Determination of disulfiram and metabolites from biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of disulfiram, diethyldithiocarbamate, diethyldithiocarbamate methyl ester, carbon disulfide, and diethylamine from a single sample of plasma or urine. The analytical procedure is based on a quantitative stepwise extraction of disulfiram and diethyldithiocarbamate methyl ester, or the conversion of diethyldithiocarbamic acid, carbon disulfide and diethylamine to diethyldithiocarbamate methyl ester for chromatographical determination. The procedure is specific, precise and simple. The application of the analytical methods developed for the determination of disulfiram and the various metabolites in plasma from mice given disulfiram intraperitoneally or humans given Antabuse orally is illustrated.     

Isocratic separation of ginsenosides by high-performance liquid chromatography on a diol column at subambient temperatures

An improved high-performance liquid chromatographic method for separation of a number of ginsenosides has been developed. The influence of temperature (from 0 to 25°C) on the retention and separation of the ginsenosides was studied by applying a binary mobile phase (acetonitrile/water, 82:18 v/v) and a diol column (LiChrospher 100 Diol). The column temperature is one of the more important parameters for the retention and separation of the components investigated. Selected thermodynamic parameters, including changes of enthalpy (ΔH°) and entropy (ΔS°), were estimated from linear van’t Hoff plots, and possible retention mechanisms were discussed. Moreover, the best separation conditions were selected based on optimization criteria including maximum retention time (t _{R max}), minimum resolution (R _{s min}), and relative resolution product (r). Temperature regions close to 14°C offered the highest selectivity and almost equal distribution of the ginsenosides peaks across the chromatogram. Under such isocratic conditions, excellent separation of chromatographic standards and selected ginseng samples was achieved in less than 16 min.     

Chiral separation of lipoxygenase metabolites utilizing high-performance liquid chromatography

An isocratic, reversed-phase HPLC assay has been developed for the separation of the enantiomers of four lipoxygenase metabolites, without the need for a derivatization step. Separation of the enantiomers was studied on a polysaccharide type chiral stationary phase column. Upon determination of suitable mobile phase composition, the assay was evaluated at various temperatures. In all cases the R enantiomer eluted before the S enantiomer. The best separations were observed at 0 degrees C.     

Determination of some benzodiazepines and metabolites in serum, urine and saliva by high-performance liquid chromatography

The performance of a number of liquid--solid systems, consisting of mixtures of buffers (0.05 M) and methanol as mobile phase and methyl-silica as stationary phase, were investigated with respect to their use in the separation of 1,4-benzodiazepines by reversed-phase high-performance liquid chromatography with UV detection at 254 nm. Phase system selectivities and column efficiencies were determined. A nomogram is presented from which the chromatographic parameters can be calculated. A complete separation of nine benzodiazepines within 12 min has been achieved, using methyl-silica as the stationary phase and 50% methanol as the eluent. The results were applied to the development of a method for the determination of therapeutic levels of diazepam and its metabolites in human serum, urine and saliva. The first step in the analysis, the extraction of diazepam and its metabolites from serum and urine, was also investigated and good recoveries were achieved. A low detection limit (0.2 ng) and high precision were obtained. The concentrations of diazepam and its metabolites in human serum, urine and saliva were determined after both single and multiple oral doses of diazepam (and oxazepam).     

Determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography.

A method for the identification and determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography was developed. Tannic acid and four phenolic compounds, namely gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid, were successfully extracted from the biological fluids by using ethyl acetate at acidic conditions. Gallic acid, pyrogallol and 4-O-methylgallic acid were found in the sheep urine, gallic acid, 4-O-methylgallic acid and ellagic acid in plasma, and gallic acid and ellagic acid in abomasal fluid after abomasal dosing of tannic acid. Tannic acid was found in the plasma apart from the abomasal fluid into which it was administered. The concentrations of tannic acid, gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid in plasma, abomasal fluid and urine were measured. This method could be applied to measurement of other hydrolysable tannins and their phenolic metabolites in biological materials.     

Pyrimidine nucleoside phosphorylase assay by automated high-performance liquid chromatography

A new assay for pyrimidine nucleoside phosphorylase is reported. This method utilizes an isocratic reversed-phase high-performance liquid chromatographic system for separation of nucleosides and bases. Product detection is accompanied by ultraviolet monitoring and radioactive flow detection. Use of an automated sample injector allows for the analysis of a series of samples, with data recorded onto a microprocessor-based cassette recorder. Data can then be downloaded into computer memory. The velocity of uridine phosphorylase (E.C. 2.4.2.3) was a linear function of enzyme concentration. The Michaelis constant for uridine at pH 8.0 was found to be in close agreement with the value obtained by a thin-layer chromatographic assay method.     

The separation of nonapeptides by reversed-phase high-performance liquid chromatography.

The separation properties of five nonapeptides on commercial reversed-phase materials have been investigated and the effects of pH, salt concentration and solvent composition have been studied. With appropriate variation of the pH and salt concentration in the mobile phase, it is possible to resolve all of the peptides investigated and their by-products. Mixtures of water and organic solvents (acetonitrile, dioxan, methanol and n-propanol) have been used. The choice of the organic solvent does not strongly influence the separation pattern. The simplicity, speed and quality of the separations and the favourable detection limits (ca. 30 ng) at 220 nm render this technique suitable to routine quantitative analysis.     

Resolution of RS-abscisic acid and the separation of abscisic acid metabolites from plant tissue by high-performance liquid chromatography

Attempts to resolve the enantiomers of racemic abscisic acid (ABA) by high-performance liquid chromatography on a chiral stationary-phase column were unsuccessful. However, reduction of RS-methyl ABA (RS-Me-ABA) with sodium borohydride generates a new chiral centre and one of the two isomeric products, the RS-Me-1',4'-cis-diol of ABA, was separated into its enantiomers by high-performance liquid chromatography on an optically active Pirkle column. High-performance liquid chromatography on a mu Bondapak C18 column separated the metabolites and conjugates of [2-14C]ABA fed to tomato shoots. The resolution method was used to measure the relative proportions of R and S enantiomers in the free acid liberated from conjugates of ABA.     

Analysis of natural corticosteroids in adrenal extracts and in biological fluids by high-performance liquid chromatography

A liquid chromatographic procedure is described for the analysis of the principal natural corticosteroids in extracts of adrenal glands. Microparticulate silicic acid columns and gradients of methanol in chloroform are used: conditions are described for the quantitative analysis of the single principal steroidal components of adrenal extracts for pharmaceutical use and of adrenal extracts of rats. In the last case, the use of a 5-micron silica column with the appropriate gradient allows the determination of corticosterone and of 18-hydroxydeoxycorticosterone, which were identified by means of mass spectrometry on their eluates. A single analysis can be performed on the extract of 15 mg of rat adrenal tissue. For the last type of analysis, isocratic conditions on a 10-micron LiChrosorb Diol column are also described. The application of the gradient elution procedure to the analysis of steroidal compounds in human plasma is also described.     

Detection and separation of mitoxantrone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method using ion-pair chromatography on reversed-phase C18 material was developed. After sample clean-up on XAD columns, mitoxantrone at concentrations below 1 ng/ml in serum and 0.2 ng/ml in urine were measurable with a coefficient of variation of less than 9.3% at a wavelength of 658 nm. Four metabolites were separated in urine. The two major metabolites co-chromatographed with the synthesized mono- and dicarboxylic acid derivatives of mitoxantrone. The method allowed the measurement of mitoxantrone and its metabolites in serum up to more than one week and in urine up to four weeks after administration of the drug.     

Chiral separation of atropine by high-performance liquid chromatography

The separation and quantitation of the enantiomers and also the determination of the enantiomeric purity are now current and indispensable tasks for the pharmaceutical analysis. Among the various techniques, liquid chromatography remains the best modality owing to several advantages. High speed, sensitivity, and reproducible results make LC the method of choice in almost all laboratories. Phases that contain alpha1-acid glycoprotein as chiral selector are suitable for separation of charged and uncharged enantiomers with widely different structure. Atropine is widely used as parasympatolytic, anticholinergic and antiemetic drugs. It is one of the preferred antidote for immediate management of toxicity associated with nerve agents. Stereoselective separation was achieved with a prepacked alpha1-acid glycoprotein column without any derivatization procedure. The liquid chromatography system is coupled to mass spectrometry with an atmospheric pressure chemical ionization interface in the positive-ion mode. The chromatographed analytes are detected in selective ion monitoring after optimisation using factorial experimental design. Small amount of enantiomeric composition can be evaluated either by MS or by UV spectrometry (less than 5%).     

Facile extraction of berberine from different plants,separation, and identification by thin-layer chromatography,high-performance liquid chromatography,and biological evaluation against Leishmaniosis

The extraction of berberine was carried out from Berberis vulgaris, Berberis aquifolium, and Hydrastis canadensis plants using ethanol and water (70:30, v/v). The extracted berberine was characterized by ultraviolet-visible and Fourier-transform infrared spectroscopy. The purity of berberine was ascertained by thin-layer chromatography using n-propanol-formic acid-water (95:1:4) and (90:1:9) solvents. hR_f values were in the range of 44–49 with compact spots (diameter 0.2–0.4 cm). HPLC was carried out using ammonium acetate buffer and acetonitrile in gradient mode with Zodiac (4.6 × 150 mm, 3 μm) column. The flow rate was 1.0 mL/min and detection was at 220 nm. The values of separation and resolution factors of the standard and the extracted berberine were in the range of 1.13–1.16 and 1.40–1.71, respectively. A comparison has shown that both thin-layer chromatography and high-performance liquid chromatography (HPLC) methods found applications in different situations and requirements. The extracted berberine samples were used to treat Leishmaniosis and the results showed better activity of berberine in comparison to the standard drug Amphotericin B. Briefly, the reported research is a novel and may be used to extract berberine from plants, separation and identification of berberine by thin layer chromatography and HPLC and to treat Leishmaniosis.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C18 and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5-21.5 nmol/ml for NEF and 0.4-9.5 nmol/ml for metabolites in serum and 4-86 nmol/ml for NEF and 8-190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Complete separation of urinary metabolites of paracetamol and substituted paracetamols by reversed-phase ion-pair high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic procedure has been developed for the separation of thirteen urinary metabolites of the analgesic drug paracetamol. The method involved the use of radially compressed columns packed with octadecylsilica with a particle diameter of 5 micron. Metabolites were chromatographed by linear gradient elution using an ion-pair solvent system composed of tetrabutylammonium hydroxide and Tris buffered to pH 5.0 with phosphoric acid, and acetonitrile as the organic solvent. Analyses can be performed at the rate of three per hour. This method enables the direct identification of sulphate and glucuronide conjugates of 3-thiomethylparacetamol and 3-thiomethylparacetamol sulphoxide which have only previously been detected following enzyme hydrolysis of urine samples. The application of this fully optimised separation to the study of the metabolism of substituted paracetamols is also discussed.     

Rapid separation of creatine, phosphocreatine and adenosine metabolites by ion-pair reversed-phase high-performance liquid chromatography in plasma and cardiac tissue.

A rapid ion-pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous detection of creatine, phosphocreatine, hypoxanthine, inosine, adenosine, AMP, ADP, ATP, 8-azaguanine, 2-chloroadenosine, and 2'-O-methyladenosine. This method has proven useful for measuring changes in nucleotide concentrations in both heart tissue and plasma samples. Separation of the compounds of interest is achieved in less than 8 min with re-equilibration in 7 min, making the total run time 15 min. Separation is performed on a 3-microns Ultrasphere ODS column employing tetrabutylammonium phosphate as the ion-pair agent and dipotassium hydrogenphosphate as the counter ion. The accuracy, rapid separation, and re-equilibration time make this method particularly useful for the routine analysis of a large number of samples.     

Sanguinarine levels in biological samples by high-performance liquid chromatography.

A high-performance liquid chromatographic method is presented for the analysis of the benzophenanthridine alkaloid, sanguinarine, found in plant extracts. The method is demonstrated to be applicable to analyzing samples such as saliva and gingival crevicular fluid for sanguinarine following a simple acidified methanolic extraction step. The method utilizes an ethyl silane column with acidic and basic ion-pairing reagents in the mobile phase with a limit of detection of 3 ng of sanguinarine in a sample.