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Biomedical subjects

Tyge Greibrokk

Publications and source records attributed to Tyge Greibrokk.

At least 19 recordsLinked to original sources
PubMed2006

Evaluation of reversed phase columns designed for polar compounds and porous graphitic carbon in "trapping" and separating neurotransmitters.

Quantification of neurotransmitters as biologically active analytes in neurological samples is of high interest for studying their effect on multiple targets. This work is part of a strategy involving two-dimensional liquid chromatography (2D LC) system with mass spectrometry (MS) detection. The concept of the on-line LC system is the coupling of reversed phase liquid chromatography (RPLC, the second separation dimension) to ion-exchange chromatography (IEC, the first dimension). Our objective in this study is to find the appropriate second dimension column, ensuring that samples of neurotransmitters are refocused and separated on it. Silica-based columns designed specifically to retain polar compounds were tested in LC conditions and compared with results obtained with a porous graphitic carbon (PGC, Hypercarb) column. These polar embedded, polar endcapped, and high-density alkyl chain columns successfully separated analytes in question using mobile phase systems with high percentage of water, or even pure water. Only Hypercarb column provided efficient retention of the most polar neurotransmitters and could be used for trapping and preconcentrating the compounds without rapid breakthrough.

Chromatography, Ion Exchange↗
PubMed2006

Limitations of porous graphitic carbon as stationary phase material in the determination of catecholamines.

A fast and sensitive capillary liquid chromatography (cLC) column-switching method with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection for the simultaneous determination of dopamine (D), epinephrine (E), norepinephrine (NE) and serotonin (SE) was pursued. A sample volume of 100 microl was loaded with a mobile phase containing 0.1% pentafluoropropionic acid (PFPA) as ion-pairing agent on a 25 mm x 0.32 mm (i.d.) 5 microm Hypercarb column. A water-acetonitrile (AcN) gradient with 0.1% acetic acid (AcOH) backflushed the compounds onto a 34 mm x 0.32 mm (i.d.) 5 microm Hypercarb analytical column. However, during a series of analyses, oxidation of the catecholamines (CAs) was observed. This was suspected to be due to the loading mobile phase composition and precluded the usefulness of this method even though the achievable detection limit was in the range of 0.75-3.0 ng/ml. The combination of the porous graphitic carbon (PGC) material and the fluorinated strong acids which were required to get enough retention for preconcentration of large volumes cannot be used for easily oxidized compounds as the CAs.

Catecholamines↗
PubMed2006

Two-dimensional capillary liquid chromatography: pH gradient ion exchange and reversed phase chromatography for rapid separation of proteins.

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4,000 A) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pH-fractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.

Animals↗
PubMed2006

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry.

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.

Chromatography, High Pressure Liquid↗
PubMed2006

Short communication controlling LC-SPE-NMR systems.

There are several stages of the LC-SPE-NMR process that should be monitored closely to ensure an efficient isolation and concentration of the target analyte, for instance analyte break-through and compound transfer from the LC-SPE to the NMR probe. In this study, analyte break-through monitoring was performed with a UV detector and a mass spectrometer placed after the SPE unit. Easy break-through was a problem when attempting multiple trapping of various compounds using C18 SPE cartridges with the original commercial system. However, on lowering the flow rate over the SPE system and using SPE cartridges packed with porous carbon, the number of trappings possible increased five-fold. To increase control over the on-line SPE-NMR transfer, a gradient pump-UV system was used to elute compounds trapped on an SPE to an NMR probe. The analyte band was placed in the active volume of the probe by a stop-flow mechanism. The modified LC-SPE system was also coupled with off-line NMR analysis for determination of a degradation product of the insecticide monuron, present in the low ppm range.

Journal Article↗
PubMed2006

Determination and removal of impurities in 2-D LC-MS of peptides.

Problems occurring during operation of a 2-D LC-MS system for separation and identification of neuropeptides, such as contamination of the used salts and column bleed, are described. When using polysulfoethyl aspartamide, which is widely used as a strong cation exchange stationary phase in the first dimension, interfering peaks were observed in the second-dimension reversed-phase chromatograms. The observed peaks, found to be caused by column bleeding, had abundance above the threshold value and influenced the quality of the analyses. The origin of the peaks was verified and appropriate measures are proposed. Additionally, peaks caused by polyethylene glycols (PEGs), covering approximately 5 min of feasible chromatographic time in every fraction, were observed. The commercial ammonium formate salts used to prepare the first-dimension mobile phase were found to contain PEG impurities, and in subsequent work the salt solutions were prepared from formic acid and ammonia to avoid any additional contaminations.

Animals↗
PubMed2006

Epimerization and stability of two new cis-benzo[a]pyrene tetrols by the use of liquid chromatography-fluorescence and mass spectrometry.

Quantitative determination of the hydrolysis products from proteins and DNA gives valuable information regarding the reactive metabolite that forms the protein and DNA adduct. Quantification of protein-benzo[a]pyrene (BP) adducts represents a more sensitive method than quantification of BP-DNA adducts. The aim of the present study was to identify two hydrolysis products from BP-derived protein adducts found in vitro and in vivo in a previous study. Male Wistar rats were injected i.p. with BP, and serum albumin was isolated and subjected to acid hydrolysis at 70 degrees C for 3 h. The hydrolysate was subjected to LC separation, and fractions of the two unknown compounds were collected. The molecular masses of the two unknown compounds were in accordance with being tetrols as judged by LC electrospray mass spectrometry. The fragmentation patterns were characteristic of tetrols with formation of the molecular ion and the loss of water molecules. In addition, the compounds were subjected to acid hydrolysis at 70 degrees C with 0.1 M HCl for 3 h. We observed that two of the known tetrols epimerized to the two unknown tetrols and vice versa. This is probably a characteristic epimerization involving not only position C(10)-OH but also another site like position C(7)-OH. The in vivo findings of the two unknown adducts are probably the result of the formation of BPDE III in the metabolism of BP. These two tetrols must then have the C(7)-OH and C(8)-OH groups in a cis position.

Animals↗
PubMed2006

Temperature effects in liquid chromatography.

Temperature has a large effect on retention, selectivity, and column efficiency and has long been accepted as an important parameter in liquid chromatography (LC). Despite this fact, temperature has not been very actively utilized in the past, mainly because of reported stability problems of the most commonly used stationary phases. However, more interest in the application of temperature for retention control has come of late because of the trend of miniaturization in chromatography and the availability of temperature-stable stationary phases. This work gives an overview of temperature effects on retention and selectivity in chromatography, especially on reversed-phase columns. Instrumental requirements, especially with respect to performing temperature gradient elution, are discussed and applications on both conventional-sized analytical columns and capillary columns are included.

Chromatography, Liquid↗
PubMed2005

Comparison of advanced oxidation processes and identification of monuron photodegradation products in aqueous solution.

The photodegradation of monuron (3-(4-chlorophenyl)-1,1-dimethylurea) in aqueous solutions under simulated solar irradiation has been conducted by different advanced oxidation processes (UV/H(2)O(2), UV/H(2)O(2)/Fe(2+), UV/H(2)O(2)/TiO(2), UV/TiO(2), dark H(2)O(2)/Fe(3+)). The degradation rates were always higher for the homogeneous catalysis in photo-Fenton reactions (UV/H(2)O(2)/Fe(2+)) compared to the heterogeneous photocatalytic systems (TiO(2)/UV and UV/H(2)O(2)/TiO(2)). Optimal concentrations of Fe(2+) and H(2)O(2) for the abatement of the herbicide in the photo-Fenton system were found to be 1 mM Fe(II) and 10 mM H(2)O(2). Several intermediary products were identified using large volume injection micro-liquid chromatography with UV detection (mu-LC-UV), mu-LC-MS and GC-MS techniques and a degradation mechanism has been proposed.

Catalysis↗
PubMed2005

Combined solid-phase extraction and 2D LC-MS for characterization of the neuropeptides in rat-brain tissue.

A comprehensive two-dimensional capillary liquid chromatographic (2D LC) method has been established for determination of neuropeptides in rat brain tissue. Rats were exposed to different levels of stress before sacrificing and the aim of this study was to design a powerful separation and detection technique capable of characterizing differences between cerebral neuropeptide expression as a function of stress level. Rat brain samples were homogenized and subjected to clean-up by solid-phase extraction (SPE) on both a reversed-phase (C(18)) and a weak cation-exchange (CBA) cartridge. The samples were divided in two fractions (A and B) depending on retention on the CBA column. Subsequently, 50 microL of the sample were injected on to a strong cation exchanger (SCX) at a mobile phase pH of 3, which enabled preconcentration of positively charged compounds. The trapped compounds were eluted using step gradients of ammonium formate in water-ACN (90:10, v/v). Before enrichment in the second dimension, the eluate from the first dimension was diluted with water containing 0.1% TFA. The compounds eluting from the first dimension were trapped in the second dimension using a dual precolumn system consisting of two short capillary columns packed with Kromasil C(18), 10 microm particles. Subsequently, the trapped compounds were backflushed on to a 10 cm long, 320 microm I.D. analytical column packed with Kromasil C(18) 3.5 microm particles, on which they were efficiently separated. Detection was performed using an ion-trap mass spectrometer (ITMS) in both the MS and the MS-MS mode. Comparison of base-peak chromatograms (BPC) from MS analysis of stressed and non-stressed rats clearly revealed several differences in neuropeptide expression. The MS-MS data obtained combined with Mascot software were employed for peptide identification.

Animals↗
PubMed2005

Determination of benzo[a]pyrene tetrols by column-switching capillary liquid chromatography with fluorescence and micro-electrospray ionization mass spectrometric detection.

The present work displays capillary liquid chromatographic column switching methodology tailored for determination of benzo[a]pyrene tetrol isomers in biological matrices using on-line fluorescence and micro-electrospray ionization mass spectrometric detection. A well-established off-line crude solid phase extraction procedure was used in order to make the method compatible with several biological matrices. The solid phase extraction eluates were evaporated to dryness, redissolved in 1.0 ml methanol:water (10:90, v/v), loaded onto a 0.32 mm I.D. x 40 mm 5 microm Kromasil C(18) pre-column for analyte enrichment and back-flushed elution onto a 0.30 mm I.D. x 150 mm 3.5 microm Kromasil C(18) analytical column. The samples were loaded with a flow rate of 50 microl min(-1) and the tetrols were separated at a flow rate of 4 microl min(-1) with an acetonitrile:10 mM ammonium acetate gradient from 10 to 90%. A sample loading flow rate up to 50 microl min(-1) was allowed. The fluorescence excitation and emission were set to 342 and 385 nm, respectively, while mass spectrometric detection of the benzo[a]pyrene tetrols was obtained by monitoring their [M - H](-) molecular ions at m/z 319. The method was validated over the concentration range 0.1-50 ng ml(-1) benzo[a]pyrene tetrols in a cell culture medium with 100 microl injection volume, fluorescence detection and the first eluting tetrol isomer as model compound, resulting in a correlation coefficient of 0.993. The within-assay (n= 6) and between-assay (n= 6) precisions were determined to 2.6-8.6% and 3.8-9.6%, respectively, and the recoveries were determined to 97.9-102.4% within the investigated concentration range. The mass limit of detection (by fluorescence) was 3 pg for all the tetrol isomers, corresponding to a concentration limit of detection of 30 pg ml(-1) cell culture medium. The corresponding mass spectrometric mass limits of detection were 4-10 pg, corresponding to concentration limits of detection of 40-100 pg ml(-1) cell culture medium.

Carcinogens↗
PubMed2005

Determination of bradykinin and arg-bradykinin in rat muscle tissue by microdialysis and capillary column-switching liquid chromatography with mass spectrometric detection.

Quantification of bradykinin peptides in limited amounts of rat muscle tissue dialysate has been performed using a packed capillary LC-ESI-TOF-MS method. The micro dialysate samples (450 microL) with added internal standard were loaded onto a 1 mm x 5 mm loading column packed with 5 microm Kromasil C18 particles by a carrier solution of 0.1% formic acid in ACN/water (5:95, v/v) at a flow rate of 250 microL/min for online preconcentration of the analytes. Back-flushed elution onto a 150 mm x 0.5 mm Zorbax C18 column packed with 5 microm particles was conducted using a linear solvent ACN/H2O gradient containing 0.1% formic acid. (Tyr8)-bradykinin was used as an internal standard and was added to the dialysis sample prior to injection. Baseline separation of bradykinin, arg-bradykinin and (tyr8)-bradykinin was achieved within 10 min. Positive ESI was performed in the m/z range of 200-1300. The method was validated in the range 0.2-1.0 ng/mL dialysate, yielding correlation coefficients of 0.995 and 0.990 for bradykinin and arg-bradykinin, respectively. The within-assay and between-assay precisions were between 4.3-9.6% and 6.2-10.6%, respectively. Both arg-bradykinin and bradykinin were detected in dialysate from rat muscle tissue, at concentrations of 0.1 and 0.4 ng/mL for bradykinin and arg-bradykinin, respectively, confirming the presence of arg-bradykinin in rat muscles.

Animals↗
PubMed2005

Reaction of glyoxal with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, cytidine, thymidine, and calf thymus DNA: identification of DNA adducts.

Glyoxal (ethanedial) is an increasingly used industrial chemical that has been found to be mutagenic in bacteria and mammalian cells. In this study, the reactions of glyoxal with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, cytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The nucleoside adducts were isolated by reversed-phase liquid chromatography and characterized by their UV absorbance and 1H and 13C NMR spectroscopic and mass spectrometric features. The reaction with 2'-deoxyguanosine gave one adduct, the previously known 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one adduct. The reaction of 2'-deoxyadenosine with glyoxal resulted in the formation of a previously not reported N6-(hydroxyacetyl)-2'-deoxyadenosine adduct. In the reaction of glyoxal with 2'-deoxycytidine and cytidine at neutral conditions and 37 degrees C, 5-hydroxyacetyl pyrimidine derivatives were obtained. When the cytidine reaction was performed at pH 4.5 and 50 degrees C, the 5-hydroxyacetyl derivative of uridine was formed through deamination of cytidine-glyoxal. Adducts in the thymidine reaction could not be detected. In the reaction of glyoxal with calf thymus DNA, the 2'-deoxyguanosine-glyoxal and 2'-deoxyadenosine-glyoxal adducts were obtained, the former being the major adduct.

Cytidine↗
PubMed2004

Temperature-programmed packed capillary liquid chromatography coupled to evaporative light-scattering detection and electrospray ionization time-of-flight mass spectrometry for characterization of high-molecular-mass hindered amine light stabilizers.

High-molecular weight-hindered amine light stabilizers (HMW-HALSs) are of utmost importance in modern polyolefin stabilization technology and in-depth knowledge about their chemical composition, particularly the oligomers, is essential for development of new and more efficient stabilizers. In the present study, the applicability of temperature-programmed packed capillary LC coupled to miniaturized ELSD and positive mode ESI-TOF-MS for analysis of HMW-HALSs is demonstrated through extensive characterization of two state-of-the-art stabilizers, i.e., HALS-1 and HALS-2. Both stabilizers were individually separated on a 320 microm i.d. x 35 cm long Hypersil 3 microm ODS-100 column using a temperature program from 30 to 120 degrees C and a quaternary mixture of ethylacetate, acetonitrile, triethylamine (TEA) and acetic acid (45.0:44.9:10.0:0.1 (v/v/v/v)) as the mobile phase. The effect of using various amounts of ethylacetate, acetonitrile and triethylamine in the mobile phase on the chromatographic separation is demonstrated. Furthermore, the LC-ESI-TOF-MS analyses revealed that HALS-1 (oligomeric) was highly complex and consisted of at least five different mass series, while HALS-2, which was assumed to be monomeric, contained two different mass series. Chemical structures for nearly all species of both stabilizers are proposed.

Amines↗
PubMed2004

Isoelectric point separation of proteins by capillary pH-gradient ion-exchange chromatography.

In the present work, isoelectric point (pl) separation of proteins by pH-gradient ion-exchange chromatography (IEC) on packed capillary columns is demonstrated. The development of a miniaturized flow-through pH probe for reliable pH monitoring of the column effluent, which was an important technical challenge for adapting this technique to capillary dimensions, was solved by designing a low microliter per minute flow rate housing to a commercially available micro pH probe. Highly linear outlet pH-gradients within the pH range 8.5-4.0 were obtained when applying simple inexpensive buffers consisting solely of piperazine, N-methylpiperazine and imidazole on 10 cm x 0.32 mm i.d. fused silica capillaries packed with anion-exchange poly(styrene divinylbenzene)-based macroporous materials, i.e. 10 microm Mono P from Amersham Biosciences and 10 microm PL-SAX from PolymerLabs. Furthermore, when using a pH-gradient from 6.8 to 4.3, both columns were able to baseline separate the A and B genetic variants of beta-lactoglobulin, which differ with two amino acid residues only, but the PL-SAX column provided almost a two-fold decrease in peak widths compared to the Mono P column. The influence of varying the buffer concentration, injection volume and column temperature on the peak widths and resolution of the beta-lactoglobulins was investigated, e.g. a 100 microl sample of dilute beta-lactoglobulins was injected directly on the column with practically no increase in peak width as compared to what obtained with conventional injection volumes. Finally, a pH-gradient from 6.8 to 4.3 was used to separate proteins in skimmed bovine milk on the PL-SAX column. The milk was simply diluted 1:10 (v/v) with water and filtrated before injection.

Chromatography, Ion Exchange↗
PubMed2004

Studies on the long-term thermal stability of particulate and monolithic poly(styrene-divinylbenzene) capillaries in reversed-phase liquid chromatography.

In the present study, the long-term high-temperature (>80 degrees C) and temperature programming stability of fused silica capillaries packed with 5 microm PLRP-S 300 A and monolithic PS-DVB capillaries (both 180 microm id x 6 cm) under reversed-phase conditions has been examined. In isothermal mode, the columns were defined as temperature-stable when a less than 10% change in apparent retention factors (k) and a less than 20% change in "retention time/peak width"-factors (n) of the probe solutes (proteins) were observed after passing 7,500 void volumes of effluent through the columns (about 100 h operation). According to these criteria, the PLRP-S and monolithic capillaries were defined temperature-stable at 100 and 130 degrees C, respectively. Furthermore, when continuously running temperature programs between 50 degrees C and the upper temperature limit determined in isothermal mode, virtually no change in k or n were observed on neither of the columns after running more than 35,000 void volumes or 1,600 temperature programs. Additionally, temperature-programmed reversed-phase separations of proteins on both types of capillaries are demonstrated and discussed.

Journal Article↗
PubMed2004

Sensitive biomonitoring of phthalate metabolites in human urine using packed capillary column switching liquid chromatography coupled to electrospray ionization ion-trap mass spectrometry.

A rapid and sensitive method for the determination of the phthalate monoesters monoethyl phthalate (MEP), monobutyl phthalate (MBP), monobenzyl phthalate (MBzP) and monoethylhexyl phthalate (MEHP), in human urine, using packed capillary column liquid chromatography coupled to electrospray quadrupole-ion trap mass spectrometry (ESI-QITMS(n)) has been developed. Sample volumes of 200 microL of deconjugated and diluted urine were loaded onto a precolumn of 30 mmx0.32 mm I.D. packed with Hypercarb 5 microm particles, using a sample carrier consisting of acetonitrile/water (15/85, v/v, adjusted to pH 2 using HCl) with a flow rate of 20 microL/min. Backflushed elution onto a 100 mmx0.32 mm I.D. analytical column packed with 5 microm Hypercarb particles was conducted using a tetrahydrofuran/water gradient where both solvents contained 10 mM ammonium acetate, at a flow rate of 4 microL/min. Determination of the monophthalates was achieved within 8 min. Ionization was performed in the negative mode and the analytes were observed as [M-H](-) at m/ z=193.1, 221.1, 255.1 and 277.0 for MEP, MBP, MBzP and MEHP, respectively. Quantification was performed in the multiple reaction monitoring (MRM) mode monitoring the fragments at m/ z=121.1, 177.0, 183.0 and 233.0 for MEP, MBP, MBzP and MEHP, respectively. The method was validated over the concentration range 2.5-125 ng/mL in pretreated urine samples, corresponding to 25-1250 ng/mL untreated urine, yielding correlation coefficients in the range 0.996-0.999. The within-assay ( n=6) and between-assay ( n=6) repeatabilities were in the range 4.0-18% and 4.8-15% RSD, respectively. The mass limits of detection were in the range 32-70 pg, corresponding to concentration limits of detection of 1.6-3.5 ng/mL of untreated urine.

Chromatography, Liquid↗