Structure Elucidation of 2,4-Dinitrophenylhydrazone Derivatives of Carbonyl Compounds in Ambient Air by HPLC/MS and Multiple MS/MS Using Atmospheric Chemical Ionization in the Negative Ion Mode

Ion trap multiple fragmentation mass spectrometry (MS(n)()) combined with high-performance liquid chromatography (HPLC) has been used for the structure elucidation and identification of 2,4-dinitrophenylhydrazone derivatives of carbonyl compounds in ambient air samples. Atmospheric pressure chemical ionization in the negative ion mode was the most suitable detection method. Different measures are described to decrease the MS background originating from the HPLC system. Low-picogram quantities were detectable in extracted mass chromatograms generated from full-scan records. Fragment ions produced by MS/MS allowed identification of substructures of the carbonyls. Detailed fragmentation paths were studied by MS(3) to MS(4) using reference compounds. A fragmentation scheme was established which enabled a structure confirmation and identification with 1-10 ng by HPLC/MS/MS. The identification of a compound coeluting with n-pentanal-DNPH and of a dimerization byproduct are given as examples.     

Improved tert-butyldimethylsilylation gas chromatographic/mass spectrometric detection of nerve gas hydrolysis products from soils by pretreatment of aqueous alkaline extraction and strong anion-exchange solid-phase extraction

In the analysis of tert-butyldimethylsilyl derivatives (IBDMS) of alkyl methylphosphonic acids (RMPA) and methylphosphonic acid (MPA), from soils by gas chromatography/mass spectrometry (GC/MS), the detection yields are generally low, due to the suppression of TBDMS derivatization by the soil matrix components and the adsorption of RMPA and MPA to the soils. An ion-exchange pretreatment of the aqueous soil extract can be used to overcome the former factor by removing interfering compounds. A pretreatment method is described for improving the detection yields due to the latter factor, using an alkaline extraction procedure. The recovery was estimated quantitatively using capillary electrophoresis. The soil samples tested included volcanogenous immature soils and showed a low aqueous extraction recovery and GC/MS detection yields. The inclusion of sodium hydroxide in the extraction solvent dramatically increased the recovery. Using a 0.1 M sodium hydroxide solution, the recovery was in excess of 68%. Interfering components were removed from the alkaline soil extract by solid-phase extraction of the acids on a silica-based strong anion exchanger. The alkaline soil extract was neutralized with hydrofluoric acid and applied to the cartridge in the fluoride form. After washing with water, MPA and RMPA could be eluted with methanolic ammonia nearly quantitatively. Using the established pretreatment method, MPA and RMPA were detected from all the soil samples in more than 67% yield.     

Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS

A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation methods.     

Accelerated solvent extraction followed by on-line solid-phase extraction coupled to ion trap LC/MS/MS for analysis of benzalkonium chlorides in sediment samples

Benzalkonium chlorides (BACs) were successfully extracted from sediment samples using a new methodology based on accelerated solvent extraction (ASE) followed by an on-line cleanup step. The BACs were detected by liquid chromatography/ion trap mass spectrometry (LC/MS) or tandem mass spectrometry (MS/MS) using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the high sensitivity offered by the ion trap MS/MS. The effects of solvent type and ASE operational variables, such as temperature and pressure, were evaluated. After optimization, a mixture of acetonitrile/water (6:4 or 7:3) was found to be most efficient for extracting BACs from the sediment samples. Extraction recoveries ranged from 95 to 105% for C12 and C14 homologues, respectively. Total method recoveries from fortified sediment samples, using a cleanup step followed byASE, were 85% for C12BAC and 79% for C14BAC. The methodology developed in this work provides detection limits in the subnanogram per gram range. Concentrations of BAC homologues ranged from 22 to 206 microg/kg in sediment samples from different river sites downstream from wastewater treatment plants. The high affinity of BACs for soil suggests that BACs preferentially concentrate in sediment rather than in water.     

Identification and quantification of cardiac glycosides in blood and urine samples by HPLC/MS/MS.

Cardiac glycosides (CG) are of forensic importance because of their toxicity and the fact that very limited methods are available for identification of CG in biological samples. In this study, we have developed an identification and quantification method for digoxin, digitoxin, deslanoside, digoxigenin, and digitoxigenin by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS). CG formed abundant [M + NH4]+ ions and much less abundant [M + H]+ ions as observed with electrospray ionization (ESI) source and ammonium formate buffer. Under mild conditions for collision-induced dissociation (CID), each [M + NH4]+ ion fragmented to produce a dominant daughter ion, which was essential to the sensitive method of selected reaction monitoring (SRM) quantification of CG achieved in this study. SRM was compared with selected ion monitoring (SIM) regarding the effects of sample matrixes on the methodology. SRM produced lower detection limits with biological samples than SIM, while both methods produced equal detection limits with CG standards. On the basis of the HPLC/MS/MS results for CG, we have proposed some generalized points for conducting sensitive SRM measurements, in view of the property of analytes as well as instrumental conditions such as the type of HPLC/MS interface and CID parameters. Analytes of which the molecular ion can produce one abundant daughter ion with high yield under CID conditions may be sensitively measured by SRM. ESI is the most soft ionization source developed so far and can afford formation of the fragile molecular ions that are necessary for sensitive SRM detection. Mild CID conditions such as low collision energy and low pressure of collision gas favor production of an abundant daughter ion that is essential to sensitive SRM detection. This knowledge may provide some guidelines for conducting sensitive SRM measurements of very low concentrations of drugs or toxicants in biological samples.     

Speciation of inorganic lead and ionic alkyllead compounds by GC/MS in prescreened rainwaters

A simple and novel screening method for lead compounds in environmental waters is proposed. The analytes, in an acetic medium, are sorbed on a C60 fullerene column as diethyldithiocarbamate complexes and subsequently eluted with isobutyl methyl ketone (IBMK), the lead being determined by flame atomic absorption spectrometry. The screening method acts as filter and indicates whether the target analytes are present above or below the detection limit of the method (0.5 ng/mL), giving no false positives. Positive samples were speciated by GC/MS, using a flow system similar to that of the screening method. Lead(II) and ionic di- and trialkyllead are derivatized with a Grignard reagent. Both methods use similar chemical and flow variables except for the eluent, IBMK in the screening method and n-hexane in the speciation method. The GC/MS speciation method is very sensitive; it exhibits a linear range of 0.02-5 ng/mL, and detection limits of 1-4 ng/L are achieved. Also, its repeatibility, as RSD, is less than 6%. The method was applied to the speciation of Pb2+ and di- and trialkyllead in positive prescreened rainwater samples from three different urban areas.     

Development of a solid-phase extraction-HPLC/single quadrupole MS method for quantification of perfluorochemicals in whole blood

A method for the determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) simultaneously with 10 closely related perfluorochemicals (PFCs) in human whole blood was developed and validated. PFOS and PFOA are used in various applications, for example, as surfactants and plastic additives, and are subject to environmental and health research due to their persistence. The main part of the data on PFCs in human blood is from serum samples, analyzed mainly by ion pair extraction followed by high-performance liquid chromatography (HPLC) and negative electrospray (ESI) tandem mass spectrometry (MS/MS). The analytical method developed here is suitable for human whole blood and involves solid-phase extraction (SPE) and HPLC negative electrospray single quadrupole mass spectrometry (HPLC/ES-MS). A whole blood aliquot was treated with formic acid and extracted on a octadecyl (C18) SPE column. The PFCs were isolated with methanol, and quantification was performed using single quadrupole mass spectrometry and perfluoroheptanoic acid as internal standard. Validation was performed in the range 0.3-194 ng/mL with recovery between 64 and 112% and limit of detection in the 0.1-0.5 ng/mL range for 11 of the 12 PFCs studied. We applied this method to 20 whole blood samples collected in 1997-2000 from the Swedish population in the ages 24-72. Eleven of the 12 PFCs were detected, and they were quantitatively and qualitatively confirmed using triple quadrupole LC/MS/MS analysis. PFOS, perfluorooctanesulfonamide, perfluorohexanesulfonate, PFOA and perfluorononanoic acid were quantified in all samples. In addition, perfluorohexanoic acid, perfluorodecanoic acid, perfluorodecanesulfonate, perfluoroundecanoic acid, perfluorododecanoic acid, and perfluorotetradecanoic acid were detected in some samples. This study shows that SPE and single quadrupole MS can be applied for extraction and quantification of PFCs in human whole blood, resulting in selectivity and low detection limits.     

MS/MS methodology to improve subcellular mapping of cholesterol using TOF-SIMS

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype C60(+)/quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum.     

Characterization of microorganisms and biomarker development from global ESI-MS/MS analyses of cell lysates

The capability for sensitive and accurate identification of microorganisms has potential applications that include the monitoring of industrial bioprocessing operations, food safety analyses, disease diagnosis, and detection of potential biological hazards. Efforts based upon matrix-assisted laser desorption/ionization mass spectrometry to detect and identify specific microorganisms have been actively pursued for several years. We report a new method being developed to select useful biomarkers for the identification of microorganisms based upon electrospray ionization (ESI)-ion trap mass spectrometry. Crude cell lysates are processed using a recently developed dualmicrodialysis device and then directly infused into an ion trap MS. The low ESI flow rate and precursor ion accumulation capability of the ion trap MS enables high-sensitivity MS/MS analyses. Precursor ions are automatically selected and analyzed using tandem MS (MS/MS) to produce "global" MS/MS surveys and processed to yield two-dimensional MS/MS spectral displays. Such global MS/MS surveys are demonstrated for Escherichia coli lysates. The distinctive MS/MS spectral patterns can be used to identify mass spectrometric-detected species useful as biomarkers, which then provide a basis for confident microorganism identification. The results presented demonstrate the application of this method for the identification of microorganisms, as well as for detection of bacteriophage MS2 in the presence of a large excess of E. coli.     

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.     

Analysis of perchlorate in water by reversed-phase LC/ESI-MS/MS using an internal standard technique

A new method was developed for the analysis of perchlorate in water by using reversed-phase liquid chromatograhy/electrospray ionization-mass spectrometry/mass spectrometry (LC/ESI-MS/MS) in the negative ESI mode. Selective and sensitive perchlorate detection was obtained by monitoring the 35ClO4- --> 35ClO3- and 37ClO4- --> 37ClO3- mass transitions. The 35ClO4- --> 35ClO3- transition was quantitated against the internal standard oxygen-labeled sodium perchlorate (NaCl18O4). Sample pretreatment for the removal of major common anions and dissolved metal ions along with internal standard quantitation sufficiently compensated for ion suppression caused by the matrix. The 37ClO4- --> 37ClO3- transition was examined to provide additional specificity. The method sensitivity, accuracy, and precision were investigated by analyzing fortified blank samples, field samples, and performance evaluation samples. The results (1.01-13.5 microg/L) for the proficiency evaluation samples differed from the certified values (1.04-14.1 microg/L) by 3-18%. The developed reversed-phase LC/ESI-MS/MS method was rapid, accurate, and reproducible. The calculated method detection limits were 0.007 microg/L for deionized reagent water and 0.009 microg/L for synthesized reagent water, respectively. The minimum reporting limit was conservatively set to 0.05 microg/L.     

Simultaneous analysis of multiple classes of antibiotics by ion trap LC/MS/MS for assessing surface water and groundwater contamination

Solid-phase extraction (SPE) and liquid chromatography in combination with ion trap mass spectrometry (LC/MS/MS) conditions were optimized for the simultaneous analysis of 13 antibiotics belonging to multiple classes and caffeine in 3 different water matrixes. The single-cartridge extraction step was developed using a reversed-phase cartridge, resulting in recoveries for the 14 compounds ranging from 71 to 119% with relative standard deviations of 16% or lower. The analytes were separated in one chromatographic run, and the SPE-LC/MS/MS detection limits ranged from 0.03 to 0.19 microg/L. The SPE procedure was validated in groundwater, surface water, and wastewater. The analysis of samples from each of the three water matrixes revealed clindamycin (1.1 microg/L) in surface water and multiple antibiotics in wastewater (0.10-1.3 microg/L). The use of identification points to unambiguously assign the identity of antibiotics in various water matrixes was applied to an ion trap data-dependent scanning method, which simultaneously collects full scan and full scan MS/MS data for the unequivocal identification of target analytes.     

Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS

Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of approximately 10(3)) 15-microm-i.d. capillary liquid chromatography separations (i.e., using nanoLC, approximately 20 nL/min mobile-phase flow rate at the optimal linear velocity of approximately 0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., approximately 100 times larger than FTICR MS), corresponding to a sensitivity of approximately 7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.     

Enabling MALDI-FTICR-MS/MS for high-performance proteomics through combination of infrared and collisional activation

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a central tool for proteomic analysis, yet the singly protonated tryptic peptide ions produced by MALDI are significantly more difficult to dissociate for tandem mass spectrometry (MS/MS) than the corresponding multiply protonated ions. In order to overcome this limitation, current proteomic approaches using MALDI-MS/MS involve high-energy collision-induced dissociation (CID). Unfortunately, the use of high-energy CID complicates product ion spectra with a significant proportion of irrelevant fragments while also reducing mass accuracy and mass resolution. In order to address the lack of a high-resolution, high mass accuracy MALDI-MS/MS platform for proteomics, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and a recently developed MS/MS technique termed CIRCA (for combination of infrared and collisional activation) have been applied to proteomic analysis. Here, CIRCA is shown to be suitable for dissociating singly protonated tryptic peptides, providing greater sequence coverage than either CID or infrared multiphoton dissociation (IRMPD) alone. Furthermore, the CIRCA fragmentation spectra are of sufficient quality to allow protein identification based on the MS/MS spectra alone or in concert with the peptide mass fingerprint (PMF). This is accomplished without compromising mass accuracy or mass resolution. As a result, CIRCA serves to enable MALDI-FTICR-MS/MS for high-performance proteomics experiments.     

High-throughput identification of in-gel digested proteins by rapid, isocratic HPLC/MS/MS

The high sensitivity and accuracy of mass spectrometry for identifying proteins has led to an explosive expansion of proteomics research, necessitating rapid procedures for HPLC/MS/MS analysis. Current HPLC/MS/MS analysis usually relies on elution of peptides from the HPLC column with a gradient that takes a total of 45-70 min for each cycle, limiting sample throughput and the speed of protein identification. Here we report a simple method for high-throughput protein identification, using isocratic, either methanol- or acetonitrile-based buffer systems, HPLC elution into an LTQ mass spectrometer. This procedure allows each cycle of highly sensitive HPLC/MS/MS analysis to be completed in 5 min, thus boosting the efficiency of HPLC/MS/MS analysis 9-14-fold. Using this method, each operator can acquire HPLC/MS/MS data for 96 in-gel proteolytic digests in one 8-h working day. The method can easily be implemented in any laboratory with an LTQ mass spectrometer. This protocol should find wide application in mass spectrometry laboratories that require high-throughput analysis but are limited by inefficient use of machine time.     

Determination of total polychlorinated n-alkane concentration in biota by electron ionization-MS/MS

Electron ionization (EI) tandem mass spectrometry (MS/MS) allowed the fast determination of the total concentration of short- and medium-chained polychlorinated n-alkanes (PCAs) in biota. EI fragment ions common to all PCAs could be identified. Collision-induced dissociations (CIDs) were carried out by ion trap and triple quadrupole EI-MS/MS. CIDs of m/z 91 --> 53 (limit of detection (LOD) 0.15 ng/microL), 102 --> 65 (LOD = 0.2 ng/microL), and 102 --> 67 (LOD = 0.1 ng/microL) were applied for the determination of the total short- and medium-chain PCA concentration in pooled fish liver samples (North Sea dab, cod, flounder) from the North Sea and from the Baltic Sea using both MS technologies. Total PCA concentrations were in the range of 88-607 ng/g. Accuracy was controlled with spiked samples and deviated not more than 15% from expected values.     

Comparison of MALDI-TOF-MS and HPLC-ESI-MS/MS for Endopeptidase Activity-Based Quantification of Anthrax Lethal Factor in Serum

Diagnosing and treating anthrax at the earliest stage of disease is critical. We developed a method to diagnose anthrax at early stages of infection by detecting anthrax lethal factor (LF) at the attomol/mL level in plasma or serum. This method uses antibody capture and quantification of LF endoproteinase activity by isotope dilution matrix-assisted laser-desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Many public health laboratories do not use MALDI-TOF-MS; thus, we have adapted the LF method for detection by electrospray ionization (ESI) tandem MS (MS/MS), which allowed comparison of both MS platforms for LF quantification. Calibration curves were linear from 0.05-2.5 ng/mL when measured after 2 h and from 0.005-1.0 ng/mL after 18 h incubation time. The limit of detection was 0.005 ng/mL using a 200 μL sample. The coefficient of variation for quality control samples was 6-12% for both MS platforms. Samples used to perform cross-validation included 158 serum samples from a study in rabbits exposed to anthrax spores by inhalation. Some were treated with anthrax immune globulin before exposure. Concentrations measured by ESI-MS/MS matched those by MALDI-TOF-MS with p = 0.99 (r(2) = 0.997) and -0.25% mean relative difference (±9% standard deviation). This study shows that isotope dilution MALDI-TOF-MS is a robust and precise quantitative MS platform.     

Colloidal graphite-assisted laser desorption/ionization MS and MS(n) of small molecules. 2. Direct profiling and MS imaging of small metabolites from fruits

Due to a high background in the low-mass region, conventional MALDI is not as useful for detecting small molecules (molecular masses <500 Da) as it is for large ones. Also, spatial inhomogeneity that is inherent to crystalline matrixes can degrade resolution in imaging mass spectrometry (IMS). In this study, colloidal graphite was investigated as an alternative matrix for laser desorption/ionization (GALDI) in IMS. We demonstrate its advantages over conventional MALDI in the detection of small molecules such as organic acids, flavonoids, and oligosaccharides. GALDI provides good sensitivity for such small molecules. The detection limit of fatty acids and flavonoids in the negative-ion mode are in the low-femtomole range. Molecules were detected directly and identified by comparing the MS and MS/MS spectra with those of standards. Various fruits were chosen to evaluate the practical utility of GALDI since many types of small molecules are present in them. Distribution of these small molecules in the fruit was investigated by using IMS and IMS/MS.     

Mu-trap for the SALDI-MS screening of organic compounds prior to LC/MS analysis

A procedure for rapidly screening and quantitatively analyzing organic molecules is presented, in which a miniaturized solid-phase extraction (SPE) cartridge containing 0.6 mg of graphitized carbon black (the GCB-mu-trap) is used for sample pretreatment. Then surface-assisted laser desorption ionization time-of-flight mass spectrometry (SALDI-TOF-MS) screening is followed by liquid chromatography/mass spectrometry (LC/MS) for robust quantitative analysis of samples containing analytes of interest. Liquid samples with volumes up to 100 mL were extracted using the GCB-mu-trap, and SALDI screening was performed by transferring a few particles of the GCB 4 sorbent from the mu-trap onto a stainless steel plate. Analytes were then simply ionized and desorbed by irradiating the GCB 4 particles without any further pretreatment. GCB 4 was found to be an excellent surface for the SALDI analysis of small molecules, providing spectra with very clean backgrounds. The small size of the cartridge (micropipet filter tip) results in enrichment of the analytes on a small surface area, affording low SALDI-TOF-MS detection limits. Furthermore, the removal of just a few particles from the mu-trap does not significantly affect the subsequent quantitative determination. This approach offers considerable reductions in analytical costs by eliminating unnecessary SPE-LC/MS analyses.