Biomedical subjects

W J Underberg

Publications and source records attributed to W J Underberg.

At least 37 records · Page 2Linked to original sources

PubMed1998

Analytical techniques used to study the degradation of proteins and peptides: physical instability.

The physical instability of proteins and peptides as well as the various analytical techniques used to study the various aspects of physical instability have been reviewed. Physical instability of proteins and peptides involve changes in secondary, tertiary and quaternary structures of these compounds. After a general introduction of the subject the literature data of these changes and their analytical aspects have been summarized in a Table.

Chemistry Techniques, Analytical↗

PubMed1998

Derivatization in capillary electrophoresis.

In recent years capillary electrophoresis (CE) has been developed into a versatile separation technique, next to gas and liquid chromatography (LC), well suited for the determination of a wide variety of e.g., pharmaceutical, biomedical and environmental samples. The main advantages of CE over chromatographic separation techniques are its simplicity and efficiency. It is well recognized, however, that the sensitivity and selectivity of the detection are relatively weak points of CE. One way to overcome these limitations is the conversion (derivatization) of the analytes into product(s) with more favourable detection characteristics. Although, in principle, almost any detection mode can be combined with a derivatization procedure, in practice, fluorescence monitoring is favoured in most cases. This paper aims to give a short overview on the various reagents that can be used for pre-, post- and on-column derivatization in CE. First, a short introduction is given on CE as an analytical technique, followed by a discussion of the pros and cons of the various modes of derivatization, a comparison of derivatizations in CE with derivatizations in LC, the principles of fluorescence and prerequisites for a good fluorophore and the potential of using diode lasers in combination with a labelling procedure. With respect to the derivatization reagents the emphasis is on the labelling of amino, aldehyde, keto, carboxyl, hydroxyl and sulfhydryl groups.

Amines↗

PubMed1998

Development and validation of transient isotachophoretic capillary zone electrophoresis for determination of peptides.

Although capillary zone electrophoresis (CZE) is known for its high resolution power and low mass detection limits, the concentration detection limits are rather poor when ultraviolet absorbance detection is used. To overcome this limitation, several on-column transient isotachophoresis (tITP) protocols have been developed and validated for the determination of both cationic and anionic model peptides, separately. Using this preconcentration method, up to 72% of the capillary can be filled with sample solution, without any loss in resolution. Thus, without any modification of the hardware set-up, the sensitivity is increased about two orders of magnitude. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility is observed in the 20 to 100 ng/mL concentration range. For the anionic peptides (N-t-Boc-Pentagastrin and two related peptides), a tITP method was developed using a dynamically coated capillary. The coating was prepared by adding Fluorad FC-135 to the leading electrolyte buffer. In this way a positively charged bilayer was formed on the inside of the capillary, producing an electroosmotic flow towards the outlet using reversed polarity conditions. In this way, acceptable analysis times were achieved. Using the developed tITP method, up to 72% of the capillary can be filled with sample solution as well. The anionic peptides are separated even better than when using CZE conditions. Linearity and reproducibility in the 20-100 ng/mL range proved to be excellent.

Angiotensin II↗

PubMed1998

Oxidation of recombinant methionyl human granulocyte colony stimulating factor.

The oxidation of methionine residues in recombinant methionyl human granulocyte colony stimulating factor with hydrogen peroxide has been investigated. Kinetic data of the oxidation were obtained by using reversed phase-high performance liquid chromatography. The stability-indicating capability of this system was confirmed with micellar electrokinetic capillary chromatography. In the pH range 1.9-7.5, the kobs value for the oxidation process is constant. Above pH 7.5, kobs tends to increase with increasing pH. In the pH range 1.9-11.8, four oxidation products were detected in RP-HPLC. Mass spectrometric analysis revealed that one mono-, one di- and two trioxidation products were formed. Using the cyanogen bromide cleavage method the nature of the oxidation products was determined. The mono-oxidation product is the protein with Met121 oxidized, while the dioxidation product has oxidized Met121 and Met126 residues. The trioxidation products are the proteins with Met121, Met126 and Met137 or Met0, Met121 and Met126 oxidized.

Amino Acid Sequence↗

PubMed1998

Degradation kinetics of three gonadorelin analogues: developing a method for calculating epimerization parameters.

PURPOSE: To develop a method for calculating epimerisation parameters, find out if the kinetics of the independent reactions can be established, and elucidate primary structure-chemical degradation relationships in the degradation kinetics of three gonadorelin analogues. METHODS: The influences of pH, temperature, and buffer concentration on the degradation of the three gonadorelin analogues buserelin, goserelin, and triptorelin were investigated using RP-HPLC. A method was developed to calculate epimerisation and hydrolysis rate constants independently. RESULTS: Explicit structure-degradation mechanism relations were found in the degradation of all three compounds. The L-serine residue was found to be involved in both a solvent-catalysed backbone hydrolysis and a hydroxyl-catalysed epimerisation whereas, the O-tertiary butyl D-serine residue was only involved in proton-catalysed ether hydrolysis. The kinetics of identical reactions in different analogues were generally comparable. CONCLUSIONS: The degradation of the gonadorelin analogues is located at a relatively small number of chemical residues and prediction of the degradation mechanisms and kinetics of other peptides with similar structural elements appears to be possible.

Buffers↗

PubMed1997

Chromatographic analysis of the enantiomers of ifosfamide and some of its metabolites in plasma and urine.

The enantiomers of the cytostatic drug ifosfamide and the two metabolites 2- and 3-dechloroethylifosfamide were isolated from plasma and urine by liquid-liquid extraction with ethyl acetate, resolved on a Chirasil-L-val gas chromatographic column and detected by a nitrogen-phosphorus-selective flame ionisation detector. Resolution of the racemic compounds for identification purposes was also accomplished with high-performance liquid chromatography on a chiral column. The validated gas chromatographic method was suitable to determine the total concentrations and the enantiomeric composition of ifosfamide and its dechloroethylated metabolites in plasma and urine samples from treated patients. Some metabolic preferences in the metabolism of ifosfamide were found.

Adult↗

PubMed1997

Pre-, on- and post-column derivatization in capillary electrophoresis.

This survey gives a short overview of the various reagents and procedures that can be used for pre-, post- and on-column derivatization in capillary electrophoresis. First there is an introduction about capillary electrophoresis as an analytical technique; this is followed by a discussion of the pros and cons of the various modes of derivatization and a comparison with liquid chromatography. In the following paragraphs the reagents for a number of functional groups are discussed. The emphasis is on derivatization of the amino group. Most of the information on the reagents and derivatization procedures is listed in tables together with information on the detection mode, analytes, sensitivity and samples. In addition to the amino group, information is given on labeling of aldehyde, keto, carboxyl, hydroxyl and sulfhydryl groups.

Amino Acids↗

PubMed1997

Determination of 4-hydroxyifosfamide in biological matrices by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the determination of 4-hydroxyifosfamide, a metabolite of ifosfamide, in plasma of cancer patients. The analyte is derivatized to 7-hydroxyquinoline, which can be detached fluorimetrically. The calibration graph is linear in the concentration range 0.05-25 microM, the limit of detection being 40 nM. Any inference from acrolein, another metabolite of ifosfamide, was ruled out. 4-Hydroxyifosfamide is very unstable in plasma and a stabilization procedure by adding citric acid has been developed. Thus treated, the samples were stable for 4 days. Analysis of a patient's plasma samples revealed that the 4-hydroxifosfamide concentration did not exceed 10 microM.

Cerebellar Neoplasms↗

PubMed1997

Degradation kinetics of methacrylated dextrans in aqueous solution.

The kinetics of the hydrolysis of glycidyl methacrylate derivatized dextran (dex-MA), hydroxyethyl methacrylate derivatized dextran (dex-Hema, and hydroxyethyl methacrylate (HEMA) were systematically investigated in aqueous solution in the H0/pH range of -1.8 to 10.4 at 37 degrees C. The degradation products were quantified with reversed-phase HPLC and used to calculate the residual amount of dextran-bound methacrylate esters. In all compounds the degradation reactions follow first-order kinetics, the rate constants being susceptible to both specific acid and specific base catalysis. The reaction rate constant was independent of both the dex-MA concentration and the degree of substitution. The log Kobs-pH profiles can be divided into three parts: a proton-catalyzed, a solvent-catalyzed, and a hydroxyl-catalyzed section. At high acidities, dex-HEMA and HEMA are equally stable, but about seven times less stable than dex-MA. At alkaline pH, the order of stability is HEMA > dex-MA > dex-HEMA. This demonstrates that at alkaline pH dex-HEMA is predominantly degraded by hydrolysis of the carbonate ester, whereas at low pH, hydrolysis of the methacrylate ester is the main degradation route of this compound.

Buffers↗

PubMed1996

Analysis of ifosforamide mustard, the active metabolite of ifosfamide, in plasma.

Ifosforamide mustard is the active metabolite of ifosfamide, a cytostatic drug. In this study a sensitive and selective method for the analysis of ifosforamide mustard in plasma is described. The method consists of direct derivatisation of ifosforamide mustard in plasma with diethyldithiocarbamate and subsequent solid-phase extraction of the resulting derivative. The analysis of the derivatisation product was performed by high-performance liquid chromatography with UV detection. The calibration graph was linear in the concentration range 0.45-45 microM and the minimum detectable concentration was 0.45 mumol. The samples were stabilised by addition of semicarbazide and sodium chloride. A patient's plasma sample was analysed by means of the described method. The ifosforamide mustard concentration was 2.3 microM.

Adult↗

PubMed1996

Degradation kinetics of gonadorelin in aqueous solution.

The degradation kinetics of gonadorelin were investigated systematically with reversed-phase high-performance liquid chromatography. The stability-indicating properties of this system were checked with photodiode array detection and by comparison with capillary zone electrophoretic analysis. Influences of gonadorelin concentration, pH, temperature, buffer ions, and ionic strength on the degradation kinetics were studied. The pH-log Kobs profile can be divided into three parts, a proton, a solvent, and a hydroxyl-catalyzed section, with different degradation products. These degradation products were characterized by mass using LC-MS. Gonadorelin is most stable at pH 5-5.5 with a half-life of 70 days at 70 degrees C. The overall degradation rate constant as a function of the temperature under acidic and alkaline conditions obeys the Arrhenius equation. The gonadorelin concentration and the concentrations of acetate, phosphate, borate, and carbonate buffer have no influence on the decomposition rate of the analyte. Increasing ionic strength led to higher Kobs at pH 2 and lower Kobs at pH 9, but influences were relatively small.

Buffers↗

PubMed1995

Structural identification of the degradation products of the antitumor peptide antagonist [Arg6,D-Trp7,9,MePhe8]substance P (6-11).

The basic hexapeptide antagonist [Arg6,D-Trp7,9,MePhe8]-substance P (6-11) was degraded in acid and alkaline media. In acid solution, only one degradation product is found whereas in alkaline solution at least six products are formed. These compounds were analytically characterized and structurally identified by reversed-phase high-performance liquid chromatography, capillary electrophoresis, liquid chromatography/mass spectrometry, fast atom bombardment tandem mass spectrometry, optical rotation analysis, and chiral gas chromatography. The product formed in acidic solution is the terminally deamidated antagonist [Arg6,D-Trp7,9,MePhe8]substance P (6-11); this product was also found in alkaline degradation mixtures. Other important degradation products originate from racemization of the amino acid residue L-Met, formation of ornithine from Arg, and the oxidation of Met to its sulfoxide form.

Amino Acid Sequence↗

PubMed1995

Degradation kinetics of antagonist [Arg6, D-Trp7,9, MePhe8]-substance P [6-11] in aqueous solutions.

Antagonist [Arg6, D-Trp7,9, MePhe8]-substance P {6-11} was subjected to a systematic stability study in which kinetic parameters were obtained for the degradation of this hexapeptide under several well-defined conditions. The influences of pH, temperature, ionic strength, buffer concentration, and initial concentration of the peptide on the reaction rate constant, kobs, were investigated with a stability-indicating reversed-phase high-performance liquid chromatographic system. From the pH-log kobs degradation profile, obtained at 63 degrees C, it appears that antagonist [Arg6, D-Trp7,9, MePhe8]-substance P {6-11} shows its maximum stability around pH 4.2. The half-life at this pH and temperature is 150 days. In both the hydroxyl- and proton-catalyzed parts of the pH-log kobs degradation profile, the influence of temperature was investigated and Arrhenius plots were constructed. The activation energies in both parts were comparable; however, the frequency factor in the hydroxyl-catalyzed part was 3.3 x 10(4) times higher than in the proton-catalyzed part. Eyring analysis of the data reveals that in both acidic and alkaline media the overall degradation was endotherm (delta H++ as well as delta G++ positive between 273 and 373 degrees K) and the entropy was negative. Increasing ionic strengths in acidic media causes an increase in kobs, while in alkaline media the kobs decreases with increasing ionic strength. Increasing buffer concentrations of acetate, phosphate, and carbonate led to an increase of kobs values. Drug concentrations up to 1 mg/ml at pH 10.8 and constant temperature and ionic strength have no influence on the overall degradation rate. At higher concentrations, above 1 mg/ml, kobs decreases.(ABSTRACT TRUNCATED AT 250 WORDS)

Buffers↗

PubMed1994

Reversed-phase high-performance liquid chromatography and capillary electrophoresis in the stability study of the neuropeptide growth factor antagonist [Arg6,D-Trp7,9,MePhe8]-substance P (6-11): a comparative study.

Reversed-phase high-performance liquid chromatography and capillary zone electrophoresis are widely used in protein and peptide analysis. Degradation of the basic peptide [Arg6,D-Trp7,9,MePhe8]-substance P (6-11) (antagonist G) was monitored with reversed-phase high-performance liquid chromatography, free capillary zone electrophoresis, and capillary zone electrophoresis with a capillary cationic coating. Capillary zone electrophoresis with a dynamically coated capillary provided better separation between antagonist G and its degradation products (formed at pH/Hv 13) than high-performance liquid chromatography and free zone capillary electrophoresis. Rate constants of the alkaline degradation of antagonist G measured with reversed-phase high-performance liquid chromatography and capillary zone electrophoresis with a dynamic coated capillary wall are similar whereas the values measured with free zone capillary electrophoresis are lower. Rate constants for the degradation of antagonist G in acidic media are comparable for the three techniques. It is concluded that capillary zone electrophoresis using a dynamic coating with Fluorad is the most suited of the above-mentioned techniques in analyzing antagonist G and its degradation products.

Amino Acid Sequence↗

PubMed1994

Short-term, combined use of paracetamol and zidovudine does not alter the pharmacokinetics of either drug.

OBJECTIVE: To investigate a possible pharmacokinetic interaction between zidovudine and paracetamol. METHODS: Six patients with AIDS took both zidovudine (AZT; 1000-1200 mg/day) and paracetamol (500 mg every 6 h) for 7 days. Pharmacokinetic monitoring was performed on day 0 (AZT alone) and after 7 days of combined use of paracetamol and AZT. RESULTS: Combined use of paracetamol and AZT did not result in a significant change in any of the calculated pharmacokinetic parameters of AZT or its primary metabolite AZT-glucuronide. In addition, paracetamol pharmacokinetic parameters at day 7 did not differ from those usually reported in the literature. CONCLUSION: Short-term, combined use of paracetamol and AZT does not lead to a change in the pharmacokinetics of either AZT or paracetamol. The effect of long-term use of this combination remains unknown. A recent case report of AZT-induced paracetamol hepatotoxicity [Am J Med 1992;93: 94-96] indicates that clinicians should still be aware of potential drug toxicity when prescribing both AZT and paracetamol.

Acetaminophen↗

PubMed1993

High-performance liquid chromatographic determination of m-iodobenzylguanidine in urine of cancer patients.

An accurate, sensitive, reproducible and selective HPLC assay is presented for the quantitative determination of m-iodobenzylguanidine (MIBG) in human urine. The sample pretreatment involves a solid-phase extraction on Bakerbond SPE cyano columns. The HPLC systems consists of a muBondapak C18 column and 25 mM ammonium phosphate (pH 3.0)-acetonitrile (75:25, v/v) as the mobile phase. Detection is performed by UV absorbance at 254 nm. Log y vs. log x is the response function that yielded the smallest sum of percent relative concentration residuals over the whole concentration range of the assay (0.2-100 micrograms/ml). The excretion profile of MIBG in urine of a three-year old patient is shown.

3-Iodobenzylguanidine↗

PubMed1993

Kinetics and mechanism of the degradation of 1a-acetylmitomycin C in aqueous solution.

The acid-catalyzed degradation of mitomycin C is supposed to be governed, to a certain extent, by the protonation status of the aziridine nitrogen in the molecule as well as the protonation degree of the opened aziridine function in a key intermediate species, formed during mitomycin degradation. In order to obtain information about the contribution of the protonation degrees of these functions in controlling the degradation processes, we investigated the degradation of 1a-acetylmitomycin C in acidic aqueous solutions. In the presence of 0.001 mol/l phosphate buffers five 1-hydroxy and mono-acetyl mitosenes are formed, whereas in 1.0 mol/l acetate buffers a total of eight products could be identified, two of them being diacetyl mitosenes. Over the whole pH range studied the formation of 1,2-Z-mitosenes prevails, indicating that, contrary to mitomycin C, a pH-independent factor controls the ultimate 1,2-stereochemistry.

Buffers↗

PubMed1993

Determination of chloroacetaldehyde, a metabolite of oxazaphosphorine cytostatic drugs, in plasma.

A derivatization high-performance liquid chromatographic method with ultraviolet detection to monitor the plasma concentration of chloroacetaldehyde, a neurotoxic metabolite of oxazaphosphorine drugs, is presented. To prevent the rapid degradation of chloroacetaldehyde, the plasma samples are stabilized with formaldehyde. The method is linear in the concentration range 1-250 nmol/ml. Blood samples from a patient who was treated with a ten-day continuous infusion of ifosfamide were assayed. The chloroacetaldehyde concentrations did not exceed 10 nmol/ml.

Acetaldehyde↗