Analysis of polar organic compounds using charge exchange ionization and membrane introduction mass spectrometry

Charge exchange ionization in conjunction with membrane introduction mass spectrometry provides a sensitive method for the detection of polar volatile organic compounds and semivolatile compounds in air. Sample introduction into an ion trap mass spectrometer was accomplished with a hollow fiber silicone membrane assembly. Atmospheric oxygen, which diffuses through the membrane, was used as the charge exchange reagent. Chemical ionization parameters were optimized using methyl ethyl ketone (2-butanone) standards in air. Several other oxygen-containing compounds, including acetone (2-propanone), methyl isobutyl ketone (4-methyl-2-pentanone), propanal, isopropyl alcohol (2-propanol), cyclohexanol, dimethyl sulfoxide (sulfinylbismethane), 2-(diethylamino)ethanol, and dimethyl methylphosphonate were analyzed with this technique. This method was used to obtain mass spectra for a variety of classes of compounds and produced a 4-20-fold improvement in response for all of the polar compounds we examined when compared to signal obtained from electron ionization.     

Combining drop-to-drop solvent microextraction with gas chromatography/mass spectrometry using electronic ionization and self-ion/molecule reaction method to determine methoxyacetophenone isomers in one drop of water

A novel analytical technique termed drop-to-drop solvent microextraction (DDSME) was developed to determine three methoxyacetophenone isomers in one drop of water, which were then detected by gas chromatography/mass spectrometry using electronic ionization mass spectrometry for quantification analysis and self-ion/molecule reaction/tandem mass spectrometry for isomer differentiation. The best optimum parameters for the DDSME technique were as follows: extraction time, 5 min; using toluene as the extraction solvent; volume of extraction solvent, 0.5 microL and no salt addition. The advantages of this method are rapidity, convenience, ease of operation, simplicity of the device, and extremely little solvent and sample consumption. The limit of detection (LOD) for this technique was 1 ng/mL. The relative standard deviation was less than 2.6% (n = 5). The linear range of the calibration curve of DDSME is from 0.01 to 5 microg/mL with correlation coefficient (r2) of >0.954. In the comparison of the LOD of DDSME with other sample pretreatment methods including liquid/liquid extraction (LLE), single-drop microextraction (SDME), solid-phase microextraction (SPME), and liquid-phase microextraction (LPME) using a dual gauge microsyringe with hollow fiber methods, this method shows much better in sensitivity than the LLE (25 ng/mL) and it is compatible with SDME (0.5 ng/mL), SPME (0.5 ng/mL), and LPME using a dual gauge microsyringe with a hollow fiber (1 ng/mL). However, DDSME was more convenient than the LPME using a dual gauge microsyringe with a hollow fiber method and much lower cost than the SPME technique.     

Charge reduction electrospray mass spectrometry

A new mass spectrometric technique, charge reduction electrospray mass spectrometry (CREMS), allowing the analysis of complex mixtures of biological molecules is described. The charge state of ions produced by electrospray ionization may be reduced in a controlled manner to yield predominantly singly charged ions through reactions with bipolar (i.e., both positively and negatively charged) ions generated using a 210Po alpha particle source. The electrospray-generated multiply charged ions undergo charge reduction in a "neutralization chamber" positioned before the entrance nozzle to the mass spectrometer. The ions are detected using a commercial orthogonal electrospray time-of-flight mass spectrometer, although the neutralization chamber can be adapted to virtually any mass analyzer. The CREMS results obtained exhibit a signal intensity drop-off with increasing oligonucleotide size similar to that observed with matrix-assisted laser desorption/ionization mass spectrometry. Proton-transfer reactions were found to be responsible for reducing charge on proteins and oligonucleotides in both positive and negative ion mode.     

Development of multi-ESI-sprayer, multi-atmospheric-pressure-inlet mass spectrometry and its application to accurate mass measurement using time-of-flight mass spectrometry

The atmospheric pressure sampling nozzle (orifice, heated capillary, or inlet) of a high mass accuracy time-of-flight mass spectrometer (TOF-MS) was modified by replacing its single nozzle with multiple atmospheric pressure nozzles. This allowed multiple streams of liquids to be introduced into the MS in parallel (an electrosprayer for each nozzle), with minimum analyte interactions between the streams. The chemical contents of all liquid streams were analyzed concurrently using a single mass spectrometer. To obtain a higher mass accuracy by providing internal reference on each scan (acquisition) and to evaluate the suitability of TOF-MS for molecular-formula confirmation, a dual-ESI-sprayer, dual-nozzle version of this design was used. The accurate masses of tens of organic compounds in the mass range of 200-3000 Da were measured, and the results were compared with those obtained using dual-sprayer, single-nozzle TOF-MS. A significant improvement in mass accuracy was observed when the former technique was used. Comparison between the mass accuracy using dual-ESI-sprayer, dual-nozzle TOF-MS and that obtained using a double-focusing mass spectrometer operating under chemical ionization (CI) and fast atom bombardment (FAB) shows the suitability of the technique for elemental-composition confirmation. Approximately 85% of samples analyzed had mass errors of less than 5 ppm, and the other 15% had mass errors less than 8 ppm. Using a high-performance liquid chromatography (HPLC) as a device for introduction of one liquid stream (sample) and a syringe pump as a device for introduction of the second liquid stream (reference standard), the accurate mass of a tryptic digest of cytochrome c was measured. The range of mass errors was from -6.1 ppm to +3.6 ppm, a significant improvement over our previously reported mass accuracy for this digest using single-nozzle TOF-MS. The interactions between analytes in the liquid streams also were investigated using a variety of sample-introduction and nozzle-design combinations, including single-ESI-sprayer, single-nozzle; dual-ESI-sprayer, single-nozzle; dual-ESI-sprayer, Y-shaped inlet; and dual-ESI-sprayer, dual-inlet. The results demonstrated that the dual-ESI-sprayer, dual-inlet design provides reference peaks on every acquisition with minimum analyte-reference interaction and, therefore, higher consistent mass accuracy.     

Ambient mass spectrometry with a handheld mass spectrometer at high pressure

The first coupling of atmospheric pressure ionization methods, electrospray ionization (ESI) and desorption electrospray ionization (DESI), to a miniature hand-held mass spectrometer is reported. The instrument employs a rectilinear ion trap (RIT) mass analyzer and is battery-operated, hand-portable, and rugged (total system: 10 kg, 0.014 m(3), 75 W power consumption). The mass spectrometer was fitted with an atmospheric inlet, consisting of a 10 cm x 127 microm inner diameter stainless steel capillary tube which was used to introduce gas into the vacuum chamber at 13 mL/min. The operating pressure was 15 mTorr. Ions, generated by the atmospheric pressure ion source, were directed by the inlet along the axis of the ion trap, entering through an aperture in the dc-biased end plate, which was also operated as an ion gate. ESI and DESI sources were used to generate ions; ESI-MS analysis of an aqueous mixture of drugs yielded detection limits in the low parts-per-billion range. Signal response was linear over more than 3 orders of magnitude. Tandem mass spectrometry experiments were used to identify components of this mixture. ESI was also applied to the analysis of peptides and in this case multiply charged species were observed for compounds of molecular weight up to 1200 Da. Cocaine samples deposited or already present on different surfaces, including currency, were rapidly analyzed in situ by DESI. A geometry-independent version of the DESI ion source was also coupled to the miniature mass spectrometer. These results demonstrate that atmospheric pressure ionization can be implemented on simple portable mass spectrometry systems.     

Headspace membrane introduction mass spectrometry for trace level analysis of VOCs in soil and other solid matrixes

A new MIMS-derived technique, headspace membrane introduction mass spectrometry (HS-MIMS), is described for direct trace level analysis of volatile organic compounds (VOCs) in soil and other dry or wet solid matrixes. A silicone membrane interface is placed about 15 cm from the ion source, and a closed airspace (headspace) is created by connecting a toggle valve to the 1/4 in. tubing that connects the membrane interface to the ion source. For the VOC analysis, the headspace is evacuated and the solid sample vessel is heated to 90 degrees C. The VOCs are rapidly desorbed from the sample, pervaporated through the membrane, and preconcentrated for 4 min in the evacuated headspace. Then, the toggle valve is opened and the trapped VOCs are released into the ion source region of a quadrupole mass spectrometer. By electron ionization and selected-ion monitoring, a relatively sharp and intense peak is obtained and used for quantification. The HS-MIMS analysis shows excellent linearity and reproducibility and detection limits for many VOCs typically of 50-100 ng/kg (ppt).     

Zeptomole-sensitivity electrospray ionization--Fourier transform ion cyclotron resonance mass spectrometry of proteins

Methods are being developed for ultrasensitive protein characterization based upon electrospray ionization (ESI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The sensitivity of a FTICR mass spectrometer equipped with an ESI source depends on the overall ion transmission, which combines the probability of ionization, transmission efficiency, and ion trapping in the FTICR cell. Our developments implemented in a 3.5 tesla FTICR mass spectrometer include introduction and optimization of a newly designed electrodynamic ion funnel in the ESI interface, improving the ion beam characteristics in a quadrupole-electrostatic ion guide interface, and modification of the electrostatic ion guide. These developments provide a detection limit of approximately 30 zmol (approximately 18,000 molecules) for proteins with molecular weights ranging from 8 to 20 kDa.     

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.     

Mass spectrometry and tandem mass spectrometry of citrus limonoids

Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring structure. CAD of the adduct ion [M + H + NH3]+ of limonoid glucosides produced the aglycone moiety corresponding to each glucoside. The combination of mass spectrometry and tandem mass spectrometry provides a powerful technique for identification and characterization of citrus limonoids.     

Tissue imaging using nanospray desorption electrospray ionization mass spectrometry

Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.     

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.     

Simple coupling of gas chromatography to electrospray ionization mass spectrometry

A simple method for direct coupling of gas chromatography (GC) with electrospray ionization mass spectrometry (ESI/MS) has been developed. The outlet of the GC capillary column was placed between the ESI needle and the atmospheric pressure ionization (API) source of a mass spectrometer. The ionization occurs via dissolution of neutral compounds into the charged ESI droplet followed by ion evaporation or via a gas-phase proton transfer reaction between a protonated solvent molecule and an analyte. The mass spectra of organic volatile compounds showed abundant protonated molecules with little fragmentation, being very similar to those produced by normal liquid ESI. The quantitative performance of the system was evaluated by determining the limit of detection (LOD), linearity ( r (2)), and repeatability (RSD). The GC-ESI/MS method was shown to be stable, providing high sensitivity and good quantitative performance.     

Trace analysis of ethanol, MTBE, and related oxygenate compounds in water using solid-phase microextraction and gas chromatography/mass spectrometry

Solid-phase microextraction (SPME) and gas chromatography/mass spectrometry have been combined for trace-level determination of very polar compounds in water, including the widely used gasoline oxygenates ethanol and methyl tert-butyl ether (MTBE). A relatively simple extraction method using a divinylbenzene/Carboxen/poly(dimethylsiloxane) SPME fiber was optimized for the routine analysis of ethanol and MTBE in groundwater and reagent water. A sodium chloride concentration of 25% (w/w) combined with an extraction time of 25 min provided the greatest sensitivity while maintaining analytical efficiency. Replicate analyses in fortified reagent and groundwater spiked with microgram per liter concentrations of ethanol and MTBE indicate quantitative and reproducible recovery of these and related oxygenate compounds. Method detection limits were 15 microg L(-1) for ethanol, 1.8 microg L(-1) for tert-butyl alcohol, 0.038 microg L(-1) for tert-amyl methyl ether, 0.025 microg L(-1) for ethyl-tert-butyl ether, and 0.008 microg L(-1) for MTBE.     

Off-line solid-phase microextraction and capillary electrophoresis mass spectrometry to determine acidic pesticides in fruits

A method based on solid-phase microextraction (SPME) and capillary electrophoresis/mass spectrometry (CE/ MS) is described for determining simultaneously five acidic pesticides (o-phenylphenol, ioxynil, haloxyfop, acifluorfen, picloram) in fruits. The CE device is coupled to an electrospray interface by a commercial sheath-flow adapter. Emphasis is placed on fulfillment of the speed and sensitivity requirements. The best separation is achieved using 32 mM ammonium formate/acid formic buffer at pH 3.1, with a working voltage of 25 kV. The MS detection of the five pesticides was performed in negative ionization mode. Full-scan spectra with base peaks corresponding to [M-H]- were obtained except for acifluorfen, which gives [M-H-CO2]- as most abundant ion. Compared with the conventional EC-UV, the limits of detection were lower for acifluorfen, haloxyfop, ioxynil, and picloram, by a factor of 20, 20, 50, and 2, respectively. Extraction involved fruit sample homogenization with an acetone-water solution (5:1), filtration, and acetone evaporation prior to fiber extraction. SPME conditions such as time, pH, ion strength, stationary phase of the fiber, sample matrix, and desorption solvents were examined. The recovery of the analytes ranged from 7 to 94%, and the relative standard deviation was between 3 and, 13%. The method was found to be linear between 0.02 and 500 mg kg(-1) with correlation coefficients ranging from 0.992 to 0.997. The limits of quantification were from 0.02 to 5 mg kg(-1). The optimized method was successfully applied to the analysis of acid pesticides in fruit samples.     

Real-time analysis of methanol in air and water by membrane introduction mass spectrometry.

We present results for the near-real-time, on-line detection of methanol in both air and water using membrane introduction mass spectrometry (MIMS). In these experiments, we compare the sensitivity of a poly(dimethylsiloxane) (PDMS) membrane and an allyl alcohol (AA) membrane to the detection of methanol. In MIMS, the membrane serves as the interface between the sample and the vacuum of the mass spectrometer. Membrane-diffused water was used as the reagent ion (H3O+) for chemical ionization of methanol in an ion trap mass spectrometer. Linear calibration curves have been obtained for methanol using both PDMS and AA membranes. For PDMS, detection limits of methanol are 14 ppmv and 5 ppm in air and water, respectively. For AA, detection limits are 3.3 ppmv and 2 ppm in air and water, respectively. We demonstrate that the sensitivity of the analysis can be altered by the chemistry of the membrane. When the AA membrane is used, the sensitivity of MIMS is enhanced over that of PDMS by a factor of 8.5 for methanol in air and by a factor of 23.4 for methanol in water.     

Breaking the pumping speed barrier in mass spectrometry: discontinuous atmospheric pressure interface

The performance of mass spectrometers with limited pumping capacity is shown to be improved through use of a discontinuous atmospheric pressure interface (DAPI). A proof-of-concept DAPI interface was designed and characterized using a miniature rectilinear ion trap mass spectrometer. The interface consists of a simple capillary directly connecting the atmospheric pressure ion source to the vacuum mass analyzer region; it has no ion optical elements and no differential pumping stages. Gases carrying ionized analytes were pulsed into the mass analyzer for short periods at high flow rates rather than being continuously introduced at lower flow rates; this procedure maximized ion transfer. The use of DAPI provides a simple solution to the problem of coupling an atmospheric pressure ionization source to a miniature instrument with limited pumping capacity. Data were recorded using various atmospheric pressure ionization sources, including electrospray ionization (ESI), nano-ESI, atmospheric pressure chemical ionization (APCI), and desorption electrospray ionization (DESI) sources. The interface was opened briefly for ion introduction during each scan. With the use of the 18 W pumping system of the Mini 10, limits of detection in the low part-per-billion levels were achieved and unit resolution mass spectra were recorded.     

Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer

The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction. It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags). Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity. Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search. This uncertainty, however, is overcome by MS/MS analysis of the peptides. Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein. This procedure is thus, highly sensitive, accurate, and rapid. In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling. Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1.     

Comprehensive gas chromatography-time-of-flight mass spectrometry using soft and selective photoionization techniques

The hyphenation of gas chromatography and mass spectrometry (GC/MS) revolutionized organic analysis. In GC/MS coupling, usually electron impact ionization is applied, and molecules are identified by their fragment pattern. Although mass spectrometry in principle is a separation method, it is used predominantly as a spectrometric technique. However, if soft (i.e., fragmentation-free) ionization techniques are applied, the inherent separation character of MS is emphasized, which has similarities to a GC boiling point separation. By combining polar column GC separation and fast soft ionization time-of-flight mass spectrometry technology, a comprehensive separation of complex petrochemical samples can be obtained (GC x MS approach). Compounds of comparable physical-chemical properties are characteristically grouped together in a two-dimensional retention time-m/z representation. This resembles the separation characteristics of comprehensive two-dimensional gas chromatography (GC x GC) and, thus, represents a novel multidimensional separation approach. In this work, a gas chromatograph equipped with a polar separation column was coupled to a home-built laser ionization time-of-flight mass spectrometer. Laser-based, single-photon ionization was used for universal soft ionization and resonance-enhanced multiphoton ionization for selective ionization of aromatic compounds. A novel capillary-jet inlet system was used for the coupling. Multidimensional comprehensive analysis of complex petrochemical hydrocarbon samples using gas chromatography coupled to mass spectrometry with soft and selective photo ionization sources is first demonstrated.     

Characterization of currently marketed heparin products: reversed-phase ion-pairing liquid chromatography mass spectrometry of heparin digests

Here we report results from the analyses by enzymatic digestion and reversed-phase ion-pairing liquid chromatography mass spectrometry (RPIP-LC-MS) of active pharmaceutical ingredient (API) unfractionated heparins (UFHs) from six different manufacturers and one USP standard sample. We employed a reverse phase ion-pairing chromatography method using a C(18) column and hexylamine as the ion-pairing reagent with acetonitrile gradient elution to separate disaccharides generated from the digestion of the heparins by lyase I and III (E.C. 4.2.2.7 and 4.2.2.8) before introduction into an ion-trap mass spectrometer by an electrospray ionization (ESI) interface. Extracted ion chromatograms (EICs) were used to determine the relative abundance of the disaccharides by mass spectrometry. Eight disaccharides were observed and a similar composition profile was observed from digests of 20 UFH samples. The compositional profile determined from these experiments provides a measure of the norm and range of variation in "good" heparin to which future preparations can be compared. Furthermore, the profile obtained in the RPIP-LC-MS assay is sensitive to the presence of the contaminant, oversulfated chondroitin sulfate A (OSCS), in heparin.