Journal of Chromatography B (v.789, #2)

News Section (N1-N2).

In order to assess occupational exposure of hospital personnel involved in the preparation and administration of antineoplastic drugs, biological and environmental monitoring are essential to identify the main exposure routes and to quantify potential health risks. If workplace contamination cannot be completely avoided, it is of utmost importance to reduce exposure to the lowest possible levels. To this aim, not only do education and training of the exposed subjects play an important role, but accurate standardized sampling techniques and analytical methods are also required. A critical overview of the most significant methods available in the literature is presented and their value is discussed, especially with respect to their sensitivity and specificity. In addition, attention is given to validation procedures and, consequently, to their reliability. The results from the most important surveys carried out at hospital departments are also discussed, with a view to improving both monitoring strategies and moreover working conditions.
Keywords: Antineoplastic drugs;

Highly sensitive quantification of vancomycin in plasma samples using liquid chromatography–tandem mass spectrometry and oral bioavailability in rats by Nobuhito Shibata; Makoto Ishida; Yarasani Venkata Rama Prasad; Weihua Gao; Yukako Yoshikawa; Kanji Takada (211-218).
We developed a highly sensitive liquid chromatography–tandem mass spectrometry assay (LC–MS–MS) for a glycopeptide antibacterial drug, vancomycin (VCM), in rat plasma. After precipitating 100 μl of plasma with 300 μl of 10% trifluoroacetic acid–methanol (2:1, v/v), the supernatant was diluted with 300 μl of distilled water and was passed through a filter. LC–MS–MS equipped with electrospray ionization in the positive ion mode used a pair of ions at 725/144 m/z for VCM in the multiple reaction-monitoring mode with a sample injection volume of 20 μl. The calibration curve had a linear range from 0.01 to 20 μg/ml when linear least square regression was applied to the concentration versus peak area plot. The drug in the sample was detected within 5 min. Precision, accuracy and limit of quantitation indicated that this method was suitable for the quantitative determination of VCM in rat plasma. Using this method, we defined for the first time that the oral bioavailability of VCM in rats was 0.069%. This method can be applied to basic pharmacokinetic and pharmaceutical studies in rats.
Keywords: Vancomycin;

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for confirmatory analysis of 17β-boldenone (17β-BOL), 17α-boldenone (17α-BOL) and androsta-1,4-diene-3,17-dione (ADD) in bovine urine was developed. [2H2]17β-Testosterone (17β-T-d2) was used as the internal standard. Sample preparation involved enzymatic hydrolysis and purification on a C18 solid-phase extraction column. Chromatographic separation of the analytes was obtained using an RP-C18 HPLC column. LC–MS–MS detection was carried out with an atmospheric pressure chemical ionisation (APCI) source equipped with a heated nebulizer (HN) interface operating in the positive ion mode. For unambiguous hormone confirmation, three analyte precursor–product ion combinations were monitored during multiple-reaction monitoring (MRM) LC–MS–MS analysis. Overall recovery (%), repeatability (relative standard deviations, RSD, %) and within-laboratory reproducibility (RSD, %) ranged from 92.2 to 97.7%, from 6.50 to 2.94% and from 13.50 to 5.04%, respectively, for all analytes. The limit of quantification in bovine urine was 0.20 ng ml−1 for 17β-BOL and ADD and 0.50 ng ml−1 for 17α-BOL. The validated method was successfully applied for determination of 17β-BOL, 17α-BOL and ADD in a large number of bovine urine samples collected within the national Official Residue Control Program.
Keywords: Boldenone; Androsta-1,4-diene-3,17-dione;

Caffeine is metabolised in humans primarily by cytochromes P450 1A2 and 2A6, xanthine dehydrogenase/oxidase, and N-acetyltransferase 2. The activities of these enzymes show a large variation due to genetic polymorphisms and/or induction by xenobiotics. Ratios of different caffeine metabolites in urine or other body fluids are frequently used to characterise the individual/actual activity of these enzymes. The common analytical method involves extensive sample preparation, followed by HPLC–UV. The presence of numerous other UV-absorbing chemicals in body fluids affects the sensitivity and selectivity of this method. We have developed an HPLC–electrospray-MS–MS method for the determination of 11 caffeine metabolites and two internal standards after a simple, extractionless preparation. Blank urine, obtained after 5 days on a methylxanthine-free diet, contained small amounts of some caffeine metabolites, but no other components producing any confounding signals. Eleven metabolites and internal standards were recovered at 90 to 110% after addition to the blank urine (0.1 to 2.5 μM in the final sample involving a 20-fold dilution of urine) in the 0.1–2.5 μM concentration range. Other metabolites, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU), were detected with similar recovery and precision, but required higher concentrations (3 to 30 μM). AFMU was completely converted into AAMU by a short alkalisation of urine. The method was explored in six healthy individuals after consuming coffee (4 mg caffeine per kg body mass). These experiments demonstrated the simplicity, high sensitivity and selectivity of the method under conditions used for phenotyping.
Keywords: Caffeine; Methylxanthines;

A simple and sensitive column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the quantification of perospirone, a serotonin and dopamine antagonist, and its metabolite ID-15036 in human plasma. The test compounds were extracted from 2 ml of plasma using chloroform–hexane (30:70, v/v) and the extract was injected into a column I (TSK-PW precolumn, 10 μm, 35×4.6 mm I.D.) for clean-up and column II (C18 STR ODS-II analytical column, 5 μm, 150×4.6 mm I.D.) for separation. The peak was detected using a fluorescence detector set at Ex 315 nm and Em 405 nm, and the total time for a chromatographic separation was ∼30 min. The method was validated for the concentration range from 0.1 to 100 ng/ml. Mean recoveries were 97% for perospirone and 96% for ID-15036. Intra- and inter-day relative standard deviations were less than 2.8 and 5.3% for perospirone and 2.4 and 4.4% for ID-15036, respectively, at the concentration range from 0.3 to 30 ng/ml. This method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.
Keywords: Perospirone;

An HPLC–ESI-MS–MS method was developed to quantify in human urine fourteen aromatic acids known as metabolites of dietary polyphenols. These metabolites were determined simultaneously in a single 20-min chromatographic analysis with multiple reaction monitoring detection. The inter- and intra-day precisions, calculated from quality control samples were 8.8 and 5.3%, respectively, and the mean accuracy was 2.3%. The method was tested on urine samples collected from one healthy volunteer who consumed a polyphenol-rich diet for 3 days. Increased levels of several aromatic acid metabolites were observed, demonstrating that the method can be used to detect changes in the excretion of microbial metabolites induced by the consumption of polyphenol-containing foods in humans.
Keywords: Polyphenols;

Determination of polycyclic aromatic hydrocarbons in milk samples by high-performance liquid chromatography with fluorescence detection by Naoya Kishikawa; Mitsuhiro Wada; Naotaka Kuroda; Syuzo Akiyama; Kenichiro Nakashima (257-264).
This paper describes a high-performance liquid chromatographic (HPLC) method for the determination of polycyclic aromatic hydrocarbons (PAHs) in milk samples. The method involves a liquid–liquid extraction procedure after saponification of milk samples with sodium hydroxide. Reproducible determination with highly sensitive detection was attained by HPLC with fluorescence detection using 1,2-bis(9-anthryl)ethane as an internal standard. The detection limits of 12 kinds of PAHs ranged from 1.3 to 76 ng/kg milk at a signal/noise ratio of 3. By the proposed method, the presence of 12 and 11 kinds of PAHs could be confirmed in commercial milk and human milk samples, respectively. The average concentrations of total PAHs (mean±SD, μg/kg) were found to be 0.99±0.37 for commercial milk (n=14), 2.01±0.30 for infant formula (n=3) and 0.75±0.47 for human milk (n=51). High correlation coefficients between the concentrations of total PAHs and triglyceride were observed for commercial milk (r=0.659) and human milk (r=0.645).
Keywords: Polynuclear aromatic hydrocarbons;

Biocompatible affinity adsorbents prepared from macroporous glycidyl methacrylate–triallyl isocyanurate copolymer (GT) has been used for removing circulating immune complexes (CICs). In this work, adsorption of circulating immune complexes on GT-based affinity adsorbents has been studied by using batch and hemoperfusion studies. In batch mode, the equilibrium adsorption level was determined to be a function of the contact time, temperature, initial CIC concentration and adsorbent dosage. In animal hemoperfusion trials, removal of CICs is efficient and rapid. IgG, IgM and complement C3, C4 are minimally affected. There are negligible decreases in RBC, WBC, PLT and HB. Acid–base equilibrium, electrolytes and plasma proteins also are minimally affected.
Keywords: Circulating immune complexes; Glycidyl methacrylate; Triallyl isocyanurate;

Liquid chromatography–tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals by Gaëlle Henneré; François Becher; Alain Pruvost; Cécile Goujard; Jacques Grassi; Henri Benech (273-281).
This study was aimed to apply an LC–MS–MS method previously developed for intracellular nucleoside reverse transcriptase inhibitors-triphosphate (NRTI-TPs) to the determination of natural deoxyribonucleotides (dNTPs) in human peripheral blood mononuclear cells. The LC–MS–MS method was directly used in assay of dATP and dTTP. Interferences by ribonucleotides (rNTPs) prevented direct application to the two other analytes: dGTP and dCTP. A periodate oxidation procedure was therefore optimized to remove rNTPs from the cell medium in order to quantitate dCTP and dGTP. The determination of the intracellular ratio of NRTI-TP/dNTP in HIV-infected patients now involves use of the same chromatographic system for simultaneous assay of several analytes.
Keywords: Deoxyribonucleotide triphosphate; Nucleosides;

“Metabonomics” has in the past decade demonstrated enormous potential in furthering the understanding of, for example, disease processes, toxicological mechanisms, and biomarker discovery. The same principles can also provide a systematic and comprehensive approach to the study of food ingredient impact on consumer health. However, “metabonomic” methodology requires the development of rapid, advanced analytical tools to comprehensively profile biofluid metabolites within consumers. Until now, NMR spectroscopy has been used for this purpose almost exclusively. Chromatographic techniques and in particular HPLC, have not been exploited accordingly. The main drawbacks of chromatography are the long analysis time, instabilities in the sample fingerprint and the rigorous sample preparation required. This contribution addresses these problems in the quest to develop generic methods for high-throughput profiling using HPLC. After a careful optimization process, stable fingerprints of biofluid samples can be obtained using standard HPLC equipment. A method using a short monolithic column and a rapid gradient with a high flow-rate has been developed that allowed rapid and detailed profiling of larger numbers of urine samples. The method can be easily translated into a slow, shallow-gradient high-resolution method for identification of interesting peaks by LC–MS/NMR. A similar approach has been applied for cell culture media samples. Due to the much higher protein content of such samples non-porous polymer-based small particle columns yielded the best results. The study clearly shows that HPLC can be used in metabonomic fingerprinting studies.

In order to better understand the cytotoxic effects of trichloroethylene (TCE) and its metabolites in TCE-induced carcinogenicity, it is necessary to determine the molecular species in biological samples. We have developed an efficient gas chromatography–mass spectrometry assay for the quantitative analysis of trichloroethylene alcohol and acetate. This method utilizes a simple esterification procedure, and a single liquid–liquid extraction with hexane–dichloromethane (1:1) that allows >90% recovery of the metabolites, followed by gas chromatography–mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the toxic TCE metabolites. The utility of the assay is demonstrated through the analysis of TCE metabolites in urine from rats administered with TCE. The limit of quantitation (precision and accuracy<20%) was 1.7 ng/ml for TCE alcohol and 2.3 ng/ml for TCE acetate.
Keywords: Trichloroethylene;

Simplified sample workup obviating protein precipitation and eluent evaporation commonly employed in earlier reports using chloroformate-mediated derivatization of aminothiols prior to mass spectrometric (MS) detection is presented. The reduction of disulfides in plasma is accomplished with dithiothreitol within minutes. A simultaneous derivatization with ethyl chloroformate (ECF) and extraction of derivatives into organic phase takes place within seconds. Along with S-amino acids, also aromatic amino acids can be determined during a 5-min run. Gas chromatography with flame ionization detection (GC–FID) proved to be sensitive enough to reach plasma homocysteine levels. A prerequisite for a reliable quantitation was fulfilled under the given conditions. Intra-assay precision was <5%, recoveries from spiked plasma complete (101.2%), detection and quantitation limits for homocysteine came to <1 and 3 μmol/l. Our results were in full agreement with those obtained by liquid chromatography (r=0.999 for homocysteine and 0.987 for cysteine), and were close to two homocysteine immunoassays (r=0.991 and 0.939, respectively).
Keywords: Homocysteine; Cysteine; Methionine; Aromatic amino acids;

Application of multiplexed capillary electrophoresis with laser-induced fluorescence (MCE–LIF) detection for the rapid measurement of endogenous extracellular signal-regulated protein kinase (ERK) levels in cell extracts by Jian Tu; LaShonda N Anderson; Jian Dai; Kevin Peters; Andrew Carr; Paula Loos; Danielle Buchanan; James J Bao; Changsheng Liu; Kenneth R Wehmeyer (323-335).
Multiplexed (96-lane) capillary electrophoresis with laser-induced fluorescence (MCE–LIF) detection was used for the rapid analysis of extracellular signal-regulated protein kinase (ERK) levels from in vitro cell extracts. The levels of ERK enzyme in cell extracts were determined by monitoring the conversion of a fluorescent-labeled peptide substrate to a phosphorylated fluorescent-labeled peptide product using MCE–LIF. The incorporation of a fluorescent internal standard was found to improve the precision of the analysis. The enzyme assay conditions including substrate concentration, reaction time and enzyme linear range were rapidly optimized using the MCE–LIF approach for both direct and immunoprecipitation-based ERK assays. The levels of ERK from in vitro cell extracts stimulated with angiopoietin 1 (Ang1*) were determined using the MCE–LIF approach. The advantages of MCE–LIF for developing and applying enzyme assays, as well as the figures of merit for the direct and immunoprecipitation ERK assays, are discussed.
Keywords: Protein kinase; Enzymes;

Galantamine is an acetylcholinesterase inhibitor, recently approved for the treatment of mild-to-moderate Alzheimer’s disease. To allow a higher throughput of samples, a new bioanalytical method for the determination of galantamine in human plasma was developed and validated. A stable isotope labelled internal standard was used. Sample preparation consisted of a simple one-step liquid–liquid extraction with toluene. The extracts were analysed with positive ion TurboIonspray tandem mass spectrometry (LC–MS–MS). The method was validated in the 1–500-ng/ml range. The accuracy, precision, selectivity, lower limit of quantification, upper limit of quantification, linearity and extraction recovery were evaluated, as well as the stability of the compound in plasma, blood, methanol and 2% BSA solutions under different conditions. The method proved very rugged during the analysis of large numbers of samples from clinical trials.
Keywords: Galantamine;

Validation of a method for quantification of ketobemidone in human plasma with liquid chromatography–tandem mass spectrometry by Matilda Lampinen; Ulf Bondesson; Elisabeth Fredriksson; Mikael Hedeland (347-354).
A liquid chromatography tandem mass spectrometry (LC–MS–MS) method for determination of the analgesic aminophenol ketobemidone in human plasma is presented. Two preparation methods for plasma samples containing ketobemidone were compared, liquid–liquid extraction (LLE) and solid-phase extraction (SPE). Both methods showed good precision (n=10), 1.7% and 2.9%, respectively (0.04 μM) and 1.1% and 2.5%, respectively (0.14 μM). The accuracy was 98% and 103%, respectively (0.04 μM) and 105% and 99%, respectively (0.14 μM). Ketobemidone could be quantified at 0.43 nM, with a relative standard deviation of 17.5% (n=19) using LLE and 18.6% (n=10) using SPE. This level was an order of magnitude lower than earlier reported quantification limits. Quantitative data from plasma samples analyzed with LC–MS–MS were in good agreement with those obtained by gas chromatography with chemical ionization mass spectrometry (GC-CI/MS). This indicates that LC–MS–MS is a good alternative method to GC–MS as it is more sensitive and time-consuming derivatization can be avoided.
Keywords: Ketobemidone;

Simultaneous liquid chromatographic assessment of thiamine, thiamine monophosphate and thiamine diphosphate in human erythrocytes: a study on alcoholics by Rosanna Mancinelli; Mauro Ceccanti; Maria Soccorsa Guiducci; Guido Francesco Sasso; Gemma Sebastiani; Maria Luisa Attilia; John Paul Allen (355-363).
An isocratic HPLC procedure for the assessment of thiamine (T), thiamine monophosphate (TMP) and thiamine diphosphate (TDP) in human erythrocytes is described. Several aspects of the procedure make it suitable for both clinical and research purposes: limits of detection and quantification of 1 and 2.5 nmol/l, respectively, recovery of 102% on average (range 93–112%), intra- and inter-day precisions within 5 and 9%, respectively, total elution time 15 min. This analytical methodology was applied to a case-control study on erythrocyte samples from 103 healthy subjects and 36 alcohol-dependent patients at risk of thiamine deficiency. Mean control values obtained were: T=89.6±22.7 nmol/l, TMP=4.4±6.6 nmol/l and TDP=222.23±56.3 nmol/l. T and TDP mean values of alcoholics were significantly lower than those of control cases: T=69.4±35.9 nmol/l (P<0.001) and TDP=127.4±62.5 nmol/l (P<10−5). The diagnostic role of TDP was evaluated and a significant role for thiamine was established in the study of alcohol related problems.
Keywords: Thiamine;

Determination of polymyxin E1 in rat plasma by high-performance liquid chromatography by Dennis J Gmur; Charles R Bredl; Sharon J Steele; Shaopei Cai; Donald R VanDevanter; Pasqua A Nardella (365-372).
A precise and accurate HPLC assay for polymyxin E1 in rat and dog plasma has been validated. Samples and standards are extracted from plasma with a 96-well C8 extraction disk plate. Sample extracts are derivatized with dansyl chloride, and polymyxin E1 derivative is quantitated on a C8 column by HPLC with fluorescence detection. The assay is linear in the range of 0.050–5.00 μg/ml for polymyxin E1. The precision and accuracy of polymyxin E1 plasma assay was well within the recommended limits set in the FDA Guidance for Bioanalytical Method Validation. Polymyxin E1 stability in rat and dog plasma for 24 h at room temperature and through three freeze–thaw cycles was demonstrated.
Keywords: Polymyxin E1;

Simple and sensitive determination of five quinolones in food by liquid chromatography with fluorescence detection by Macarena Ramos; Angela Aranda; Elena Garcia; Thea Reuvers; Henny Hooghuis (373-381).
A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of five different quinolones: enrofloxacin, ciprofloxacin, sarafloxacin, oxolinic acid and flumequine in pork and salmon muscle. The method includes one extraction and clean-up step for the five quinolones together which are detected in two separated HPLC runs by means of their fluorescence. The proposed analytical method involves homogenizing of the tissue sample with 0.05 M phosphate buffer, pH 7.4 and clean-up by Discovery DS-18 cartridges. For chromatographic separation a Symmetry C18 column is used in two different runs: (1) ciprofloxacin, enrofloxacin and sarafloxacin with acetonitrile–0.02 M phosphate buffer pH 3.0 (18:82) as mobile phase and the detector at excitation wavelength: 280 nm and emission wavelength 450 nm; and (2) oxolinic acid and flumequine with acetonitrile–0.02 M phosphate buffer pH 3.0 (34:66) as mobile phase and excitation wavelength: 312 nm and emission wavelength: 366 nm. Detection limit was as low as 5 ng g−1, except for sarafloxacin which had a limit of 10 ng g−1. Standard curves using blank muscle tissues spiked at different levels showed a good linear correlation coefficient, r 2 higher than 0.999 for all quinolones.
Keywords: Quinolones;

Quantitative determination of free intracellular α-keto acids in neutrophils by Jörg Mühling; Markus Fuchs; Marie E Campos; Jens Gonter; Jörg M Engel; Armin Sablotzki; Thilo Menges; Stefan Weiss; Marius G Dehne; Matthias Krüll; Gunter Hempelmann (383-392).
For the first time, a procedure is described for the quantitative analysis of free α-keto acid content in human neutrophils (PMNs) relative to single cell number by reversed-phase fluorescence high-performance liquid chromatography. The procedure is minimally invasive and is unsurpassed in the quality of PMN separation, ease of sample preparation as well as sample stability. This method can satisfy the rigorous demands for an ultra-sensitive, comprehensive and rapid intracellular α-keto acid analysis in particularly for the surveillance of severe diseases as well as cellular or organ dysfunction.
Keywords: α-Keto acids;

Improved quantitative detection of 11 urinary phthalate metabolites in humans using liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry by Manori J Silva; Nicole A Malek; Carolyn C Hodge; John A Reidy; Kayoko Kato; Dana B Barr; Larry L Needham; John W Brock (393-404).
Phthalates are widely used as industrial solvents and plasticizers, with global use exceeding four million tons per year. We improved our previously developed high-performance liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometric (HPLC–APCI-MS/MS) method to measure urinary phthalate metabolites by increasing the selectivity and the sensitivity by better resolving them from the solvent front, adding three more phthalate metabolites, monomethyl phthalate (mMP), mono-(2-ethyl-5-oxohexyl)phthalate (mEOHP) and mono-(2-ethyl-5-hydroxyhexyl)phthalate (mEHHP); increasing the sample throughput; and reducing the solvent usage. Furthermore, this improved method enabled us to analyze free un-conjugated mono-2-ethylhexyl phthalate (mEHP) by eliminating interferences derived from coelution of the glucuronide-bound, or conjugated form, of the mEHP on measurements of the free mEHP. This method for measuring phthalate metabolites in urine involves solid-phase extraction followed by reversed-phase HPLC–APCI-MS/MS using isotope dilution with 13C4 internal standards. We further evaluated the ruggedness and the reliability of the method by comparing measurements made by multiple analysts at different extraction settings on multiple instruments. We observed mMP, monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mEHP, mEHHP and mEOHP in the majority of urine specimens analyzed with DEHP-metabolites mEHHP and mEOHP present in significantly higher amounts than mEHP.
Keywords: Phthalates;

Liquid chromatographic determination of carvedilol in human plasma by P Ptáček; J Macek; J Klı́ma (405-410).
A high-performance liquid chromatographic method for the quantitation of carvedilol in human plasma is presented. The method is based on protein precipitation with methanol, concentration of the supernatant by evaporation and reversed-phase chromatography with fluorimetric detection. The separation was performed on a Develosil 3 μm ODS 100×4.6 mm I.D. column and the mobile phase consisted of acetonitrile-30 mM potassium dihydrogenphosphate buffer, pH 2 (30:70 v/v). With only 250 μl of plasma used for sample preparation, the limit of quantitation 1.3 ng/ml was achieved. Dihydroergocristine mesylate was used as the internal standard. The between-day precision expressed by relative standard deviation was less than 6% and inaccuracy does not exceed 3%. The assay was used for pharmacokinetic studies.
Keywords: Carvedilol;

A simple method for determination of perphenazine by capillary zone electrophoresis with amperometric detection is described. The optimum conditions of separation and detection are 1.50×10−3 mol/l Na2B4O7−1.0×10−3 mol/l NaOH (pH 9.9) for the buffer solution, 18 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, and 0.80 V versus saturated calomel electrode for the detection potential, respectively. The limit of detection is 5.0×10−8 mol/l or 44 amol (S/N=3). The linear range of the calibration curve is 1.00×10−7 to 1.00×10−4 mol/l. The relative standard deviation is 1.5% for the migration time and 2.9% for the electrophoretic current at peak maximum. The method is applied to the determination of perphenazine in human urine.
Keywords: Perphenazine;

Determination of dialysate creatinine by micellar electrokinetic chromatography by Ewa Poboży; Anna Radomska; Robert Koncki; Stanisław Gła̧b (417-424).
Micellar electrokinetic chromatography with UV absorbance detection has been applied for fast and selective determination of creatinine in samples of postdialysate fluid. Optimization of the method was performed, with the best results being obtained using a 30 mM borate–100 mM sodium dodecyl sulphate background electrolyte, pH 9, with the detector set at 235 nm and an applied voltage of 17 kV across a fused-silica capillary of 67 cm/75 μm I.D. The linear range of the technique was over 2 orders of magnitude (5–1000 μM). The developed analytical procedure is useful for the monitoring of clinical hemodialysis treatment, because creatinine levels in real undiluted samples of postdialysate range from 80 to 350 μM. The separation system allows the analysis of about six to seven samples of spent dialysate per hour in almost real time. The determinations are not influenced by other components of dialysate fluid nor by other surrogates extracted from patient blood. The results of analysis using the developed procedure and the kinetic spectrophotometric Jaffe method conventionally used in clinical settings for creatinine determination are fully comparable. Successful clinical evaluation of the analytical system was performed. The developed system is useful for bloodless estimation of bioanalytical parameters of hemodialysis sessions such as creatinine–time profiles and total creatinine removal. Both these parameters are important in clinical models of hemodialysis therapy.
Keywords: Creatinine;

A method for determination of glutathione (GSH) in single human hepatocarcinoma (HH) cells was described by capillary zone electrophoresis with electrochemical detection at a gold/mercury amalgam micro-disk electrode. When HH cells were washed with the running buffer instead of physiological buffer saline, only one electrophoretic peak for GSH is depicted on the electropherograms of single HH cells. When electroosmotic injection of 0.01 mol/l NaOH for lysing the cell introduced into the capillary, the lysis time can be shorten to 5 s. The whole cell injection and no need of derivatization reaction lead more accurate and precise results. The average amount of GSH in an individual HH cell is 22.3±5.8 fmol (mean±standard deviation), which is consistent with that of its homogenate.
Keywords: Glutathione;

Capillary electrophoresis-based method to quantitate DNA–protein interactions by Mario F Fraga; Esteban Ballestar; Manel Esteller (431-435).
A novel, rapid and simple capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) has been developed for the quantitative study of protein–DNA interactions. This method is particularly useful for the study of basic proteins, the most common of the DNA-interacting proteins. To avoid protein stickiness to the capillary walls we have introduced the use of neutral polyacrylamide that requires the use of reverse polarity. Under these conditions, excellent separation of DNA and protein–DNA complexes was obtained without the requirement of a gel matrix, thereby allowing the easy and reliable quantification of protein–DNA affinities. Analysis of the affinities of histones H2B and H4 for a synthetic oligo have been used to demonstrate the reproducibility and accuracy of this method. We have observed that H4 has a higher affinity for DNA than H2B, with half saturation fractions lying in the micromolar range.
Keywords: DNA; Proteins; Histone;

In humans, diclofenac is metabolised to 4′-hydroxy (OH), 3′-OH and 5-OH metabolites. The polymorphic CYP2C9 is involved in the metabolism of diclofenac to 4′-OH diclofenac and 3′-OH diclofenac. The aim of the present study was to develop a high-performance liquid chromatographic method to simultaneously measure diclofenac and its metabolites in urine, suitable for metabolic studies. After liquid–liquid extraction the compounds were separated in a reversed-phase column and measured by ultraviolet absorption at 282 nm. For all compounds intra-day and inter-day variations were less than 7%, and the limits of quantitation were 0.25 mg/l. No analytical interference with endogenous compounds was found. The relationship between diclofenac metabolic ratios among different CYP2C9 genotypes is reported. The CYP2C9∗3/∗3 subject had the highest diclofenac/4′-OH ratios. However no difference was found between CYP2C9∗2/∗2 and ∗1/∗1 genotypes. The chromatographic method developed was sensitive and reliable for the measurement of diclofenac and its metabolites simultaneously in human urine, and is suitable for use in diclofenac metabolism studies.
Keywords: Diclofenac; Hydroxydiclofenac; CYP2C9 genotypes;