High-performance liquid chromatographic determination of levodropropizine in human plasma with fluorometric detection

The present describes a new high-performance liquid chromatographic method with fluorescence detection for the analysis of levodropropizine [S-(−)-3-(4-phenylpiperazin-1-yl)-propane-1,2-diol] (Levotuss), an anti-tussive drug, in human serum and plasma. A reversed-phase separation of levodropropizine was coupled with detection of the native fluorescence of the molecule, using excitation and emission wavelengths of 240 nm and 350 nm respectively. The analytical column was packed with spherical 5 μm poly(styrene-divinylbenzene) particles and the mobile phase was 0.1 M NaH₂PO₄ pH 3-methanol (70:30, v/v), containing 0.5% (v/v) tetrahydrofuran. For quantitation, p-methoxylevodropropizine was used as the internal standard. Samples of 200 μl of either serum or plasma were mixed with 200 μl of 0.1 M Na₂HPO₄ pH 8.9 and extracted with 5 ml of chloroform-2-propanol (9:1, v/v). The dried residue from the organic extract was redissolved with distilled water and directly injected into the chromatograph. The limit of detection for levodropropizine, in biological matrix, was about 1–2 ng/ml, at a signal-to-noise ratio of 3. The linearity was satisfactory over a range of concentrations from 3 to 1000 ng/ml (r² = 0.99910); within-day precision tested in the range 5–100 ng/ml as well as day-to-day reproducibility proved acceptable, with relative standard deviations better than 1% in most cases. Interferences from as many as 91 therapeutic or illicit drugs were excluded.     

High-performance liquid chromatographic determination of α-keto acids

A high-performance liquid chromatographic method has been developed for the determination of eight kinds of α-keto acids. These acids were derivatized with o-phenylenediamine into 2-quinoxalinol derivatives, which were extracted into chloroform. The quinoxalinol derivatives were separated by reversed-phase high-performance liquid chromatography using a 250 mm × 2.1 mm I.D. column packed with LiChrosorb RP-8 (5 μm). This method could be satisfactorily applied to urine samples without any prepurification.     

High-performance liquid chromatographic determination of α-keto acids in human urine and plasma

A high-performance liquid chromatographic method has been developed for the determination of α-keto acids in human urine and plasma. These acids were prepurified using a column of hydrazide gel and derivatized with o-phenylenediamine into 2-quinoxalinol derivatives, which were extracted into ethyl acetate. The 2-quinoxialinol derivatives were separated by reversed-phase paired-ion chromatography using a 250 × 4 mm-i.d. column packed with LiChrosorb RP-8 (5 μm). This method is sensitive, selective, and reproducible. The α-keto acids in urine and plasma from normal individuals were determined.     

High-performance liquid chromatographic determination of vertilmicin in rat plasma using sensitive fluorometric derivatization

A sensitive and reliable high-performance liquid chromatographic method was developed for the determination of vertilmicin in rat plasma. Derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by C(18) reversed-phase chromatography allowed the fluorimetric detection of vertilmicin. Optimal conditions for the derivatization of vertilmicin are described. The limit of quantification was 0.02 mg/L. The pharmacokinetics of vertilmicin was studied in 24 rats following intramuscular injection (i.m.) of different doses (4, 8, 16, 32 mg/kg of body weight). The pharmacokinetic parameter values were estimated by use of 3P97 program. In this study, we assessed the dose proportionality of vertilmicin after single intramuscular injection doses and obtained new information on the pharmacokinetics of the compound.     

High-performance liquid chromatographic determination of beta-phenylethylamine in human plasma with fluorescence detection

A simple and highly sensitive method for the determination of beta-phenylethylamine in human plasma is investigated. The method employs high-performance liquid chromatography with fluorescence detection. beta-Phenylethylamine and p-methylbenzylamine (internal standard) in human plasma are isolated by cation-exchange chromatography on a Toyopak SP cartridge and then converted into the corresponding fluorescent derivatives with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives are separated within 30 min on a reversed-phase column, TSK gel ODS-120T, with isocratic elution, and detected fluorometrically. The detection limit of beta-phenylethylamine is 0.3 pmol/ml in plasma (S/N = 3).     

High-performance liquid chromatographic assays with fluorometric detection for mivacurium isomers and their metabolites in human plasma

Two high-performance liquid chromatographic assays coupled with fluorometric detection have been developed for the determination of mivacurium isomers (trans-trans, cis-trans and cis-cis) and their monoester and alcohol metabolites in human plasma. A novel solid-phase extraction procedure allowed good recovery of the mivacurium isomers (mean 98%) and their monoester metabolites (mean 83%), whereas the alcohol metabolites were analyzed after direct precipitation of plasma proteins. For all analytes, these assays proved to be sensitive (LOQ 3.9–15.6 ng/ml), reproducible (C.V. < 15%) and accurate (>94%) over the therapeutic range of concentrations of mivacurium and its metabolites. These two methods were applied successfully to a pharmacokinetic study of mivacurium after a bolus dose of 0.15 mg/kg in anesthetized patients.     

Improved high-performance liquid chromatographic determination with amperometric detection of α-amanitin in human plasma based on its voltammetric study

A sensitive and simple method is described for the selective determination in human plasma of α-amanitin, the most poisonous and prevalent toxin in the lethal fungi of species Amanita, using high-performance liquid chromatography with amperometric detection. After an extraction of plasma with disposable C₁₈ silica cartridges, the extracts were separated by isocratic reversed-phase chromatography using a macroporous poly(styrene—divinylbenzene) column and a mobile phase of 0.05 M phosphate buffer—acetonitrile (91:9) at the apparent pH of 9.5. Amperometric detection was performed by applying an oxidation potential as low as +350 mV (vs. Ag/AgCl) to a glassy carbon electrode, in a thin-layer flow-cell. The linear range for α-amanitin was 3–200 ng/ml, and the relative limit of detection in plasma was 2 ng/ml at a signal-to-noise ratio of 2. The intra-assay precision was evaluated at levels of 10 and 200 ng/ml; the coefficients of variation were 4.5 and 2.6% (n=5), respectively. Inter-assay coefficients of variation were 6.5 and 4.2% (n=5) for the same concentrations of toxin. These analytical conditions have been chosen on the basis of a preliminary in batch cyclic voltammetric investigation of α-, β- and γ-amanitins, which has allowed their oxidation process to be clarified and the pH dependence of their oxidation potentials to be determined. All three amanitins are oxidized at the same potential values, and adsorption onto the electrode surface of both reactant and products was found in all cases. This adsorption did not affect the signal recorded for α- and γ-amanitins at the amperometric detector, and for β-amanitin a stronger adsorption for the anodic product was found, which leads to a marked positive shift of the potential required for the oxidation of this isomer in the amperometric detector cell.     

High-performance liquid chromatographic determination of 4,4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone in plasma

An analytical method has been developed for the determination of 4,4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone (I, trade name A-007) in plasma. Plasma samples are primed with the internal standard, 2,2′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone (II), deproteinized with acetonitrile, centrifuged and filtered prior to assay. The components are then separated on a reversed-phase column with retention times of 4.4 and 6.0 min for I and II, respectively. Ultraviolet detection at 365 nm was employed and little interference with the analyte or the internal standard was noted from other plasma components. This method has been applied to the plasma of rats and monkeys dosed for pharmacokinetic and toxicity studies.     

High-performance liquid chromatographic determination of indoleamines, dopamine, and norepinephrine in rat brain with fluorometric detection

A high-performance liquid chromatography-fluorescence procedure for the determination of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan, dopamine, and norepinephrine has been developed. The method uses an ion-pairing system on an Ultrasphere ODS (5-microns) column with detector wavelength settings of excitation at 290 nm and emission at 330 nm. The procedure has been used to quantitate these indoleamines and catecholamines in rat brain tissue after homogenization in a perchloric acid solution; an aliquot of this solution is injected directly onto the HPLC column. Column sensitivities range from 6.1 pmol for tryptophan to 1.1 pmol for 5-hydroxytryptamine.     

High-performance liquid chromatographic determination of pentamidine in plasma

This report describes the analysis of pentamidine by isocratic reversed-phase high-performance liquid chromatography (HPLC) using a commercially available compound (melphalan) as the external standard. Previously described assays use ion-pairing HPLC, an internal standard (hexamidine) that is not readily available, and require a relatively large sample size. In the present assay, pentamidine was extracted from plasma using solid-phase extraction and was analyzed using a C₁₈ column and a mobile phase containing 18% acetonitrile, 2% methanol, 0.2 M ammonium acetate and 0.5% triethylamine. The identity of the eluting peaks was verified using a diode array detector. The extraction yield of pentamidine was 82%. The limit of detection was 8.6 ng/ml with a sample size of 100 μl. The inter-day and intra-day coefficients of variation ranged between 0.3% and 10% with an average of 5%. This method was applied to study the pharmacokinetics of pentamidine in rodents.     

High-performance liquid chromatographic determination of fluconazole in plasma

A high-performance liquid chromatographic method with ultraviolet absorbance detection at 260 nm was developed for the analysis of fluconazole in plasma. The method involves sample clean-up by liquid-liquid extraction. The proposed technique is reproducible, selective, reliable and sensitive. Calibration standards were prepared in the range 1.25-20 mg/l. The limit of quantitation was 0.4 mg/l. The coefficients of variation were 5% between measurements of a single extract injected in duplicate, and 7% between two extractions of spiked samples at the same concentrations. The separation between fluconazole and endogenous substances was satisfactory. This method was designed in order to minimise the risk of interference from substances that could be co-administered to critically ill patients undergoing hemodiafiltration. With a run time below 5 min, the present method is rapid and easy to use for later clinical studies, as well as for routine monitoring.     

High-performance liquid chromatographic determination of ganciclovir in plasma

A rapid, selective and sensitive isocratic reversed-phase high-performance liquid chromatographic method for the determination of ganciclovir in plasma samples was developed. This method, which was applied to the analysis of plasma ganciclovir from heart transplant patients under ganciclovir therapy for cytomegalovirus infections, represents a suitable analytical tool for drug monitoring and pharmacokinetic investigations.     

High-performance liquid chromatographic determination of ambroxol in human plasma

Ambroxol has been determined in biological fluids using a rapid and sensitive high-performance liquid chromatographic method. The samples prepared from plasma by liquid—liquid extraction were analysed on reversed-phase silica gel by competing-ion chromatography with ultraviolet detection. The method was applied to the determination of ambroxol levels in twelve healthy volunteers after oral administration of 90 mg of ambroxol in tablets of Mucosolvan and Ambrosan.     

High-performance liquid chromatographic determination of tramadol in human plasma

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.     

High-performance liquid chromatographic and gas—liquid chromatographic determination of diazepam and nordiazepam in plasma

A one-step method for extraction of diazepam, nordiazepam, and internal standard into toluene is followed by chromatographic separation and detection with either dual-wavelength high-performance liquid chromatography or electron-capture gas—liquid chromatography. Agreement between the two methods was excellent for diazepam (r = 0.99, n = 38) and good for nordiazepam (r = 0.96, n = 79) over a concentration range that included subtherapeutic, therapeutic, and toxic plasma levels.     

High-performance liquid chromatographic determination of thiopentene enantiomers in sheep plasma

An HPLC method was developed to determine the plasma concentrations of R(+)- and S(−)-thiopentone for pharmacokinetic studies in sheep. The method required separation of the thiopentone enantiomers from the corresponding pentobarbitone enantiomers which are usually present as metabolites of thiopentone. Phenylbutazone was used as an internal standard. After acidification, the plasma samples were extracted with a mixture of ether and hexane (2:8). The solvent was evaporated to dryness and the residues were reconstituted with sodium hydroxide solution (pH 10). The samples were chromatographed on a 100 mm × 4 mm I.D.. Chiral AGP-CSP column. The mobile phase was 4.5% 2-propanol in 0.1 M phosphate buffer (pH 6.2) with a flow-rate of 0.9 ml/min. This gave k′ values of 1.92, 2.92, 5.71, 9.30 and 11.98 for R(+)-pentobarbitone, S(−)-pentobarbitone, R(+)-thiopentone, S(−)-thiopentone, and phenylbutazone, respectively. At detection wavelength of 287 nm, the limit of quantitation was 5 ng/ml for R(+)-thiopentone and 6 ng/ml for S(−)-thiopentone. The inter-day coefficients of variation at concentrations of 0.02, 0.1 and 8 μg/ml were, respectively, 4.8, 4.4 and 3.5% for R(+)-thiopentone and, respectively, 5.0, 4.3 and 3.9% for S(−)-thiopentone (n = 6 each enantiomer). At the same concentrations, the intra-day coefficients of variation from six sets of replicates (measured over six days) were, respectively, 8.0, 8.0 and 8.8% for R(+)-thiopentene and 8.8, 7.4 and 9.6% for S(−)-thiopentone. Linearity over the standard range, 0.01–40 μg/ml, was shown by correlation coefficients> 0.998. This method has proven suitable for pharmacokinetic studies of thiopentone enantiomers after administration of rac-thiopentone in human plasma also and would be suitable for pharmacokinetic studies of the pentobarbitone eantiomers.