Thiol-ene micropillar array electrospray ionization platform for zeptomole level bioanalysis.

A micropillar array electrospray ionization (µPESI) platform fabricated from thiol-enes with 56 individual polyethylene glycol coated µPESI chips for bioanalytical mass spectrometry is introduced. Bioanalysis capability is shown by measurement of a protein, a protein digest and a cell lysate sample. The thiol-ene polyethylene glycol (PEG) coated µPESI chip allows the use of a wide range of aqueous-organic solvent compositions and provides a detection limit at 60 zeptomole level (6 × 10-20 mol) for a peptide standard.

Atmospheric pressure photoionization-mass spectrometry and atmospheric pressure chemical ionization-mass spectrometry of neurotransmitters.

A group of five neurotransmitters with different properties was analyzed using atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The sensitivity of the techniques for the analytes was tested in six solvents and in positive and negative ion modes. APPI was found to be superior in sensitivity for all the compounds in both positive and negative ion modes. In positive ion mode, water/methanol/formic acid was found to be the best solvent, whereas in negative ion mode, water/methanol/ammonium hydroxide performed best. Detection limits using APPI were between 2.5-250 fmol, depending on the compound. The sensitivity was best for the neurosteroids dehydroepiandrosterone and beta-estradiol, and acetylcholine (LOD 2.5-10 fmol).

Poly(dimethylsiloxane) electrospray devices fabricated with diamond-like carbon-poly(dimethylsiloxane) coated SU-8 masters.

This study presents coupling of a poly(dimethylsiloxane) (PDMS) micro-chip with electrospray ionization-mass spectrometry (ESI-MS). Stable electrospray is generated directly from a PDMS micro-channel without pressure assistance. Hydrophobic PDMS aids the formation of a small Taylor cone in the ESI process and facilitates straightforward and low-cost batch production of the ESI-MS chips. PDMS chips were replicated with masters fabricated from SU-8 negative photoresist. A novel coating, an amorphous diamond-like carbon-poly(dimethylsiloxane) hybrid, deposited on the masters by the filtered pulsed plasma arc discharge technique, improved significantly the lifetime of the masters in PDMS replications. PDMS chip fabrication conditions were observed to affect the amount of background peaks in the MS spectra. With an optimized fabrication process (PDMS curing agent/silicone elastomer base ratio of 1/8 (w/w), curing at 70 degree C for 48 h) low background spectra were recorded for the analytes. The performance of PDMS devices was examined in the ESI-MS analysis of some pharmaceutical compounds and amino acids.

Preparation of porous n-type silicon sample plates for desorption/ionization on silicon mass spectrometry (DIOS-MS).

This study focuses on porous silicon (pSi) fabrication methods and properties for desorption ionization on silicon mass spectrometry (DIOS-MS). PSi was prepared using electrochemical etching of n-type silicon in HF-ethanol solution. Porous areas were defined by a double-sided illumination arrangement: front-side porous areas were masked by a stencil mask, eliminating the need for standard photolithography, and backside illumination was used for the backside ohmic contact. Backside illumination improved the uniformity of the porosified areas. Porosification conditions, surface derivatizations and storage conditions were explored to optimize pSi area, pore size and pore depth. Chemical derivatization of the pSi surfaces improved the DIOS-MS performance providing better ionization efficiency and signal stability with lower laser energy. Droplet spreading and drying patterns on pSi were also examined. Pore sizes of 50-200 nm were found to be optimal for droplet evaporation and pore filling with the sample liquid, as measured by DIOS efficiency. With DIOS, significantly better detection sensitivity was obtained (e.g. 150 fmol for midazolam) than with desorption ionization from a standard MALDI steel plate without matrix addition (30 pmol for midazolam). Also the noise that disturbs the detection of low-molecular weight compounds at m/z < 500 with MALDI could be clearly reduced with DIOS. Low background MS spectra and good detection sensitivity at the 100-150 fmol level for pharmaceutical compounds were achieved with DIOS-MS.

Electrospray mass and tandem mass spectrometry identification of ozone oxidation products of amino acids and small peptides.

Aqueous ozonation of the 22 most common amino acids and some small peptides were studied by electrospray mass (ESI-MS) and tandem mass spectrometry. After 5 min of ozonation only His, Met, Trp, and Tyr form oxidation products clearly detectable by ESI-MS. For His, the main oxidation product is formed by the addition of three oxygen atoms, His + 30; for Met and Tyr by the addition of one oxygen atom, Met + O and Tyr + O, and for Trp by the addition of two oxygen atoms, Trp + 20. Ozone oxidation occurs rapidly, products are already detected after 30 s of ozonation, and the reactivity order is Met > Trp > Tyr > His. The structures of the oxygen addition products were investigated by electrospray product ion mass spectra, and by comparing these spectra to those of protonated intact amino acids, and when available, to those of model compounds. His + 30 was assigned as 2-amino-4-oxo-4-(3-formylureido)butanoic acid (1) formed by oxidation of the His imidazole ring, Met + O as methionine sulfoxide (2), Trp + 20 as N-formylkynurenine (4), and Tyr + O as a mixture of dihydroxyphenylalanines (7 and 8). Ozonation of peptides show that the same number of oxygen atoms are added as expected from the ozonation of the free amino acids. The product ion mass spectra of both the protonated intact peptides, MH+, and the main ozonation products (M + nO)H+ (n = 1-3) revealed b and y type ions as the main fragments, which allow one to assign the type and location of modified amino acid in the model peptides.

Fragmentation of protonated O,O-diethyl O-aryl phosphorothionates in tandem mass spectral analysis.

The gas-phase ion chemistry of protonated O,O-diethyl O-aryl phosphorothionates was studied with tandem mass spectrometric and ab initio theoretical methods. Collision-activated dissociation (CAD) experiments were performed for the [M+H](+) ions on a triple quadrupole mass spectrometer. Various amounts of internal energy were deposited into the ions upon CAD by variation of the collision energy and collision gas pressure. In addition to isobutane, deuterated isobutane C4D10 also was used as reagent gas in chemical ionization. The daughter ions [M+H-C2H4](+) and [M+H-2C2H4](+) dominate the CAD spectra. These fragments arise via various pathways, each of which involves γ-proton migration. Formation of the terminal ions [M+H-2C2H4-H2O](+), [M+H-2C2H4-H2S](+), [ZPhOH2](+), [ZPhSH2](+), and [ZPhS](+) [Z = substituent(s) on the benzene ring] suggests that (1) the fragmenting [M+H](+) ions of O,O-diethyl O-aryl phosphorothionates have protons attached on the oxygen of an ethoxy group and on the oxygen of the phenoxy group; (2) thiono-thiolo rearrangement by aryl migration to sulfur occurs; (3) the fragmenting rear-ranged [M+H](+) ions have protons attached on the oxygen of an ethoxy group and on the sulfur of the thiophenoxy group. To get additional support for our interpretation of the mass spectrometric results, some characteristics of three protomers of O,O-diethyl O-phenyl phosphorothionate were investigated by carrying out ab initio molecular orbital calculations at the RHF/3-21G* level of theory.

Fragmentation of protonated O,O-dimethyl O-aryl phosphorothionates in tandem mass spectral analysis.

A study was carried out on the fragmentation of 12 protonated O,O-dimethyl O-aryl phosphorothionates by tandem quadrupole mass spectrometry. Some of the studied compounds are used in agriculture as pesticides. Energy-resolved and pressure-resolved experiments were performed on the [M + H](+) ions to investigate the dissociation behavior of the ions with various amounts of internal energy. On collisionally activated dissociation, the [M + H](+) ions decompose to yield the [M + H - CH3OH](+), (CH3O)2PS(+) (m/z 125), and (CH3O)2PO(+) (m/z 109) ions as major fragments. The ions [M + H - CH3OH](+) and (CH3O)2PS(+) probably arise from the [M + H](+) ions of the O,O-dimethyl O-aryl phosphorothionates with the proton on the sulfur or on the oxygen of the phenoxy group. The origin of the hydroxy proton of the methanol fragment was in many cases, surprisingly, the phenyl group and not the reagent gas. This was confirmed by using deuterated isobutane, C4D10, as reagent gas in Cl. The fragment ions (CH3O)2PO(+) and [ZPhS](+) are the results of thiono-thiolo rearrangement reaction. The precursor ion for the ion (CH3O)2PO(+) arises from most compounds upon chemical ionization, whereas the precursor ion for the ion [ZPhS](+) arises only from a few compounds upon chemical ionization. The observed fragments imply that several sites carry the extra proton and that these sites get the proton usually upon ionization. The stability order and some characteristics of three protomers of O,O-dimethyl O-phenyl phosphorothionate were investigated by ab initio calculations at the RHF/3-21G* level of theory.

Mass spectrometric and tandem mass spectrometric behavior of nitrocatechol glucuronides: a comparison of atmospheric pressure chemical ionization and electrospray ionization.

The mass spectrometric (MS) and tandem mass spectrometric (MS/MS) behavior of six nitrocatechol-type glucuronides using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) was systematically studied, and the effect of operation parameters on the fragmentations are presented. The positive ion APCI- and ESI-MS spectra showed an intense protonated molecule and the respective negative ion spectra a deprotonated molecule with minimal fragmentation. The main fragment ions in the MS/MS spectra of the protonated and deprotonated molecules were [M + H - Glu]+ and [M - H - Glu]-, respectively, formed by the loss of the glucuronide moiety. The measured limits of detection indicated that ESI is a significantly more efficient ionization method than APCI in the negative and positive ion modes for the compounds studied. MS/MS was found to be less sensitive, but more reliable and simple than MS due to the absence of chemical noise.

Liquid chromatography/atmospheric pressure ionization-mass spectrometry in drug metabolism studies.

The study of the metabolic fate of drugs is an essential and important part of the drug development process. The analysis of metabolites is a challenging task and several different analytical methods have been used in these studies. However, after the introduction of the atmospheric pressure ionization (API) technique, electrospray and atmospheric pressure chemical ionization, liquid chromatography/mass spectrometry (LC/MS) has become an important and widely used method in the analysis of metabolites owing to its superior specificity, sensitivity and efficiency. In this paper the feasibility of LC/API-MS techniques in the identification, structure characterization and quantitation of drug metabolites is reviewed. Sample preparation, LC techniques, isotope labeling, suitability of different MS techniques, such as tandem mass spectrometry, and high-resolution MS in drug metabolite analysis, are summarized and discussed. Automation of data acquisition and interpretation, special techniques and possible future trends are also the topics of the review.

Effect of eluent on the ionization efficiency of flavonoids by ion spray, atmospheric pressure chemical ionization, and atmospheric pressure photoionization mass spectrometry.

The effect of nine different eluent compositions on the ionization efficiency of five flavonoids was studied using ion spray (IS), atmospheric pressure chemical ionization (APCI), and the novel atmospheric pressure photoionization (APPI), in positive and negative ion modes. The eluent composition had a great effect on the ionization efficiency, and the optimal ionization conditions were achieved in positive ion IS and APCI using 0.4% formic acid (pH 2.3) as a buffer, and in negative ion IS and APCI using ammonium acetate buffer adjusted to pH 4.0. For APPI work, the eluent of choice appeared to be a mixture of organic solvent and 5 mM aqueous ammonium acetate. The limits of detection (LODs) were determined in scan mode for the analytes by liquid chromatography/mass spectrometry using IS, APCI and APPI interfaces. The results show that negative ion IS with an eluent system consisting of acidic ammonium acetate buffer provides the best conditions for detection of flavonoids in mass spectrometry mode, their LODs being between 0.8 and 13 microM for an injection volume of 20 microl.

Identification of ozone-oxidation products of oxycodone by electrospray ion trap mass spectrometry.

The products of oxycodone oxidized by ozone were characterized by electrospray ionization-tandem mass spectrometry (ESI--MS/MS). Liquid Chromatography(LC)--MS analyses revealed that the main constituents in the oxidation reaction mixture included the protonated molecules m/z 316, corresponding to oxycodone, and m/z 332, m/z 348, m/z 366, corresponding to the oxidation products. ESI--MS/MS and MS(n) spectra were used to study oxycodone fragmentation in detail and to characterize the structures of oxidation products. The results show that the oxidation products were formed by addition of one or two oxygen atoms or by addition of three oxygen and two hydrogen atoms to oxycodone. The fragmentation of the oxidation products also shows that the aromatic ring oxidizes due to rupture of the C-3--C-4 bond during product formation.

Effect of nitro groups and alkyl chain length on the negative ion tandem mass spectra of alkyl 3-hydroxy-5-(4'-nitrophenoxy) and alkyl 3-hydroxy-5-(2', 4'-dinitrophenoxy) benzoates

Tandem mass spectrometric behaviour was studied for a small combinatorial library of alkyl 3-hydroxy-5-(4'-nitrophenoxy) benzoates (A1-A5) and alkyl 3-hydroxy-5-(2', 4'-dinitrophenoxy) benzoates (B1-B5). The spectra were recorded by negative ion electrospray low-energy collision induced dissociation (CID) tandem mass spectrometry. The product ion spectra of [M - H](-) of the benzoates A1-A5 are similar, as are those of benzoates B1-B5. However, the spectra of the B series compounds differ significantly from those of the A series owing to the second electron-withdrawing nitro substituent in the B compounds. In addition, the length of the alkyl chain has an effect on the fragmentation. However, both series of compounds exhibit an abundant nitrophenoxy ion formed by the loss of 3-hydroxybenzoate. This is at m/z 138 in A1-A5 and at m/z 183 in B1-B5. A precursor ion scan of the nitrophenoxy ion provides a rapid method to identify the synthesised compounds in this type of combinatorial mixture. Copyright 1999 John Wiley & Sons, Ltd.

Analysis of bisphosphonates by capillary electrophoresis-electrospray ionization mass spectrometry.

Capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) was applied to the direct identification and quantitation of clodronate and its four common impurities. The coaxial interface technique and negative ion mode were used in the detection. Ion source parameters and sheath liquid composition were optimized to produce maximum abundance of singly charged deprotonated molecules used in monitoring. In addition, the effects of electrolyte composition and instrumental parameters of CE on separation were studied. The developed method provides high separation power and specificity to bisphosphonate analysis. In quantitative analysis, the method showed good linearity (r=0.9946-0.9989), satisfactory repeatability (migration time variation: RSD=0.43-1.0%, peak area variation: RSD=2.2-9.4%) and sufficient sensitivity (detection limits: 0.08-0.22 mg ml(-1)) for identification of bisphosphonates from bulk material.

Development and validation of a capillary zone electrophoretic method for the determination of bisphosphonate and phosphonate impurities in clodronate.

Capillary zone electrophoresis with direct UV detection at low wavelength and reversed polarity was applied for the separation and quantitation of bisphosphonate and phosphonate impurities in clodronate bulk material. Polyacrylamide-coated capillaries were used to reduce the interactions between the analytes and the electric double layer of the capillary, and to minimize electroosmotic flow. Study was made of the major factors affecting the separation, i.e., pH and ionic strength of the electrolyte solution and various instrumental parameters. The developed method provided reproducible separations of clodronate and related impurities (between-day precision of migration times: RSD < 2.3%, 275 runs). Acceptable validation results in the impurity quantitation range of 0.5-7.5 microg ml(-1) (corresponding to 0.1-1.5% of clodronate working concentration) were obtained in specificity, within-day and between-day precision, accuracy and linearity.

Determination of mono- and sesquiterpenes in water samples by membrane inlet mass spectrometry and static headspace gas chromatography.

A membrane inlet mass spectrometric (MIMS) method is presented and compared with a static headspace gas chromatographic method (HSGC) for the determination of terpenes in water. The MIMS method provides a very simple and fast analysis of terpenes in water, detection limits being relatively low, from 0.2 mug l(-1) for monoterpenes to 2 mug l(-1) for geraniol. The analysis of terpenes by the HSGC (equipped with flame ionization detector, FID) method is more time-consuming and the detection limits (2 mug l(-1) for monoterpenes to 100 mug l(-1) for geraniol) are higher than with MIMS. However, the HSGC method has the advantage of determining individual mono- and sesquiterpene compounds, whereas MIMS provides only separation of different classes of terpenes. Both methods were applied to the analysis of mono- and sesquiterpenes in several condensation water samples of pulp and paper mills.

Analysis of (dichloromethylene) bisphosphonate in urine by capillary gas chromatography-mass spectrometry.

An anion exchange extraction method of bisphosphonates from urine is described. More than 90% of the (dichloromethylene) bisphosphonate (Cl2MBP, clodronate) was recovered from urine. The extracted bisphosphonates were trimethylsilylated and analysed with capillary gas chromatography-mass spectrometry (GC/MS). The mass spectrometric techniques used were electron ionization (EI), ammonia chemical ionization (CI), ammonia CI tandem mass spectrometry and methane negative chemical ionization (NCI). The limit of detection of Cl2MBP was 25 pg/injection in the NCI/selective ion recording (SIR)-mode. At 100 ng ml-1 of Cl2MBP the precision of the whole assay method was 17.9% (N = 6). The NCI/SIR technique offers a sensitive and highly selective method for the quantitation of Cl2MBP in urine.

Desorption atmospheric pressure photoionization with polydimethylsiloxane as extraction phase and sample plate material.

Desorption atmospheric pressure photoionization (DAPPI) is an ambient ionization technique for mass spectrometry (MS) that can be used to ionize polar as well as neutral and completely non-polar analytes. In this study polydimethylsiloxane (PDMS) was used as a solid phase extraction sorbent for DAPPI-MS analysis. Pieces of PDMS polymer were soaked in an aqueous sample, where the analytes were sorbed from the sample solution to PDMS. After this, the extracted analytes were desorbed directly from the polymer by the hot DAPPI spray solvent plume, without an elution step. Swelling and extracting the PDMS with a cleaning solvent prior to extraction diminished the high background in the DAPPI mass spectrum caused by PDMS oligomers. Acetone, hexane, pentane, toluene, diisopropylamine and triethylamine were tested for this purpose. The amines were most efficient in reducing the PDMS background, but they also suppressed the signals of low proton affinity analytes. Toluene was chosen as the optimum cleaning solvent, since it reduced the PDMS background efficiently and gave intensive signals of most of the studied analytes. The effects of DAPPI spray solvents toluene, acetone and anisole on the PDMS background and the ionization of analytes were also compared and extraction conditions were optimized. Anisole gave a low background for native PDMS, but toluene ionized the widest range of analytes. Analysis of verapamil, testosterone and anthracene from purified, spiked wastewater was performed to demonstrate that the method is suited for in-situ analysis of water streams. In addition, urine spiked with several analytes was analyzed by the PDMS method and compared to the conventional DAPPI procedure, where sample droplets are applied on PMMA surface. With the PDMS method the background ion signals caused by the urine matrix were lower, the S/N ratios of analytes were 2-10 times higher, and testosterone, anthracene and benzo[a]pyrene that were not detected from PMMA in urine, were observed in the MS spectrum.

Identification of trichothecenes by thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography-mass spectrometry.

Thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography-mass spectrometry are compared for the identification of six trichothecenes. Thermospray spectra of the trichothecenes exhibit only a very abundant ammonium adduct ion. Plasmaspray, which provides a more energetic ionization process than thermospray, produces some fragment ions in addition to an abundant ammonium adduct ion. The spectra obtained by dynamic fast-atom bombardment exhibit a protonated molecule, a glycerol adduct ion and numerous fragment ions formed by the losses of functional groups as neutrals in various combinations. Thermospray and plasmaspray are suitable only for monitoring of the trichothecenes, whereas dynamic fast-atom bombardment is suitable for monitoring and for structure characterization.

Characterization of trichothecenes by tandem mass spectrometry using reactive collisions with ammonia.

The use of low-energy (7 eV Elab) reactive collisions with ammonia provides a highly specific method for the characterization of trichothecenes since the collisions between the protonated molecule and ammonia not only produce ions formed by collisionally activated dissociation but ammonium adduct ions and ions formed by substitution reactions. The good detection limits (20-300 pg) and good quantitative reproducibility (relative standard deviations are 5-10%) mean that the use of reactive collisions with ammonia is also very suitable for monitoring and quantitative analyses.

Use of M-series retention index standards in the identification of trichothecenes by electron impact mass spectrometry.

A method for the reliable identification of a series of trichothecenes as their trifluoroacetate esters in porridge flake samples is described. The esters were separated by gas chromatography and identified from their retention times relative to n-alkyl-bis(trifluoromethyl)phosphine sulphide (M-series) retention index standards and their electron impact mass spectra. The relative retention times offer an independent identification method by which the reliability of the identification can be improved. The mass spectra and the relative retention times were obtained from the same gas chromatographic-mass spectrometric run. Detection limits were of the order to 0.005-0.05 mg/kg. All the flake samples including oats (ten of fourteen samples studied) contained deoxynivalenol (0.01-0.2 mg/kg) and one oat flake sample contained HT-2 toxin (0.008 mg/kg). The other trichothecenes monitored were not found.