Fosfomycin determination in serum by capillary zone electrophoresis with indirect ultraviolet detection

Capillary zone electrophoresis with indirect ultraviolet detection was used for the determination of fosfomycin in serum. Running buffer consisted of a mixture of 200 mM sodium borate with 10 mM phenylphosphonic acid used as ultraviolet absorbing background electrolyte. Relationships between the pH of the buffer and the efficiency of the separation (migration times and selectivities) or the sensitivity of detection were investigated. The method was then validated over a 10–100 μg ml⁻¹ concentration range to be applied to further therapeutic drug monitoring. The choice of ethylphosphonic acid as internal standard is discussed. The specificity and the linearity of the technique are demonstrated. The inter-day precision was satisfactory with a relative standard deviation of less than 2%. Accuracy was calculated with a standard error near 0.5 and 18% for 100 and 10 μg ml⁻¹, 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.     

Analysis of trace amino acid neurotransmitters in hypothalamus of rats after exhausting exercise using microdialysis

A simple but effective coupling of microdialysis and capillary electrophoresis with laser induced fluorescence detection technique was applied to analysis of amino acid neurotransmitters in the hypothalamus of rats after acute exhausting exercise. The separation of amino acids was achieved using an uncoated fused-silica capillary (57 cm×75 μm I.D.) with a buffer of 10 mM disodium tetraborate at pH 10 and an applied voltage of 12.5 kV. The detection limit was 10⁻¹⁰ M for each amino acid. It is sufficiently sensitive and rapid for the determination of amino acids in a 5-μl Microdialysate. In comparison to pre-exercise, a significant increase in the levels of six hypothalamic amino acids (arginine, glycine, lysine, glutamic acid, alanine, γ-amino-n-butyric acid) was found after exercise. These results demonstrate that the increase of metabolic amino acids in the hypothalamus of rats can be induced by exhausting exercise and suggests that amino acid neurotransmitters may play functional roles in the central effects of exercise.     

High-performance liquid chromatographic determination of cocaine and its metabolites in serum microsamples with fluorimetric detection and its application to pharmacokinetics in rats

A sensitive, selective and simple HPLC method with fluorimetric detection is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 μl). Chromatographic separation is achieved on a Hypersil BDS C₁₈ column (100×2.1 mm, 5 μm) with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.6, containing 1.0·10⁻⁴ M tetrabutylammonium phosphate (14:10:76, v/v/v). The detection limit (0.5 ng/ml) for all the compounds, using direct fluorometric detection operated at excitation and emission wavelengths of 230 and 315 nm, respectively, was approximately five-times lower than that of using a UV detector operated at 235 nm. The effects of ratio of 2-propanol to chloroform in extraction solvents on the recovery and precision for cocaine and its metabolites were systematically examined. The method was used to study the pharmacokinetics of cocaine after administration of intravenous 2 mg/kg and oral 20 mg/kg doses.     

Determination of non-protein-bound iron in human synovial fluid by high-performance liquid chromatography with electrochemical detection

Non-protein-bound iron in human synovial fluid was determined using high-performance liquid chromatography with electrochemical detection. The procedure was based on the separation of the iron—diethylenetriaminepentaacetic acid (DPTA) complex formed directly on a chromatographic column containing an anion-exchange resin followed by electrochemical detection. The method enabled more than 0.1 μM Fe(III) to be determined with an injection volume of 10 μl. A mixture of synovial fluid, 20 μM DTPA and acetate buffer was incubated in the presence and absence of superoxide (O⁻₂) generated by a xanthine—xanthine oxidase system and was ultrafiltered through a 30 000 molecular mass cut-off filter. No iron was detected in the ultrafiltrate at physiological pH. However, the presence of iron was observed in the ultrafiltrate at low pH, and O⁻₂ and decreased pH, iron may be released into the synovial fluid.     

Determination of the halothane metabolites trifluoroacetic acid and bromide in plasma and urine by ion chromatography

Halothane (CF₃CHClBr), a widely used volatile anesthetic, undergoes extensive biotransformation in humans. Oxidative halothane metabolism yields the stable metabolites trifluoroacetic acid and bromide which can be detected in plasma and urine. To date, analytical methodologies have either required extensive sample preparation, or two separate analytical procedures to determine plasma and urine concentrations of these analytes. A rapid and sensitive method utilizing high-performance liquid chromatography-ion chromatography (HPLC-IC) with suppressed conductivity detection was developed for the simultaneous detection of both trifluoroacetic acid and bromide in plasma and urine. Sample preparation required only ultrafiltration. Standard curves were linear (r²≥0.99) from 10 to 250 μM trifluoroacetic acid and 2 to 5000 μM bromide in plasma and 10 to 250 μM trifluoroacetic acid and 2 to 50 μM bromide in urine. The assay was applied to quantification of trifluoroacetic acid and bromide in plasma and urine of a patient undergoing halothane anesthesia.     

Gas chromatography–electron-capture detection of urinary methylhippuric acid isomers as biomarkers of environmental exposure to xylene

Methylhippuric acid isomers (MHAs), urinary metabolites of xylenes, were determined, after clean-up by C18-SPE and esterification with hexafluoroisopropanol and diisopropylcarbodiimide, by GC with ECD detection, on an SPB-35 capillary column (30 m, 0.32 mm I.D., 0.25 μm film thickness, β=320). S-benzyl-mercapturic acid was used for internal standardization. Chromatographic conditions were: oven temperature 162°C, for 14.2 min; ramp by 30°C/min to 190°C, for 3.5 min; ramp by 30°C/min to 250°C, for 4 min; helium flow rate: 1.7 ml/min; detector and injector temperature: 300°C. The sample (1 μl) was injected with a split injection technique (split ratio 5:1). MHA recovery was >95% in the 0.5–20 μmol/l range; the limit of detection was <0.25 μmol/l; day-to-day precision, at 2 μmol/l, was Cv<10%. Urinary MHAs were determined in subjects exposed to different low-level sources of xylenes: (a) tobacco smoking habit and (b) BTX urban air pollution (airborne xylene ranging from 0.1 to 3.7 μmol/m³). Study (a) showed a significant difference between urinary MHA median excretion values of nonsmokers and smokers (4.6 μmol/l vs. 8.1 μmol/l, p<0.001). Study (b) revealed a significant difference between indoor workers and outdoor workers (4.3 μmol/l vs. 6.9 μmol/l, p<0.001), and evidenced a relationship between MHAs (y, μmol/mmol creatinine) and airborne xylene (x, μmol/m³) (y=0.085+0.34x; r=0.82, p<0.001, n=56). Proposed biomarkers could represent reliable tools to study very low-level exposure to aromatic hydrocarbons such as those observed in the urban pollution due to vehicular traffic or in indoor air quality evaluation.     

Measurement of plasma uracil using gas chromatography–mass spectrometry in normal individuals and in patients receiving inhibitors of dihydropyrimidine dehydrogenase

A sensitive gas chromatographic–mass spectrometric method is described for reliably measuring endogenous uracil in 100 μl of human plasma. Validation of this assay over a wide concentration range, 0.025 μM to 250 μM (0.0028 μg/ml to 28 μg/ml), allowed for the determination of plasma uracil in patients treated with agents such as eniluracil, an inhibitor of the pyrimidine catabolic enzyme, dihydropyrimidine dehydrogenase. Calibration standards were prepared in human plasma using the stable isotope, [¹⁵N₂]uracil, to avoid interference from endogenous uracil and 10 μM 5-chlorouracil was added as the internal standard.     

Comparison of anion-exchange and ion-modified reversed-phase liquid chromatography for the determination of S-sulfocysteine

A dual Hg–Au amalgam electrode is used to detect S-sulfocysteine (SSC) in this study. There exist two main components in the acetonitrile (ACN) rat brain extracts, namely, Cl⁻ and GSSG (oxidized glutathione), that are active in our detection system (GSH is not extracted in ACN). Two strong anion-exchange columns from different companies were used to separate the samples under different conditions, but SSC and Cl⁻ were not separated at the optimum detection pH of 5.2. The signal from Cl⁻ was greatly decreased by lowering the potential at the downstream electrode, though it cannot be completely eliminated. While a silver cartridge removed Cl⁻ from micromoles to several millimoles without any negative effect on the SSC signal in aqueous standards, a large negative peak which interferes with SSC detection was unfortunately introduced when a silver cartridge was applied to brain tissue samples. However, SSC and Cl⁻ in the samples are successfully separated by ion-modified reversed-phase LC in acetate buffer at the optimum detection pH (5.2). The separation conditions are 20 mM acetic acid, 2% methanol, 0.5 mM cetyltrimethylammonium p-toluene sulfonate (CTMA) (pH 5.2). Most importantly, the sensitivity of SSC under the optimum separation conditions is not sacrificed. The detection limit is 8 nM (20 μl injected).     

Determination of ceftazidime in plasma using high-performance liquid chromatography and electrochemical detection: Application for individualizing dosage regimens in elderly patients

This study describes a sensitive HPLC–electrochemical detection method for the analysis of ceftazidime, a third-generation cephalosporin, in human plasma. The extraction procedure involved protein precipitation with 30% trichloroacetic acid. The separation was achieved on a reversed-phase column (250×4.6 mm I.D., 5 μm) packed with C₁₈ Kromasil with isocratic elution and a mobile phase consisting of acetonitrile–25 mM KH₂PO₄–Na₂HPO₄ buffer, pH 7.4 (10:90, v/v). The proposed analytical method is selective, reproducible and reliable. The assay has a precision of 0.2–15.1% (C.V.) in the range of 5–200 μg ml⁻¹. (corresponding to 0.5 to 20 ng of ceftazidime injected onto the column), and is optimised for assaying 50 μl of plasma. The extraction recovery from plasma was approximately 100%. The method was highly specific for ceftazidime and there was no interference from either commonly administered drugs or endogenous compounds. This assay was used to measure ceftazidime in elderly patients for therapeutic drug monitoring.     

Monitoring of met-enkephalin in vivo with 5-min temporal resolution using microdialysis sampling and capillary liquid chromatography with electrochemical detection

A method based on microdialysis sampling and capillary liquid chromatography with electrochemical detection that allows in vivo monitoring of met-enkephalin with 5-min temporal resolution is described. Sampling was achieved using a concentric microdialysis probe made from polycarbonate membrane material with a 20 kDa cut-off. This probe had an in vitro relative recovery for met-enkephalin of 63% at a dialysis flow-rate of 0.6 μl/min. Separations were performed using 7 cm×25 μm I.D. fused-silica capillary columns packed with 5 μm Alltima C₁₈ particles. A carbon fiber microelectrode was used as the detector electrode. The mass detection limit for met-enkephalin with this system was 40 amol. With on-column preconcentration, up to 2 μl of sample could be loaded onto the column resulting in concentration detection limits as low as 20 pM for met-enkephalin. Direct injection of dialysate, collected at 5-min intervals, allowed determination of met-enkephalin concentrations in the rat globus pallidus under basal and K⁺-induced depolarization conditions.     

Determination of idarubicin and idarubicinol in rat plasma using reversed-phase high-performance liquid chromatography and fluorescence detection

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of idarubicin and idarubicinol in rat plasma. Blood samples were analyzed from 16 rats which had received an intravascular dose of 2.25 mg kg⁻¹ idarubicin. After deproteinization with acetonitrile, the separation was performed with a LiChrospher 100 RP-18 column (5 μm), using fluorescence detection (excitation: 485 nm/emission: 542 nm). The mean recovery was 95.6% for idarubicin and 90.7% for idarubicinol, respectively. The detection limit was 0.25 ng ml⁻¹ using an injection volume of 50 μl. Daily relative standard deviation (RSD) was 3.2% (10 ng idarubicin/ml, n=10) and 4.4% (10 ng idarubicinol/ml, n=10).     

Determination of reduced and oxidized homocysteine and related thiols in plasma by thiol-specific pre-column derivatization and capillary electrophoresis with laser-induced fluorescence detection

A new sensitive and rapid capillary electrophoresis (CE) assay for measuring reduced and oxidized thiols in human plasma has been developed. To prevent oxidation of the thiols, whole blood was immediately centrifuged after collection and the plasma proteins were precipitated with perchloric acid. The reduced thiols in the supernatant were derivatized quantitatively at 25°C, pH 7.5 with a fluorescent reagent, fluorescein-5-maleimide (FM). The total plasma concentration of thiols, including the fraction coupled to proteins, was assayed after an initial reduction of the disulfide linkage in plasma with dithiothreitol. The separation of FM-thiols was performed in an acetonitrile/10 mM sodium phosphate–50 mM SDS buffer [25:75 (v/v); pH 7.0] using a fused-silica capillary (57 cm×75 μm I.D.) at 45°C. A 3-mW argon-ion laser (λ_ex 488 nm/λ_em 520 nm) was employed for FM-thiol detection. With the electric field of 530 V/cm, the time needed for the separation of FM-homocysteine, FM-glutathione and FM-N-acetylcysteine was less than 8 min. The lower limit of detection was 3 μM for the total thiols and 10 nM for the reduced thiols. The method was applied to the determination of homocysteine levels in plasma from patients with end-stage renal disease.     

Dual microcolumn immunoassay applied to determination of insulin secretion from single islets of Langerhans and insulin in serum

A dual microcolumn immunoassay (DMIA) was developed and applied to determination of insulin in biological samples. The DMIA utilized a protein G capillary column (150 μm I.D.) with covalently attached anti-insulin to selectively capture and concentrate insulins in a sample. Insulins retained in the capillary immunoaffinity column were desorbed and injected onto a reversed-phase capillary column (150 μm I.D.) for further separation from interferences such as cross-reactive antigens and non-specifically adsorbed sample components. Bovine, porcine and rat insulin all cross-reacted with the antibody and could be determined simultaneously. Using a UV absorbance detector, the dual microcolumn system had a detection limit of 10 fmol or 20 pM for 500-μl sample volumes. The DMIA system was used to measure glucose-stimulated insulin secretion from single rat islets of Langerhans. Because of the separation in the second dimension, both rat I and rat II insulin could be independently determined. The system was also evaluated for determination of insulin in serum. Using microcolumns instead of conventional HPLC columns resulted in several advantages including use of less chromatographic material and improved mass detection limit.     

Separation of near-infrared fluorescent conjugates of dATP and related compounds by capillary electrophoresis

Capillary electrophoresis was examined as a means for the separation and quantitation of deoxyadenosine triphosphate (dATP) and other nucleotides that were labeled with the near-infrared fluorescent dye IRD700 or related tags. Under the final optimized conditions the labeled dATP was separated from several possible impurities, including the unconjugated forms of IRD700 and dATP, as well as dADP, dAMP and their corresponding IRD700 conjugates. The assay was performed under two sets of conditions. First, the sample was injected onto a 50 cm×75 μm I.D. fused-silica capillary at 25 kV in the presence of a pH 9.5, 140 mM borate running buffer. The resulting peaks were monitored at both 254 and 680 nm, where the latter wavelength was used to identify any species that contained the IRD700 label. A second injection was then performed under the same conditions but with a fixed concentration of dTTP now being added to the running buffer; this resulted in the formation of a complex between the dTTP and any dATP, dADP or dAMP-containing components, which changed their rates of migration and allowed them to be differentiated from unconjugated IRD700 or dye contaminants. Only 6 nl of a 1:10 diluted sample were required per analysis. The limit of detection at this injection volume was approximately 1.0 μM (or 6·10⁻¹⁵ mol for a 6-nl injection) for each monitored component. The linear range extended up to at least 80 μM. The analysis time was 20 min per injection and the day-to-day precision was ±2–3%. The same method was also found to be useful in examining related conjugates, such as those based on the dye IRD40.     

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.     

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%.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

High-performance liquid chromatographic procedure for the quantitative determination of paclitaxel (Taxol) in human plasma

An isocratic high-performance liquid chromatographic method has been developed and validated for the quantitative determination of paclitaxel (Taxol^®), a novel antimitotic, anticancer agent, in human plasma. The analysis required 0.5 ml of plasma, and was accomplished by detection of the UV absorbance of paclitaxel at 227 nm following extraction and concentration. The method involved extraction of paclitaxel from plasma, buffered with 0.5 ml of 0.2 M ammonium acetate (pH 5.0), onto 1-ml cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the mobile phase, acetonitrile-methanol-water (4:1:5, v/v/v) containing 0.01 M ammonium acetate (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5 μm column. The retention time of paclitaxel was 10 min. The validated quantitation range of the method was 10–1000 ng/ml (0.012–1.17 μM) of paclitaxel in plasma. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of clinical study samples. The observed recovery for paclitaxel was 83%. Epitaxol, a biologically active stereoisomer, and baccatin III, a degradation product, were also chromatographically separated from taxol by this assay. The method was applied to samples from a clinical study of paclitaxel in cancer patients, providing a pharmacokinetic profiling of paclitaxel.     

High-performance anion-exchange chromatography of ultraviolet-absorbing constituents of human urine

A 100-μl urine sample was chromatographed on a column packed with a strongly basic macroreticular anion-exchange resin (Diaion CDR-10, 5– μm diameter with a nominal 35% cross linkage). The elution was performed with a linear acetate gradient from 0 to 6.0 M at an average flow-rate of 0.72 ml/min and at an average pressure of 104 kg/cm². The relative standard deviation of retention times and peak height was ± 4% or less. The properties of the macroreticular anion-exchange resin, the effect of the particle size, the pH of acetate buffers, and the effect of the flow-rate of the eluent on the separation were investigated. Thirty three components of urine were then resolved and named.