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J F Stobaugh

Publications and source records attributed to J F Stobaugh.

At least 37 records · Page 2Linked to original sources
PubMed1992

Characterization of sulphoalkyl ether derivatives of beta-cyclodextrin by capillary electrophoresis with indirect UV detection.

A capillary electrophoresis method which characterizes the degrees of substitution of heterogeneous sulphoalkyl ether beta-cyclodextrin derivatives is described. The separation is based on the different electrophoretic mobilities observed from changes in the overall charge of the molecule as a result of substitution. Individual peaks of the electropherogram then provide a measure of each degree of substitution of the present beta-cyclodextrin. Detection of these beta-cyclodextrin derivatives is performed by indirect UV detection.

Benzoates↗
PubMed1991

Reversed-phase ion-pair liquid chromatography of the angiotensins.

The isocratic reversed-phase liquid chromatography of the angiotensins and a number of their synthetic analogues is described. Complete separation of 10 out of 12 peptides was achieved through a solvent optimization strategy with a total analysis time of about 20 min. The retention behavior of the angiotensins studied was described in terms of the hydrophobic contribution of their amino acid residues; there was good correlation between predicted and experimental retention for those peptides that were retained by a common mechanism. However, because ion-pair chromatography was required for good peak symmetry, retention was substantially modulated by the presence of acidic and basic residues. The limit of detection of these peptides was 3-5 pmol by UV absorbance at 214 nm. For those peptides containing a primary amino group the detection limit was improved by two orders of magnitude by fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde/cyanide to the corresponding N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives. The contribution of the CBI ring system to retention was also investigated.

Amino Acid Sequence↗
PubMed1990

Stereoselective pharmacokinetics and metabolism of the enantiomers of cyclophosphamide. Preliminary results in humans and rabbits.

[R(+),S(-)]-Cyclophosphamide [(R,S)-CP] is an anticancer drug, containing a chiral phosphorous atom, which is prepared and used clinically as the racemic mixture. A new high-performance liquid chromatographic assay suitable for pharmacokinetic studies of CP enantiomers in plasma has been reported recently by this laboratory (Reid et al., Anal Chem 61: 441-446, 1989). Briefly, the assay involves ethyl acetate extraction of CP enantiomers from plasma followed by derivatization to diastereomers in a two-step process utilizing chloral and (+)-naproxen acid chloride. Chromatographic analysis was performed on a reversed phase (ODS) column with detection at 232 nm. In the present study, preliminary results on the applicability of this assay to pharmacokinetic studies are presented. Several rabbits were used to compare the influence of i.p., i.v., and oral routes of administration on the stereoselective disposition of (R,S)-CP. Following i.p. administration, S-CP was cleared faster than R-CP. Following oral administration, only R-CP was detectable in plasma, while i.v. administration resulted in minor or no stereoselective disposition. These results indicated that there was a marked stereoselective metabolism of the S-CP enantiomer, with the i.p. and oral routes producing the greatest differences due to first-pass metabolism. Incubation of rabbit-liver microsomes with (R,S)-CP demonstrated that the monooxygenase system can exhibit marked stereoselectivity in its metabolism of CP. The ratio of R-CP to S-CP in the incubation medium increased during the incubation period from 1:1 initially to 4.5:1 after 60 min. The results from the experiments with rabbits indicate that the first-pass metabolism of this drug is highly stereoselective; in contrast, cancer patients who had received (R,S)-CP as an i.v. infusion showed no stereoselectivity in the elimination of the enantiomers. Pharmacokinetic studies with cancer patients, receiving (R,S)-CP as an oral dose, are in progress in order to determine if stereoselective first-pass metabolism of this drug also occurs in humans.

Administration, Oral↗
PubMed1990

Determination of the anticancer drug, 15-deoxyspergualin, in plasma ultrafiltrate by liquid chromatography and precolumn derivatization with naphthalene-2,3-dicarboxaldehyde/cyanide.

An alternative analytical method for the determination of 15-deoxyspergualin in plasma is described. The drug was initially separated from the plasma matrix by ultrafiltration and a precolumn derivatization step was performed with naphthalene-2,3-dicarboxaldehyde in the presence of sodium cyanide to yield the fluorescent N-substituted 1-cyanobenz[f]isoindole (CBI) derivative. The CBI derivative was separated and quantitated by reversed-phase chromatography using an ODS Hypersil column and mobile phase of KH2PO4 (0.1 M)-H3PO4-acetonitrile (48:0.8:52, v/v/v) containing dodecyl sodium sulphate (18 mM). The excitation and emission wavelengths for the fluorescence detector were 420 and 490 nm, respectively. The peak height was linearly related to drug concentration over the range from 5 ng ml-1 (10 nM) to 10 micrograms ml-1 (20 microM) in phosphate buffer (0.1 M, pH 7.0), spiked plasma ultrafiltrate and ultrafiltrate obtained from spiked plasma. Measurements could be made with a relative standard deviation of 4.5% or less in phosphate buffer (0.1 M, pH 7.0), 6.1% or less in spiked plasma ultrafiltrate and 12% or less in ultrafiltrate obtained from spiked plasma.

Antibiotics, Antineoplastic↗
PubMed1990

Chemiluminescence: a sensitive and versatile method for analyte detection.

The generation of light from the oxidation of oxalic esters with hydrogen peroxide has been applied to the detection of luminescent materials. In order to improve the efficiency of this method, which is less than 0.1%, and to enhance the selectivity for target analytes, an in-depth investigation of the oxalate ester-hydrogen peroxide reaction has been conducted. A kinetic model has been developed based on the effects of catalysts, reagents and reaction conditions for maximum light production. Application of the model to liquid chromatography through the "time-dependent emission window" concept affords a predictable maximum sensitivity for selected analytes. Application to the detection and quantitation of met- and leu-enkephalins which have been labelled with naphthalene-2,3-dicarboxyaldehyde/cyanide provides support for this methodology. Other bioanalytical applications are presented.

Humans↗
PubMed1990

Fluorogenic derivatization of peptides with naphthalene-2,3-dicarboxaldehyde/cyanide: optimization of yield and application in the determination of leucine-enkephalin spiked human plasma samples.

Initial attempts to derivatize the alpha-amino site of several tripeptides with naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN) resulted in poor yields of the expected N-substituted 1-cyanobenz[f]isoindole (CBI) products. Examination of the CBI-formation mechanism, in conjunction with knowledge of the general structure-reactivity properties of the tripeptides, led to the recognition of a competing non-productive reaction pathway. Through the use of model reactions and the isolation and structural elucidation of a predicted side-product the viability of the competing pathway was confirmed. From an understanding of the key features of both the productive and non-productive reaction pathways, a rational approach for the optimization of CBI-derivative yield was proposed and confirmed experimentally. This information led, in turn, to the development of HPLC methodology suitable for the determination of leu-enkephalin spiked into human plasma; fluorescence detection was used in conjunction with leu-enkephalin amide as the internal standard. The method enabled leu-enkephalin to be determined at a concentration of 0.31 nmol ml-1 with an error of less than 4% for 25 pmol injected.

Chemical Phenomena↗
PubMed1990

Multidimensional liquid chromatography of opioid peptides: fluorogenic labelling, retention prediction and separation optimization.

The ultra-trace analysis of opioid peptides in biological samples can be achieved by multidimensional liquid chromatography with pre-column fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion. However, in order to take full advantage of the high sensitivity possible with detectors based on laser-induced fluorescence or chemiluminescence, each component of the analytical method must be carefully optimized. In this study, strategies are presented for the prediction of retention time and the optimization of separations of derivatized opioid peptides in multidimensional LC systems.

Acetonitriles↗
PubMed1989

Multi-dimensional high-performance liquid chromatography of opioid peptides following pre-column derivatization with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion. Preliminary results on the determination of leucine- and methionine-enkephalin-like fluorescence in the striatum region of the rat brain.

The reversed-phase high-performance liquid chromatography of three synthetic opioid peptides, 5leucine-enkephalin, 5methionine-enkephalin and [D-2alanine]-5methionine enkephalin, has been studied after their pre-column fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide to the corresponding 1-cyanobenz[f]isoindole (CBI) derivatives. The chromatographic properties of the three synthetic CBI-peptides were characterized using three different stationary phases, ODS Hypersil, CPS Hypersil and Spherisorb Phenyl, eluted with mobile phases containing various concentrations of methanol, tetrahydrofuran or acetonitrile in 26 mM trifluoroacetic acid, adjusted to pH 3.5. The data obtained using single chromatographic columns were used to design a multi-dimensional system in which the three synthetic CBI-peptides of interest were transferred as a single fraction from one column to a second. The first column served to separate the peptides from the majority of the material in the samples, and the second column was used to separate the three CBI-peptides from each other. The best separation was achieved in which the first column was Spherisorb Phenyl and the second column was ODS Hypersil. Both columns were eluted with a mobile phase of 45% acetonitrile (v/v) in 26 mM trifluoroacetic acid (pH 3.5) at a flow-rate of 1.0 ml/min. The method has been applied to the determination of leucine- and methionine-enkephalin-like fluorescence in the striatum of the rat brain.

Animals↗
PubMed1989

Analysis of 5-fluorouracil in plasma by precolumn derivatization with 4-bromomethyl-7-methoxycoumarin, followed by multi-dimensional high-performance liquid chromatography.

An assay for 5-fluorouracil (5-FU) has been developed that utilizes a double extraction with ethyl acetate, followed by precolumn derivatization with 4-bromo-methyl-7-methoxycoumarin. The reaction mixture was quenched with 5% acetic acid, extracted with hexane, and analyzed by multi-dimensional high-performance liquid chromatography. Derivatized 5-FU was injected into a cyanopropyl column and a heart cut containing the analyte was then switched to an octadecyl column and quantitated by fluorescence detection. The assay had a limit of detection of 0.5 ng 5-FU/ml plasma and was linear to 20 micrograms/ml. It was shown to be free of interferences from the other anticancer agents commonly used in combination with 5-FU. This assay should have the sensitivity needed to measure the low levels that occur after low-dose, continuous infusion of 5-FU.

Animals↗
PubMed1989

Liquid chromatographic determination of cyclophosphamide enantiomers in plasma by precolumn chiral derivatization.

On the basis of reactions described in the synthetic literature, a two-step chiral derivatization sequence was developed for the anticancer agent cyclophosphamide (CP). The sequence involves amidoalkylation of CP with anhydrous chloral containing 1% dimethylformamide followed by acylation of the resulting secondary alcohol with a chiral carboxylic acid chloride, (+)-6-methoxy-alpha-methyl-2-naphthaleneacetyl chloride, to form a diastereomeric pair. Derivatized (-)-CP and (+)-CP exhibited retention times of 17.2 and 20.7 min, respectively, when chromatographed on Hypersil ODS with acetonitrile/phosphate buffer (pH 6.8) as the mobile phase. Preparation of the individual diastereomers from enantiomerically pure CP enabled correlation of the chromatographically observed peaks with a particular enantiomer. Various aspects of the overall assay methodology have been systematically investigated (derivatization solvents, temperatures, reaction time, and work-up procedure) and optimized on the scale required for trace analysis in biological fluids. Calibration curves were established for each enantiomer in spiked human plasma over the therapeutically relevant concentration range of 0.99-49.94 micrograms/mL.

Chemical Phenomena↗
PubMed1989

High-performance liquid chromatography of 5-fluorouracil after derivatization with 4-bromomethyl-7-methoxycoumarin. Characterization of the derivative and the use of column switching for the improvement of resolution and the enhancement of sensitivity.

The derivatization of 5-fluorouracil with 4-bromomethyl-7-methoxycoumarin has been reported previously; however, the structure of the derivative was not confirmed. The synthesis and purification of the 5-FU derivative is described along with the spectroscopic (MS and NMR) determination that it is labelled at both heterocyclic nitrogens as expected. A column switching HPLC system is also presented which consists of primary separation on a cyanopropyl column followed by a final separation on an ODS column with fluorescence detection. This system removes all interferences from the derivatization system and has a limit of detection for the pure derivative of less than 50 fmol (injection volume = 100 microliters).

Chromatography, High Pressure Liquid↗
PubMed1989

Suitability of DTAF as a fluorescent labelling reagent for direct analysis of primary and secondary amines--spectral and chemical reactivity considerations.

DTAF has been used successfully to prepare fluorescent labelled reagents for fluorescence polarization immunoassays. Its applicability as a derivation reagent for direct fluorescence analysis of primary and secondary amines was evaluated. DTAF was shown to have spectral properties that closely resemble those of fluorescein and that are apparently insensitive to the presence of the triazine nucleus. Spectrally determined pKa values also closely resemble those of fluorescein and other amino aryl-s-triazines. DTAF is prone to hydrolytic degradation with the rate of reaction increasing with increasing pH, until a pH value is reached at which ionization of the amine bridging the two aromatic nuclei occurs; at this pH the rate reaches a plateau value. Both primary and secondary amines react efficiently with DTAF, and the reactivity increases with the increasing basicity of the amine reactants. The reaction is pH-dependent, proceeding most efficiently at pH values at which both the "bridging amine" of DTAF and the amine substrate are unionized. Methyl substituted secondary amines were consistently more reactive than the corresponding primary amine, but further imposition of steric bulk about the amine nitrogen significantly reduced the reactivity of amines toward DTAF. In cases where such steric bulk is minimal. DTAF appears to be a suitable fluorescent labelling reagent for direct analytical applications.

Amines↗
PubMed1988

High-performance liquid chromatography of bis[1,2-bis-(diphenylphosphino)ethane]gold(I) chloride, a potential antineoplastic agent.

The chromatography of [Au(dppe)2]+ (I), a potential antineoplastic drug, was studied on a variety of stationary phases (ODS Hypersil, PLRP-S, Partisil SAX and Partisil SCX) using aqueous mobile phases containing 60% acetonitrile, 15% tetrahydrofuran and various electrolytes. The effects of both the concentration (0-20 mM) and the nature of the electrolytes, added to the mobile phase, on the chromatography of I were investigated. A wide variety of electrolytes were investigated in which the hydrophobicity of both the anion and the cation were varied. The analyte of interest was found to be unretained by the like-charged Partisil SAX column. On the other hand, I was retained on the Partisil SCX by an ion-exchange mechanism and retention could be controlled by manipulating the electrolyte composition of the mobile phase. I was retained on the two reversed-phase materials by a mixture of solvophobic and electrostatic interactions but, under the conditions studied, the latter mechanism was the dominant one. The retention of I on the two reversed-phase materials was influenced much more by the nature and concentration of the cation added to the mobile phase than it was by the nature and concentration of the anion. Therefore, manipulation of the nature and concentration of the cationic species in the mobile phase appears to afford the most useful means of manipulating the retention of I, and presumably its analogues, on reversed-phase columns.

Antineoplastic Agents↗
PubMed1988

Chemical derivatization as a strategy to enhance detectability of agents used in cancer management.

The bioanalysis of drugs used in the management of cancer is often complicated by the lack of selectivity and sensitivity. Chemical derivatization of these drugs prior to their chromatographic analysis represents a viable strategy to improve chromatographic resolution and to enhance detectability. This review provides examples of how this approach can meet these objectives. Derivatization of racemic cyclophosphamide with a chiral acylating agent, following hydroxyalkylation to introduce a reactive centre into the molecule, provides the basis for its stereospecific analysis. The analysis of dianhydrogalactitol is described, in which diethyldithiocarbamate is used as a nucleophilic derivatizing agent that improves chromatographic behaviour and analytical sensitivity. The final example that is described is the design and preparation of improved fluorogenic reagents (o-phthalaldehyde analogues) for the derivatization of peptides and application of these reagents to the trace analysis of leu-enkephalin in plasma.

Journal Article↗
PubMed1986

Aspects of the stability of isoindoles derived from the reaction of o-phthalaldehyde-ethanethiol with primary amino compounds.

The stability of a series of fluorescent isoindoles formed under analytical conditions following the reaction of o-phthalaldehyde (OPA) and ethanethiol (ET) with a series of primary amines is reported. Increasing the bulk and degree of substitution of the isoindole N-substituent resulted in substantial increases in isoindole stability. The effects of excess reagents on isoindole stability is examined and OPA is observed to accelerate isoindole degradation whilst ET provides a stabilizing effect. Comparison with previously reported data involving the use of 2-mercaptoethanol revealed that ET clearly forms the more stable isoindole derivatives, i.e. a minimum of five-fold improved stability based on the time for 10% degradation to occur. Identification of the major degradation product together with kinetic data suggests that degradation proceeds via autoxidation.

Journal Article↗
PubMed1985

Rational design and evaluation of improved o-phthalaldehyde-like fluorogenic reagents.

Evidence was presented suggesting that the fluorescent isoindole produced by reaction of o-phthalaldehyde (OPA), ethanethiol, and primary amine was formed by initial imine formation followed by conversion to an alpha-alkylaminobenzylsulfide and subsequent ring closure to form the isoindole nucleus. This mechanism suggested that the minimum structural requirement for condensation to an isoindole was an o-diacyl benzene in which one of the carbonyl groups was aldehydic. A major drawback of OPA as an analytical reagent is the limited stability of the fluorescent 1,2-disubstituted isoindole. Since isoindole instability is related to autoxidation at C-3, the use of o-(formyl) arylketones as alternatives to OPA is attractive in increasing the lifetime of the fluorescent species in that such reagents would form 1,2,3-trisubstituted isoindoles. Two compounds, o-acetylbenzaldehyde (OAB) and o-benzoylbenzaldehyde (OBB), were synthesized and evaluated as potential fluorogenic reagents. Both formed fluorescent products. The rate of formation of isoindole from the latter was too slow to make it of practical analytical value; however, OAB formed isoindoles with t1/2 less than 10 s and offered markedly improved stability over that observed with OPA.

Aldehydes↗