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.

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.

Adjusting mobility scales of ion mobility spectrometers using 2,6-DtBP as a reference compound.

Performance of several time-of-flight (TOF) type ion mobility spectrometers (IMS) was compared in a joint measurement campaign and their mobility scales were adjusted based on the measurements. A standard reference compound 2,6-di-tert butylpyridine (2,6-DtBP) was used to create a single peak ion mobility distribution with a known mobility value. The effective length of the drift tube of each device, considered here as an instrument constant, was determined based on the measurements. Sequentially, two multi-peaked test compounds, DMMP and DIMP, were used to verify the performance of the adjustment procedure in a wider mobility scale. By determining the effective drift tube lengths using 2,6-DtBP, agreement between the devices was achieved. The determination of effective drift tube lengths according to standard reference compound was found to be a good method for instrument inter-comparison. The comparison procedure, its benefits and shortcomings as well as dependency on accuracy of literature value are discussed along with the results.

Utilization of a multimembrane inlet and a cyclic sudden sampling introduction mode in membrane inlet mass spectrometry.

Sudden sampling introduction into a membrane inlet mass spectrometer (MIMS) considerably improves the selectivity of the membrane inlet and is therefore applicable even for compounds with low permeabilities through a silicone membrane. In this study the basics of cyclic non-steady-state sudden increase sample injection were studied using a three-membrane inlet and a portable sector double-focusing mass spectrometer. The operational parameters of the inlet system providing the most efficient enrichment of volatile organic compounds (VOCs) in air were defined. Simulation of the diffusion process following sudden sample introduction into the three-membrane inlet was also carried out. Experimental testing of the three-membrane inlet system with the cyclic sudden sample injection mode for benzene, toluene, styrene, and xylene in air was performed. The simulation and the experimental results demonstrated that, when this mode is used, the VOCs/nitrogen relative enrichment factor of samples introduced into the mass spectrometer equipped with a three-membrane inlet is increased by a factor of approximately 10(5) compared with a direct introduction method. This effect may be used to decrease detection limits of compounds obtained with mass spectrometry to decrease matrix flow through the inlet at the same detection limits.

Feasibility of atmospheric pressure desorption/ionization on silicon mass spectrometry in analysis of drugs.

The feasibility of atmospheric pressure desorption/ionization on silicon mass spectrometry (AP-DIOS-MS) for drug analysis was investigated. It was observed that only compounds with relative high proton affinity are efficiently ionized under AP-DIOS conditions. The limits of detection (LODs) achieved in MS mode with midazolam, propranolol, and angiotensin II were 80 fmol, 20 pmol, and 1 pmol, respectively. In MS/MS mode the LODs for midazolam and propranolol were 10 fmol and 5 pmol, respectively. The good linearity (r(2) > 0.991), linear dynamic range of 3 orders of magnitude, and reasonable repeatability showed that the method is suitable for quantitative analysis.

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.

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.

N-in-one determination of retention factors for drugs by immobilized artificial membrane chromatography coupled to atmospheric pressure chemical ionization mass spectrometry.

Immobilized artificial membrane (IAM) chromatography is widely used in drug discovery for ranking the absorption properties of drug candidates. In this work an IAM chromatography method using atmospheric pressure chemical ionization mass spectrometric detection (IAM/APCI-MS) was developed for the determination of log k(IAM) values for a mixture of compounds (9-in-one). Values were calculated from isocratic runs (0, 10, 20, 30, 35% acetonitrile) in both positive and negative modes. Good correlation (r(2) = 0.97) was achieved for n-in-one results obtained with ammonium acetate buffer and mass spectrometry, compared with the traditional method involving single compound analysis with phosphate buffered saline and an ultraviolet detector. A gradient elution method providing fast determination of relative log k(IAM) values in a single IAM/APCI-MS run was demonstrated for the same compounds.

Purge-and-membrane mass spectrometry, a screening method for analysis of VOCs from soil samples.

Purge-and-membrane mass spectrometry (PAM-MS) is a combination of dynamic headspace sampling and membrane extraction. A new and simple purge-and-membrane sampler is introduced and its basic testing results for the analysis of VOCs in soil samples are reported. Soil moisture had no effect on desorption times in the case of sand, but the desorption times increased when the content of organic matter in the soil sample (garden soil) increased. The longest desorption times were measured with dry garden soil samples. For both types of samples, minor differences in desorption peak areas were observed between 10 and 20% moisture. Detection limits of the VOCs varied in the range 2-150 microg/kg, depending on the soil type. Good linearity (correlation coefficient > 0.990) was observed in the range 0.5-50 mg/kg. Aging of the spiked soil samples had only a slight effect on desorption peak areas for samples stored at 5 degrees C up to two weeks, but after six months of storing, differences were observed between dry sand and moistened garden soil. In both cases, peak areas were diminished. On average, 46% of compounds could be desorbed from the aged sand and 86% from the aged garden soil. The modified vapor fortification method was used in preparing standard soil samples, which were analyzed by static headspace gas chromatography (HSGC) and PAM-MS. Some authentic soil samples were also analyzed using both of these techniques. Many of the vapor fortification samples and the authentic samples were also analyzed in another laboratory by HSGC. The agreement between the methods and the laboratories was generally good.

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.

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.

Analysis of residual solvents in pharmaceuticals with purge-and-membrane mass spectrometry.

A method using purge-and-membrane mass spectrometry (PAM-MS) was developed for the analysis of residual solvents in pharmaceutical products. The method combines dynamic headspace and membrane inlet mass spectrometry. The limits of detection for the compounds studied, benzene, toluene, chloroform, 2-pentene and 2-methyl- and 3-methylpentane, were 0.05-0.1 mg/kg. In quantitative analysis the method showed good linearity (r(2) > 0.998) and acceptable within-day (RSD = 7.9-18%) and between-day (RSD = 6.8-10%) repeatability. The PAM-MS method combined with the custom-made Solver program was compared with a method using purge-and-trap gas chromatography/mass spectrometry (P&T-GC/MS) for identification of residual solvents from authentic samples. The results showed that PAM-MS/Solver provides reliable identification of the main volatile organic compounds (VOCs) in the pharmaceuticals, but VOCs with low concentrations (below 0.5 mg/kg) were better identified by P&T-GC/MS. Other advantages of the PAM-MS method were short analysis times and non-requirement for pre-treatment of samples.

Quantification of anions and cations in environmental water samples. Measurements with capillary electrophoresis and indirect-UV detection.

The aim of this study was to validate two separation methods for determination of inorganic anions and cations from natural waters with capillary electrophoresis (CE) by using indirect-UV detection. The research is related to method development for screening of groundwater samples obtained in site investigations for spent fuel of the Finnish nuclear industry. In CE analysis, anions were separated in pyromellitic acid (pH 7.7) in the order bromide, chloride, sulphate, nitrite, nitrate, fluoride and dihydrogenphosphate. Cations were separated at pH 3.6 after anions using an 18-crown-6-ether solution. In these analyses, ammonium migrated first followed by potassium, calcium, sodium and magnesium. The concentrations of the ions in the natural water samples were calculated by using two or three calibration curves made using reference solutions at concentration levels of 0.5-250 mg/l. The repeatabilities of the anion and cation methods were tested using laboratory-made reference sample mixtures with high and low salt concentrations. The limits of quantification in the analyses were between 0.02 and 0.1 mg/l, depending on the ion. Concentrations of ions tested in natural waters varied from a few milligrams to tens of grams per litre.

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.

Determination of phenolic compounds in water using membrane inlet mass spectrometry.

Two membrane inlet mass spectrometric (MIMS) methods for determining phenolic compounds in water are described and compared, namely direct analysis and analysis after acetylation of the phenolic compounds. Direct analysis of phenolic compounds in water is a very simple and rapid method and detection limits are relatively low (from 30 mug 1(-1) for phenol to 1000 mug 1(-1) for 4-nitrophenol). Analysis of phenolic compounds after aqueous acetylation is also a very simple and rapid method, and the detection limits are even two orders of magnitude lower than in the direct analysis. For example the detection limit of phenol acetate is 0.5 mug 1(-1) and that of 4-nitrophenol is 10 mug 1(-1). The acetylation method was also tested in the analysis of phenolic compounds from contaminated surface water samples.

Comparison of different methods for the determination of volatile organic compounds in water samples.

The aim of this work was to compare the characteristics of three methods, membrane inlet mass spectrometry (MIMS), purge-and-trap gas chromatography-mass spectrometry (P&T) and static headspace gas chromatography (HSGC), for the determination of volatile organic compounds in water samples as used in routine analysis. The characteristics examined included linear dynamic ranges, detection limits of selected environmentally hazardous volatile organic compounds (e.g. toluene, benzene and trichloroethene) in water, required analysis time and reproducibility of the analytical methods. The MIMS and P&T methods had the lowest detection limits for all the tested compounds, ranging from 0.1 to 5 mug 1(-1). Linear dynamic ranges using the MIMS method were about four orders of magnitude and using the P&T method about two orders of magnitude. Detection limits of the HSGC method were 10-100 times higher than those of the other two methods, but the linear dynamic ranges were larger, even up to six orders of magnitude. The analysis time per sample was shortest for the MIMS method, from 5 to 10 min, and ranged around from 35 to 45 min for the HSGC and P&T methods. The reproducibilities of the methods were of the same order of magnitude, in the range of 1-13%. Agreement between the analytical results obtained for spiked samples and for environmental water samples by the three different methods was very good.

A Cryotrap Membrane Introduction Mass Spectrometry System for Analysis of Volatile Organic Compounds in Water at the Low Parts-per-Trillion Level.

Detection of volatile organic compounds (VOCs) in aqueous solution at low parts-per-trillion (ppt) levels is accomplished using a very simple and efficient on-line preconcentration cryotrap membrane introduction mass spectrometry (CT-MIMS) system. The conventional MIMS probe is modified so that the membrane interface is placed about 15 cm away from the ion source. A U-shaped trap tube is then inserted between the membrane interface and the ion source. Cryotrapping is performed with liquid nitrogen for 15 min, followed by fast heating at ∼15 °C s(-)(1), which thermally releases the condensed VOCs almost at once into the ion source region of a quadrupole mass spectrometer. By applying electron ionization and a selective ion monitoring scan mode, a very sharp and intense peak is obtained. The performance of the CT-MIMS system was compared with that of conventional MIMS, and after reaching the best conditions for the trapping and heating cycles, an improvement factor in signal intensity of about 100 was observed for a series of VOCs. The extraordinary sensitivity of CT-MIMS system allows VOCs to be detected at very low concentrations, detection limits being typically on the order of 10-20 ppt. The results also show excellent linearity and reproducibility for the system.

Analysis of aldehydes and ketones from beer as O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine derivatives.

O-(2,3,4,5,6-pentafluorobenzyl)Hydroxylamine (PFBOA) was used as a derivatization reagent for carbonyl compounds in beer. Derivatization was carried out in aqueous solution without extraction or concentration of the sample. The effects of antifoam agent, reaction time and pH on the reaction efficiency were studied. Antifoam RD, a silicone polymer-based antifoam reagent, was the best antifoaming agent since it did not cause interferences. Reaction time studies showed that the yield of aldehydes increased up to 12 hr and then decreased slightly. The yield of 3-hydroxybutanone, a test compound for ketones, increased throughout the 48 hr test period. The natural pH of beer (ca. 4.5) was favourable for the determination of carbonyl compounds as PFBOA derivatives. Higher pH values caused yield losses and some compounds, such as butanedione, 2,3-pentanedione and 5-hydroxymethyl-2-furfural, could not be measured at all in neutral or basic conditions. Carbonyl compounds were identified by GC-MS, using three different ionization techniques, electron impact ionization, chemical ionization, and negative chemical ionization. The formation of the protonated molecules by ammonia chemical ionization and formation of the negative molecular ions and [M - HF](-.) ions by negative chemical ionization permitted reliable identification of the various carbonyl compounds studied. Sixteen carbonyl compounds from the 32 standard compounds were identified in beer and 11 of the most significant were quantitated using GC-ECD. Reproducibility of quantitation for beer samples was good, the relative standard deviations varied between 2.7 and 6.7 %. The estimated detection limits of the PFBOA derivatives of the carbonyl compounds in beer varied in the range of 0.01-1 microg/dm(3).

Analysis of acrolein and acrylonitrile in aqueous solution by membrane introduction mass spectrometry.

Acrolein and acrylonitrile can be quantified directly at low levels in aqueous solution using membrane introduction mass spectrometry. Electron impact was used to generate positively charged ions and electron capture of the O-(2,3,4,5,6-pentafluorobenzyl)hydroxyl amine (PFBOA) derivative was used to generate negatively charged ions of acrolein in aqueous solutions. The origins of all ions in the mass spectra and product MS/MS spectra recorded using both ionization methods were assigned and a reaction scheme is given which accounts for the fragmentation of the PFBOA derivative. Detection limits were measured using multiple reaction monitoring in both the methods. With electron capture detection, acrolein could be detected without preconcentration at 10 ppb levels. Electron impact ionization and multiple reaction monitoring both allowed the measurement of acrylonitrile at levels as low as 10 ppb.