Isotope dilution high-performance liquid chromatography/tandem mass spectrometry method for quantifying urinary metabolites of atrazine, malathion, and 2,4-dichlorophenoxyacetic acid.

We have developed an isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for quantifying the urinary metabolites of the pesticides atrazine, malathion, and 2,4-dichlorophenoxyacetic acid (2,4-D). Urine samples are extracted with an organic solvent, and the organic fraction is concentrated. The concentrate is then analyzed using HPLC/MS/MS. The limits of detection for the metabolites are less than 0.5 microgram/L (parts per billion) in 10 mL of urine, with a high degree of accuracy and precision.     

Speciation and identification of organoselenium metabolites in human urine using inductively coupled plasma mass spectrometry and tandem mass spectrometry

A method for speciation and identification of organoselenium metabolites found in human urine samples using high performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS) and tandem mass spectrometry (MS/MS) is described. Reversed-phase chromatographic separation was used for sample fractionation with the ICP-MS functioning as an element-selective detector, and six distinct selenium-containing species were detected in a human urine sample. Fractions were then collected and analyzed using a triple quadrupole mass spectrometer with electrospray ionization and collision-induced dissociation to obtain structural information. The first two fractions were identified specifically as selenomethionine and selenocystamine, estimated to be present at approximately 11 and 40 ppb, respectively. To the best of our knowledge, this is the first time these two metabolites have been positively identified in human urine.     

Single-photon ionization quadrupole mass spectrometry with an electron beam pumped excimer light source

The application of soft ionization methods for mass spectrometry (MS), such as single-photon ionization (SPI) using vacuum ultraviolet (VUV) light, provides powerful analytical instrumentation for real-time on-line monitoring of organic substances in gaseous matrixes. A compact and mobile quadrupole mass spectrometer (QMS) system using a novel electron beam pumped rare gas VUV lamp for SPI has been developed for on-line analysis of organic trace compounds (ppb concentrations). The VUV radiation of the light source is employed for SPI in the ion source of the QMS. The concept of the interfacing of the VUV light source with the QMS is described and the SPI-QMS is characterized. On-line detection limits down to 50 ppb for benzene, toluene, and m-xylene were achieved. The instrument is well suited for continuous measurements of aromatic and aliphatic trace compounds and can therefore be used for on-line monitoring of trace compounds in dynamically fluctuating process gases. First measurements of gas standards, petrochemical samples, and on-line monitoring of automotive exhaust are presented.     

Identification of organic acids in secondary organic aerosol and the corresponding gas phase from chamber experiments

Organic acids in the gas and aerosol phase from photooxidation of 1,3,5-trimethylbenzene in the presence of 300 ppb propene and 300 ppb NOx in smog chamber experiments were determined using a wet effluent diffusion denuder/aerosol collector coupled to ion chromatography (IC) with conductivity detection. Behind the IC, the samples were collected using a fraction collector, for identification of unresolved/unidentified organic acids with IC-mass spectrometry (MS). In total, 20 organic acids were found with MS of which 10 were identified. The organic acids identified offline by IC-MS were then further quantified based on the online IC data. The identification was additionally confirmed with gas chromatography-mass spectrometry. At the maximum aerosol concentration, organic acids comprised 20-45% of the total aerosol mass. The method has a detection limit of 10-100 ng/m3 for the identified carboxylic acids.     

Simultaneous quantitative analysis of metabolites using ion-pair liquid chromatography-electrospray ionization mass spectrometry

We have developed an analytical method, consisting of ion-pair liquid chromatography coupled to electrospray ionization mass spectrometry (IP-LC-ESI-MS), for the simultaneous quantitative analysis of several key classes of polar metabolites, like nucleotides, coenzyme A esters, sugar nucleotides, and sugar bisphosphates. The use of the ion-pair agent hexylamine and optimization of the pH of the mobile phases were critical parameters in obtaining good retention and peak shapes of many of the above-mentioned polar and acidic metabolites that are impossible to analyze using standard reversed-phase LC/MS. Optimum conditions were found when using a gradient from 5 mM hexylamine in water (pH 6.3) to 90% methanol/10% 10 mM ammonium acetate (pH 8.5). The IP-LC-ESI-MS method was extensively validated by determining the linearity (R2 > 0.995), sensitivity (limit of detection 0.1-1 ng), repeatability, and reproducibility (relative standard deviation <10%). The IP-LC-ESI-MS method was shown to be a useful tool for microbial metabolomics, i.e., the comprehensive quantitative analysis of metabolites in extracts of microorganisms, and for the determination of the energy charge, i.e., the cellular energy status, as an overall quality measure for the sample workup and analytical protocols.     

Qualitative and quantitative analysis of tumor cell metabolism via stable isotope labeling assisted microfluidic chip electrospray ionization mass spectrometry

In this work, a stable isotope labeling assisted microfluidic chip electrospray ionization mass spectrometry (SIL-chip-ESI-MS) platform for qualitative and quantitative analysis of cell metabolism was developed. Microfluidic cell culture, drug-induced cell apoptosis analysis, and cell metabolism measurements were performed simultaneously on the specifically designed device. MCF-7 cells were cultivated in vitro and exposed in anticancer agent (genistein and genistein-d(2)) for cell-based drug assay. A dual-isotopic labeling was presented for effective qualitative analysis of multiplex metabolites. Interestingly, three coeluting pairs of isotopomers appeared with an m/z difference of two. Despite complex biological matrixes, they can be easily recognized and identified by chip-ESI-MS/MS, which significantly facilitates candidate biomarker discovery. The quantitative performance of this system was evaluated using genistein as a model drug by means of stable isotope dilution analysis. The linear equation obtained is y = 0.06x - 3.38 × 10(-3) (R(2) = 0.995) at the dynamic range from 0.5 to 40 μM. The detection limit is 0.2 μM. The method shows an excellent stability of 2.2% relative standard deviation (RSD) and a good repeatability of 5.5% RSD. Our results have successfully demonstrated the capability of selective and quantitative analysis of cell-based drug absorption and metabolites with high stability, sensitivity, and repeatability on the chip-ESI-MS system. Consequently, the present device shows promise as a high-throughput, low-cost, and online platform for cell metabolism studies and drug screening processes.     

Shotgun metabolomics approach for the analysis of negatively charged water-soluble cellular metabolites from mouse heart tissue

A shotgun metabolomics approach using MALDI-TOF/TOF mass spectrometry was developed for the rapid analysis of negatively charged water-soluble cellular metabolites. Through the use of neutral organic solvents to inactivate endogenous enzyme activities (i.e., methanol/chloroform/H2O extraction), in conjunction with a matrix having minimal background noise (9-amnioacridine), a set of multiplexed conditions was developed that allowed identification of 285 peaks corresponding to negatively charged metabolites from mouse heart extracts. Identification of metabolite peaks was based on mass accuracy and was confirmed by tandem mass spectrometry for 90 of the identified metabolite peaks. Through multiplexing ionization conditions, new suites of metabolites could be ionized and "spectrometric isolation" of closely neighboring peaks for subsequent tandem mass spectrometric interrogation could be achieved. Moreover, assignments of ions from isomeric metabolites and quantitation of their relative abundance was achieved in many cases through tandem mass spectrometry by identification of diagnostic fragmentation ions (e.g., discrimination of ATP from dGTP). The high sensitivity of this approach facilitated the detection of extremely low abundance metabolites including important signaling metabolites such as IP3, cAMP, and cGMP. Collectively, these results identify a multiplexed MALDI-TOF/TOF MS approach for analysis of negatively charged metabolites in mammalian tissues.     

Structural characterization of metabolites of the X-ray contrast agent iopromide in activated sludge using ion trap mass spectrometry

Identification of degradation products of environmental contaminants is a challenging task because not only are they present in very low concentrations but they are also mixed with complex matrixes that interfere with detection. This work illustrates a simple approach using ion trap mass spectrometry combined with H/D-exchange experiments to elucidate the structures of iopromide metabolites formed during biodegradation in activated sludge. Iopromide is an X-ray contrast agent that has been detected frequently in effluents of wastewater treatment plants and in surface waters due to its persistence and high usage. Three metabolites produced by oxidation of the primary alcohols (forming carboxylates) on the side chains of iopromide were identified in a batch reactor with mixed liquor from a conventional activated sludge. Derivatization of the carboxylic acid to form a methyl ester and interpretation of the MS2 data of this derivative aided in the confirmation of the identities of these metabolites. Furthermore, one metabolite formed by dehydroxylation at the two side chains was identified in a batch reactor with mixed liquor from a nitrifying activated sludge. The MS2 fragmentation pattern of iopromide and its metabolites revealed that the iodinated ring remains intact and that minor transformations in the structure occur during biodegradation of iopromide in biological wastewater treatment plants.     

Ultra performance liquid chromatography isotope dilution tandem mass spectrometry for the absolute quantification of proteins and peptides

A selective, rapid, and sensitive 12.7-min ultra performance liquid chromatography-isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) method was developed and compared to conventional high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-ID/MS/MS) for the absolute quantitative determination of multiple proteins from complex matrixes. The UPLC analysis was carried out on an Acquity UPLC ethylene-bridged hybrid (BEH) C18 reversed-phase column (50 x 2.1 mm i.d., 1.7-microm particle size) with gradient elution at a flow rate of 300 microL/min. For the HPLC separation, a similar gradient profile on a reversed-phase C18 column with dimensions of 150 x 1.0 mm at a flow rate of 30 microL/min was utilized. The aqueous and organic mobile phases were 0.1% formic acid in water and acetonitrile, respectively. Detection was performed on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. Linear calibration curves were obtained in the concentration range of 10-90 fmol/microL. Relative standard deviation values equal to or less than 6.5% were obtained by the UPLC-ID/MS/MS method, thus demonstrating performance equivalent to conventional HPLC-ID/MS/MS for isotope dilution quantification of peptides and proteins. UPLC provides additional dimensions of rapid analysis time and high-sample throughput, which expands laboratory emergency response capabilities over conventional HPLC.     

Capillary column switching restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry system for simultaneous and direct analysis of drugs in biofluids

Capillary online restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry (RAM-LC-ESI-MS/MS) for direct analysis of drugs and metabolites spiked in biological fluids was developed. Using a column switching setup it was possible to perform effective sample preparation and analysis of raw biological fluids (plasma and urine) without matrix effects in the electrospray mass spectrometric detection step. The peak focusing efficiency of the extraction column was more effective in backflush compared to foreflush mode. The system was able to concentrate diminished samples of polar drugs and their metabolites reaching quantifiable results as low as 1 ng/mL utilizing a sample volume of only 333 nL of biofluids. New column hardware was developed to circumvent clogging problems experienced with plasma injections. The glass fiber filter frit, which is commonly used, was replaced with a short piece of 20 microm i.d. fused silica capillary. The extraction columns were able to handle up to 60 injections and showed a high loading capacity, making the saturation of the MS detector the limiting factor on the linear dynamic range. The simultaneous separation and detection of 10 drugs and metabolites was obtained in 8 min of analysis, including the online sample preparation and enrichment step.     

Single-neuron analysis using CE combined with MALDI MS and radionuclide detection

Capillary electrophoresis (CE) has been combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and radionuclide detection to assay mass-limited biological samples. Nanovial sampling techniques enable injections into the CE capillary from 50 to 150-nL volume samples; after the separation, nanoliter fraction collection combines the CE effluent with a MALDI matrix and minimizes sample spreading, thus allowing both MALDI MS and radionuclide detection on the CE fractions. MALDI MS complements the elution time information of CE by providing accurate molecular mass data, and radionuclide detection provides zeptomole limits of detection with quantitative information. While MALDI MS detects all fully processed peptides at sufficient concentration, culturing the neuron in media containing 35S-Met provides selective radionuclide detection of newly synthesized methionine-containing peptides. The analysis and detection of the expected neuropeptides and hormones in a single 40-microm bag cell neuron from Aplysia californica with CE/MALDI MS/radionuclide detection demonstrates the ability of this hyphenated approach to work with chemically complex mass-limited samples.     

Method for simultaneous imaging of endogenous low molecular weight metabolites in mouse brain using TiO2 nanoparticles in nanoparticle-assisted laser desorption/ionization-imaging mass spectrometry

We report the detection of a group of endogenous low molecular weight metabolites (LMWM) in mouse brain (80-500 Da) using TiO(2) nanoparticles (NPs) in nanoparticle-assisted laser desorption/ionization-imaging mass spectrometry (Nano-PALDI-IMS) without any washing and separation step prior to MS analysis. The identification of metabolites using TiO(2) NPs was compared with a conventional organic matrix 2,5-dihydroxybenzoic acid (DHB) where signals of 179 molecules were specific to TiO(2) NPs, 4 were specific to DHB, and 21 were common to both TiO(2) NPs and DHB. The use of TiO(2) NPs enabled the detection of a higher number of LMWM as compared to DHB and gold NPs as a matrix. This approach is a simple, inexpensive, washing, and separation free for imaging and identification of LMWM in mouse brain. We believe that the biochemical information from distinct regions of the brain using a Nano-PALDI-IMS will be helpful in elucidating the imbalances linked with diseases in biomedical samples.     

Integration of knowledge-based metabolic predictions with liquid chromatography data-dependent tandem mass spectrometry for drug metabolism studies: application to studies on the biotransformation of indinavir

Despite recent advances in the application of data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) to the identification of drug metabolites in complex biological matrixes, a prior knowledge of the likely routes of biotransformation of the therapeutic agent of interest greatly facilitates the detection and structural characterization of its metabolites. Thus, prediction of the [M + H]+ m/z values of expected metabolites allows for the construction of user-defined MS(n) protocols that frequently reveal the presence of minor drug metabolites, even in the presence of a vast excess of coeluting endogenous constituents. However, this approach suffers from inherent user bias, as a result of which additional "survey scans" (e.g., precursor ion and constant neutral loss scans) are required to ensure detection of as many drug-related components in the sample as possible. In the present study, a novel approach to this problem has been evaluated, in which knowledge-based predictions of metabolic pathways are first derived from a commercial database, the output from which is used to formulate a list-dependent LC/MS(n) data acquisition protocol. Using indinavir as a model drug, a substructure similarity search on the MDL metabolism database with a similarity index of 60% yielded 188 "hits", pointing to the possible operation of two hydrolytic, two N-dealkylation, three N-glucuronidation, one N-methylation, and several aromatic and aliphatic oxidation pathways. Integration of this information with data-dependent LC/MS(n) analysis using an ion trap mass spectrometer led to the identification of 18 metabolites of indinavir following incubation of the drug with human hepatic postmitochondrial preparations. This result was accomplished with only a single LC/MS(n) run, representing significant savings in instrument use and operator time, and afforded an accurate view of the complex in vitro metabolic profile of this drug.     

饮料中营养强化剂的检测

本文将样品经氨水水解后利用提取剂提取出脂肪酸经甲基化反应后,用气相色谱-质谱联用仪测定出食品中添加的脂肪酸类营养强化剂,采用保留时间和MS/SCAN方法来定性,利用选择离子定量。本检测方法前处理简单快捷,易于操作,方法重现性良好,其RSD可达0.82%～1.89%;回收率可达91.2%～103.1%;灵敏度高,检测限为0.1mg/kg。     

Comparative study of photodissociation and surface-induced dissociation by laser desorption Fourier transform mass spectrometry.

Photodissociation (PD) and surface-induced dissociation (SID) are compared for structural analysis of several nonvolatile compounds analyzed by laser desorption Fourier transform mass spectrometry (LD/FTMS). SID and PD of a porphyrin and two metalloporphyrins were investigated using a variety of experimental conditions. Optimum structural information is obtained from PD when parent ions are irradiated for relatively long times (10-30 s) using 575-nm radiation and short times (0.5-1 s) using 308- or 388-nm radiation. Shorter irradiation times in the visible region resulted in less efficient production of structurally significant product ions, while longer times in the ultraviolet region produced more nonspecific fragment ions, apparently at the expense of more structurally significant fragment ions. SID conversion efficiencies for the porphyrins are estimated for collision energies from 25 to 360 eV, with maximum conversion efficiency found using 62- and 115-eV collision energies for the two porphyrins studied. Results from a concurrent study on the combined use of PD and SID for MS/MS/MS are discussed in the context of these results. The MS3 ion spectra generated by the two dissociation techniques differ more significantly than MS2 product ion spectra. These data suggest some general guidelines for MSn studies of nonvolatile compounds analyzed by LD/FTMS, employing PD and SID for ion activation.     

Metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode MALDI time-of-flight mass spectrometry

Metabolites in islets of Langerhans and Escherichia coli strain DH5-alpha were analyzed using negative-mode, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For analysis of anionic metabolites by MALDI, 9-aminoacridine as the matrix yielded a far superior signal in comparison to alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydrobenzoic acid, 2,4,6,-trihydroxyacetophenone, and 3-hydroxypicolinic acid. Limits of detection for metabolite standards were as low as 15 nM for GDP, GTP, ADP, and ATP and as high as 1 muM for succinate in 1-muL samples. Analysis of islet extracts allowed detection of 44 metabolites, 29 of which were tentatively identified by matching molecular weight to compounds in METLIN and KEGG databases. Relative quantification was demonstrated by comparing the ratio of selected di- and triphosphorylated nucleotides for islets incubated with different concentrations of glucose. For islets at 3 mM glucose, concentration ratios of ATP/ADP, GTP/GDP, and UTP/UDP were 1.9 +/- 1.39, 1.12 +/- 0.50, and 0.79 +/- 0.35 respectively, and at 20 mM glucose stimulation, the ratios increased to 4.13 +/- 1.89, 5.62 +/-4.48, and 4.30 +/- 4.07 (n = 3). Analysis was also performed by placing individual, intact islets on a MALDI target plate with matrix and impinging the laser directly on the dried islet. Direct analysis of single islets allowed detection of 43 metabolites, 28 of which were database identifiable. A total of 43% of detected metabolites from direct islet analysis were different from those detected in islet extracts. The method was extended to prokaryotic cells by analysis of extracts from E. coli. Sixty metabolites were detected, 39 of which matched compounds in the MetaCyc database. A total of 27% of the metabolites detected from prokaryotes overlapped those found in islets. These results show that MALDI can be used for detection of metabolites in complex biological samples.     

Screening and selective quantification of illicit drugs in wastewater by mixed-mode solid-phase extraction and quadrupole-time-of-flight liquid chromatography-mass spectrometry

For the first time, a mixed-mode solid-phase extraction with fractionation of basic analytes from neutral and acidic species during cartridge elution and liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) was combined for the quantitative determination of 24 illicit drugs and metabolites in urban sewage samples. The effects of several sample preparation and instrumental parameters in the sensitivity and selectivity of the quantitative method are thoroughly discussed. Under final working conditions, recoveries above 63% and 82% were attained for all species in raw and treated sewage, respectively; whereas, the limits of quantification of the method, defined for a signal-to-noise of 10 (S/N = 10), ranged from 2 to 50 ng L(-1). Sequential elution of mixed-mode cartridges allowed a significant reduction of matrix effects observed during electrospray ionization of basic drugs versus those measured for hydrophilic balance reversed-phase sorbents and the same mixed-mode polymer without fractionated elution. Analysis of raw wastewater samples confirmed the ubiquity of cocaine (COC), benzoylecgonine (BE), and 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in this matrix. The capability of the above methodology to identify new illicit drugs and/or metabolites in sewage samples is also discussed. With this aim, a two step strategy is proposed. First, high-resolution MS chromatograms, acquired throughout each chromatographic run, are automatically searched against an in-house built database, a reduced list of candidate drugs is generated, and the corresponding extracted ion chromatograms are obtained. In a further LC run, the tandem mass spectrometry (MS/MS) spectra of unknown peaks are acquired using different collision energies and compared with those existing in public libraries, or interpreted, to assign the unknown peak to one of the previously selected candidates.     

Parallel electrophoretic depletion, fractionation, concentration, and desalting of 96 complex biological samples for mass spectrometry

The preparation of complex biological samples for high-throughput mass spectrometric analyses remains a significant bottleneck, limiting advancement of the capabilities of mass spectrometry (MS) and ultimately limiting development of novel clinical assays. The removal of interfering species (e.g., salts, detergents, and buffers), concentration of dilute analytes, and the reduction of sample complexity are required in order to maximize the quality of resultant MS data. This study describes a novel sample preparation method that makes use of electrophoresis to prepare complex biological samples for high-throughput MS analysis. The method provides for integration of key sample preparation steps, including depletion, fractionation, desalting, and concentration. The prepared samples are captured onto a monolithic reversed-phase capture target that can be analyzed directly by a mass spectrometer. Up to 96 individual samples are simultaneously prepared for MS analysis in under 1 h. For standard proteins added to serum, this method provides femtomole level sensitivity and reproducible label-free detection (coefficient of variation <30%). This study demonstrates that this electrophoretic sample preparation system permits high-throughput sample preparation for mass spectrometric analysis of complex biological samples, such as serum, plasma, and tissue extracts.     

Detection, confirmation, and quantification of staphylococcal enterotoxin B in food matrixes using liquid chromatography--mass spectrometry

Although immunoassay-based methods are sensitive and widely used for measuring protein toxins in food matrixes, there is a need for methods that can directly confirm the molecular identity of the toxin in situations where immunoassay tests yield a positive result. A method has been developed that uses mass spectrometry to identify a protein toxin, staphylococcal enterotoxin B (SEB), in a model food matrix, apple juice. The approach employs ultrafiltration to remove low molecular weight components from the sample, after which the remaining high molecular weight fraction, containing the protein, is digested with trypsin. The tryptic fragments are separated from residual biopolymers and analyzed by liquid chromatography-electrospray mass spectrometry. The background is still sufficiently complex that tandem mass spectrometry (MS/MS) is used to confirm the identity of target peptides. Limits of detection are 80 ng of SEB for MS and 100 ng for full scan MS/MS, using a tryptic fragment as the analytical target. Lower detection limits can be obtained using selected ion monitoring and multiple reaction monitoring. The presence of SEB can be confirmed at concentrations as low as 5 parts-per-billion by increasing the size of the sample to 10 mL. The method is applicable to the detection of SEB in other water-soluble food matrixes.