Determination of thiamphenicol in human plasma by micellar electrokinetic capillary chromatography

A micellar electrokinetic chromatographic method is described for the determination of thiamphenicol in human plasma. The plasma sample was basified by adding K₂HPO₄ and was then extracted with ethyl acetate. After the solvent was evaporated, the residue was reconstituted in water. Approximately 40 nl of the solution were injected hydrodynamically. The running buffer was 20 mM borate (pH 9.2) containing 40 mM sodium dodecyl sulfate and 10% acetonitrile. The applied voltage was 18 kV and the detector wavelength was set at 195 nm. On-column sample stacking was achieved during the analysis to enhance the sensitivity; the limit of quantitation was 0.1 μg/ml. Linearity was over the range of 0.2 to 10 μg/ml. Recovery was 93.7±3.3%, the intra-day precision and accuracy was 99.6±2.8%; the inter-day precision and accuracy was 98.4±3.4%. The concentration of thiamphenicol in human plasma from eight volunteers was measured after administering thiamphenicol capsules orally.     

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 μm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5±2.8% with a linear range of 0.1–100 μg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

High-performance liquid chromatography assay of rifapentine in human serum

A high-performance liquid chromatographic method for the determination of rifapentine in human serum was developed. The method utilized a Spherisorb C₁₈ column, ultraviolet detection (336 nm), rifampin as internal standard and a calibration curve (C = 7.010 A_s/A_in ± 0.156, R = 0.999) with reproducibility studies which yield a coefficient of variation (C.V.) of intra-day and inter-day assays lower than 10%. The average recovery of rifapentine from serum in the concentration range of 0.5 to 30 μg/ml was 92.93 ± 9.704%.     

Determination of urinary 5-hydroxytryptophol by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the routine determination of elevated urinary levels of the serotonin metabolite 5-hydroxytryptophol (5-HTOL) is described. Urine samples were treated with β-glucuronidase, and 5-HTOL was isolated by solid-phase extraction on a small Sephadex G-10 column prior to injection onto an isocratically eluted C₁₈ reversed-phase column. Detection of 5-HTOL was performed electrochemically at +0.60 V vs. Ag/AgCl. The limit of detection was ca. 0.05 μM, and the intra-assay coefficients of variation were below 6% with urine samples containing 0.2 and 2.1 μM 5-HTOL and a standard solution of 2.0 μM (n = 5). The recovery of 5-HTOL after the sample clean-up procedure was close to 100%. A good correlation (r² = 0.97; n = 12) was obtained between the present method and a sensitive and specific gas chromatographic—mass spectrometric method. The total (free plus conjugated) 5-HTOL levels in urine were normally below 0.2 μM, but after an acute dose of alcohol they increased to 0.5–15 μM.     

Determination of the angiotensin-converting enzyme inhibitor lisinopril in urine using solid-phase extraction and reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method is described for assaying lisinopril in human urine. Urine (1 ml) containing lisinopril and enalaprilat (internal standard) was acidified with 10 μl of 6 M nitric acid, passed through a Sep-Pak C₁₈ cartridge and eluted with 3 ml of 10% acetonitrile, followed by 6 ml of distilled water. The separations were carried out using a μBondapak C₁₈ column with a mobile phase comprising acetonitrile (60 ml), methanol (10 ml) and tetrahydrofuran (10 ml) in 15 mM phosphate buffer (920 ml) at pH 2.90. Separations were performed at 40°C and detection was at 206 nm. Standard calibration plots of lisinopril in urine were linear (r> 0.998) and recovery was greater than 64%. The lowest quantifiable concentration was 0.5 μg/ml. Within-day and between-day imprecision (coefficient of variation) ranged from 2.51% to 9.26%, and inaccuracy was less than 8.3%.     

Determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in human urine

A method for simultaneous determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in urine is described. These compounds are metabolites of N-methyl-2-pyrrolidone, a powerful and widely used organic solvent. 5-Hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide were purified from urine by adsorption to a C₈ solid-phase extraction column and then elution by ethyl acetate–methanol (80:20). After evaporation, the samples were derivatised at 100°C for 1 h by bis(trimethylsilyl)trifluoroacetamide. Ethyl acetate was then added and the samples were analysed by gas chromatography–mass spectrometry in the electron impact mode. The extraction recovery for 5-hydroxy-N-methylpyrrolidone was about 80% while that for 2-hydroxy-N-methylsuccinimide was about 30%. The intra-day precision for 5-hydroxy-N-methylpyrrolidone was 2–4% and the between-day precision 4–21% (4 and 60 μg/ml). The intra-day precision for 2-hydroxy-N-methylsuccinimide was 4–8% and the between-day precision 6–7% (2 and 20 μg/ml). The detection limit was 0.2 μg/ml urine for both compounds. The method is applicable for analysis of urine samples from workers exposed to N-methyl-2-pyrrolidone.     

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC–MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1000 μg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1±13.5 μg/l for 2-ethylhexanoic acid, 104.8± 80.6 μg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2± 389.5 μg/l for 2-ethyl-3-oxohexanoic acid.     

Measurement of unbound caffeic acid in rat blood by on-line microdialysis coupled with liquid chromatography and its application to pharmacokinetic study

To monitor the levels of caffeic acid in rat blood, an on-line microdialysis system coupled with liquid chromatography was developed. The microdialysis probe was inserted into the jugular vein/right atrium of male Sprague-Dawley rats. Caffeic acid (100 mg/kg, i.v.) was then administered via the femoral vein. Dialysates were automatically injected onto a liquid chromatographic system via an on-line injector. Samples were eluted with a mobile phase containing methanol–100 mM monosodium phosphoric acid (35:65, v/v, pH 2.5). The UV detector wavelength was set at 320 nm. The detection limit of caffeic acid was 20 ng/ml. The in vivo recoveries of the microdialysis probe for caffeic acid at 0.5 and 1 μg/ml were 48.34±2.68 and 47.64±3.43%, respectively (n=6). Intra- and inter-assay accuracy and precision of the analyses were ≤10% in the range of 0.05 to 10 μg/ml. Pharmacokinetics analysis of results obtained using such a microdialysis–chromatographic method indicated that unbound caffeic acid in the rat fitted best to a biexponential decay model.     

Routine determination of flumequine in kidney tissue of pig using automated liquid chromatography

A high-performance liquid chromatographic assay is described as a routine analytical method for the determination of flumequine (FLU) and its hydroxylated metabolite (OH-FLU) in pig kidney tissue. Kidney samples (2 g) containing FLU and OH-FLU were extracted by liquid-liquid extraction with ethyl acetate (10 ml). Analytical separations were performed by reversed-phase HPLC with fluorometric detection at 252 nm excitation and 356 nm emission under gradient conditions. The mobile phase was acetonitrile-2.7·10⁻³ M oxalic acid in water (pH 2.5). The assay is specific and reproducible within the flumequine range of 0.050–2.5 μg/g and recovery at 0.050 μg/g was 94.8%.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in rat hepatic microsome after chiral derivatization with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate

A reversed-phase high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat hepatic microsome has been developed. Racemic atenolol was extracted from alkalinized rat hepatic microsome by ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and evaporated using a gentle stream of air. Atenolol racemic compound was derivatized with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate at 35°C for 30 min to form diastereomers. After removal of excess solvent, the diastereomers were dissolved in phosphate buffer (pH 4.6)–acetonitrile (50:30). The diastereomers were separated on a Shimadzu CLC-C18 column (10 μm particle size, 10 cm×0.46 cm I.D.) with a mobile phase of phosphate buffer–methanol–acetonitrile (50:20:30, v/v) at a flow-rate of 0.5 ml/min. A UV–VIS detector was operated at 254 nm. For each enantiomer, the limit of detection was 0.055 μg/ml (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) was 0.145 μg/ml (RSD <10%). In the range 0.145–20 μg/ml, intra-day coefficients of variation were 1.0–7.0% and inter-day coefficients of variation were 0.4–16.5% for each enantiomer. The assay was applied to determine the concentrations of atenolol enantiomers in rat hepatic microsome as a function of time after incubation of racemic atenolol.     

Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 μM and the calibration curve was linear at least up to 5 μM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 μM and from 3.5 to 6.7 at 5 μM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 μM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.     

Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at −20°C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 μl) on a 100-mg C₁₈-endcapped cartridge. Matrix components were eliminated with 750 μl of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 μl of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 μl of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4–20 μg/ml and 2–150 μg/ml for plasma and urine, respectively. THe extraction recovery of temozolomide was 86–90% from plasma and 103–105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37°C. An acidic pH (<5–6) shoul be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.     

Determination of amphotericin B in human serum by liquid chromatography

A rapid reversed-phase high-performance liquid chromatographic method with a 30-mm long column is described for assaying amphotericin B in serum. After deproteinization of serum samples with methanol, the supernatant was injected onto a reversed-phase C₁₈ column, using 2.5 mM Na₂EDTA-acetonitrile (70:30, v/v) as the mobile phase. Amphotericin B was eluted at 1.5 min. Calibration plot of the peak area against concentration was linear from 0.05 to 25 μg/ml (C.V. of 3%). Within-day and day-to-day imprecision (C.V.) ranged between 1.33% and 3.61%. The application was evaluated in 55 serum samples from patients treated with amphotericin B.     

Small blood volumes from children for quantitative sotalol determination using high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method using fluorescence detection has been developed for sotalol determination in small plasma samples of children and newborns with limited blood volume. In sample sizes of 100 μl of plasma, sotalol was extracted using an internal standard and solid-phase extraction columns. Chromatographic separation was performed on a Spherisorb C₆ column of 150×4.6 mm I.D. and 5 μm particle size at ambient temperature. The mobile phase consisted of acetonitrile–15 mM potassium phosphate buffer (pH 3.0) (70:30, v/v). The excitation wavelength was set at 235 nm, emission at 300 nm. The flow-rate was 1 ml/min. Sotalol and the internal standard atenolol showed recoveries of 107±8.9 and 97±8.1%, respectively. The linearity range for sotalol was between 0.07 and 5.75 μg/ml, the limit of quantitation 0.09 μg/ml. Precision values expressed as percent relative standard deviation of intra-assay varied between 0.6 and 13.6%, that of inter-assay between 2.4 and 14.4%. Accuracy varied between 86.1 and 109.8% (intra-assay) and 95.4 and 103.3% (inter-assay). Other clinically used antiarrhythmic drugs did not interfere. As an application of the assay, sotalol plasma concentrations in a 6-year-old child with supraventricular tachycardia treated with oral sotalol (3.2 mg/kg per day) are reported.     

Sensitive high-performance liquid chromatographic determination with fluorescence detection of phenol and chlorophenols with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a labeling reagent

A sensitive HPLC method for the determination of phenol and chlorophenols was developed. The fluorescence labeling reaction of phenols with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) was completed in 30 min at 60°C. The separation of DIB-derivatives of five representative phenols, i.e., phenol, o-, p-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, was achieved within 35 min with an ODS column using isocratic elution. The detection limits of these DIB derivatives at a signal-to-noise ratio (S/N) of 3 were in the range of 0.024 to 0.08 μM (0.12–0.45 pmol/20 μl injection). Twelve kinds of DIB derivatives with phenols containing mono-, di-, tri-, tetra- and penta-chlorophenol were also well separated within 208 min by changing the elution conditions. The derivatives were stable for at least for 24 h when they were placed at room temperature in the dark. The proposed method was applied to the assay of human urine samples and free and total phenol were determined. The relative standard deviations (RSDs) of the proposed method for within and between-day assay were <7.0% and <14.2%, respectively. The average concentrations of free and total phenol found in urine (n=6) were 4.3±2.5 and 29.5±14.0 μM, respectively.     

High-performance liquid chromatographic determination of modafinil and its two metabolites in human plasma using solid-phase extraction

A simple procedure for the simultaneous determination of modafinil, its acid and sulfone metabolites in plasma is described. The assay involved an extraction of the drug, metabolites and internal standard from plasma with a solid-phase extraction using C₁₈ cartridges. These compounds were eluted by methanol. The extract was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was redissolved in 250 μl of mobile-phase and a 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile-phase (26%, v/v acetonitrile in 0.05 M orthophosphoric acid buffer adjusted to pH 2.6) at a flow-rate of 1.1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 225 nm. Intra-day coefficients of variation ranged from 1.0 to 2.9% and inter-day coefficients from 0.9 to 6.1%. The limits of detection and quantitation of the assay were 0.01 μg/ml and 0.10 μg/ml respectively.     

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in sub-microliter samples of rat airway surface liquid (ASL), a thin (10–30 μm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (1–3 μl per cm² of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 μM for nitrate and 30 μM for nitrite (S/N=3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102±12 μM in rat ASL and 70±1.0 μM in rat plasma, whereas nitrite was 83±28 μM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387±16 μM, and to 377±88 μM in plasma. The concentration of nitrite increased to 103±7.0 μM for ASL and 138±17 μM for plasma.     

Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 × 2.1 mm I.D., 5 μm Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate—acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 μl sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean ± S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 ± 1.2% and 91.4 ± 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.     

Analysis of cysteine and N-acetylcysteine in human plasma by high-performance liquid chromatography at the basal state and after oral administration of N-acetylcysteine

A high-performance liquid chromatographic method for the determination of free reduced cysteine and N-acetylcysteine in human plasma at the basal state and after oral administration of N-acetylcysteine is described. The method is based on acid-catalysed conversion of plasma thiols to the corresponding S-nitroso derivatives by excess of nitrite and their subsequent cation-pairing RP-HPLC with detection at 333 nm. Recovery rates of cysteine and N-acetylcysteine added to human plasma were 94.6 and 99.6%, respectively. Inter- and intra-day precision were below 6%. In healthy humans (n=5), free reduced cysteine was determined to be (mean±S.E.) 10.0±0.96 μM. No N-acetylcysteine was detected in plasma of these subjects above the limit of detection (e.g. 170 nM). The method was successfully applied to a pharmacokinetic study on orally administered N-acetylcysteine to healthy volunteers.