Toxicological screening with formula-based metabolite identification by liquid chromatography/time-of-flight mass spectrometry

An analytical procedure was evaluated for the comprehensive toxicological screening of drugs, metabolites, and pesticides in 1-mL urine samples by TurboIon spray liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) in the positive ionization mode and continuous mass measurement. The substance database consisted of exact monoisotopic masses for 637 compounds, of which an LC retention time was available for 392. A macroprogram was refined for extracting the data into a legible report, utilizing metabolic patterns and preset identification criteria. These criteria included +/-30 ppm mass tolerance, a +/-0.2-min window for absolute retention time, if available, and a minimum area count of 500. The limit of detection, determined for 90 compounds, was <0.1 mg/L for 73% of the compounds studied and >1.0 mg/L for 6% of the compounds. For method comparisons, 50 successive autopsy urine samples were analyzed by this method, and the results confirmed by gas chromatography/mass spectrometry (GC/MS). Findings for parent drugs were consistent with both methods; in addition, LC/TOFMS regularly revealed apparently correct findings for metabolites not shown by GC/MS. Mean and median mass accuracy by LC/TOFMS was 7.6 and 5.4 ppm, respectively. The procedure proved well-suited for tentative identification without reference substances. The few false positives emphasized the fact that all three parameters, exact mass, retention time, and metabolite pattern, are required for unequivocal identification.     

Use of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry to investigate pesticide residues in fruits

In this paper, the potential of coupling liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF) for the determination of pesticides in a variety of fruit samples (orange peel and flesh, banana skin and flesh, strawberry and pear) has been explored. The quantitative application at residue levels has been proven for two insecticides (buprofezin and hexythiazox), which were satisfactorily determined at three concentration levels, 0.1, 1, and 5 mg/kg, obtaining a suitable linearity range (correlation coefficient>0.99) of more than 2 orders of magnitude. Satisfactory recoveries have been obtained for both compounds at the three levels tested in all sample matrices, with lowest calibration levels (LCL) of 0.075 and 0.01 mg/kg. The excellent potential of QTOF for identification purposes is illustrated by the high number of identification points (IPs) earned, up to 21, at the highest concentration of 5 mg/kg, or between 11 and 21 at the 0.1 and 1 mg/kg levels. The application of LC-QTOF MS to real samples revealed the presence of several positives at concentrations close to the LCL, all of which were confirmed with more than 11 IPs. The potential of QTOF for elucidation of nontarget analytes has also been demonstrated by the finding of one transformation product (TP) of buprofezin in a banana skin sample. This TP was identified by obtaining the full scan product ion spectra at different collision energies with acceptable accurate mass deviation. The work performed in this paper illustrates the suitability and excellent confirmatory potential of LC-QTOF MS for pesticides residues analysis in food samples.     

Exact mass measurements for confirmation of pesticides and herbicides determined by liquid chromatography/time-of-flight mass spectrometry

The accuracy and precision of exact mass measurements are determined using positive ions formed in the electrospray of 10 nonvolatile or thermally unstable carbamate, urea, and thiourea pesticides and herbicides. Environmentally significant approximately 7-ng quantities of the analytes were separated with microbore liquid chromatography, and the exact mass measurements were made in real time with a benchtop time-of-flight mass spectrometer. The positive ion electrospray mass spectra of the analytes generally consist of one or a few ions which are usually adducts of the molecule with a proton, a sodium ion, or an ammonium ion. Fragment ions and the rich mass spectra typical of electron ionization (EI) are generally not produced in the soft electrospray ionization process. Confirmation of the identity of a nonvolatile pesticide or herbicide depends largely on the masses of the few ions formed and the retention time, which can vary with chromatography conditions. Identifications of these analytes in environmental or other samples are less certain than identifications of volatile pesticides determinated by gas chromatography and EI mass spectrometry. The benchtop time-of-flight mass spectrometer was equipped with an electrostatic mirror, and resolving powers of 3500-5000 were routinely obtained and used for these exact mass measurements. This type of mass spectrometer is significantly less costly and complex than other types of mass spectrometers with exact mass measurement capabilities. The mean errors from three replicate exact mass measurements of the 10 test analytes were in the range of 0-5.4 parts-per-million. Potential interferences from substances with similar exact masses were evaluated.     

Bioassay-directed identification of estrogen residues in urine by liquid chromatography electrospray quadrupole time-of-flight mass spectrometry

A new approach to the search for residues of known and unknown estrogens in calf urine is presented. Following enzymatic deconjugation and solid-phase extraction, a minor part of the samples is screened for estrogen activity using a recently developed rapid reporter gene bioassay. The remainder of the bioactive extracts is analyzed by gradient liquid chromatography (LC) with, in parallel, bioactivity and mass spectrometric detection via effluent splitting toward a 96-well fraction collector and an electrospray quadrupole time-of-flight mass spectrometer (QTOFMS). The LC fractions in the 96-well plate are used for the detection of estrogen activity using the bioassay. The biogram obtained features a 20-s time resolution, and the suspect well numbers can be easily correlated with the LC/QTOFMS retention time. The mass spectral data from the thus assigned relevant parts of the chromatograms are background subtracted, followed by accurate mass measurement, element composition calculation, and identification. The method allows estrogen activity detection and identification of (un)known estrogens in urine at the 1-2 ng/L level, in compliance with current residue analysis performance for hormone abuse in cattle. The applicability of this LC/bioassay/QTOFMS approach for the identification of estrogens in real-life samples is demonstrated by the analysis of several calf urine samples, and preliminary data from a pig feed sample.     

Large scale pesticide multiresidue methods in food combining liquid chromatography--time-of-flight mass spectrometry and tandem mass spectrometry

Liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) are powerful and complementary techniques that can independently cover the majority of the challenges related with pesticide residue food control. The sequential combination of both systems benefits from their complementary advantages and assists to increase the performance and to simplify routine large scale pesticide multiresidue methods. The proposed approach consists of three stages: (1) automated pesticide screening by LC-TOFMS; (2) identification by LC-TOFMS accurate mass measurements; and (3) confirmation and quantitation by LC-MS/MS. We have developed a fast comprehensive (identification/confirmation + quantitation) automated screening method for 100 target pesticides in crops. In the first stage, a set of data including m/z accurate mass windows (within 20 mDa width) and retention time is obtained (using a standard solution containing all the targeted pesticides) in order to build the automated screening procedure, which is created automatically by assigning retention time and the m/z mass window for each target pesticide. Samples are then analyzed, and the method enables the screening and preliminary identification of the species first by retention time and m/z mass window, followed by subsequent identification (only if positive results) by LC-TOFMS accurate mass measurements. After that, final confirmation of the positive findings using two MRM transitions and accurate quantitation is performed by LC-MS/MS using a hybrid triple quadrupole linear ion trap (QqLIT) mass spectrometer. In addition, the use of this QqLIT instrument also offers additional advantageous scanning modes (enhanced product ion and MS3 modes) for confirmatory purposes in compounds with poor fragmentation. Examples of applications to real samples show the potential of the proposed approach, including the detection of nonselected "a priori" compounds as a typical case of retrospective evaluation of banned or misused substances.     

Recognition of trypsin autolysis products by high-performance liquid chromatography and mass spectrometry

Potential artifactual contributions are assessed in high-pressure liquid chromatograms and fast atom bombardment mass spectra from autolysis of different preparations of the widely used protease trypsin. Both commercially supplied and laboratory-purified samples were examined. Bovine pancreatic trypsin (1 mg/mL) was found to be completely destroyed in 2 h at pH 8.5, degraded to a complex mixture of small peptides which were characterized by their molecular weights. Some identifications were supported by sequencing by tandem mass spectrometry or by mass spectrometric analysis of the mixture resulting from a single Edman degradation. Autolysis of porcine pancreatic trypsin produced a completely different set of peptides. Five sites of hydrolysis at asparagine residues in bovine trypsin were also identified.     

Applications of Hadamard transform to gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry

Successful application of the Hadamard transform (HT) technique to gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) is described. Novel sample injection devices were developed to achieve multiple sample injections in both GC and LC instruments. Air pressure was controlled by an electromagnetic valve in GC, while a syringe pump and Tee connector were employed for the injection device in LC. Two well-known, abused drugs, 3,4-methylenedioxy-N-methylamphetamine (MDMA) and N, N-dimethyltryptamine (DMT), were employed as model samples. Both of the injection devices permitted precise successive injections, resulting in clearly modulated chromatograms encoded by Hadamard matrices. After inverse Hadamard transformation of the encoded chromatogram, the signal-to-noise (S/N) ratios of the signals were substantially improved compared with those expected from theoretical values. The S/N ratios were enhanced approximately 10-fold in HT-GC/MS and 6.8 in HT-LC/MS, using the matrices of 1023 and 511, respectively. The HT-GC/MS was successfully applied to the determination of MDMA in the urine sample of a suspect.     

Determination of fluorescence-labeled asparaginyl-oligosaccharide in glycoprotein by reversed-phase ultraperformance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

Eight fluorescence reagents, i.e., DBD-F, NBD-F, DNS-Cl, NDA, PSC, FITC, Fmoc-Cl, and DMEQ-COCl, which are reactive to an amino functional group, were tested for the labeling of asparaginyl-oligosaccharides in a glycoprotein. Although the optimal reaction conditions and the fluorescence maximal wavelengths were different for each reagent, the highly sensitive fluorescence detection at the femtomole level of Disialo-Asn (a representative asparaginyl-oligosaccharide) was obtained from the labeling utilizing these reagents. Among them, PSC was the most reliable reagent in terms of detection sensitivity (approximately 3 fmol, signal-to-noise ratio of 5 (S/N = 5) on the chromatogram). However, the structural information could not be obtained from the fluorescence detection. Thus, the on-line determination of a real sample was carried out by UPLC-ESI-TOF-MS. The detection limit of the PSC-labeled Disialo-Asn by selected-ion chromatography was 58 fmol (S/N = 5). When the proposed procedure was applied to the determination of oligosaccharides in ovalbumin, 15 species of PSC-labeled oligosaccharides possessing Man, GlcNAc, and Gal units were identified from the UPLC-ESI-TOF-MS. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Based on the ovalbumin results, the proposed labeling with PSC followed by UPLC-ESI-TOF-MS detection seems to be useful for the on-line asparaginyl-oligosaccharide analysis.     

Liquid chromatography/time-of-flight mass spectrometry with high-speed integrated transient recording

High-performance liquid chromatography (HPLC) has been interfaced to a time-of-flight mass spectrometer. The interface is a continuous flow probe and ions are desorbed from the liquid matrix by energetic ion bombardment. Pulsed and delayed ion extraction from the source permits the use of broad ionization times, results in the production of analog signals in each time-of-flight cycle, and provides both energy and spatial focusing. A high-speed integrated transient recording system has been developed and is also reported. This instrument is the prototype for development of a high-speed, high-mass range LC detector with high duty cycle. Its performance is demonstrated for the separation of several mixtures of small peptides.     

Application of liquid chromatography/quadrupole-linear Ion trap mass spectrometry and time-of-flight mass spectrometry to the determination of pharmaceuticals and related contaminants in wastewater

This paper describes an enhanced liquid chromatography-mass spectrometry (LC-MS) strategy for the analysis of a selected group of 56 organic pollutants in wastewater. This group comprises 38 pharmaceuticals and 10 of their most frequent metabolites, 6 pesticides, and 2 disinfectants. The LC-MS methodology applied is based in the use of a hybrid triple-quadrupole linear ion trap mass spectrometer (QTRAP) in combination with time-of-flight mass spectrometry (TOF-MS). The join application of both techniques provided very good results in terms of accurate quantification and unequivocal confirmation. Quantification was performed by LC-QTRAP-MS operating under selected reaction monitoring (SRM) mode in both positive and negative electrospray ionization. Unequivocal identification was provided by the acquisition of three SRM transitions per compound in most of the cases and by LC-TOF-MS analysis, which allows obtaining accurate mass measurements of the identified compounds with errors lower than 2 ppm. Additionally, the use of TOF-MS permits retrospective analysis, since the full spectrum is recorded at all times with a high sensitivity. Thus, review of recorded chromatograms looking for new compounds or transformation products suspected to be present in the samples is feasible allowing one to increase the scope of the method along the monitoring program. The analytical performance of the quantitative LC-QTRAP-MS method was evaluated in effluent wastewater samples. Linearity of response over 3 orders of magnitude was demonstrated for most compounds (R(2) > 0.99). Method limits of detection were between 0.04 and 50 ng L(-1). Finally, the methodology was successfully applied to a monitoring study intended to characterize wastewater effluents of six sewage treatment plants in Spain. The presence of most of compounds was detected at concentrations ranging from 9 ng L(-1) (atrazine) to 15 microg L(-1) (paraxanthine).     

Quantitative detection of trace systemins in Solanaceous plants by immunoaffinity purification combined with liquid chromatography/electrospray quadrupole time-of-flight mass spectrometry

Systemins are a class of systemic wound polypeptide hormones that play a central role in mediating defenses against pest attacks and other woundings. It has been desired to develop a sensitive and accurate determination method to monitor trace systemins in plants for the better understanding of molecular mechanisms of the polypeptide hormones. A superior method for accurate identification and quantitative determination of trace systemins in Solanaceous plants is described in this work, which is based on immunoaffinity column (IAC) purification and enrichment followed by liquid chromatography online coupled to electrospray quadrupole time-of-flight mass spectrometry (LC/ESI QTOF MS). The specific antitomato systemin polyclonal antibody had been produced and immobilized on a CNBr-activated Sepharose stationary phase. The prepared IAC was utilized for the extraction and enrichment of tomato systemin (TomSys), potato systemin (PotSys I and PotSys II), pepper systemin (PepSys), and nightshade systemin (NishSys) from Solanaceous plants. Subsequent identification and determination by LC/ESI QTOF MS revealed that the IAC enables efficient and specific enrichment of PotSys I, PotSys II, and especially TomSys. Under the optimized conditions, the developed method was successfully applied in the determination of TomSys in tomato leaves and PotSys I and PotSys II in potato leaves, and it offers detection limits (LODs, S/N =3) of 29, 72, and 135 pg/g and mean recoveries of 92.9%, 56.7%, and 34.8% at three concentrations (1.0, 2.0, and 4.0 ng/g) for TomSys, PotSys I, and PotSys II, respectively.     

Data analysis tool for comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

Data processing and identification of unknown compounds in comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC/TOFMS) analysis is a major challenge, particularly when large sample sets are analyzed. Herein, we present a method for efficient treatment of large data sets produced by GC×GC/TOFMS implemented as a freely available open source software package, Guineu. To handle large data sets and to efficiently utilize all the features available in the vendor software (baseline correction, mass spectral deconvolution, peak picking, integration, library search, and signal-to-noise filtering), data preprocessed by instrument software are used as a starting point for further processing. Our software affords alignment of the data, normalization, data filtering, and utilization of retention indexes in the verification of identification as well as a novel tool for automated group-type identification of the compounds. Herein, different features of the software are studied in detail and the performance of the system is verified by the analysis of a large set of standard samples as well as of a large set of authentic biological samples, including the control samples. The quantitative features of our GC×GC/TOFMS methodology are also studied to further demonstrate the method performance and the experimental results confirm the reliability of the developed procedure. The methodology has already been successfully used for the analysis of several thousand samples in the field of metabolomics.     

Automated quantitative analysis of complex lipidomes by liquid chromatography/mass spectrometry

Recent advances in mass spectrometry have revolutionized the analysis of lipid compositions of cells and other biomaterials by simplifying the analytical protocol dramatically and by increasing the sensitivity of detection by several orders of magnitude. However, the throughput of the published mass spectrometric methods is severely limited by data analysis, which requires extensive operator involvement. Consequently, we have developed an automated method that allows unattended identification and quantification of lipid molecular species of all the major lipid classes from a two-dimensional chromatographic/mass spectrometric data set. More than 100 polar lipid species could be automatically quantified from different biological samples with good accuracy and reproducibility. The response was linear over approximately 3 orders of magnitude with the equipment used, and approximately 35 samples could be analyzed in a day. This method makes high-throughput lipidomics feasible in biology, biotechnology, and medicine.     

Analysis of alkenone unsaturation indices with fast gas chromatography/time-of-flight mass spectrometry

Extensively purified C37 alkenone references and mixtures thereof were analyzed by gas chromatography/flame ionization detection (GC/FID) and fast gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS), to establish the latter as an alternative, fast, and reliable analysis method for alkenone unsaturation indices (U(k')(37)). This index is a tool for past sea surface temperature reconstructions with extensive use in paleoclimate and paleoceanographic research. TOF-MS was chosen because of its unique capability to acquire full-range spectra at high data rates (up to 500 spectra s(-1)) and to produce homogeneous spectra across a gaschromatographic peak, allowing faster separations than conventional GC/MS and the employment of enhanced peak deconvolution algorithms. Analysis time per sample could be reduced to run times of <10 min, i.e., by a factor of approximately 10 compared to conventional GC/FID (90-100 min) methods. However, %@mt;sys@%%@ital@%%@bold@%U%@reset@%%@rsf@%%@sx@%37%@be@%%@ital@%k%@rsf@%'%@sxx@%%@mx@% values from GC/TOF-MS showed deviations from those obtained by GC/FID, resulting from sensitivity differences between the C37:2 and C37:3 alkenone when analyzed by GC/TOF-MS. A solution to this bias is presented by determining compound-specific linear response factor equations to derive sensitivity ratios (SR) that allow conversion of GC/TOF-MS values into calibrated GC/FID data. Using alkenone mixtures of known composition and a variety of samples from natural environments, the applicability of this approach is demonstrated.     

Identification of phosphorylation sites in N-linked glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Glycan phosphorylation is a significant feature of complex carbohydrate chemistry and glycobiology. For example, N-linked glycans containing mannose-6-phosphate (Man-6-P) residues play a key role as targeting signals for the transport of proteins from the Golgi apparatus to lysosomes. Structural information on Man-6-P glycans involved in transport of proteins is usually obtained using nuclear magnetic resonance (NMR) spectroscopy. However, an alternative and simple method with comparable accuracy is desirable because large amounts of samples and special techniques are required for structural analysis using NMR. Recently, postsource decay (PSD) fragment spectra obtained by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) have provided critical information on complex carbohydrates. Since few Man-6-P-containing glycans are commercially available, very little information has been collected on the mass spectrometry of phosphorylated glycans. In this report, four kinds of phosphorylated glycans labeled with 2-aminopyridine (PA) were purified from yeast mannan, and their PSD spectra were measured in the positive ion mode. The phospho-6-O-mannose monoester linkages (PO3H-Man) in glycans are stable, although cleavage of the mannose-1-phosphate linkage (Man-alpha-1-PO3H) occurs readily. Fragment ions indicated the presence of the alpha-1,3-branching chain of an N-linked high-mannose-type glycan, and characteristic fragmentation patterns were observed for phosphorylated glycans. On the basis of the MALDI-PSD spectra, we deduced fragmentation rules for phosphorylated N-glycans that will be valuable for distinguishing the position of phosphorylation.     

Determination of hyperforin in mouse brain by high-performance liquid chromatography/tandem mass spectrometry

Hyperforin is one of the essential active ingredients of St. John's wort extract, which is used as an antidepressant for mild to moderately severe depressions. In vitro and in vivo data as well as several clinical studies and meta analyses have confirmed the pharmacological effect of treatment with hyperforin-containing preparations. However, little is known about the brain availability of hyperforin until now. Accordingly, a highly sensitive and selective LC/MS method for this purpose was developed and validated. This method proved suitable for the determination of hyperforin in mouse brain, after oral administration of hyperforin sodium salt and St. John's wort extract. This method involves liquid-liquid extraction of hyperforin with ethyl acetate followed by separation with rapid reversed-phase high-performance liquid chromatography and tandem mass spectrometry detection using electrospray ionization. Excellent linearity was obtained for the entire calibration range from 0.25 to 10 ng/mL (corresponding to 2.5-100 ng/g brain tissue concentration, calculated with the factor derived from sample processing) with an average coefficient of correlation of 0.9992. The recovery of hyperforin from mouse brain homogenates was between 71.4 and 75.3% with a relative standard deviation of less than 3%. Validation assays for the lower limit of quantitation yielded an accuracy of 5.8%. Intraday accuracy and precision for the developed method were between 4.6 and 10.6% and 4.3-8.4%, respectively, while the interday parameters varied between 6.7 and 12.2% for accuracy and 2.0-5.0% for precision. After the method validation, hyperforin brain levels in mice, treated with 15 mg/kg hyperforin (either as the sodium salt or as 5% St. John's wort extract), were investigated. The average concentration of hyperforin found for the sodium salt group was 28.8+/-10.1 ng/g of brain (n = 8), which was somewhat higher than the hyperforin concentration of 15.8+/-10.9 ng/g of brain (n = 8), determined in the extract-treated group. This method is robust, selective, and highly sensitive and represents an appropriate tool to further prove the occurrence and distribution of hyperforin in mouse brain.