Microemulsion electrokinetic chromatography with on-line atmospheric pressure photoionization mass spectrometric detection

In the present paper we report, for the first time, the successful on-line coupling of microemulsion electrokinetic chromatography (MEEKC) with mass spectrometric (MS) detection using an atmospheric pressure photoionization interface. Microemulsions (MEs) including mostly volatile ingredients and classical MEs based on nonvolatile buffer components and sodium dodecyl sulfate (SDS) as surfactant were compared with respect to their compatibility with MS detection. The investigations performed revealed that MEs with up to 3% SDS and buffers containing sodium borate can be employed without significant suppression of the MS signals. A test mixture of nine substances could be separated by MEEKC using a ME consisting of 0.8% octane, 2% SDS, 6.6% butanol, and 90.6% of 20-mmol ammonium hydrogencarbonate buffer (pH 9.5). Operating the MS instrument in the MS(2) mode provided improved signal/noise ratios for analytes leading to characteristic MS-MS transitions. Thereby, limits of detection ranging between 0.5 (carbamazepine) and 5 microg mL(-1) (phenacetin) could be obtained.     

Infrared laser ablation atmospheric pressure photoionization mass spectrometry

In this paper we introduce laser ablation atmospheric pressure photoionization (LAAPPI), a novel atmospheric pressure ion source for mass spectrometry. In LAAPPI the analytes are ablated from water-rich solid samples or from aqueous solutions with an infrared (IR) laser running at 2.94 μm wavelength. Approximately 12 mm above the sample surface, the ablation plume is intercepted with an orthogonal hot solvent (e.g., toluene or anisole) jet, which is generated by a heated nebulizer microchip and directed toward the mass spectrometer inlet. The ablated analytes are desolvated and ionized in the gas-phase by atmospheric pressure photoionization using a 10 eV vacuum ultraviolet krypton discharge lamp. The effect of operational parameters and spray solvent on the performance of LAAPPI is studied. LAAPPI offers ~300 μm lateral resolution comparable to, e.g., matrix-assisted laser desorption ionization. In addition to polar compounds, LAAPPI efficiently ionizes neutral and nonpolar compounds. The bioanalytical application of the method is demonstrated by the direct LAAPPI analysis of rat brain tissue sections and sour orange (Citrus aurantium) leaves.     

Determination of 16 phthalate metabolites in urine using automated sample preparation and on-line preconcentration/high-performance liquid chromatography/tandem mass spectrometry

We developed an on-line solid-phase extraction (SPE) method, coupled with isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) and with automated sample preparation, to simultaneously quantify 16 phthalate metabolites in human urine. The method requires a silica-based monolithic column for the initial preconcentration of the phthalate metabolites from the urine and a silica-based conventional analytical column for the chromatographic separation of the analytes of interest. It uses small amounts of urine (100 microL), is sensitive (limits of detection range from 0.11 to 0.90 ng/mL), accurate (spiked recoveries are approximately 100%), and precise (the inter- and intraday coefficients of variation are <10%). The method is not labor intensive, and, because pretreatment of the urine samples was performed automatically using an HPLC autosampler, involves minimal sample handling, thus minimizing exposure to hazardous chemicals. The method was validated on spiked, pooled urine samples and on urine samples from 43 adults with no known exposure to phthalates. The high sensitivity and high throughput (HPLC run time, including the preconcentration step, is 27 min) of this analytical method combined with the ease of use and effective automated sample preparation procedure make it suitable for large epidemiological studies to evaluate the prevalence of human exposure to phthalates.     

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.     

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.     

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.     

Atmospheric pressure photoionization mass spectrometry of fullerenes

Atmospheric pressure photoionization (APPI) was evaluated for the analysis of fullerenes. An important response improvement was found when using toluene mediated APPI in negative mode if compared with other atmospheric pressure ionization (API) sources (electrospray and atmospheric pressure chemical ionization). Fullerene APPI negative mass spectra were dominated by the isotopic cluster of the molecular ion, although isotopic patterns for M+1, M+2, and M+3 ions showed higher than expected relative abundances. These discrepancies are explained by the presence of two isobaric ions, one due to (13)C and the other due to the addition of hydrogen to a double bond of the fullerene structure. Triple quadrupole tandem mass spectrometry, ultrahigh resolution mass spectrometry, and accurate mass measurements were used to confirm these assignments. Additionally, cluster ions M+16 and M+32 were characterized following the same strategy. Ions due to the addition of oxygen and alkyl additions were attributed to the presence of methanol in the mobile phase. For the fast chromatographic separation of fullerenes (less than 3.5 min), a sub-2 μm C18 column and isocratic elution (toluene/methanol, 45:55 v/v) was used. Highly selective-selected ion monitoring (H-SIM) mode (mass resolving power, >12,500 fwhm) was proposed monitoring the two most intense isotope ions in the [M](-?) cluster. Method limits of quantitation down to 10 pg L(-1) for C(60) and C(70) fullerenes and between 0.75 and 5.0 ng L(-1) for larger fullerenes were obtained. Finally, the ultrahigh performance liquid chromatography (UHPLC)-APPI-MS method was used to analyze fullerenes in river and pond water samples.     

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.     

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

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

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.     

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).     

Determination of monosaccharide anhydrides in atmospheric aerosols by use of high-performance liquid chromatography combined with high-resolution mass spectrometry

A novel method for determination of the monosaccharide anhydrides (MAs) levoglucosan, mannosan, and galactosan in atmospheric aerosols has been developed. The method is based on solvent extraction of aerosol filter samples and chemical analysis performed with high-performance liquid chromatography (HPLC) combined with time-of-flight high-resolution mass spectrometry. The MAs were separated on two series connected 2.1 mm x150 mm reversed-phase HPLC columns and identified by negative electrospray ionization mass spectrometry using the m/z 113, 129, and 161 as monitoring ions. The limit of quantification (LOQ) at S/N 10 ranges between 20 and 40 pg for the three isomers. The LOQ can easily be improved. Samples from Elverum (urban background) and Oslo (urban) in Norway were used in validation experiments. Tetrahydrofuran was found to be the most efficient extraction solvent. The choice of solvent is of crucial importance to minimize adsorption to the glassware. Interactions between the MAs and active sites on the quartz filter surface are observed.     

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

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

Determination of ethylenethiourea in crops using particle beam liquid chromatography/mass spectrometry.

Several food crops were analyzed for residues of ethylenethiourea (ETU), a suspect thyroid and liver carcinogen present in EBDC fungicides, using a commercial particle beam (PB) LC/MS method. The PB/LC/MS detection limits for ETU in crops (5 ppb, 1.25 ng) are comparable to those obtained by LC with electrochemical detection. Spectra obtained from crop samples containing as little as 5 ng of ETU were matched with the NBS library reference EI spectrum. Isotopically labeled ETU was used as an internal standard for quantitation and determination of recoveries. No enhancement of molecular ion signal intensity from unlabeled ETU was observed upon coelution with the isotopically labeled variant. This MS method permits detection of ETU with increased selectivity without compromising sensitivity.     

Separation and identification of twelve catechins in tea using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry

A method has been developed for the direct microscale determination of 12 catechins in green and black tea infusions. The method is based on liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS). Standard catechin mixtures and tea infusions were analyzed by LC/APCI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of eight major catechins and caffeine in tea were established based on LC retention times and simultaneously recorded mass spectra. In addition, monitoring of the catechin-specific retro Diels-Alder fragment ion at m/z 139 throughout the chromatogram provided a unique fingerprint for catechin content in the samples that led to the identification of four minor chemically modified catechin derivatives in the infusions. This report is the first to describe the comprehensive determination of all 12 reported catechins in a single analysis. The utility of LC/APCI-MS for providing routine separation and identification of catechins at femtomole to low-picomole levels without extraction or sample pretreatment, and its potential as a standard analytical tool for the determination of polyphenols in natural products and biological fluids, are discussed.     

Analytical methodology for TEX86 paleothermometry by high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry

The TEX86 is a recently proposed paleothermometer through which ancient seawater temperatures of up to 120 My ago can be reconstructed. It is based on the relative distribution of glycerol dibiphytanyl glycerol tetraethers as measured by high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS). The aim of this study was to examine and improve several analytical aspects in the determination of this important proxy in environmental matrices. Comparison of TEX86 analysis using single ion mode (SIM) and mass scanning (m/z 950 to 1450) detection, respectively, revealed that SIM is up to 2 orders of magnitude more sensitive and that the TEX86 can be determined with a reproducibility of +/-0.004 or +/-0.3 degrees C using this method. Comparison of TEX86 values obtained with two different HPLC/APCI-MS set-ups revealed no significant differences. In addition, analysis of TEX86 of extracts obtained by Soxhlet, ultrasonic, and accelerated high-pressure extraction techniques also showed no significant differences between the methods. Our results suggest that TEX86 analysis by HPLC/APCI-MS is robust and can be determined with analytical errors comparable to those of other temperature proxies.     

Generation and identification of reactive metabolites by electrochemistry and immobilized enzymes coupled on-line to liquid chromatography/mass spectrometry

The detection of reactive metabolites using conventional in vivo and in vitro techniques is hampered because the intermediately formed reactive species are prone to covalent binding to cellular macromolecules. Therefore, the application of improved methods is required. The on-line coupling of an electrochemical reactor and horseradish peroxidase immobilized on magnetic microparticles with liquid chromatography/mass spectrometry (EC/LC/MS or HRP/LC/MS) allows the direct detection of reactive metabolites of the model compounds amodiaquine, amsacrine, and mitoxantrone, which are all known for readily binding to cellular macromolecules after metabolization by cytochrome P450. EC/LC/MS and HRP/LC/MS experiments were compared to rat liver microsome incubations and proved to be valuable complementary methods since reactive quinone, quinone imine, and quinone diimine species could be detected directly and not only after trapping with glutathione. Furthermore, N-dealkylation and N-oxidation of amodiaquine were successfully simulated by electrochemical oxidation reactions, as well as the formation of an aldehyde. Therefore, EC/LC/MS and HRP/LC/MS are promising tools for the identification of both reactive and stable metabolites in drug development.