Biomedical subjects

I S Krull

Publications and source records attributed to I S Krull.

At least 19 recordsLinked to original sources

PubMed1992

9-Fluoreneacetyl-tagged, solid-phase reagent for derivatization in direct plasma injection.

We describe here a resin-based derivatization reagent, containing a 9-fluoreneacetyl tag on a controlled-pore substrate, for direct injection analysis of amphetamine in plasma. On-line, pre-column derivatization was performed by direction injection of diluted plasma sample into an sodium dodecyl sulfate-containing mobile phase. Amphetamine was trapped in the hydrophobic derivatization column and derivatized at elevated temperature by the activated solid-phase reagent. The derivatized 9-fluoreneacetyl amphetamide was separated by reversed-phase high-performance liquid chromatography with a step gradient and determined by fluorescence detection. The synthesis scheme, characterization, and optimization of the derivatization conditions for the solid-phase reagent are described. The method was evaluated by reproducibility tests and single blind spiking analysis. This solid-phase reagent combined with a surfactant containing mobile phase provided a sensitive and simple procedure for on-line derivatization in direct injection analysis of biological fluids.

Amphetamine↗

PubMed1992

Capillary electrophoresis: the promise and the practice.

The field of capillary electrophoresis has undergone rapid development over the past 10 years. Many advances have been made, but there are still some problems that must be addressed before the technique can reach its full potential.

Amino Acid Sequence↗

PubMed1992

Interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry for analysis of proteins.

Molecular weights (MWs) of different proteins were determined by interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry (IEC-LALLS). A high-performance strong cation-exchange column was used to elute proteins using fast (5 min) and conventional (15-30 min) gradients. The eluted proteins were characterized on-line by determining their MWs using LALLS. The specific refractive index (RI) increment (dn/dc) and the RI of the solvent used over the gradient range were determined off-line and used to calculate the absolute weight-average MWs. Four proteins, ribonuclease A, alpha-chymotrypsinogen A, trypsinogen and beta-lactoglobulin A (beta-LACT) were studied. Accurate MWs were obtained for all the proteins using fast and conventional gradients, except for beta-LACT, which aggregated as a function of the gradient employed. The degree of aggregation of beta-LACT increased as the rapidity of the gradient was increased over a fixed gradient range. This study indicated that it is possible to separate and characterize proteins rapidly using IEC-LALLS.

Chromatography, Ion Exchange↗

PubMed1992

Comparison of 9-fluorenylmethoxycarbonyl and 9-fluoreneacetyl-tagged silica-based derivatization reagents in high-performance liquid chromatography.

Two silica reagents based on a 4-hydroxy-3-nitrobenzoyl backbone were synthesized and characterized with 9-fluorenylmethoxycarbonyl (FMOC) and 9-fluoreneacetyl (FA) tags. These reagents were tested by derivatization of primary and secondary amines. Derivatization conditions such as temperature, time and triethylamine catalyst were tested. The FA-tagged silica reagent showed better performance than the FMOC-tagged silica reagent by a comparison of derivatization efficiencies, stabilities of reagents, and blank reagent interferences with derivatization. Finally, cadaverine and an aliphatic amine mixture were analysed using the FA-tagged reagent by pre-column, off-line derivatization and fluorescence detection.

Acetates↗

PubMed1991

Electrochemical detection of proteins in high-performance liquid chromatography using on-line, postcolumn photolysis.

Direct detection of proteins in high-performance liquid chromatography electrochemistry (LCEC) is difficult. By using on-line, postcolumn photolysis, proteins now can be detected by LCEC at microgram per milliliter levels. The compatibilities of size exclusion chromatography (SEC), reversed-phase chromatography (RPC), ion-exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC) with photolysis-electrochemical detection is described for proteins together with the analytical figures of merit. Inherent from the advantages of electrochemical detection, the method is sensitive and selective.

Chromatography, High Pressure Liquid↗

PubMed1991

Use of Ce(IV) oxidizing agent for the derivatization of polycyclic aromatic hydrocarbons for liquid chromatography-electrochemical detection.

Polycyclic aromatic hydrocarbons (PAHs) were oxidized with Ce(IV), and the resulting quinones were determined by reductive-mode liquid chromatography-electrochemical detection. This oxidation is a rapid, automatable step, involving of Ce(IV) reagent to the PAH sample and cleaning up the derivative with C18 solid-phase extraction. Using a C18 analytical column and a 2-propanol-phosphate buffer as the mobile phase, detection limits were in the ppb range for naphthalene, phenanthrene and anthracene, with linearity over 3-5 orders of magnitude. Method validation was performed by addition of the PAHs to tap water and determining the levels by reference to a calibration curve. The three PAHs can be simultaneously derivatized and determined under the same chromatographic conditions. Analysis of a motor-oil sample is also shown.

Anthracenes↗

PubMed1991

Quantitation of insulin injection by high-performance liquid chromatography and high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was evaluated as a potential technique for the regulatory analysis of commercial dosage forms of insulin. A comparison was made to a liquid chromatographic analysis presently being proposed as an official monograph in the United States Pharmacopeia. The salient points of this comparison were accuracy, precision and ease of use. Both authentic (i.e. single blind, spiked) samples and commercial pharmaceutical formulations (injections) were examined. Chromatographic analyses of both commercial formulations and authentic samples were characterized by good precision, with accuracy being supported by results from authentic (spiked) samples. Conventional HPCE (by which is meant a non-micellar electrolyte used with an uncoated, unmodified fused-silica capillary) achieved reasonable accuracy, but less than impressive precision, when applied to authentic samples. When used for commercial formulations, this type of HPCE did not produce a level of accuracy suitable for regulatory purposes, even with the use of an internal standard.

Animals↗

PubMed1991

Determination of pituitary and recombinant human growth hormone molecular weights by modern high-performance liquid chromatography with low angle laser light scattering detection.

The determination of molecular weight for pituitary and recombinant human growth hormone (p-hGH/Crescormon and r-hGH/Protropin) has been performed. This has involved on-line coupling of size-exclusion chromatography (SEC) and gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A 5-microns, 300 A, Delta-bond octyl column was used. Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for p-hGH and r-hGH. Known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data has been obtained for both proteins under conventional RP-HPLC gradient elution conditions. SEC data of both hGHs were found to be concentration, mobile phase, and column dependent for the particular analyses. Both medium- and high-resolution SEC-LALLS studies were performed, and all of these determinations further confirmed our RP-HPLC results. On-line LALLs provides certain advantages in identifying aggregates that may be present, even in medium-resolution SEC, where incomplete resolution occurs. The on-line coupling of modern RP-HPLC for biopolymers with LALLS detection represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus higher-order aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.

Chromatography, Gel↗

PubMed1991

Solid-phase reagent containing the 3,5-dinitrophenyl tag for the improved derivatization of chiral and achiral amines, amino alcohols and amino acids in high-performance liquid chromatography with ultraviolet detection.

A solid-phase reaction technique is described for improved derivatization of aliphatic amines, amino alcohols and amino acids. A polymeric activated ester is used for the immobilization of the 3,5-dinitrobenzoyl group, which can then be used for derivatizations of strong or weak nucleophiles, while avoiding solution-phase derivatization conditions. The reagent is easily prepared and can be regenerated after use to attain its original reactivity. The resulting chromatograms are free of system peaks due to excess derivatizing reagent, and sample handling is kept to a minimum. The reagent can be used in conjunction with both reversed- and normal-phase chromatography and can be used for off-line gas chromatographic or high-performance liquid chromatographic (HPLC) derivatizations. In addition, the reagent can be used on-line for derivatization in HPLC. Since the labelling reagent is a strong pi-acid, chiral substrates can be derivatized and separated on a Pirkle-type pi-donor column. The confirmation and quantitation of amphetamine in urine was accomplished using a polymer containing two labelling moieties, p-nitrobenzoyl and 3,5-dinitrobenzoyl. The derivatization and separation of chiral and achiral amines, amino alcohols and amino acids is described.

Alcohols↗

PubMed1991

A mixed-bed, multi-derivatization approach using polymeric reagents for derivatizations of amines in high performance liquid chromatographic detection.

Immobilized, polymeric reagents containing covalently attached tagging groups have been synthesized and reacted individually, off-line or on-line, pre-column in high performance liquid chromatographic (HPLC) detection. These reagents have also been combined into a single, mixed-bed reactor, useful for simultaneously preparing several derivatives from a single analyte, all at the same time. Each derivative possesses different chromatographic and detection properties, dependent on the nature of the original polymeric reagent containing the immobilized, tagging species. These particular reagents were designed to impart Ultraviolet/fluorescence, Ultraviolet/electrochemistry (oxidative/reductive or oxidative-hv-electrochemistry) to the final derivatives. Variations in the amounts/ratios of polymeric reagents contained in a single mixed-bed reactor will lead to varying ratios of the final derivatives. These can be predicted knowing the approximate reactivity of each polymeric reagent, percent derivatizations, and overall rates for each reagent towards a given substrate. In this first example of mixed-bed, polymeric reagents for improved derivatization approaches in chromatography, emphasis has been placed on simple amines or amine-like analytes. Multiple derivatives can be effectively used to improve the identification of an unknown analyte in a complex sample matrix, as well as to improve the detectability of that analyte. As one real world application, amphetamine in human urine was quantitated via on-line derivatizations with a mixed-bed reactor. With the least sampling work-up, the resulting sample solutions were directly injected into the on-line derivatization HPLC system for quantitation. The method was validated by single blind spiking experiments. The precision and accuracy were acceptable.

Amphetamine↗

PubMed1991

A polymeric reagent for derivatization of weak nucleophiles in HPLC-UV.

Microporous and macroporous polystyrene beads are modified to contain various activated centers to which are attached a detector-sensitive label useful for analytical-scale derivatization of nucleophilic species. The effect of surface area, porosity, and percent cross-linkage of the polymeric support containing an o-nitro activated ester is investigated. The polymers are characterized by a loading determination to calculate the amount of active acylating reagent incorporated per gram of dry polymer. The kinetics of reaction with substrates of varying steric hindrance vs. solvent strength are determined with the optimized polymeric support and a second polymeric reagent is synthesized based on that support. The second polymer contains a hydroxybenzotriazole functionality. This polymer has increased electrophilicity and is used to prepare a much stronger acylating reagent than the polymeric o-nitrobenzophenol when labeled with 3,5-dinitrobenzoyl chloride. The polymeric hydroxybenzotriazole (HoBTA-DNB) is able to derivatize much weaker nucleophiles such as alcohols, thiols, and phenols at room temperature in less than 30 seconds.

Alcohols↗

PubMed1991

Coated capillaries and additives for the separation of proteins by capillary zone electrophoresis and capillary isoelectric focusing.

Strategies reported for the separation of proteins in capillary zone electrophoresis and capillary isoelectric focusing are reviewed. The strategies are grouped into two categories: coated capillaries and buffer/sample additives. Success attained with each case and also, more importantly, the limitations of the methodology are discussed. Recent results from our own laboratory in the area of capillary isoelectric focusing in uncoated, fused silica capillaries using additives are summarized. The advantages and disadvantages of coated columns vs. additives are delineated.

Electrophoresis↗

PubMed1990

Determination of aromatic and sulfur-containing amino acids, peptides, and proteins using high-performance liquid chromatography with photolytic electrochemical detection.

Aromatic amino acids, sulfur-containing amino acids, peptides containing such constituents, and proteins can now be detected in high-performance liquid chromatography by the use of on-line, postcolumn, continuous photolytic derivatization with electrochemical (HPLC-hv-EC) detection. The overall approach is a very simple, reproducible, rapid, and fully automatable approach for the determination of certain amino acids, peptides, and proteins with excellent selectivity, sensitivity, and linearities of response. Dual-electrode response ratios, lamp-on/lamp-off behavior, and chromatographic capacity factors all contribute to the enhanced selectivity of the overall HPLC-hv-EC determination for these particular classes of bioorganics and biopolymers. The analytical figures of merit, chromatography detection, and method validation approaches have all been optimally derived and demonstrated reproducible. Applications of the basic methodology to real-world samples are demonstrated and validated.

Amino Acids↗

PubMed1990

Complete on-line determination of biopolymer molecular weight via high-performance liquid chromatography coupled to low-angle laser light scattering, ultraviolet, and differential refractive index detection.

An optically modified high-performance liquid chromatography refractive index detector was developed to allow complete on-line determinations for biopolymer molecular weights. On-line concentration, refractive index, specific refractive index increment (dn/dc2)mu, and Rayleigh factor were determined under flow injection analysis (FIA) and size exclusion chromatography (SEC) conditions using low-angle laser light scattering, ultraviolet, and modified refractive index detection. This instrumental system is capable of determining absolute on-line molecular weights. The error and time requirements involved in conventional methodologies for proteins have been reduced. Sample quantities have been reduced from 150 to 200 mg, in conventional off-line methods, to less than 2 mg for on-line FIA and 0.5 mg for on-line SEC, if mass absorptivities (a) are known. Otherwise, the determination of a will be the most sample-demanding step, requiring about 3 mg of the pure protein. On-line measurements of (dn/dc2)mu are in good agreement with traditional off-line values established at Donnan equilibrium (usually within 5%). In addition, this technique provides true injected mass as determined by the UV detector, after chromatographic exposure where losses may occur, which is then used in the calculation of biopolymer molecular weight.

Calibration↗

PubMed1990

Polymeric dimethylaminopyridinium reagents for derivatization of weak nucleophiles in high-performance liquid chromatography-ultraviolet/fluorescence detection.

This paper introduces a novel polymeric dimethylaminopyridinium 9-fluorenyl-methoxycarbonyl reagent for off-line derivatizations of weak nucleophiles in high-performance liquid chromatography. The method of synthesis and characterization of the polymeric reagent via loading determinations is presented and discussed. Derivatization conditions (solvent, time, and temperature) for primary and secondary alcohols were optimized. As one application, off-line derivatizations of 2-chloro-1-propanol, a potential carcinogen in foodstuffs, were carried out with this polymeric reagent with single-blind and standard addition techniques. A specific sample treatment procedure was also developed. The accuracy and precision of the method were determined and data were statistically evaluated.

1-Propanol↗

PubMed1990

Determination of biopolymer (protein) molecular weights by gradient elution, reversed-phase high-performance liquid chromatography with low-angle laser light scattering detection.

The determination of molecular weights for certain proteins has been performed. This has involved the on-line coupling of gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A new 1.5-micron, non-porous, Monosphere RP-C8 column has been used in order to perform fast and conventional RP-HPLC gradients (5-45 min). Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for ribonuclease A, lysozyme, and bovine serum albumin. Standard mixtures of known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data have been obtained for all three proteins, but only under certain, conventional reversed-phase gradient elution conditions. Between 5-10 min of fast gradient elution, each protein appears to exhibit unusual Mw values, suggestive of aggregate formations. Methods have been developed to define the nature of such aggregates. The on-line coupling of modern RP-HPLC for biopolymers with LALLS represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.

Acetonitriles↗

PubMed1990

Determination of inorganic anions by flow injection analysis and high-performance liquid chromatography combined with photolytic-electrochemical detection.

Post-column, on-line photolytic derivatization in liquid chromatography with electrochemical detection for some inorganic anions is described. Flow injection analysis and ion-pair reversed-phase chromatography followed by photolysis and electrochemical detection were used for the determinations of anions. Several operation conditions, such as mobile phase, lamp used for the photolysis, flow-rate, and applied potential, have been optimized for the determinations. Analytical figures of merit were determined. Method validation was carried out by the analysis of single blind, spiked samples. Inherent from the advantages of electrochemical detection in liquid chromatography, the method is of high sensitivity and selectivity for anion analysis.

Anions↗

PubMed1990

Post-column detection by on-line photolysis-electrochemistry in liquid chromatography; application to the determination of phenylalanine in human urine.

On-line, post-column photochemical derivatization for liquid chromatography-electrochemical detection (LC-hv-EC) has been employed for the analysis of phenylalanine in urine. The method is highly sensitive and selective. The quantitative results show good precision and reproducibility. It can be used easily and has the clinical use of diagnosing phenylketonuria disease.

Chromatography, Liquid↗