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Biomedical subjects

D Westerlund

Publications and source records attributed to D Westerlund.

At least 37 records · Page 2Linked to original sources
PubMed1996

Direct injection of large volumes of plasma in a column-switching system for the analysis of local anaesthetics. I. Optimization of semi-permeable surface precolumns in the system and characterization of some interference peaks.

Possibilities for accomplishing direct injection of large volumes (500 microliters) of plasma samples into a column-switching HPLC system were investigated. A new format of precolumn containing a semi-permeable surface (SPS) support (1 cm x 1 cm) was used for the sample clean-up and trace enrichment and was combined with a Kromasil C18 column for the final separation. A stable chromatographic system with respect to the separation selectivity and separation time was constructed and evaluated. The main parameters were the hydrophobicity of the SPS column, pH of the eluents, concentration of the organic modifier in the eluents and the detection wavelength. Two main interference peaks that were eluted in front of the ropivacaine peak were systematically characterized by varying the loading conditions for the SPS precolumn. The SPS column could tolerate large volumes (< or = 500 microliters) of plasma injections with a total volume of more than 50 ml. The developed system is stable, which permits the detection of 30 ng/ml ropivacaine in human plasma.

Amides↗
PubMed1996

Capillary zone electrophoresis and micellar electrokinetic chromatography, with taurodeoxycholate as micellar agent, of protein kinase A peptide substrates.

The separation of protein kinase A peptide substrates with the general formula -X-Arg-Arg-Ala-Ser-Y-, where X and Y may be the same or different amino acids, was studied by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Taurodeoxycholate (TDC) was used as the micellar agent. CZE was effective in separating a peptide series differing in the number of amino acids, but not for a series with a difference in the terminating amino acid. For the latter series, MEKC generally gave a higher selectivity, but some of the peptide pairs were more easily separated by CZE, demonstrating the complementary character of the two techniques. The efficiency of the MEKC system was typically < 50% of that of CZE, but its higher selectivity generally outbalanced the lower efficiency regarding resolution. The distribution of the peptides to the micelles was studied by determination of retention factors. Electrostatic and hydrophobic forces were found to be determining factors in the distribution; the most highly charged basic peptides were most heavily distributed, and for peptides with the same charge those containing more hydrophobic amino acids were more strongly distributed. The contribution of some structural features to the distribution degree was also determined.

Amino Acid Sequence↗
PubMed1996

Indirect UV detection of carbohydrates in capillary zone electrophoresis by using tryptophan as a marker.

A rapid and sensitive method for the analysis of sugars was developed by capillary zone electrophoresis (CZE) with indirect UV detection. A mixture of nine mono- and oligosaccharides was separated in about 20 minutes on a fused silica capillary in a highly alkaline (pH > 12) background electrolyte (BGE). With tryptophan as the marker detected at 280 nm the femtomole level detection of sugars was possible. The system was optimized by studies on the buffer pH, the marker concentration, as well as the inner diameter of the capillary. The indirect detection of sugars was also performed with a cationic marker, N-benzylcinchonidium chloride (BCDC). However, the detection sensitivity was much lower in comparison to the tryptophan marker system.

Buffers↗
PubMed1995

Performance of amino-silylated fused-silica capillaries for the separation of enkephalin-related peptides by capillary zone electrophoresis and micellar electrokinetic chromatography.

Enkephalin-related peptides were separated at low pH in a capillary with covalently bonded aminopropyl groups. The peptides are electrostatically repelled from the capillary surface and much higher efficiencies and faster separations were achieved compared to separations using uncoated capillaries. At low pH the amino groups are protonated, which results in reversed electroosmosis. The influence of voltage and ionic strength on the mobility and the separation efficiency was studied. The repeatability of migration times within one day was very good with relative standard deviations of 0.3-0.7%. Increasing the pH decreased the electroosmosis, eventually turning towards the cathode in the pH range 5-6; the separation performance, however, was lower at higher pH. Neutral and anionic micellar agents were added to the background electrolyte at different concentrations; the enkephalins had weak association with the neutral micellar agents but were distributed to the anionic taurodeoxycholic acid (TDC) micelles, giving rise to changes in separation selectivities. Very high efficiencies were obtained for peptides with a low distribution to the TDC micelles, while the efficiencies were impaired for those with a strong association with the micelles, which may indicate a slow mass transfer in the association process.

Amino Acid Sequence↗
PubMed1995

Capillary electroseparations of enkephalin-related peptides and protein kinase A peptide substrates.

The separations of enkephalin-related peptides and protein kinase A peptide substrates, with the common structural feature -Arg-Arg-X-Ser-Val-, were studied in micellar electrokinetic chromatography (MEKC) systems and compared with the capillary zone electrophoresis (CZE) mode. The influence of the magnitude and the direction of the electroosmotic flow on the selectivity was studied. Reversed electroosmosis was obtained by adding a hydrophobic amine, dimethyldodecylamine, to the background electrolyte; the amine forms cationic micelles with a low critical micelle concentration (0.3 mM). The neutral micellar agent, Brij 35, competes with the amine for adsorption sites on the capillary surface decreasing the reversed electroosmosis. In such a system, mixed cationic micelles are formed to which the peptides were not distributed at low pH, but an improved resolution was obtained due to the effects on electroosmosis. In systems containing the less hydrophobic amine dimethyloctylamine, in which probably no mixed micelles are formed, an improved separation of protein kinase A peptide substrates was obtained due to distribution to Brij 35 micelles. In separations of enkephalins, a high pH gave very low efficiencies due to surface-analyte interactions, and the best CZE separations were obtained at low pH. Changes in migration order were observed in the pH range 2-3, possibly due to differences in peptide pKa values or conformation changes of the peptides. The enkephalins were only to a small extent distributed to the Brij 35 micelles, but this improved the separation at pH 2 compared to the CZE mode.

Amines↗
PubMed1994

Determination of amide-type local anaesthetics by direct injection of plasma in a column-switching high-performance liquid chromatographic system using a pre-column with a semipermeable surface.

A high-performance liquid chromatographic method using column switching was applied to the direct determination of two local anaesthetics, ropivacaine and bupivacaine, in human plasma. The method is intended to be used in a combined LC-GC system; here only the LC-part is described. After addition of internal standard, the samples were filtered and directly injected into a semipermeable surface (SPS) pre-column where the analytes were strongly retained and separated from many endogenous compounds by a short washing step. The retained analytes were transferred by a buffered methanol phase from the pre-column into a carbonaceous HPLC column and they were detected by UV detection at 254 nm. The SPS pre-column could withstand numerous (> 200) direct injections of plasma samples (10 microliters). The method has a detection limit of 8.2 ng and requires a total assay time of 15 min per plasma sample. Quantitative recoveries were obtained over the range 3.3-114 micrograms/ml with inter-day precisions of 1.6-5.2% (C.V.).

Amides↗
PubMed1993

Capillary electroseparations of some di-, tri-, and tetrapeptides and enkephalin-related peptides.

Electrophoretic mobilities and capacity factors for short peptides were determined in micellar electrokinetic systems (MEKC) using UV detection at 210 nm. On the basis of determined capacity ratios, taurodeoxycholic acid was found to be a selective micellar agent for peptides. Structural features other than hydrophobicity were found to be decisive for the distribution of the peptides to the micellar phase. Basic peptides of enkephalin-type containing arginine were more highly distributed to the micelles than others, indicating the importance of electrostatic forces in the distribution mechanism. In systems with polyacrylamide-coated capillaries, the micellar velocity toward the anode is larger than the electroosmosis, and conditions permitting splitting of a series of related peptides, i.e., some peptides migrating with the micelles and others toward the cathode resulting in infinite selectivities, were developed. Separation by micellar electrokinetic chromatography is demonstrated to be a suitable alternative for peptides that are difficult to separate by capillary zone electrophoresis.

Amino Acid Sequence↗
PubMed1992

Separation of (R)- and (S)-naproxen using micellar chromatography and an alpha 1-acid-glycoprotein column: application for chiral monitoring in human liver microsomes by coupled-column chromatography.

A column-switching system for fast determination of (R)- and (S)-naproxen in liver microsomes has been developed. The centrifuged sample was injected directly onto a pre-column with octadecylcoated silica. The retained analytes were then directed to an alpha 1-AGP column using a mobile phase composed of phosphate buffer (pH 6.5), dimethylocytylamine (30 mM) and the nonionic surfactant, Tween 20 (40 g/l). The method gave high absolute recoveries and good repeatabilities: 99.6% (1.7% relative standard deviation) and 94.9% (2.4% R.S.D.) for the (R)- and (S)-naproxen, respectively. The use of a surfactant in combination with an aliphatic amine in the mobile phase involves reduced retention times with retained enantioselectivity. Furthermore, the presence of the surfactant makes it possible to inject biological samples directly into the chromatographic system.

Chemistry Techniques, Analytical↗
PubMed1992

Separation of glucuronides from urine by coupled-column separation using underivatized silica as precolumn.

Glucuronides are separated from urine by coupled-column separations (CCSs). The fraction containing the glucuronide(s) is transferred on-line from a silica precolumn to the analytical column (octadecyl derivatized silica), enriched, and separated by ion-pair chromatography. The retention and selectivity on the precolumn are controlled by pH, buffer components, organic modifier, and ion-pair agent. After the injection of filtered urine samples, glucuronides with different chemical properties can be separated. The total analysis of morphine-3-glucuronide and morphine-6-glucuronide is accomplished in less than six minutes, with UV detection at 210 nm.

Buffers↗
PubMed1989

Determination of phosphonoformate (foscarnet) in biological fluids by ion-pair reversed-phase liquid chromatography.

Bioanalytical liquid chromatographic methods for the determination of phosphonoformate (foscarnet) have been developed. Biological fluids, after simple pre-treatment (ultrafiltration and/or treatment with charcoal), were injected into a reversed-phase liquid chromatographic system with electrochemical detection. Foscarnet was retained as an ion pair with tetrahexylammonium; addition of pyrophosphate was necessary in order to obtain an acceptable peak. This additive could also be used for the fine regulation of the retention to achieve the necessary selectivity.

Chromatography, High Pressure Liquid↗
PubMed1989

Effects of system peaks in a coupled column system for noscapine and its metabolites.

System peaks created in a coupled column system, after transfer of the precolumn mobile phase to the analytical column have been investigated. Co-elution of an analyte with a system peak may affect the chromatographic performance of the analyte. The possibility of using the peak compression effect, in order to increase the detectability of the compounds noscapine, narcotoline and cotarnine, respectively, is studied. Three different mobile phases for the analytical column have been tested, the first contained a co-ion, the second a counter-ion and the last both a co-ion and a counter-ion. A compressed noscapine peak is obtained not only when it is eluted in a positive gradient of the co-ion in the first system, but also in a positive gradient of the counter-ion in the second system. Slightly compressed narcotoline and cotarnine peaks are obtained in the third system as long as they eluted before the deficiency peak of the counter-ion.

Chromatography, Liquid↗
PubMed1988

Determination of noscapine and its metabolites in plasma by coupled-column liquid chromatography.

Noscapine, narcotoline and cotarnine were quantified in deproteinized plasma samples by using a coupled-column liquid chromatographic system. The drug and the metabolites were first separated into two groups on a short polar precolumn (-CN) with an acidic mobile phase, containing a low content of acetonitrile. The metabolites were transferred to a hydrophobic analytical column (C18) and separated with a mobile phase containing a counter ion and a co-ion in an acidic buffer with an high acetonitrile content. Noscapine was transferred to another hydrophobic analytical column (C18) with a mobile phase containing a counter ion in an acidic buffer with an high acetonitrile content. Ultraviolet detection at 310 nm was used for all three compounds. The limits of quantitation were 9 ng/ml for noscapine, 13 ng/ml for cotarnine and 20 ng/ml for narcotoline. The within-day precisions were better than 6% (relative standard deviation), and the absolute recoveries were above 82%.

Alkaloids↗
PubMed1988

Effects of system peaks in ion-pair reversed-phase liquid chromatography for noscapine and metabolites.

System peaks were generated in an ion-pair reversed-phase system by co-injection of an alkylsulphate with the analytes. The acidic mobile phase contained acetonitrile and an aliphatic tertiary amine as a co-ion. The retention time of the system peak was regulated by the concentration and hydrophobicity of the co-ion and the alkylsulphate. The peak performance of the analytes was affected by co-elution with a system peak. Both peak distortions and improvements appeared, and the principles for the latter could be applied in a dual-column system involving trace enrichment and column switching.

Amines↗
PubMed1987

Determination of N-acetylcysteine, intact and oxidized, in plasma by column liquid chromatography and post-column derivatization.

N-Acetylcysteine in plasma may exist as intact N-acetylcysteine (NAC) or be oxidized to disulphides, either as a dimer or mixed with other thiol-containing compounds. To prevent oxidation of NAC, whole blood was immediately centrifuged after collection and the plasma proteins were precipitated with perchloric acid. NAC was measured by direct injection of the supernatant into the chromatographic system and the oxidized forms, coupled to small sulphides (ONACS), were determined after reductive cleavage of all NAC disulphides in the supernatant with dithiothreitol before injection. The total plasma concentration of the compound, i.e., including the fraction coupled to proteins (ONACP), was assayed after an initial reduction of the disulphide linkages in plasma. After subsequent precipitation of proteins, the supernatant was directly injected. The chromatographic system was a reversed-phase column (C18) with an acidic mobile phase. After a fast (less than 6 s) post-column reaction with pyrenemaleimide, NAC was detected by fluorimetry. The contribution to the band broadening by the reactor was about 10%. The limit of quantification of NAC in plasma was 240 nM, with an intra-assay precision of 14%.

Acetylcysteine↗
PubMed1987

New method for the measurement of eosinophil migration.

A new application of the Boyden chamber method for the measurement of eosinophil migration, without the need of eosinophil isolation, has been developed. A cell suspension containing a mixture of granulocytes, neutrophils to the greater part, was used. The eosinophils were identified by staining their granules with Chromotrope 2R. The method made it possible to study the migration of eosinophils from normal individuals without eosinophilia. Control experiments demonstrated that the chemotactic and chemokinetic response of eosinophils in the granulocyte mixture was in accordance with the response of isolated eosinophils from the same donor. Normal eosinophils demonstrated a significant chemotactic response to C5f, platelet-activating factor (PAF), leukotriene B4 (LTB4), and f-meth-leu-phe (f-MLP). Furthermore, PAF was demonstrated to be significantly more eosinophil chemotactic than neutrophil chemotactic.

Caseins↗
PubMed1985

Automated determination of amoxycillin in biological fluids by column switching in ion-pair reversed-phase liquid chromatographic systems with post-column derivatization.

Amoxycillin, a polar aminopenicillin, is rather unstable in biological fluids. Degradation can be prevented by fast sample pretreatment and storage at -70 degrees C or below. After pH adjustment, it is stable in biological fluids for over 16 h. The samples were handled by automated chromatography overnight. The chromatographic system consisted of a small guard column, two analytical columns separated by a switching valve, a post-column reactor and a fluorescence detector. The chromatographic events and the calculation of results were handled by a computing integrator. The chromatography was based on ion-pairing principles. An efficient clean-up of the biological fluids was obtained by a heart cut from the first column, where the neutral mobile phase contained hexyl sulphate. In the second column the organic anion was exchanged for a large quaternary ammonium compound; amoxycillin was then retained as an ion pair. The composition of the mobile phases had to be designed carefully in order to avoid a disturbance of the chromatographic performance on the last column. An adequate selectivity and sensitivity was obtained by a post-column derivatization with fluorescamine. Detection limits were 10 and 25 ng/ml for plasma and urine, respectively, and the inter-assay precisions at low levels (350 and 2000 ng/ml for plasma and urine, respectively) were ca. 5% (R.S.D.).

Amoxicillin↗
PubMed1985

Oral cyclacillin interacts with the absorption of oral ampicillin, amoxycillin, and bacampicillin.

The relative bioavailabilities of single oral doses of ampicillin, amoxycillin, and bacampicillin were compared with and without concomitant administration of a six-times higher molar dose of cyclacillin. As the absorption of cyclacillin has been shown to involve a capacity-limited transport system in animals, it was selected as the reference compound for the study. The treatments were given to 14 fasting volunteers using a randomized, complete crossover design. The drugs in plasma and urine were determined by liquid chromatography. Renal clearance was 17%, 10% and 19% lower when ampicillin, amoxycillin, and bacampicillin were given together with cyclacillin. Consequently, differences in the relative bioavailability were based on urinary recoveries assuming constant non-renal clearance. When amoxycillin was given with cyclacillin there was a 67% delay in the time of the plasma peak concentration, and an 8% lower urinary recovery than when it was given alone. There was a 50% and 33% delay in the tmax of ampicillin and bacampicillin when combined with cyclacillin; the urinary recovery of ampicillin in the combination was 10% lower but that of bacampicillin was similar. There was also a 20% delay in the tmax of cyclacillin when combined with amoxycillin. The differences in renal clearance indicate an interaction in the renal elimination of the drugs, but the effect was probably not the explanation for the marked shift in time of the absorption of these rapidly absorbed drugs. The results support the existence of a capacity-limited transport system for aminopenicillins in the human gut.

Administration, Oral↗