Biomedical subjects

Tapio Kotiaho

Publications and source records attributed to Tapio Kotiaho.

At least 19 recordsLinked to original sources

PubMed2006

Evaluation of cocktail approach to standardise Caco-2 permeability experiments.

The purpose of this study was to investigate the suitability and reliability of n-in-one approach using FDA suggested compounds for standardising Caco-2 permeability experiments. Special attention was paid to the evaluation of rank order correlation and mechanistic insights of compound permeability. Transport studies with antipyrine, metoprolol, ketoprofen, verapamil, hydrochlorothiazide, ranitidine, mannitol and fluorescein were performed in 12- and 24-well formats, as single compounds and in cocktails under iso-pH 7.4 and pH-gradient (pH 5.5 vs. 7.4) conditions. Compounds were quantified using n-in-one LC/MS/MS analysis. The cocktail-dosing proved to be a feasible method to determine the permeability of the Caco-2 cell line and to introduce external standards for permeability tests. Even though sink conditions were lost in cocktail experiments for highly permeable compounds, the rank order of compound permeability and the classification to low and high permeability compounds remained unchanged between single and cocktail studies and permeability values of 12- and 24-well formats were directly comparable. Under pH-gradient conditions the margin between high and low permeability compounds was narrower due to the lower permeability (higher fraction of ionisation) of basic molecules. Of the compounds studied, antipyrine, metoprolol, hydrochlorothiazide and mannitol are suitable for evaluation and standardisation purposes of passive permeability, while fluorescein would function as paracellular marker under iso-pH 7.4. As efflux activity may vary between cell batches, verapamil is a useful marker for P-glycoprotein.

Biological Transport, Active↗

PubMed2006

Gas chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry.

An atmospheric pressure chemical ionization (APCI) microchip is presented for combining a gas chromatograph (GC) to a mass spectrometer (MS). The chip includes capillary insertion channel, stopper, vaporizer channel, nozzle and nebulizer gas inlet fabricated on the silicon wafer, and a platinum heater sputtered on a glass wafer. These two wafers are joined by anodic bonding creating a two-dimensional version of an APCI microchip. The sample from GC is directed via heated transfer line capillary to the vaporizer channel of the APCI chip. The etched nozzle forms narrow sample plume, which is ionized by an external corona discharge needle, and the ions are analyzed by a mass spectrometer. The GC-microchip APCI-MS combination provides an efficient method for qualitative and quantitative analysis. The spectra produced by microchip APCI show intensive protonated molecule and some fragmentation products as in classical chemical ionization for structure elucidation. In quantitative analysis the GC-microchip APCI-MS showed good linearity (r(2) = 0.9989) and repeatability (relative standard deviation 4.4%). The limits of detection with signal-to-noise ratio of three were between 0.5 and 2 micromol/L with MS mode using selected ion monitoring and 0.05 micromol/L with MS/MS using multiple reaction monitoring.

Anisoles↗

PubMed2006

Capillary liquid chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry.

A miniaturized nebulizer chip for capillary liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (capillary LC-microchip APCI-MS) is presented. The APCI chip consists of two wafers, a silicon wafer and a Pyrex glass wafer. The silicon wafer has a DRIE etched through-wafer nebulizer gas inlet, an edge capillary insertion channel, a stopper, a vaporizer channel and a nozzle. The platinum heater electrode and pads for electrical connection were patterned on to the Pyrex glass wafer. The two wafers were joined by anodic bonding, creating a microchip version of an APCI-source. The sample inlet capillary from an LC column is directly connected to the vaporizer channel of the APCI chip. The etched nozzle in the microchip forms a narrow sample plume, which is ionized by an external corona needle, and the formed ions are analyzed by a mass spectrometer. The nebulizer chip enables for the first time the use of low flow rate separation techniques with APCI-MS. The performance of capillary LC-microchip APCI-MS was tested with selected neurosteroids. The capillary LC-microchip APCI-MS provides quantitative repeatability and good linearity. The limits of detection (LOD) with a signal-to-noise ratio (S/N) of 3 in MS/MS mode for the selected neurosteroids were 20-1000 fmol (10-500 nmol l(-1)). LODs (S/N = 3) with commercial macro APCI with the same compounds using the same MS were about 10 times higher. Fast heat transfer allows the use of the optimized temperature for each compound during an LC run. The microchip APCI-source provides a convenient and easy method to combine capillary LC to any API-MS equipped with an APCI source. The advantages and potentials of the microchip APCI also make it a very attractive interface in microfluidic APCI-MS.

Atmospheric Pressure↗

PubMed2006

Desorption electrospray ionization mass spectrometry for the analysis of pharmaceuticals and metabolites.

The performance of desorption electrospray ionization (DESI) in the analysis of a group of pharmaceuticals and their glucuronic acid conjugates is reported. The suitability of different sprayer solvents and different surfaces was examined. In the positive ion mode, water/methanol/trifluoroacetic acid performed best, whereas, in the negative ion mode, water/methanol/ammonium hydroxide was found to be the most suitable spray solvent. Of the surfaces investigated, polymethylmethacrylate (PMMA) was found to give the best performance in terms of sensitivity. Spray solution flow rate and the distance of the sprayer tip from the surface were also found to have significant effects on the signal intensity. Analytes with basic groups efficiently formed the corresponding protonated molecules in the positive ion mode, whereas acidic analytes, such as the glucuronic acid conjugates, formed intense signals due to the deprotonated molecules in the negative ion mode. Ionization of neutral compounds was less efficient and in many cases it was achieved through adduct formation with simple anions or cations.

Molecular Structure↗

PubMed2006

New surfaces for desorption electrospray ionization mass spectrometry: porous silicon and ultra-thin layer chromatography plates.

The performance of nanoporous silicon (pSi) and ultra-thin layer chromatography (UTLC) plates as surfaces for desorption electrospray ionization (DESI) was compared with that of polymethyl methacrylate (PMMA) and polytetrafluoroethylene (PTFE), both popular surfaces in previous DESI studies. The limits of detection (LODs) and other analytical characteristics for six different test compounds were determined using all four surfaces. The LODs for the compounds were in the fmol-pmol (pg-ng) range. The LODs with the pSi surface were further improved for each of the compounds when heat was applied to the surface during sample application which gave LODs as low as or lower than those achieved with PMMA and PTFE. The UTLC plates were successfully used as a rapid means of chromatographic separation prior to DESI-MS analysis. Another advantage achieved using the newer pSi and UTLC surfaces was increased speed of analysis, associated with drying of solution-phase samples. This took place immediately at the UTLC surface and it could be achieved rapidly by gently heating the pSi surface. The presence of salts in the sample did not cause suppression of the analyte signal with any of the surfaces.

Acetylcholine↗

PubMed2006

Minimum proton affinity for efficient ionization with atmospheric pressure desorption/ionization on silicon mass spectrometry.

We performed a systematic study using a set of compounds with different proton affinities (PAs) on the ionization in atmospheric pressure desorption/ionization on silicon mass spectrometry (AP-DIOS-MS). The compounds studied included various aromatic molecules of different sizes. The PAs of these compounds were calculated using ab initio and hybrid density functional theory calculations at the B3LYP/6-31G(d) level of theory. We observed that only compounds with relatively high PAs above a threshold value of 920-950 kJ/mol were efficiently ionized as protonated molecules under AP-DIOS conditions and produced very clean mass spectra.

Adsorption↗

PubMed2005

Characterization of SU-8 for electrokinetic microfluidic applications.

The characterization of SU-8 microchannels for electrokinetic microfluidic applications is reported. The electroosmotic (EO) mobility in SU-8 microchannels was determined with respect to pH and ionic strength by the current monitoring method. Extensive electroosmotic flow (EOF), equal to that for glass microchannels, was observed at pH > or =4. The highest EO mobility was detected at pH > or =7 and was of the order of 5.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer. At pH < or =3 the electroosmotic flow was shown to reverse towards the anode and to reach a magnitude of 1.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer (pH 2). Also the zeta-potential on the SU-8 surface was determined, employing lithographically defined SU-8 microparticles for which a similar pH dependence was observed. SU-8 microchannels were shown to perform repeateably from day to day and no aging effects were observed in long-term use.

Journal Article↗

PubMed2005

Analysis of small molecules by ultra thin-layer chromatography-atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

The feasibility of ultra thin-layer chromatography atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (UTLC-AP-MALDI-MS) has been studied in the analysis of small molecules. Because of a thinner adsorbent layer, the monolithic UTLC plates provide 10-100 times better sensitivity in MALDI analysis than conventional high performance thin-layer chromatography (HPTLC) plates. The limits of detection down to a low picomole range are demonstrated by UTLC-AP-MALDI-MS. Other advantages of UTLC over HPTLC include faster separations and lower solvent consumption. The performances of AP-MALDI-MS and vacuum MALDI-MS have been compared in the analysis of small drug molecules directly from the UTLC plates. The desorption from the irregular surface of UTLC plates with an external AP-MALDI ion source combined with an ion trap instrument provides clearly less variation in measurements of m/z values when compared with a vacuum MALDI-time-of-flight (TOF) instrument. The performance of the UTLC-AP-MALDI-MS method has been applied successfully to the purity analysis of synthesis products produced by solid-phase parallel synthesis method.

Atmospheric Pressure↗

PubMed2005

Fabrication of enclosed SU-8 tips for electrospray ionization-mass spectrometry.

We describe a novel electrospray tip design for MS which is fabricated completely out of SU-8 photoepoxy. A three-layer SU-8 fabrication process provides fully enclosed channels and tips. The tip shape and alignment of all SU-8 layers is done lithographically and is therefore very accurate. Fabrication process enables easy integration of additional fluidic functions on the same chip. Separation channels can be made with exactly the same process. Fluidic inlets are made in SU-8 during the fabrication process and no drilling or other postprocessing is needed. Channels have been fabricated and tested in the size range of 10 microm x 10 microm-50 microm x 200 microm. Mass spectrometric performance of the tips has been demonstrated with both pressure-driven flow and EOF. SU-8 microtips have been shown to produce stable electrospray with EOF in a timescale of tens of minutes. With pressure driven flow stable spray is maintained for hours. Taylor cone was shown to be small in volume and well defined even with the largest channel cross section. The spray was also shown to be well directed with our tip design.

Epoxy Resins↗

PubMed2005

Analysis of amphetamines and fentanyls by atmospheric pressure desorption/ionization on silicon mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry and its application to forensic analysis of drug seizures.

The suitability of atmospheric pressure desorption/ionization on silicon mass spectrometry (AP-DIOS-MS) and matrix-assisted laser desorption ionization mass spectrometry (AP-MALDI-MS) for the identification of amphetamines and fentanyls in forensic samples was studied. With both ionization techniques, the mass spectra recorded showed abundant protonated molecules, and the background did not disturb the analysis. The use of tandem mass spectrometry (MS/MS) allowed unambiguous identification of the amphetamines and fentanyls. AP-DIOS-MS/MS and AP-MALDI-MS/MS were also successfully applied to the identification of authentic compounds from drug seizures. Common diluents and tablet materials did not disturb the analysis and compounds were unequivocally identified. The limits of detection (LODs) for amphetamines and fentanyls with AP-DIOS-MS/MS were 1-3 pmol, indicating excellent sensitivity of the method. The LODs with AP-MALDI-MS/MS were about 5-10 times higher.

Amphetamines↗

PubMed2005

Tetraalkylammonium halides as chemical standards for positive electrospray ionization with ion mobility spectrometry/mass spectrometry.

Chemical standards for positive ion mode electrospray ionization ion mobility spectrometry/mass spectrometry (ESI(+)-IMS/MS) are suggested. The low clustering tendency of tetraalkylammonium halides makes them ideal chemical standards for ESI(+)-IMS/MS. A homologous series of these compounds forms a useful external standard for instrument testing and resolution calibration of an IMS instrument. Selected homologues or a mixture of tetraalkylammonium halides can be used as mobility standards in the analytical runs. Absolute and relative reduced mobilities were calculated for C2--C8, C10 and C12 tetraalkylammonium halides. Absolute reduced mobilities in nitrogen were 1.88, 1.56, 1.33, 1.15, 1.02, 0.92, 0.84, 0.73, and 0.67 cm2 V(-1) s(-1), for C2--C8, C10 and C12 tetraalkylammonium halides, respectively. Relative reduced mobilities (relative to 2,6-di-tert-butylpyridine) for the same compounds were 1.20, 1.00, 0.855, 0.743, 0.658, 0.59, 0.54, 0.47, and 0.43, respectively.

Halogens↗

PubMed2004

Atmospheric pressure photoionization-mass spectrometry with a microchip heated nebulizer.

A novel, microfabricated heated nebulizer chip for atmospheric pressure photoionization-mass spectrometry (APPI-MS) is presented. The chip consists of fluidic and gas inlets, a mixer, and a nozzle etched onto silicon wafer that is anodically bonded to a Pyrex glass wafer, on which an aluminum heater is sputtered. A krypton discharge lamp is used as the source for 10-eV photons to initiate the photoionization process. Dopant, delivered as part of the sample solution, is used to achieve efficient ionization. The use of the microfabricated heated nebulizer with APPI in the analysis of four analytes is demonstrated, and the spectra are compared to those obtained with a conventional APPI source. Ionization in positive and negative ion modes was successfully achieved and the spectra were mainly similar to those obtained with conventional APPI, indicating that the ionization in microfabricated and conventional APPI sources takes place by the same mechanisms. The flow rates with conventional APPI are approximately 100 muL/min, whereas the microchip heated nebulizer allows the use of flow rates 0.05-5 muL/min, thus being compatible with microfluidic separation systems or micro- and nano-LC. A stable signal was demonstrated throughout a 5-h measurement, which proved the excellent stability of the micro-APPI. The same heated nebulizer chip can be used for weeks.

Atmospheric Pressure↗

PubMed2004

Microchip atmospheric pressure chemical ionization source for mass spectrometry.

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

Atmospheric Pressure↗

PubMed2004

Development of an ion mobility spectrometer for use in an atmospheric pressure ionization ion mobility spectrometer/mass spectrometer instrument for fast screening analysis.

An ion mobility spectrometer that can easily be installed as an intermediate component between a commercial triple-quadrupole mass spectrometer and its original atmospheric pressure ionization (API) sources was developed. The curtain gas from the mass spectrometer is also used as the ion mobility spectrometer drift gas. The design of the ion mobility spectrometer allows reasonably fast installation (about 1 h), and thus the ion mobility spectrometer can be considered as an accessory of the mass spectrometer. The ion mobility spectrometer module can also be used as an independently operated device when equipped with a Faraday cup detector. The drift tube of the ion mobility spectrometer module consists of inlet, desolvation, drift, and extraction regions. The desolvation, drift and extraction regions are separated by ion gates. The inlet region has the shape of a stainless steel cup equipped with a small orifice. Ion mobility spectrometer drift gas is introduced through a curtain gas line from an original flange of the mass spectrometer. After passing through the drift tube, the drift gas serves as a curtain gas for the ion-sampling orifice of the ion mobility spectrometer before entering the ion source. Counterflow of the drift gas improves evaporation of the solvent from the electrosprayed sample. Drift gas is pumped away from the ion source through the original exhaust orifice of the ion source. Initial characterization of the ion mobility spectrometer device includes determination of resolving power values for a selected set of test compounds, separation of a simple mixture, and comparison of the sensitivity of the electrospray ionization ion mobility spectrometry/mass spectrometry (ESI-IMS/MS) mode with that of the ESI-MS mode. A resolving power of 80 was measured for 2,6-di-tert-butylpyridine in a 333 V/cm drift field at room temperature and with a 0.2 ms ion gate opening time. The resolving power was shown to be dependent on drift gas flow rate for all studied ion gate opening times. Resolving power improved as the drift gas flow increased, e.g. at a 0.5 ms gate opening time, a resolving power of 31 was obtained with a 0.65 L/min flow rate and 47 with a 1.3 L/min flow rate for tetrabutylammonium iodide. The measured limits of detection with ESI-MS and with ESI-IMS/MS modes were similar, demonstrating that signal losses in the IMS device are minimal when it is operated in a continuous flow mode. Based on these preliminary results, the IMS/MS instrument is anticipated to have potential for fast screening analysis that can be applied, for example, in environmental and drug analysis.

Journal Article↗

PubMed2004

Negative ion-atmospheric pressure photoionization-mass spectrometry.

The ionization mechanism in the novel atmospheric pressure photoionization mass spectrometry (APPI-MS) in negative ion mode was studied thoroughly by the analysis of seven compounds in 17 solvent systems. The compounds possessed either gas-phase acidity or positive electron affinity, whereas the solvent systems had different polarities and gas-phase acidities and some of them positive electron affinities. The analytes that possessed gas-phase acidity formed deprotonated ions in proton transfer; in addition, fragments and solvent adducts were observed. The compounds of positive electron affinity formed negative molecular ions by electron capture or charge exchange and substitution products of form [M - X + O](-) by substitution reactions. The efficiency of deprotonation was decreased if the solvent used possessed higher gas-phase acidity than the analyte. Solvents of positive electron affinity captured thermal electrons and deteriorated the ionization of all the analytes. Also, the proportion of substitution products was affected by the solvent. Finally, the performances of negative ion APPI and negative ion APCI were compared. The sensitivity for the studied compounds was better in APPI, but the formation of substitution products was lower in APCI.

Journal Article↗

PubMed2004

Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry.

A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1-2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.

Anabolic Agents↗

PubMed2004

A new maximum likelihood approach with asymmetric residual distribution for multicomponent mass spectra analysis.

This paper proposes a new maximum likelihood approach for the deconvolution of identity and quantity of individual compounds based on the multicomponent mass spectra measured by mass spectrometry (MS). Mixture analysis of multicomponent mass spectra is, typically, based on a linear multicomponent mass spectrum model, where the compounds of the measured spectra to be solved are explicitly stated and assumed to be known. In many cases, however, the measured spectrum may contain unknown compounds that are not explicitly stated in the model and a commonly used least square (LS) solution fails. Moreover, a standard improvement over the LS method in these cases, namely the M-estimation (ME) approach, also suffers from this same problem. Our method overcomes the limitations of the LS and ME methods by modeling the effect of the unknown compound(s) to the residual of the linear model. The experimental results presented show that this new approach can separate more robustly the complex multicomponent mass spectra into their individual constituents compared to the LS and ME methods.

Air Pollutants↗

PubMed2003

Development of LC/MS/MS methods for cocktail dosed Caco-2 samples using atmospheric pressure photoionization and electrospray ionization.

Good reliability of Caco-2 permeability studies requires competent sampling and analytical methods to ensure the comparability of day-to-day experiments. In this work, two n-in-one LC/MS/MS methods based on two different ionization techniques were developed and validated for a group of reference compounds; eight of them are recommended by the Food and Drug Administration (FDA) for the evaluation of oral drug permeability. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), as an interface for quantitative LC/MS analysis was evaluated in comparison to the electrospray ionization (ESI). Generally, the validation parameters, including sensitivity, accuracy, and repeatability, were comparable for the APPI and ESI methods. The main difference was that the linear quantitative range of APPI was 3-4 orders of magnitude (r(2) >/= 0.998) whereas in ESI it was typically 2-3 orders of magnitude (r(2) >/= 0.990). By the APPI and ESI methods, the simultaneous analysis of nine highly heterogeneous compounds was achieved within 5.5-7 min, which leads to significant savings in time and cost of the analyses. The successful validation data indicate the usefulness of both the methods for the rapid and sensitive (LOD values typically </=5 nM) testing of drug transport across the Caco-2 monolayers, and after case-specific modification, the methods enable the permeability screening of new drug candidates. The applicability and reliability of both methods was demonstrated with a real batch of Caco-2 samples, each providing similar values of apparent permeability (P(app)). In addition, the individual permeabilities of the compounds in the cocktail experiments correlated closely to the ones obtained from single-compound experiments.

Journal Article↗