A fully automated nanoelectrospray tandem mass spectrometric method for analysis of Caco-2 samples

Caco-2 cells offer a means to rapidly screen permeability of drug candidates, allowing pharmaceutical companies to eliminate candidates unable to cross the intestinal barrier early in the discovery process. This screening process is typically performed by conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS), which can require time-consuming method development. An alternative to LC/MS/MS, automated nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS), is introduced. This novel approach requires an off-line ZipTip desalting step followed by automated nanoESI-MS/MS, using the NanoMate 100 and ESI Chip. In addition to reduced method development time, automated nanoESI-MS/MS also offers no carry-over between samples, low sample consumption, and ease-of-use as compared with conventional pulled-capillary nanoelectrospray. Furthermore, the infusion system described has the potential to be high-throughput. A comparison of Caco-2 samples analyzed both by LC/MS/MS and by automated nanoESI-MS/MS is presented. The permeability and recovery data of the two compounds analyzed in this study obtained from conventional LC/MS/MS and by automated nanoESI-MS/MS were in excellent agreement.     

Liquid chromatography/time-of-flight mass spectrometric analyses for the elucidation of the photodegradation products of triclosan in wastewater samples

Liquid chromatography coupled with time-of-flight mass spectrometry (LC/TOFMS) was applied for the identification of four new photodegradation products of triclosan, a major antimicrobial agent used in personal care products. Wastewater samples, spiked at 7 microg/mL with triclosan, were irradiated with natural sunlight in order to generate the photodegradation products. Aliquots of the spiked water samples were taken at different times of irradiation and compounds were isolated from the water samples by solid-phase extraction. Separation and detection of the compounds and degradation products were accomplished by LC/TOFMS, which provided highly selective information about elemental compositions. Accurate mass measurements for the four degradation products permitted postulation of proposed empirical formulae in this study. Replacement of chlorine atoms by hydroxyl groups and chlorine losses are the major degradation pathways proposed. The degradation products were formed also under environmental conditions in wastewater matrices, thus suggesting their presence in real wastewater treatment processes.     

A multiple electrospray interface for parallel mass spectrometric analyses of compound libraries

A parallel spray interface for mass spectrometry is described. This new electrospray interface enables effluent flow streams from an array of HPLC columns to be sampled independently and sequentially on a chromatographic time-scale. Unlike our previously reported parallel LC-MS interface, which incorporated a dual-sheath spray interface accommodating up to four flow streams that are sampled simultaneously, this new interface permits up to four columns to be sampled sequentially by means of a stepping motor and rotating plate assembly. Effluent flow streams from an array of four HPLC columns are connected to a parallel arrangement of electrospray needles co-axial to the mass spectrometer entrance aperture. Within the needle assembly, the individual spray tips are oriented in a circular array, where each needle is situated 90 degrees relative to one another for four-column operation. An eight-column system is described with needles spaced at 45 degree intervals. In between the needle assembly and the mass spectrometer entrance aperture is a Teflon disk with a through-hole that is mounted to a stepping motor assembly. By precisely controlling the stepping of the motor assembly, it is possible to sample each of the spray positions multiple times per second. By operating the quadrupole mass spectrometer in the single ion monitoring (SIM) mode, it was possible to acquire data at each of the spray positions during the course of the elution of compounds from the HPLC column array while maintaining both good sensitivity and peak shape. Preliminary results suggest this technique will be useful for high throughput combinatorial library analysis and profiling.     

A rapid and efficient mass spectrometric method for the analysis of explosives

The high explosives trinitrotoluene, nitroglycerine, pentaerythritol tetranitrate and hexahydro-1,3,5-trinitro-1,3,5-triazine are efficiently ionised under negative ion atmospheric pressure chemical ionisation (APCI) conditions. The limit of detection is improved, in some cases by several orders of magnitude, by complexation with chlorine demonstrating this to be a highly suitable method for enhancing the detection capabilities for explosives. The spectra produced from introduction of the analytes in a liquid matrix, with and without chlorine present, contain a number of ions that arise through secondary processes including breakdown and adduct formation. Sample introduction into an APCI source in air, via a heated-plate inlet with a supplementary feed of dichloromethane, produces improved response for the chloride adducts of the analytes and minimises their decomposition during analysis. The tandem mass spectra produced from the chloride adducts are simple. Optimisation of the trapping parameters of the ion trap detector enhances selected transitions, yields highly reproducible spectra and improves the limits of detection for MS/MS analysis.     

A method for monitoring and controlling reproducibility of intensity data in complex electrospray mass spectra: A thermometer ion-based strategy

Reproducibility in mass spectral data is important in both biomarker discovery and spectral database searching. We report a strategy, employing a series of substituted benzylpyridinium thermometer ions that can be used to monitor changes in performance of multiple aspects of an electrospray ionization source that impact the intensity axis of a spectrum. Performance attributes, which could confound even isotope-based quantification strategies, are readily assessed using a mixture of thermometer ions. Based on the observed behavior of the ions, a procedure is proposed for monitoring instrument performance and compensating for factors that affect reproducibility across both time and instruments.     

A validated liquid chromatographic/tandem mass spectrometric method for the determination of phencyclidine in microliter samples of rat serum

A liquid chromatographic/tandem mass spectrometric method is described for the determination of phencyclidine (PCP) in small volumes of rat serum (e.g. 50 microl). Samples were extracted using a mixed-mode strong cation-exchange column and then separated isocratically using a narrow-bore (2.1 mm i.d.) 3 microm Hypersil phenyl column and a mobile phase consisting of an ammonium formate buffer (pH 2.7) with 60% (v/v) methanol. Detection was accomplished using positive ion electrospray ionization in the multiple reaction monitoring mode. Mass spectra were obtained and peaks were observed at an m/z (% abundance) of 244 (100), 159 (25), and 86 (89). Tandem mass spectra were also obtained from the m/z 244 precursor ion with peaks observed at m/z 159 (100), 86 (96), and 91 (11). Optimum serum PCP sensitivity and precision were obtained at a transition of m/z 244 --> 159. Matrix-associated ion suppression did not significantly affect the accuracy (100-112%) or precision (CV < or =8%) of the assay. The lower limit of quantitation was 1 ng ml(-1) in 50 microl of serum. The method was used to study the serum pharmacokinetics of PCP in rats after an intravenous bolus dose of PCP.     

Pretreatment procedures for characterization of arsenic and selenium species in complex samples utilizing coupled techniques with mass spectrometric detection

Research interest in analyzing arsenic and selenium is dictated by their species-dependent behavior in the environment and in living organisms. Different analytical methodologies for known species in relatively simple chemical systems are well established, yet the analysis of complex samples is still a challenge. Owing to the complex matrix and low concentrations of target species that may be chemically labile, suitable pretreatment of the sample becomes a critical step in any speciation procedure. In this paper, the pretreatment procedures used for arsenic and selenium speciation are reviewed with the emphasis on the link between the analytical protocol applied and the biologically-significant information provided by the results obtained. In the first approach, the aim of pretreatment is to convert the original sample into a form that can be analyzed by a coupled (hyphenated) technique, preventing possible losses and/or species interconversion. Common techniques include different leaching and extraction modes, enzymatic hydrolysis, species volatilization, and so on, with or without species preconcentration. On the other hand, if the speciation analysis is performed for elucidation of elemental pathways and specific functions in a living system, more conscious pretreatment and/or fractionation is needed. The macroscopic separation of organs and tissues, isolation of certain types of cells, cell disruption and separation of sub-cellular fractions, as well as isolation of a specific biomolecules become important. Furthermore, to understand molecular mechanisms, the identification of intermediate—often highly instable—metabolites is necessary. Real life applications are reviewed in this work for aquatic samples, soils and sediments, plants, yeast, and urine.     

A rapid sample preparation method for mass spectrometric characterization of N-linked glycans

A rapid method for analysis of glycans of glycoproteins is presented. This method comprised deglycosylation, sample cleanup and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of glycans. The enzymatic deglycosylation of N-linked glycoproteins was enhanced in terms of speed and reproducibility using an enzyme-friendly surfactant. The released glycans were desalted using a micro-scale solid phase extraction (SPE) device packed with a hydrophilic interaction chromatography (HILIC) sorbent. Hydrophilic glycans were well retained by SPE, while salts and surfactants were removed from the sample. The glycans were eluted using 25-50 microL of solvent and analyzed directly without derivatization using MALDI-MS. MALDI quadrupole time-of-flight (Q-Tof) instrumentation was utilized for glycan profiling and structure characterization by tandem mass spectrometry (MS/MS). The presented method allows sensitive analysis of glycans benefiting from optimized deglycosylation reactions and efficient sample cleanup.     

Development and validation of a capillary high-performance liquid chromatography/electrospray tandem mass spectrometric method for the quantification of bisphenol A in air samples

Bisphenol A (BPA) is a toxic industrial chemical that affects the endocrine system even at low concentrations. A new method, based on capillary high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) analysis, has been developed to determine BPA in atmospheric samples. The method involves collection of air samples (typically 2 m(3)) on glass fiber filters, with ultrasonic extraction and sample concentration under vacuum before analysis. HPLC analysis was performed isocratically at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and MS/MS analysis in negative ion multiple reaction monitoring (MRM) mode, using BPA-d(16) as internal standard. The present method provides linear response in the range 0.007-3.5 microg/filter (R(2) > 0.999) and is characterized by high accuracy (mean bias 2%) and good reproducibility (mean RSD 5%). High sensitivity (LOD = 2 ng/m(3) based on 2 m(3) of air collected), specificity, and speed of the analysis make the present method suitable for routine determination of BPA in the atmosphere, both for ambient and personnel monitoring.     

Characterization of liquid chromatography-tandem mass spectrometry method for the determination of acrylamide in complex environmental samples

This work describes the characterization of a solid-phase extraction (SPE) and liquid-chromatography-tandem mass spectrometry-based method for the analysis of acrylamide (AA) in complex environmental waters. The method involved the SPE of AA using activated carbon, and the AA was detected with tandem mass spectrometry after separating on an ion exclusion high-performance liquid chromatography column. The method incorporated two labeled AA standards for quantification using isotope dilution and to assess absolute extraction recovery. The method was evaluated for inter- and intra-day precision and accuracy. The method was both accurate (i.e., <30 % error) and precise (i.e., <20 % relative standard deviation), with absolute extraction recoveries averaging 37 %. The mass spectrometry provided excellent sensitivity, with instrumental limits of detection and quantitation values of 23 and 75 pg, respectively. The method detection limit was determined to be 0.021 μg/L. The analysis of AA was successfully performed in real-world samples that contained total dissolved solids concentrations ranging from 23,600 to 297,000 mg/L and AA concentrations ranging from 0.082 to 1.0 μg/L.

A nanoparticle-based solid-phase extraction method for liquid chromatography-electrospray ionization-tandem mass spectrometric analysis

A solid-phase extraction (SPE) procedure with the use of superparamagnetic Fe(3)O(4) nanoparticles as extracting agent was developed for HPLC-ESI-MS/MS analysis. Four most heavily used triazine pesticides (herbicides) were taken as the test compounds. The NPs showed an excellent capability to retain the compounds tested, and a quantitative extraction was achieved within 10min under the testing conditions, i.e. 100 microL NP solution was added to 400 mL sample in a beaker with stirring. After extraction, the superparamagnetic NPs were easily collected by using an external magnet. Very importantly, analytes retained on the Fe(3)O(4) NPs could be quantitatively recovered by dissolving the NPs with an HCl solution, allowing subsequent HPLC-ESI-MS/MS quantification. A capillary HPLC-ESI-MS/MS method with the present NP-based SPE procedure was developed for the determination of triazines including atrazine, prometryn, terbutryn, and propazine. Atrazine-d(5) was used as internal standard. The method had an LOD of 10 pg/mL atrazine, and a linear calibration curve over a range from 30 pg to 50.0 ng/mL. Simultaneous determination of the four triazine pesticides in water samples taken from local lakes was demonstrated.     

Gas chromatographic/mass spectrometric and microbiological analyses on irradiated chicken

Ionizing radiation is widely used as treatment technique for food preservation. It involves among others reduction of microbial contamination, disinfestations, sprout inhibition and extension of shelf life of food. However, the commercialization of irradiated food requires the availability of reliable methods to identify irradiated foodstuffs. In this paper, we present results on the application to irradiated chicken of this method, based on the detection, in muscle and skin samples, of the peaks of ions 98 Da and 112 Da, in a ratio approximately 4:1, typical of radiation induced 2-dodecylcyclobutanones (2-DCB). Aim of the work was also to study the time stability of the measured parameters in samples irradiated at 3 and 5 kGy, and to verify the efficacy of the treatment from a microbiological point of view. Our results show that, one month after irradiation at 3 kGy, the method is suitable using the skin but not the muscle, while the measured parameters are detectable in both samples irradiated at 5 kGy. The microbial population was substantially reduced even at 3 kGy.     

A liquid chromatography/tandem mass spectrometric multi-mycotoxin method for the quantification of 87 analytes and its application to semi-quantitative screening of moldy food samples

This paper describes the extension of a previously published method based on liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) from 39 to currently 87 analytes. Besides the mycotoxins for which regulated concentrations exist, the method now comprises not only almost all mycotoxins for which standards are commercially available, but also a number of other important metabolites produced by fungi involved in food spoilage. The method is based on a single extraction step using an acidified acetonitrile/water mixture followed by analysis of the diluted crude extract. Method performance characteristics were determined after spiking breadcrumbs as model matrix at multiple concentration levels. With very few exceptions, coefficients of variation of the whole procedure of <5% and repeatabilities at the highest spiking level of <7% were obtained. Limits of detection ranged between 0.02 and 225 μg kg⁻¹. The quantitative determination of ergopeptides was disturbed by epimerization due to the acidic conditions. From the remaining 77 analytes, the apparent recoveries of nine substances deviated significantly from the CEN target range of 70–110% due to incomplete extraction and/or matrix effects. In principle, the latter can be compensated for by the application of matrix-matched calibration. The developed method was applied to 18 moldy samples (including bread, fruits, vegetables, jam, cheese, chestnuts and red wine) from private households. This study revealed the great value of the described method: 37 different fungal metabolites were identified at concentrations of up to 33 mg kg⁻¹, and some of these have never been reported before in the context of moldy food products. Figure ESI (+) MS/MS chromatogram (total ion current of all MRMs) of a sample of moldy dark bread     

Direct mass spectrometric detection of trace explosives in soil samples

The detection of explosives in soil is of great significance in public security programmes and environmental science. In the present work, a ppb-level method was established to directly detect the semi-volatile explosives, RDX and TNT, present in complex soil samples. The method used thermal sampling technique and a direct current atmospheric pressure glow discharge source mounted with a brass cylinder electrode (9 mm × 4.6 mm i.d./5.6 mm o.d.) to face the samples, requiring no sample pretreatment steps such as soil extraction (about ten hours). It was characterized by the merits of easy operation, high sensitivity and fast speed, and has been validated by real soil samples from various locations around a factory or firecracker releasing fields. It took only 5 min per sample, with the limit of detection down to 0.5 ppb (S/N = 3) trinitrohexahydro-1,3,5-triazine in soils heated at 170 °C. It is also extendable to the analysis of other volatile analytes.     

First mass spectrometric detection of boldenone in horse mane samples

Summary In order to investigate the detection of boldenone in horse mane samples, a boldenone study was conducted on two horses. The analytical procedure consisted in a hydrolysis using the Sorensen buffer, a liquid-liquid extraction using diethyl ether and a PFPA derivatization. The instrumental method was a gas chromatography sequential mass spectrometry performed on an ion trap instrument in full scan mode. The limit of detection was estimated to 1 pg mg⁻¹. The detection of boldenone in the mane was made possible for up to 12 months after administration.     

A multiple-reaction-monitoring mass spectrometric method for simultaneous quantitative analysis of five plasma apolipoproteins

A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systematically quantify the relative expressions of five candidate plasma apolipoproteins that have been previously shown to be dysregulated in neuropsychiatric disorders and cognitive dysfunction:apolipoprotein H(APOH),apolipoprotein J(APOJ),apolipoprotein A4(APOA4),apolipoprotein E(APOE),and apolipoprotein D(APOD).The peptides and transitions of each APO were carefully selected according to the tandem MS signals acquired on a TripleTOFTM 5600,followed by optimization of the declustering potential and collision energy voltages for transitions on a QTRAP 5500.Our results showed that the collision energies of mTRAQ-labeled peptides were approximately 15%–20%higher than corresponding non-labeled peptides.Through optimized transitions and parameters,we analyzed the relative abundances of the five APOs in human plasma with and without depletion of high abundant proteins.The results indicated that the MRM signals of four target APOs were significantly increased after depletion,while the MRM signal of one APO,APOD,was decreased.Furthermore,the relative abundances of the five target APOs in healthy human plasma were stable,and the ranking of these proteins according to their MS responses changed slightly.Therefore,we deduced that the rank order of the MS signals for these target proteins can be developed as a diagnostic signature for diseased plasma.     

Gas chromatographic-mass spectrometric method for the determination of selenium in biological samples.

The determination of selenium by capillary gas chromatography-mass spectrometry (GC-MS), using an enriched stable isotope 76Se as internal standard, is described. Reference values for selenium in human biological fluids (serum, red blood cells and urine) are reported. With the advent of new compact capillary GC-MS (benchtop) instruments, this method will be very simple and accurate for routine analysis.     

An electrospray mass spectrometric method for accurate mass determination of highly acid-sensitive phosphoramidites

An accurate mass determination method utilizing electrospray ionization mass spectrometry is described for analysis of several different types of phosphoramidites that are extremely acid-sensitive compounds. An earlier method, which applied a LiCl/acetonitrile system, was extended for this special application by using polymeric standards including poly(ethylene glycol) (PEG), poly(ethylene glycol) dimethyl ether (PDE) and poly(propylene glycol) (PPG). Concentrations of standards, samples and LiCl were optimized and potential impurities that affect the analyses were also investigated.