High-performance liquid chromatographic determination of chloramphenicol and four analogues using reductive and oxidative electrochemical and ultraviolet detection.

A high-performance liquid chromatographic procedure is described for the separation, quantitation and identification of chloramphenicol, dehydrochloramphenicol, nitrophenylaminopropanedione, nitrosochloramphenicol and aminochloramphenicol. An isocratic reversed-phase system with ultraviolet and electrochemical detectors in tandem was assembled and used. The system was constructed with special accommodation to enable us to use the electrochemical detector in both reductive and oxidative modes. Retention characteristics, hydrodynamic voltammograms under reductive and oxidative conditions and ultraviolet absorbance are reported. Applicability of the procedure to biological fluids was demonstrated by separation and detection of chloramphenicol after incubation with human blood.

Analysis of monobutyryl and dibutyryl derivatives of adenosine 3',5'-monophosphate in biological samples using isocratic ion pair high-performance liquid chromatography.

Adenosine 3',5'-monophosphate (cyclic AMP), its dibutyryl and monobutyryl derivatives, and a number of other naturally occurring adenine-containing compounds were separated by isocratic ion pair high-performance liquid chromatography. A mobile phase consisting of 30% methanol in 0.1 M KH2PO4 (pH 4.0) containing 1 mM tetramethylammonium hydroxide as the counterion was used to separate the butyryl derivatives. To sufficiently separate cyclic AMP from other adenine-containing compounds, a mobile phase containing 6% methanol in the same aqueous buffer plus counterion was used. Extraction of these cyclic nucleotides from deproteinized biological samples using disposable reverse-phase extraction columns is described. This not only eliminated lipophilic contaminants, but also served to concentrate the samples. The outlined procedures were used to determine the concentrations of the butyryl derivatives in lung tissue and perfusate following a 35-min lung perfusion with 100 microM N6-O2'-dibutyryl cyclic AMP. The role of this technique in the analysis of cyclic nucleotide derivatives as compared with conventional assay procedures is discussed.

High-performance liquid chromatographic analysis of iodochlorhydroxyquin and hydrocortisone in ointments and creams.

A simple isocratic, high-performance liquid chromatographic (HPLC) assay procedure was developed for the simultaneous determination of iodochlorhydroxyquin and hydrocortisone in ointments and creams using phenyl salicylate as an internal standard. Ointment samples were extracted by direct dissolution in ether. Homogeneous suspensions of the creams were prepared in the mobile phase. The samples were spiked by the addition of standard iodochlorhydroxyquin, standard hydrocortisone, and the internal standard and subsequently extracted with the mobile phase. HPLC was performed using a reverse-phase microparticulate C-18 column, a precolumn, and a UV detector set at 256 nm. A mobile phase containing methanol and 0.05 M phosphoric acid (70:30) was employed at a flow rate of 1 ml/min. The percent iodochlorhydroxyquin and hydrocortisone found to be present in eight commercial products is reported.

Identification and quantitation of a metabolite of anethol dithiolthione in rat and mouse urine using high-performance liquid chromatography.

Urine samples from rats and mice fed anethol dithiolthione (ADT) [3-(p-methoxyphenyl)-1,2-dithiol-3-thione] were analyzed using reversed-phase high-performance liquid chromatography. Urine was introduced directly on the liquid chromatograph which was modified by replacing the sample loop with a guard column. Highly polar urine components were washed off the guard column prior to chromatography. A major metabolite and the parent compound (ADT) were separated and detected using the chromatographic conditions described in this study. The metabolite was identified as desmethyl ADT. The identification was based on co-chromatography on two columns using two mobile phases and peak height ratios of the metabolite and the reference standard. Data pertaining to the pattern of excretion of ADT and desmethyl ADT in the animals studied are reported.

High-performance liquid chromatography of preparations of ribonucleic acid inactivator(s) from cupric ion and hydroquinone before and after treatment with histidine.

Preparations of viral RNA inactivator(s) produced during the cupric ion-catalyzed oxidation of hydroquinone were analyzed by high-performance liquid chromatography (HPLC) using UV and electrochemical (EC) detectors. In addition to hydroquinone and the main oxidation product (p-benzoquinone), which is known not to be the inactivator(s), the analysis showed three unidentified components (I-III). Partial UV absorption spectra of I-III were determined by HPLC with the UV detector set at various wavelengths. Components II and III, but not I, were highly unstable in the presence of L-histidine, which is an excellent chelator of cupric ion and can promptly stop ongoing viral RNA inactivation by the inactivator(s). The product p-benzoquinone was also highly unstable in the presence of L-histidine; the reaction between these two compounds (with or without copper) resulted in a cascade of products. The possibility that the inactivator(s) is II or III, or both, is discussed.

High-performance liquid chromatographic analysis of iodochlorhydroxyquin in plasma.

A simple isocratic high-performance liquid chromatographic procedure for the analysis of iodochlorhydroxyquin in human plasma is described. Protein was precipitated using perchloric acid, and the supernatant and precipitated protein fractions were extracted with ether. The ether phases were evaporated to dryness, reconstituted in mobile phase, and chromatographed. A reversed-phase microparticulate C18 column, a precolumn, and a UV detector at 256 nm were used. A mobile phase containing 80% methanol and 20% of 0.05 M phosphoric acid was employed at a flow rate of 1 ml/min. Quantitation of iodochlorhydroxyquin in the 1--15-microgram/ml range in human plasma was demonstrated with a coefficient of variance of 0.1--0.06. Hydrocortisone, which is used in combination with iodochlorhydroxyquin in ointments and creams, does not interfere in the assay.

The production of viral RNA inactivator(s) from the interaction of dihydric and trihydric phenols with cupric ion.

Eight lots of reagent-grade phenol from four companies were tested for capacity to interact with Cu2+ to produce an inactivator or inactivators of the transfective RNA obtained from poliovirions; such capacity to interact with Cu2+ is referred to as cofactor activity. Six of the lots showed cofactor activity; two did not. A review of the data on the phenol lots and of the properties of the impurity or impurities conferring cofactor activity suggested that the active impurity(ies) might be a dihydric or trihydric phenol. Commercial catechol, resorcinol, hydroquinone, orcinol and pyrogallol were tested and found active. The activity of hydroquinone was outstandingly high. Upon serial recrystallization, the activity of catechol, hydroquinone, orcinol and pyrogallol remained constant, but the activity of resorcinol decreased markedly, in stepwise fashion, showing the most of the activity of the commercial resorcinol was due to impurity(ies). Each of catechol, hydroquinone, orcinol, pyrogallol, and the commercial resorcinol was shown to react with Cu2+ to produce inactivator(s). The effective target for inactivator(s) was the RNA and not the transfection process. The kinetics of inactivator(s) production varied for the different phenols, and the inactivator activity of the incubated mixture of pyrogallol and Cu2+ was notably labile.

High-performance liquid chromatographic analysis of cocaine in human plasma.

A simple isocratic HPLC procedure for the analysis of cocaine in plasma, with or without an internal standard, is described for the first time. Basified plasma was extracted in ether, re-extracted in acetic acid, which was subsequently basified prior to the final extraction in n-hexane. The hexane extract was evaporated to dryness, reconstituted in the mobile phase and then chromatographed. A reverse-phase micro-particulate C-18 column, a pre-column, and a UV detector set at 232 nm were used. A mobile phase containing 75% methanol and 25% 0.05 mol/L potassium phosphate buffer (pH 6.6) was used at a flow rate of 0.8 mL/min. Cocaine in the range of 20 to 500 ng/mL in plasma was determined on the basis of (a) peak height and (b) ratio of peak height to that of tetracaine internal standard. On either basis a linear regression on concentration was determined. The correlation coefficients (r) were 0.993 and 0.988 for (a) and (b) respectively. Twenty-two commonly used drugs were examined for interference. Eight drugs were considered candidates for potential interference with cocaine; lidocaine and droperidol were found to interfere in actual patients' samples. Only meperidine was found to interfere with the internal standard. Cocaine was determined in plasma from patients who received cocaine and other drugs.

Inactivation of transfective poliovirion RNA by a product or products of the interaction of trace copper with an impurity or impurities in reagent-grade phenol.

A search for the cause of the inactivation of the transfectivity of the RNA from poliovirions, in the absence of a protective agent such as L-histidine, revealed that the inactivation is associated with trace contamination with copper and with an impurity or impurities in the phenol used to release the RNA from the poliovirions. Cu2+ and the impurity(ies) interact in vitro to produce a proximate inactivator or inactivators of the RNA. Phenol free or nearly free of active impurity can be prepared by steam distillation. Light is not required for formation or for action of the proximate inactivator. Addition of L-histidine to RNA undergoing inactivation promptly stops the inactivation, probably by taking copper away from the proximate inactivator.

High-performance liquid chromatographic separation and detection of catecholamines and related compounds.

Four sets of chromatographic conditions are described for the separation and identification of selected catecholamines and related chemicals by high-performance liquid chromatography. Three mobile phases, three different columns and three detection systems, including ultraviolet absorption, fluorescence and electrochemical detection are reported. The use of detection response ratios as an additional means of identification is discussed and demonstrated. Nineteen compounds were studied and the retention times and detector responses are reported.

Systematic identification of drugs of abuse II: TLC.

A limited number of spray reagents and solvent systems were selected or developed to separate and identify over 40 of the most commonly encountered drugs of abuse. A new reagent is reported, and new uses were developed for well-known reagents. A flowsheet for the systematic utilization of the spray reagents is given, and use of this sequence made it possible to to identify systematically an unknown drug using only two to four TLC plates, providing that the drug was one of the compounds investigated. This TLC system also can be used to complement and confirm results obtained from spot tests.