Rotation planar chromatography coupled on-line with atmospheric pressure chemical ionization mass spectrometry

The coupling of a rotation planar preparative thin-layer chromatography system on-line with mass spectrometry is demonstrated using a simple plumbing scheme and a self-aspirating heated nebulizer probe of a corona discharge atmospheric pressure chemical ionization source. The self-aspiration of the heated nebulizer delivers approximately 20 microL/min of the 3.0 mL/min eluate stream to the mass spectrometer, eliminating the need for an external pump in the system. The viability of the coupling is demonstrated with a three-dye mixture composed of fat red 7B, solvent green 3, and solvent blue 35 separated and eluted from a silica gel-coated rotor using toluene. The real-time characterization of the dyes eluting from the rotor is illustrated in positive ion full-scan mode. Other self-aspirating ion source systems including atmospheric pressure photoionization, electrospray ionization, and inductively coupled plasma ionization, for example, might be configured and used in a similar manner coupled to the chromatograph to expand the types of analytes that could be ionized, detected, and characterized effectively.     

Identification and quantification of flavonoids in human urine samples by column-switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry

A rapid and sensitive high-performance liquid chromatographic mass spectrometric (HPLC-MS) method is described for the determination and quantification of 12 dietary flavonoid glycosides and aglycons in human urine samples. Chromatographic separation of the analytes of interest was achieved by column-switching, using the first column (a Zorbax 300SB C-3 column) for sample cleanup and eluting the heart-cut flavonoid fraction onto the second column (a Zorbax SB C-18 column) for separation and detection by ultraviolet and atmospheric pressure chemical ionization MS using single ion monitoring in negative mode. The fragmentor voltage was optimized with regard to maximum abundance of the molecular ion and qualifier ions of the analytes. Calibration graphs were prepared for urine, and good linearity was achieved over a dynamic range of 2.5-1000 ng/mL. The inter- and intraassay coefficients of variation for the analysis of the 12 different flavonoids in quality control urine samples were 12.3% on average (range 11.0-13.7%, n = 24, reproducibility) and the repeatability of the assay were 5.0% (mean, range 0.1-14.8%, n = 12). A subset of 10 urine samples from a human dietary intervention study with high and low flavonoid content was analyzed, and the results are reported.     

High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.     

Phenyl-modified reversed-phase liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry: a universal method for the analysis of partially oxidized aromatic hydrocarbons

A new liquid chromatographic method for the efficient separation of aromatic compounds having a wide range of sizes, molecular structures, and polarities has been developed. Based on a phenyl-modified silica reversed stationary phase and a methanol-water solvent gradient, it allows the separation of mono- and polycyclic aromatic hydrocarbons (PAHs) having up to five condensed aromatic rings and partially oxidized derivatives within a single chromatographic run of 40-min duration. The applicability of the method is demonstrated using 81 reference substances (PAHs, phenols, quinones, acids, lactones, esters, etc.) and real samples of environmental, medical, and technical relevance (ozonized PAHs, lake water, human urine, diesel exhaust condensates). The retention times of the investigated aromatics exhibit a regular increase with molecular mass and a systematic decrease with increasing number and polarity of functional groups. In case of intramolecular hydrogen bonding, a positive shift of retention time provides additional structural information. The combination of chromatographic retention time with the molecular mass and structural information from mass spectrometric detection allows the tentative identification of unknown aromatic analytes at trace levels, even without specific reference substances. With atmospheric pressure chemical ionization (APCI), low detection limits and highly informative fragmentation patterns can be obtained by in-source collision-induced fragmentation in a single-quadrupole LC-APCI-MS system as applied in this study, and multidimensional MS experiments are expected to further enhance the potential of the presented method.     

Determination of nitrated phenolic compounds in rain by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

A sensitive, specific, and rapid analytical method based on liquid-liquid extraction and liquid chromatography/atmospheric pressure chemical ionization mass spectrometry has been developed for trace analysis of nitrated phenolic compounds in rain samples. Selective detection in the low nanogram-per-milliliter range was achieved on the basis of selected ion monitoring of the respective phenolate anions [M - H]-. The presence of alkylated (C1-C3) and nonalkylated nitrophenols (C0) was confirmed by their characteristic neutral loss of nitrogen dioxide upon collision-induced dissociation in tandem mass spectrometry mode. In individual rain samples, 27 C0-C3-nitrophenol isomers as well as 16 C0-C3-dinitrophenol isomers were detected. Total levels of nitro- and dinitrophenol isomers were estimated on the basis of 2-nitrophenol and 2,6-dinitrophenol. Concentrations between 0.66 and 2.0, 12-29, 12-36, and 5.5-12 microg/L were obtained for the C0-, C1-, C2-, and C3-nitrophenols, respectively. Lower levels of 0.19-1.4, 0.39-2.1, 0.053-0.55, and 0.081-0.10 microg/L were estimated for the corresponding C0-, C1-, C2-, and C3-dinitrophenols. The highest number of individual isomers was found in winter rain samples, and distinctive isomeric patterns were observed for individual samples. Mono- and dialkylated nitrophenols and non- and monoalkylated dinitrophenols represent the major part of nitro- and dinitrophenol species. Comparing the pattern of Co-, C1-, and C2-nitrophenols in rain with the corresponding Co-, C1-, and C2-benzene pattern in ambient air suggests that atmospheric oxidation and nitration processes of alkylbenzenes are favored, as compared to those of benzene.     

Microchip atmospheric pressure chemical ionization source for mass spectrometry

A novel microchip heated nebulizer for atmospheric pressure chemical ionization mass spectrometry is presented. Anisotropic wet etching is used to fabricate the flow channels, inlet, and nozzle on a silicon wafer. An integrated heater of aluminum is sputtered on a glass wafer. The two wafers are jointed by anodic bonding, creating a two-dimensional version of an APCI source with a sample channel in the middle and gas channels symmetrically on both sides. The ionization is initiated with an external corona-discharge needle positioned 2 mm in front of the microchip heated nebulizer. The microchip APCI source provides flow rates down to 50 nL/min, stable long-term analysis with chip lifetime of weeks, good quantitative repeatability (RSD < 10%) and linearity (r(2) > 0.995) with linear dynamic rage of at least 4 orders of magnitude, and cost-efficient manufacturing. The limit of detection (LOD) for acridine measured with microchip APCI at flow rate of 6.2 muL/min was 5 nM, corresponding to a mass flow of 0.52 fmol/s. The LOD with commercial macro-APCI at a flow rate of 1 mL/min for acridine was the same, 5 nM, corresponding to a significantly worse mass flow sensitivity (83 fmol/s) than measured with microchip APCI. The advantages of microchip APCI makes it a very attractive new microfluidic detector.     

Secondary ionization of chemical warfare agent simulants: atmospheric pressure ion mobility time-of-flight mass spectrometry

For the first time, the use of a traditional ionization source for ion mobility spectrometry (radioactive nickel ((63)Ni) beta emission ionization) and three alternative ionization sources (electrospray ionization (ESI), secondary electrospray ionization (SESI), and electrical discharge (corona) ionization (CI)) were employed with an atmospheric pressure ion mobility orthogonal reflector time-of-flight mass spectrometer (IM(tof)MS) to detect chemical warfare agent (CWA) simulants from both aqueous- and gas-phase samples. For liquid-phase samples, ESI was used as the sample introduction and ionization method. For the secondary ionization (SESI, CI, and traditional (63)Ni ionization) of vapor-phase samples, two modes of sample volatilization (heated capillary and thermal desorption chamber) were investigated. Simulant reference materials, which closely mimic the characteristic chemical structures of CWA as defined and described by Schedule 1, 2, or 3 of the Chemical Warfare Convention treaty verification, were used in this study. A mixture of four G/V-type nerve simulants (dimethyl methylphosphonate, pinacolyl methylphosphonate, diethyl phosphoramidate, and 2-(butylamino)ethanethiol) and one S-type vesicant simulant (2-chloroethyl ethyl sulfide) were found in each case (sample ionization and introduction methods) to be clearly resolved using the IM(tof)MS method. In many cases, reduced mobility constants (K(o)) were determined for the first time. Ion mobility drift times, flight times, relative signal intensities, and fragmentation product signatures for each of the CWA simulants are reported for each of the methods investigated.     

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.     

High-performance liquid chromatography linked to inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry for the simultaneous detection and identification of metabolites of 2-bromo-4-trifluoromethyl

The use of HPLC coupled to inductively coupled plasma mass spectrometry (ICPMS) and orthogonal acceleration time-of-flight (oa-TOF) for the profiling, identification, and quantification of metabolites in rat urine following the administration of 2-bromo-4-trifluoromethylacetanilide is described. The metabolites present in the sample were separated by reversed-phase gradient chromatography with UV-diode array detection. The bulk of the eluent (90%) from the UV detector was directed to an ICPMS where bromine-containing metabolites were detected and quantified using ICPMS. The minor portion of the eluent (10%) was taken for oa-TOFMS for identification. By these means, the metabolites were identified as sulfate and glucuronide conjugates of a ring hydroxy-substituted metabolite, a N-sulfate, a N-hydroxylamine glucuronide, and N- and N-hydroxyglucuronides.     

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.     

Profiling of acarviostatin family secondary metabolites secreted by Streptomyces coelicoflavus ZG0656 using ultraperformance liquid chromatography coupled with electrospray ionization mass spectrometry

Profiling of acarviostatin family secondary metabolites secreted by Streptomyces coelicoflavus ZG0656 was performed by means of a rapid and facile procedure using ultraperformance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC/ESI-MS). The acarviostatins were separated on a C18 UPLC column with a series of acetonitrile-aqueous ammonia gradients. The target homologues were detected using the multiple reaction monitoring mode, and the chemical structures were confirmed by analyzing the diagnostic fragment ions in their MS/MS spectra. All six known reference acarviostatins (I03, II03, II13, II23, III03, IV03) were thus identified. In addition, at least 74 acarviostatin homologues, including 65 novel compounds, were characterized. Some of the features of the novel structures included having up to five acarviosine moieties, an acarviosine moiety at the reducing terminus, or an incomplete acarviosine moiety at the nonreducing terminus. This type of investigation may be useful for researchers who study secondary metabolomics in microorganisms and plants, especially those who perform metabolic profiling of aminooligosaccharides and other natural products with similar structures.     

Two-dimensional ultra-thin-layer chromatography and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry in bioanalysis

The feasibility of ultra-thin-layer chromatography (UTLC) and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) for bioanalysis was studied with benzodiazepines as model substances in human urine. Two-dimensional (2D) UTLC was shown to be an efficient technique for the separation of benzodiazepines. Separations occurred in 4-12 min, and the separated compounds were identified by AP-MALDI-MS. The limits of detection with AP-MALDI-MS and AP-MALDI-MS/MS were in the picomole range and thus low enough for bioanalysis. The applicability of the 2D UTLC-AP-MALDI-MS was demonstrated in detection of metabolites with an authentic biological urine sample.     

Automated in-tube solid-phase microextraction coupled with liquid chromatography/electrospray ionization mass spectrometry for the determination of beta-blockers and metabolites in urine and serum samples.

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was evaluated for the determination of beta-blockers in urine and serum samples. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC/MS analyses of beta-blockers were initially performed by liquid injection onto a LC column. Nine beta-blockers tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M + H]+ were observed for all beta-blockers. The beta-blockers were separated with a Hypersil BDS C18 column using acetonitrile/methanol/water/acetic acid (15:15:70:1) as a mobile phase. To optimize the extraction of beta-blockers, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 30 microL of sample in 100 mM Tris-HCl (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary (Supelco, Bellefonte, PA). The beta-blockers extracted by the capillary were easily desorbed by mobile-phase flow, and carryover of beta-blockers was not observed. Using in-tube SPME/LC/ESI-MS with selected ion monitoring, the calibration curves of beta-blockers were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9982 (n = 18) and detection limits (S/N = 3) of 0.1-1.2 ng/mL. This method was successfully applied to the analysis of biological samples without interference peaks. The recoveries of beta-blockers spiked into human urine and serum samples were above 84 and 71%, respectively. A serum sample from a patient administrated propranolol was analyzed using this method and both propranolol and its metabolites were detected.     

Wavelet-based method for noise characterization and rejection in high-performance liquid chromatography coupled to mass spectrometry

We present a new method for rejecting noise from HPLC-MS data sets. The algorithm reveals peptides at low concentrations by minimizing both the chemical and the random noise. The goal is reached through a systematic approach to characterize and remove the background. The data are represented as two-dimensional maps, in order to optimally exploit the complementary dimensions of separation of the peptides offered by the LC-MS technique. The virtual chromatograms, reconstructed from the spectrographic data, have proved to be more suitable to characterize the noise than the raw mass spectra. By means of wavelet analysis, it was possible to access both the chemical and the random noise, at different scales of the decomposition. The novel approach has proved to efficiently distinguish signal from noise and to selectively reject the background while preserving low-abundance peptides.     

Electrochemically modulated liquid chromatography coupled on-line with electrospray mass spectrometry

Electrochemically modulated liquid chromatography (EMLC) has been coupled to an electrospray mass spectrometer. This combination takes advantage of the ability of EMLC to manipulate retention and enhance separation efficiency solely through changes in the potential applied to a conductive stationary phase, thereby minimizing complications because of possible changes in analyte ionization efficiencies when gradient elution techniques are used. Three examples are presented that demonstrate the attributes of this EMLC/electrospray mass spectrometry (ES-MS) coupling. The first two examples involve the separation of mixtures of corticosteroids or of benzodiazepines, showing the general utility of the union for eluent identification and low-level detection. The ability to identify products from on-column redox transformations is also demonstrated using the benzodiazepine mixture. The third example investigates the electrooxidation of aniline by utilizing an EMLC column as an on-line electrochemical reactor and product separator and ES-MS for detection and product identification.     

Microchip for combining gas chromatography or capillary liquid chromatography with atmospheric pressure photoionization-mass spectrometry

We present a microfabricated nebulizer chip for combining atmospheric pressure photoionization-mass spectrometry (APPI-MS) with gas chromatography (GC) or capillary liquid chromatography (capLC). The chip consists of a silicon plate and a glass plate or two glass plates. The chip includes a sample inlet channel, auxiliary gas and dopant inlet, vaporizer channel, nozzle, and platinum heater. The sample eluted from the capLC or GC is mixed with auxiliary gas and dopant (toluene) in the heated vaporizer. The chip forms a confined jet of the sample vapor, which is photoionized as it exits the chip. The analytical performance of GC- and capLC-microchip APPI-MS was evaluated with some polycyclic aromatic hydrocarbons, amphetamines, and steroids. The GC-muAPPI-MS method provides high sensitivity down to 0.8 fmol, repeatability (RSD = 7.5-14%), and linearity (r = 0.9952-0.9987). The capLC-muAPPI-MS method shows high sensitivity down to 1 fmol, good repeatability (RSD = 3.6-8.1%), and linearity (r = 0.9989-0.9992).     

Comparison of atmospheric pressure photoionization, atmospheric pressure chemical ionization, and electrospray ionization mass spectrometry for analysis of lipids

In this work, we compare the quantitative accuracy and sensitivity of analyzing lipids by atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) LC/MS. The target analytes include free fatty acids and their esters, monoglyceride, diglyceride, and triglyceride. The results demonstrate the benefits of using LC/APPI-MS for lipid analysis. Analyses were performed on a Waters ZQ LC/MS. Normal-phase solvent systems were used due to low solubility of these compounds in aqueous reversed-phase solvent systems. By comparison, APPI offers lower detection limits, generally highest signal intensities, and the highest S/N ratio. APPI is 2-4 times more sensitive than APCI and much more sensitive than ESI without mobile-phase modifiers. APPI and APCI offer comparable linear range (i.e., 4-5 decades). ESI sensitivity is dramatically enhanced by use of mobile phase modifiers (i.e., ammonium formate or sodium acetate); however, these ESI adduct signals are less stable and either are nonlinear or have dramatically reduced linear ranges. Analysis of fish oils by APPI shows significantly enhanced target analyte intensities in comparison with APCI and ESI.     

Evaluation of matrix effects in metabolite profiling based on capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry

The coupling of liquid chromatography to electrospray ionization quadrupole time-of-flight mass spectrometry can be a powerful tool for metabolomics, i.e., the comprehensive detection of low molecular weight compounds in biological systems. There have, however, been doubts about the feasibility and reliability of this approach, because LC-MS--especially with electrospray ionization--can be subject to matrix effects. We evaluated matrix effects for our metabolomics platform in three ways: (i) postextraction addition of a set of reference compounds to different complex biological matrixes to determine absolute and relative matrix effects, (ii) postcolumn infusion of two reference compounds, and (iii) mixing of two complex matrixes. Our data demonstrate that there are indeed significant absolute matrix effects when comparing highly divergent samples. However, relative matrix effects are negligible--unless extremely divergent matrixes are compared--and do not compromise the relative quantification that is aimed for in nontargeted metabolomics studies. In conclusion, employing LC-coupled ESI-QTOF-MS for metabolomics studies is feasible yet rigorous validation is necessary.