrometrie

Zusammenfassung Eine Anordnung wurde konstruiert, mit der eine sich drehende Probenoberfläche durch eine feste Gegenelektrode abgetastet werden kann. Die mittlere Eindringtiefe pro Funkenentladung liegt in der Größenordnung von Mikrometern. Nachweisgrenzen von 0,1 ppm sind möglich. Durch wiederholtes Abtasten können auch Profilanalysen durchgeführt werden.