Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2±2.7 and 99.9±2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 μg/ml, respectively.     

Quantitative liquid chromatographic analysis of anthracyclines in biological fluids

Anthracyclines are amongst the most widely used drugs in oncology, being part of the treatment regimen in most patients receiving systemic chemotherapy. This review provides a comprehensive summary of the sample preparation techniques and chromatographic methods that have been developed during the last two decades for the analysis of the 4 most administered anthracyclines, doxorubicin, epirubicin, daunorubicin and idarubicin in plasma, serum, saliva or urine, within the context of clinical and pharmacokinetic studies or for assessing occupational exposure. Following deproteinization, liquid-liquid extraction, solid phase extraction or a combination of these techniques, the vast majority of methods utilizes reversed-phase C18 stationary phases for liquid chromatographic separation, followed by fluorescence detection, or, more recently, tandem mass spectrometric detection. Some pros and cons of the different techniques are addressed, in addition to potential pitfalls that may be encountered in the analysis of this class of compounds.     

High-performance liquid chromatographic analysis of methocarbamol enantiomers in biological fluids

Methocarbamol enantiomers in rat and human plasma were quantified using a stereospecific high-performance liquid chromatographic method. Racemic methocarbamol and internal standard, (R)-(−)-flecainide, were isolated from plasma by a single-step extraction with ethyl acetate. After derivatization with the enantiomerically pure reagent (S)-(+)-1-(1-naphthyl)ethyl isocyanate, methocarbamol diastereomers and the (R)-flecainide derivative were separated on a normal-phase silica column with a mobile phase consisting of hexane—isopropanol (95:5, v/v) at a flow-rate of 1.6 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm. The resolution factor between the diastereomers was 2.1 (α = 1.24). An excellent linearity was observed between the methocarbamol diastereomers/internal standard derivative peak-area ratios and plasma concentrations, and the intra- and inter-day coefficients of variation were always <9.8%. The lowest quantifiable concentration was 0.5 μg/ml for each enantiomer (coefficients of variation of 9.8 and 8.8% for (S)- and (R)-methocarbamol, respectively), while the limit of detection (signal-to-noise ratio 3:1) was approximately 10 ng/ml. The assay was used to study the pharmacokinetics of methocarbamol enantiomers in a rat following intravenous administration of a 120 mg/kg dose of racemic methocarbamol and to evaluate plasma and urine concentrations in a human volunteer after oral administration of a 1000-mg dose of the racemate. The method is suitable for stereoselective pharmacokinetic studies in humans as well as in animal models.     

Solid-phase extraction of methadone enantiomers and benzodiazepines in biological fluids by two polymeric cartridges for liquid chromatographic analysis

The aim of this work was to present the advantages of two polymeric cartridges (Oasis HLB from Waters and Abselut Nexus from Varian) for the solid-phase extraction of methadone enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and of some benzodiazepines (diazepam, flunitrazepam, nitrazepam, oxazepam) in serum and urine in comparison with classical C18-bonded-silica cartridges or liquid extraction. After addition of serum or urine samples, these two cartridges were washed with a water-methanol mixture (95:5, v/v) and eluted with diethylether. After rapid evaporation, the residue was regenerated with mobile phase and injected either in a chiral column (Cyclobond I-2000 RSP) for methadone enantiomers and its metabolite or in a reversed-phase column (Symmetry Shield RP8) for benzodiazepines. The results showed that the chromatograms of blank serum and urine were cleaner than those obtained from classical solid-phase extraction or liquid extraction. The recoveries from these two polymeric cartridges were higher (95-102%) than those obtained by the two previous classical methods and the total time for extraction and solvent evaporation was also shorter (about 6-7 min). For methadone and benzodiazepine extraction, the use of acidic or alkaline buffer was not necessary.     

Rapid and sensitive pre-column extraction high-performance liquid chromatographic assay for propofol in biological fluids

A completely automated high-performance liquid chromatographic system is described for the determination of the phenolic anaesthetic propofol. The method is based on pre-column extraction in a closed system allowing direct injection of biological samples without any sample pretreatment. The assay is sensitive (limit of quantification is 5 ng/ml serum), reliable (the variability within a series is 2%) and rapid (results are available after 6 min).     

Liquid chromatographic determination of caffeine in biologic fluids

A high-performance liquid chromatographic procedure was developed for the determination of caffeine in various biologic fluids and coffee. A reversed-phase column and UV detection at 254 nm were used to obtain a sensitivity of 0.1 μg/ml caffeine in serum and saliva using a sample volume of 0.1 ml. Caffeine metabolites and commonly ingested xanthines do not interfere with the assay. The within-day coefficients of variation were 9.8 and 9.9% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. The day-to-day coefficients of variation were 6.8 and 6.6% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. Serum and saliva caffeine concentrations were determined following a single oral dose of coffee and an intravenous infusion of caffeine in one subject. Computer estimates of caffeine pharmacokinetic parameters in one subject are in excellent agreement with previously published values.     

High-performance liquid chromatographic analysis of sulfinpyrazone and its metabolites in biological fluids

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of sulfinpyrazone and its sulfone and p-hydroxy metabolites in plasma and urine. The method uses two different procedures for sample preparation: (1) a rapid and convenient procedure using a single extraction with 1-chlorobutane and subsequent back-extraction into sodium hydroxide solution for the analysis of sulfinpyrazone and its sulfone metabolite, and (2) a more time consuming procedure using triple extraction with ethylene dichloride, a buffer wash, and back extraction into the base for the additional analysis of the p-hydroxy metabolite. The lower limit of sensitivity for sulfinpyrazone is 50 ng/ml. Concentrations of sulfinpyrazone between 0.05 to 0.1 and 50 μg/ml were measured with an average coefficient of variation of 3.9%, ranging from 1.5 to 6.1%.     

Enantioselective analysis of atenolol in biologic fluids: comparison of liquid-liquid and solid-phase extraction methods

In this study we evaluated a liquid-liquid extraction procedure and a solid-phase extraction procedure for sample preparation for the enantioselective analysis of atenolol in plasma and urine by high-performance liquid chromatography. A Chiralcel OD-H column was used for the resolution of atenolol enantiomers with hexane-ethanol (85:15, v/v) plus 0.1% diethylamine as the mobile phase. In the liquid-liquid extraction procedure, atenolol was extracted from alkalinized body fluids with 5 ml chloroform-2-propanol (4:1, v/v). In the solid-phase extraction procedure, atenolol was isolated from plasma using a C8 column and methanol. Both extraction procedures were efficient in recovering atenolol and removing endogenous interferents. The RSDs and deviation from nominal values were lower than 10% for both within-day and between-day assays. The results show that there were no statistically significant differences in between-day variation. The t-test showed that there were no significant differences between the real concentrations and the determined concentrations. The limit of quantitation was 10 ng/ml and the linear range was 10-5,000 ng/ml for both methods. These methods can be used in pharmacokinetic studies.     

Liquid chromatographic determination of ethyl alcohol in body fluids

A high-performance liquid chromatographic technique for ethyl alcohol determination in body fluids is proposed. Ethyl alcohol is quantitatively converted into acetaldehyde-phenylhydrazone by oxidation in the presence of alcohol dehydrogenase, nicotinamide–adenine dinucleotide and phenylhydrazine. The derivative is suitable for reversed-phase liquid chromatography and ultraviolet detection at 276 nm. The limits of linearity, detection and quantification as well as accuracy and reproducibility were investigated in water, serum and whole blood. Analytical responses were linear within the 0.008 to 5 g/l range, and the limit of quantification was 0.02 g/l both in aqueous standard and in biological matrix assays. Mean analytical recovery of ethyl alcohol in blood serum averaged 98.2±4.2%, imprecision (CV%) at 0.80 g/l was 2.2%, and the limit of quantification was 0.02 g/l. Serum concentrations of persons that avoided alcoholic beverages for a week were less than the limit of quantification. Ethyl alcohol concentrations in serum and whole blood compared well with those obtained by headspace gas chromatography. This simple and reliable procedure, which was also used for a urine assay, could be suitable for validation of the screening procedures used to monitor ethanol abuse.     

High-performance liquid chromatographic determination of sotalol in biological fluids

A sensitive, selective and reproducible reversed-phase high-performance liquid chromatographic method is described for the quantification of sotalol in human serum and urine. Sotalol and the internal standard, atenolol, were extracted from alkalinized serum and urine (pH 9.0) into 1-butanol—chloroform (20:60, v/v). The organic phase was evaporated, and to the residue was added 0.1 M sulphuric acid (serum analysis) or mobile phase (urie analysis). The mobile phase consisted of 0.01 M phosphate buffer (pH 3.2) and acetonitrile (20:80, v/v) containing 3 mM n-octylsodium sulphate. The flow-rate was 1.5 ml/min. The retention times of atenolol and sotalol were 7 and 10 min, respectively. Ultraviolet detection at 226 nm made it possible to achieve a detection limit of 0.03 μmol/l.     

Quantitative gas chromatographic determination of 3-methylhistidine in biological fluids

The gas chromatographic procedure is suggested to determine 3-methylhistidine in biological fluids. The amino acid fraction containing 3-methylhistidine is separated by ion-exchange chromatography. Amino acids are transformed into N-trifluoroacetyl-O-isobutyl esters which are analyzed by the gas chromatography instrument with micropacked columns and ionization-resonance detector. The limit of the quantitative determination of 3-methylhistidine is 50 ng per a probe.     

High-performance liquid chromatographic determination of quinine in rat biological fluids

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of quinine in rat biological fluids is described. Due to its selectivity and sensitivity, the proposed method can be used in the case of such rat biological fluids as cerebrospinal fluid (CSF) and perilymph for which the accessible volumes are limited to 100 μl and 10 μl, respectively. Consequently, the assay method has been applied to the measurements of quinine concentration in rat plasma, CSF and perilymph samples.