Ion Trap Tandem Mass Spectrometry Research Articles

Phytochemical Study of the Alkaloidal Fractions of Unonopsis duckei R. E. Fr. Guided by Electrospray Ionisation Ion‐trap Tandem Mass Spectrometry

The Unonopsis genus is a promising source of aporphinoid alkaloids, substances with great biological potential. These alkaloids have a well-defined mass spectrometry fragmentation pattern that, together with previous phytochemical knowledge, can guide the isolation of alkaloids not yet described for the genus. Isolate substances not yet described in the Unonopsis genus, guided by alkaloidal profile analyses of stem barks, twigs and leaves of Unonopsis duckei using electrospray ionisation ion-trap tandem mass spectrometry (ESI-IT/MS(n) ). Methanolic extracts from stem barks, twigs and leaves were submitted to a liquid-liquid, acid-base partitioning treatment to obtain the alkaloidal fractions. These fractions were analysed by direct infusion into an ESI-IT/MS(n) system. The major alkaloids observed for each fraction were submitted to fragmentation analysis. The MS fragmentation patterns revealed the presence of alkaloids previously reported for Annonaceae, including nornuciferine, anonaine, asimilobine, liriodenine and lysicamine, known for the Unonopsis genus, as well as others that were not yet described for this genus. In this way, the proaporphine alkaloid glaziovine was isolated, as well as a mixture of the aporphine alkaloids glaucine and norglaucine, all described for the first time in the Unonopsis genus. Mass spectrometry monitoring was fundamental to prioritise the isolation of substances not yet identified for the Unonopsis genus, dismissing known compounds and simplifying the phytochemical study.

Liquid chromatography/negative electrospray ionization ion trap MS2 mass spectrometry application for the determination of microcystins occurrence in Southern Portugal water reservoirs

Microcystins (MCs) are toxins produced by cyanobacteria which are common organisms in the phytoplankton of eutrophic lakes, rivers and freshwater reservoirs. In the present work, a novel method of liquid chromatography–electrospray ion trap tandem mass spectrometry (LC/ESI/Ion trap–MS/MS), operated in the negative ionization mode, was developed for the analysis of these cyanotoxins. The method was applied to determine the amounts of total microcystins-LR, -YR and -RR in two water reservoirs in Southern Portugal, namely Alqueva and Beliche. A total of 30 water samples were analysed along 2011. Solid phase extraction (SPE) was used for sample cleaning-up and analyte enrichment. The extracted toxins were separated on a C18 column with a gradient of acetonitrile/water with 0.1% formic acid. Detection of microcystins was carried out using multiple reaction monitoring (MRM) in the negative polarity mode, as this method gave a higher selectivity. The MC-RR, YR and LR quantification limits were 17.9, 31.7 and 15.8 ng/L, respectively; quite below the limits recommended by WHO guidelines for drinking water (1 μg/L). Total MC highest concentrations were found in the warm months of June, July and September in Alqueva sampling sites, with concentrations of MC LR and RR ranging 17–344 and 25–212 ng/L, respectively, showing comparable results for MC-RR and LR and slightly lower concentration of MC-YR. Detected values for Beliche reservoir were below quantification limits.

Determination of N-nitrosamines and nicotine in air particulate matter samples by pressurised liquid extraction and gas chromatography-ion trap tandem mass spectrometry

N-nitrosamines are potentially hazardous pollutants, classified as probable carcinogenic and mutagenic by the U.S. Environmental Protection Agency (EPA). In this paper, the presence of these pollutants was studied in air samples taken at different locations of Tarragona (urban and harbour). As a result, a reliable method has been developed for determining N-nitrosamines and nicotine based on pressurised liquid extraction (PLE) and gas chromatography-(chemical ionisation) ion trap tandem mass spectrometry (GC-(CI)MS/MS). The chromatographic analysis enables the determination of these compounds in less than 13min with total separation and good resolution between the compounds. Recovery values were higher than 80% for most of the compounds and the repeatability of the method was under 18% (5ngm−3, %RSD, n=4). MDLs were between 0.1ngm−3 (NMor and NPip) and 2ngm−3 (NMEA). NMor, NPyr, NPip and nicotine were the most frequent compounds in urban and harbour samples at concentration levels between 0.3ngm−3 (NPyr) and 12.5ngm−3 (nicotine) and between 0.13ngm−3 (NPyr) and 3.8ngm−3 (nicotine), respectively.

Characterisation of Phenolic Compounds by HPLC–TOF/IT/MS in Buds and Open Flowers of ‘Chemlali’ Olive Cultivar

Plant phenolics are secondary metabolites that constitute one of the most widely occurring groups of phytochemicals that play several important functions in plants. In olive (Olea europaea L), there is not enough information about the occurrence of these compounds in buds and flowers. To conduct a comprehensive characterisation of buds and open flowers from the olive cultivar 'Chemlali'. The polar fraction of buds and open flowers was extracted using solid-liquid extraction with hydro-alcoholic solvent. Then extracts were analysed using high performance liquid chromatography (HPLC) coupled to electrospray ionisation time-of-flight mass spectrometry (ESI/TOF/MS) and electrospray ionisation ion-trap tandem mass spectrometry (ESI/IT/MS²) operating in negative ion mode. Phenolic compounds from different classes including secoiridoids, flavonoids, simple phenols, cinnamic acid derivatives and lignans were tentatively identified in both extracts. Qualitatively, no significant difference was observed between flower buds and open flowers extracts. However, quantitatively the secoiridoids presented higher percentage of total phenols in open flowers (41.7%) than in flower buds (30.5%) in contrast to flavonoids, which decreased slightly from 38.1 to 26.7%. Cinnamic acid derivatives and simple phenols did not show any change. Lignans presented the lowest percentage in both extracts with an increase during the development of the flower bud to open flower. The HPLC-TOF/IT/MS allowed the characterisation, for the first time, of the phenolic profile of extracts of 'Chemlali' olive buds and open flowers, proving to be a very useful technique for the characterisation and structure elucidation of phenolic compounds.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) in fish, seafood products and fish oil in Spain

A total of 84 samples of wild and farmed fish, cephalopods and fish oils for animal feeding, traded in Spain, were analysed for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) in 2009–2012, by gas chromatography–ion trap tandem mass spectrometry (GC-MS-MS). The method was optimised for screening at moderate costs, allowing PCDD/Fs determination at 1 pg World Health Organization–toxic equivalent quantities (WHO-TEQ) g−1 wet weight (w w) and dl-PCBs at 0.02 pg WHO-TEQ g−1 w w. Concentrations in fish and cephalopods ranged from values below the limit of detection to 1.7 pg g−1 WHO-TEQ sum PCDD/Fs and dl-PCBs, considered as safe with regard to EU legislation. Higher levels were found in cod livers (5.4–54.2) and fish oils (3.3–30.7), with one noncompliant sample in each group.

An efficient GC–IDMS method for determination of PBDEs and PBB in plastic materials

In this study, a fast, inexpensive, simple and reliable analytical method, involving pressurized solvent extraction (PSE) system and gas chromatography, coupled to an ion trap tandem mass spectrometry (GC–ITMS–MS), has been developed. It was validated for determination of 2,2′,4,4′-tetrabromodiphenyl (BDE-47), 2,2′,3,4,4′,5′,6-heptabromodiphenyl (BDE-183), 2,2′,3,3′,4,4′,5,5′,6-nonabromodiphenyl (BDE-206) and decabromodiphenyl (BDE-209) ethers and decabromobiphenyl (BB-209) in polypropylene (PP), polyethylene (PE) and acrylonitrile butadiene styrene (ABS) polymeric matrix. Certified reference materials (CRM) were used to assess the trueness of the method. Quantification of the analytes was performed by a primary method, i.e. isotope dilution mass spectrometry (IDMS). An important advantage of the method is speed. While the analysis of a single sample could take one and half hours, analyses of six samples take 5h. This is due to the automated PSE system, which allows extraction of six samples simultaneously. Automated system increases the repeatability and reduces analyst dedication and human error input. Recoveries between 79.6% and 93.7% were obtained. GC/ITMS–MS presented high selectivity by eliminating matrix effect so that the LOD values of 0.079–0.493mg/kg in polymeric matrix were obtained. The method was applied to various selected electrical and electronic products. Sum of the mass fractions of PBDE and PBB analytes in the materials was determined to be under 1000mg/kg, which is the limit defined by directives.

Confirmation and 3D profiling of anabolic steroid esters in injection sites using imaging desorption electrospray ionisation (DESI) mass spectrometry

In this study, desorption electrospray ionisation (DESI) linear ion trap tandem mass spectrometry (MSn) was applied for the confirmation and three-dimensional profiling of anabolic steroid esters in an injection site of bovine muscle. The spatial resolution of the DESI-MSn was demonstrated by scanning hormone esters and marker ink lines drawn at various distances on a microscopic slide at set distances, using an x-scanner with manual y and z adjustment. Tissue slices of bovine muscle injected with a hormone cocktail were analysed. All anabolic steroid esters could be directly detected in the sample and confirmed on the basis of identification points awarded for selected MS/MS transitions according to the performance criteria given in Commission Decision 2002/657/EC. Moreover, the injection site could be mapped by two-dimensional and three-dimensional imaging MS, showing a horizontal and vertical distribution through the muscle tissue. This DESI approach offers potential for analysis of injection sites of steroid esters from illegally treated animals; moreover, direct analysis by ambient imaging DESI-MS still allows conventional extraction and analysis of the whole tissue for further confirmatory or contra-analysis afterwards.

Optimal extraction and fingerprint analysis of Cnidii fructus by accelerated solvent extraction and high performance liquid chromatographic analysis with photodiode array and mass spectrometry detections

A confirmatory and reliable procedure has been developed for extraction and determination of Cnidii fructus by accelerated solvent extraction (ASE) and high-performance liquid chromatography coupled with photodiode array, electrospray ionisation ion trap tandem mass spectrometry and time of flight mass spectrometry (HPLC-PDA-ESI-ITMSn/TOF-MS). The determination method enabled the characterisation of sixteen bioactive components in C. fructus and quantification of three major coumarins, namely osthole, imperatorin and isopimpinellin. Response surface methodology (RSM) was employed to optimise the extraction parameters yielding the optimum conditions of ASE (extraction temperature 122°C, extraction time 5min and two static cycles). And the total contents of three major coumarins extracted by ASE under the optimum conditions was significantly higher than those by reflux and ultrasonic extraction (P<0.05) with better reproducibility. At last, the proposed method coupled with pattern recognition was applied to analysis of C. fructus from eight different regions in China.

Characterization of four Phyllanthus species using liquid chromatography coupled to tandem mass spectrometry

This paper reports a comparison of four Phyllanthus species (P. amarus, P. stipulatus, P. niruri and P. tenellus), commonly known as stone breaker, by the characterization of the chemical profile of their aqueous extracts. Such characterization was carried out using liquid chromatography coupled to ion trap tandem mass spectrometry (LC–IT-MSn) under reversed-phase gradient elution mode. The results of MS/MS and MS3 on-line experiments, using the electrospray ionization source in the positive and negative mode, are extensively discussed. Furthermore, quercetin-3-O-β-d-glucuronopyranoside was isolated in multimilligram scale from the aqueous extract of P. stipulatus and characterized by mass spectrometry and NMR. Although it is an unusual flavonol in natural products, LC–IT-MSn experiments showed it to be present also in P. amarus.

Preparation and analytical characterization of 1‐(1‐phenylcyclohexyl)piperidine (PCP) and 1‐(1‐phenylcyclohexyl)pyrrolidine (PCPy) analogues

Classic examples of psychoactive arylcycloalkylamines include ketamine and 1-(1-phenylcyclohexyl)piperidine (PCP) and many others serve as important structural templates for neuropharmacological research. The recent emergence of PCP analogues that can be obtained from internet retailers requires the implementation of appropriate monitoring strategies for harm reduction purposes. Access to analytical data plays a key part when encountering these substances, especially if reference material is not available. The present study describes the synthesis of three substituted 1-(1-phenylcyclohexyl)piperidines, (3-MeO-, 4-MeO- and 3-Me-PCP) and three substituted 1-(1-phenylcyclohexyl)pyrrolidine analogues (3-MeO-, 4-MeO- and 3-Me-PCPy). Analytical characterizations of all six arylcyclohexylamines and their primary 1-phenylcyclohexanamine intermediates included gas chromatography ion trap electron- and chemical ionization and high resolution mass spectrometry, liquid chromatography electrospray hybrid triple-quadrupole linear ion trap tandem mass spectrometry, infrared, diode array detection and (1) H and (13) C nuclear magnetic resonance (NMR) spectroscopy. Solvent (CDCl3 vs. d6 -DMSO) and protonation effects (free bases vs hydrochloride salts) were studied in order to investigate the impact on shifts and splitting patterns, for example, when attempting to assign separate axial and equatorial proton chemical shifts of NMR spectra. Differentiation between the isomeric 3-MeO-/4-MeO-PCP and PCPy analogues was feasible under mass spectral conditions. Gas chromatography analysis appeared to induce notable degradation of the 4-MeO-substituted analytes, especially when dealing with the HCl salts which led to the detection of the substituted 1-phenylcyclohex-1-ene nucleus. This phenomenon was observed to be less pronounced with the 3-MeO isomers, possibly due to the resonance properties of the para-methoxy group followed by more facile elimination of the amine.

Proteomic Analysis of Two Metabolic Proteins with Potential to Translocate to Plasma Membrane Associated with Tumor Metastasis Development and Drug Targets

Metastasis is the main cause for death of breast cancer patients. However, the underlying mechanism is still poorly understood. Plasma membrane (PM) proteins play a key role in various biological processes, especially for cell migration. In this study, we used a set of well-characterized mammary mouse cell lines, 67NR, 168FARN, 4T1, representing the metastatic progression, to study the differentially expressed membrane proteins. These proteins were analyzed by a linear ion trap tandem mass spectrometry (LTQ-MS/MS) following cell surface biotinylation and streptavidin purification. A total of 1667 membrane proteins were identified, out of which 472 were characterized as differentially expressed with at least 2-fold change and p-value < 0.01. Functional clustering of the 472 proteins revealed that 178 of them were metabolic proteins. Finally, we focused on two metabolic proteins, fatty acid synthase (FASN) and NAD(P)H steroid dehydrogenase-like protein (NSDHL), which were validated by Western blot and immunofluorescence. We found that FASN and NSDHL translocated to the plasma membrane from the intracellular compartment, and their expressions increased from 67NR to 4T1. This alteration of localization along with differential expressions might be necessary for metastasis development. Potentially, FASN and NSDHL could serve as drug targets in new antimetastasis therapy.

Differential Estimation of Isomeric 2- and 4-methoxylated Estrogens in Serum by Matrix-assisted Laser Desorption Ionization-Tandem Mass Spectrometry

As a simple and rapid method for small-molecule analysis, matrix-assisted laser desorption ionization coupled with linear ion-trap tandem mass spectrometry (MALDI-LTQ-MS/MS) was used to differentially quantify 2- and 4-methoxyestrogens in spiked serum samples. Both 2- and 4-methoxyestrogens were consistently identified using distinct analyte-matrix adduct [M+H+CHCA](+) precursor ions when a mixture of 10 mg mL(-1) α-cyano-4-hydroxycinnamic acid (CHCA) in 0.2% trifluoroacetic acid/70% acetonitrile was used as the MALDI matrix. Especially, unusual isomeric product ions from each precursor ion were characterized during MALDI-MS/MS analysis, which permitted differential estimations of isomeric 2- and 4-methoxylated estrone and 17β-estradiol. The calibration linearity was higher than 0.99 in the dynamic range, while the inter-day precision and accuracy ranged from 6.8 to 30.6%, and from 91.4 to 108.5%, respectively. The present technique could be used as a screening assay for 2- and 4-methoxyestrogens without the need for prior chromatographic separation, opening up possible applications in large-scale pharmacokinetic studies.

Sensitive Monitoring of Volatile Chemical Warfare Agents in Air by Atmospheric Pressure Chemical Ionization Mass Spectrometry with Counter-Flow Introduction

A new method for sensitively and selectively detecting chemical warfare agents (CWAs) in air was developed using counter-flow introduction atmospheric pressure chemical ionization mass spectrometry (MS). Four volatile and highly toxic CWAs were examined, including the nerve gases sarin and tabun, and the blister agents mustard gas (HD) and Lewisite 1 (L1). Soft ionization was performed using corona discharge to form reactant ions, and the ions were sent in the direction opposite to the airflow by an electric field to eliminate the interfering neutral molecules such as ozone and nitrogen oxide. This resulted in efficient ionization of the target CWAs, especially in the negative ionization mode. Quadrupole MS (QMS) and ion trap tandem MS (ITMS) instruments were developed and investigated, which were movable on the building floor. For sarin, tabun, and HD, the protonated molecular ions and their fragment ions were observed in the positive ion mode. For L1, the chloride adduct ions of L1 hydrolysis products were observed in negative ion mode. The limit of detection (LOD) values in real-time or for a 1 s measurement monitoring the characteristic ions were between 1 and 8 μg/m(3) in QMS instrument. Collision-induced fragmentation patterns for the CWAs were observed in an ITMS instrument, and optimized combinations of the parent and daughter ion pairs were selected to achieve real-time detection with LOD values of around 1 μg/m(3). This is a first demonstration of sensitive and specific real-time detection of both positively and negatively ionizable CWAs by MS instruments used for field monitoring.

Dispersive liquid–liquid microextraction for the determination of macrocyclic lactones in milk by liquid chromatography with diode array detection and atmospheric pressure chemical ionization ion-trap tandem mass spectrometry

Eprinomectin (EPRI), abamectin (ABA), doramectin (DOR), moxidectin (MOX) and ivermectin (IVM) were determined in milk samples using dispersive liquid–liquid microextraction (DLLME) and liquid chromatography with diode array detection (LC-DAD) coupled to atmospheric pressure chemical ionization in negative ion mode ion-trap tandem mass spectrometry (APCI-IT-MS/MS). Milk proteins were removed by precipitation with trichloroacetic acid and the analytes were preconcentrated using 2mL of acetonitrile containing 200μL of chloroform as extraction mixture. The effect of several parameters for the liquid–liquid microextraction efficiency was evaluated. Standard additions method was used for quantification purposes, the correlation coefficients were better than 0.9970 in all cases and the quantification limits ranged from 1.0 to 4.7ngg−1 and from 0.1 to 2.4ngg−1 when using DAD and MS, respectively. The DLLME-LC-APCI-IT-MS/MS optimized method was successfully applied to different milk samples and none of the studied analytes was detected in the samples studied. The recoveries for milk samples spiked at concentration levels ranging between 0.5 and 50ngg−1, depending on the compound, were between 89.5 and 105%, with relative standard deviations lower than 9% (n=135). Simplicity, rapidity and reliability are important advantages of the proposed method, while the sample preparation step can be regarded as environmentally friendly.

Rapid analysis of multiclass antibiotic residues and some of their metabolites in hospital, urban wastewater and river water by ultra-high-performance liquid chromatography coupled to quadrupole-linear ion trap tandem mass spectrometry

The present work describes the development of a fast and robust analytical method for the determination of 53 antibiotic residues, covering various chemical groups and some of their metabolites, in environmental matrices that are considered important sources of antibiotic pollution, namely hospital and urban wastewaters, as well as in river waters. The method is based on automated off-line solid phase extraction (SPE) followed by ultra-high-performance liquid chromatography coupled to quadrupole linear ion trap tandem mass spectrometry (UHPLC–QqLIT). For unequivocal identification and confirmation, and in order to fulfill EU guidelines, two selected reaction monitoring (SRM) transitions per compound are monitored (the most intense one is used for quantification and the second one for confirmation). Quantification of target antibiotics is performed by the internal standard approach, using one isotopically labeled compound for each chemical group, in order to correct matrix effects. The main advantages of the method are automation and speed-up of sample preparation, by the reduction of extraction volumes for all matrices, the fast separation of a wide spectrum of antibiotics by using ultra-high-performance liquid chromatography, its sensitivity (limits of detection in the low ng/L range) and selectivity (due to the use of tandem mass spectrometry) The inclusion of β-lactam antibiotics (penicillins and cephalosporins), which are compounds difficult to analyze in multi-residue methods due to their instability in water matrices, and some antibiotics metabolites are other important benefits of the method developed. As part of the validation procedure, the method developed was applied to the analysis of antibiotics residues in hospital, urban influent and effluent wastewaters as well as in river water samples.

Direct analysis of urinary 1-hydroxypyrene using extractive electrospray ionization ion trap tandem mass spectrometry

Fast detection (0.5 min) of 1-hydroxypyrene (1-OHP) in urine and hydrolyzed urine without pre-treatment has been successfully achieved by using extractive electrospray ionization ion trap tandem mass spectrometry (EESI-MS/MS) under optimized EESI and MS/MS conditions. Experimental results indicated that for MS/MS analysis the operating parameter of activation Q (AQ) was critical for 1-OHP fragmentation in collision induced dissociation (CID) experiments, and in the EESI process the primary ESI solvent was a key factor for extractive ionization of urinary 1-OHP. The limit of detection (LOD) and limit of quantification (LOQ) were 0.75 and 2.25 μM for both urine and hydrolyzed urine samples. A five-point working curve ranging from 2.29 to 22.91 μM for 1-OHP in urine or hydrolyzed urine was obtained (R2 = 0.9941 for urine and R2 = 0.9983 for hydrolyzed urine), and the relative standard deviations (RSD, n = 6) were 2.6–9.7% and 1.5–6.4%, respectively. The developed EESI-MS/MS method was validated by detecting 1-OHP in both urine and hydrolyzed urine samples. Recoveries were determined to >50% and 0.5 min was taken for each measurement, indicating that the proposed method is a promising strategy for high throughput analysis of urinary 1-OHP required for health risk assessment of exposure to polycyclic aromatic hydrocarbons (PAHs).

Simultaneous determination of aconitine, mesaconitine, hypaconitine, bulleyaconitine and lappaconitine in human urine by liquid chromatography-electrospray ionization-tandem mass spectrometry

Aconitine, hypaconitine, mesaconitine, bulleyaconitine and lappaconitine are Aconitum alkaloids that have been proved to be neurotoxic and cardiotoxic. A method with liquid–liquid extraction and liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) was developed and validated for the quantification of the five Aconitum alkaloids in human urine. Upon the successful chromatographic separation, the alkaloids were determined by ion trap tandem mass spectrometry. The precursor and major product ions of aconitine at m/z 646 → 586, mesaconitine at m/z 632 → 572, hypaconitine at m/z 616 → 556, bulleyaconitine at m/z 644 → 586 and lappaconitine at m/z 585 → 535 were monitored in positive ion mode. Linear calibration curves were generated from 0.02–5 μg mL−1 with coefficients of greater than 0.99. The method was validated with the intra-day and inter-day precisions represented by relative standard deviation of less than 7% in urine, and the recoveries from spiked urine samples varied from 89.8% to 96.4% for lappaconitine, 87.4–96.3% for hypaconitine, 81.5–90.6% for mesaconitine, 82.6–90.0% for aconitine and 82.2–88.7% for aconitine. Stability of aconitine was assessed under acidic, neutral and alkaline conditions. Aconitine hydrolysis was not observed during the urine sample pretreatment under neutral and weak acidic conditions.

A serially coupled stationary phase method for the determination of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine by liquid chromatography ion trap tandem mass spectrometry

Oxidative attack to DNA is of particular interest since DNA modifications can lead to heritable mutations. The most studied product of DNA oxidation is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). While 8-oxodG determination in blood and tissue cells is prone to artifacts, its measurement in urine employing liquid chromatography tandem mass spectrometry (LC-MS/MS) has gained more and more interest for increased reliability. LC-MS/MS can be affected by matrix effects and this is particularly true when ion trap is used as MS analyzer, due to ion accumulation in the trap and related space charge effect. In the present work, we have developed a LC-MS/MS method where the combination of cation exchange and reverse phase solid phases resulted in LC separation optimization. This together with the employment of an isotopically labeled internal standard, allowed the usage of ion trap LC-MS/MS, typically not employed for quantitative measurement in biological samples, for the measurement of 8-oxodG in urine samples from control populations.Four different urine matrices were employed for method validation. Limit of quantitation was set at least at 0.5ng/ml. While analyzing urine samples from healthy volunteers, 8-oxodG levels reported as ng/ml were statistically different comparing males with females (p<0.05, Mann Whitney test); while comparing results normalized for creatinine no statistical significant difference was found. Mean urinary 8-oxodG level found in healthy volunteers was 1.16±0.46nmol/mmol creatinine.The present method by enhancing at best the chromatographic performances allows the usage of ion trap LC-MS/MS for the measurement of 8-oxodG in urine samples from control populations.

Comparison of peptide and protein fractionation methods in proteomics

Multiple fractionation or separation methods are often combined in proteomics to improve signal-to-noise and proteome coverage and to reduce interference between peptides in quantitative proteomics. Furthermore, a given fractionation method provides additional information on the analytes, such as molecular weight, hydrophobicity or isoelectric point that can be used to improve identification, and to discover protein splice variants or large post-translational modifications. Here we describe a Taverna scientific workflow for analysis and comparison between strong cation exchange (SCX) chromatography, peptide isoelectric focusing (pIEF) and SDS-PAGE performed using robust capillary LC and ion trap tandem mass spectrometry.

Direct analysis of 4-methylimidazole in foods using paper spray mass spectrometry

Rapid qualitative and quantitative analysis of 4-methylimidazole (4-MEI) in caramel and beverage samples is demonstrated using the paper spray form of ambient ionization mass spectrometry. The minimum level of pure 4-MEI detectable using multiple reaction monitoring (MRM) in a triple quadrupole instrument was 3 pg μL⁻¹ in neat solvent and 5 pg μL⁻¹ in a matrix containing caramel. This method was used to analyze 11 caramel samples for 4-MEI. After implementing effective quality control protocols, average relative standard deviations for paper spray triple quadrupole MS were less than 15% and the linear dynamic range was three orders of magnitude. Results obtained on three different days by two different analyst groups agreed closely. An ion trap tandem MS method of approximate quantitative analysis is also described and it gave similar precision to the triple quadrupole experiment when fluctuations in ion currents were cancelled by simultaneously isolating the analyte and internal standard and fragmenting both in an ion trap simultaneous MRM experiment. As another alternative, a rapid qualitative screening method based on the use of high-resolution measurements instead of tandem mass spectrometry using an Orbitrap was also tested and found to give a detection limit of 100 pg μL⁻¹.