Journal of Chromatography B (v.843, #1)

A novel method was developed for the determination of quinolone (QN) residues such as ofloxacin, norfloxacin, ciprofloxacin and lomefloxacin by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection. The procedure was based on the chemiluminescent enhancement by QNs of the Ce(SO4)2–Ru(bpy)3 2+–HNO3 system. The separation was carried out with an isocratic elution using the mobile phase of 3:15:82 (v/v/v) acetonitrile–methanol–ammonium acetate buffer (containing 7.5 × 10−4  M TBAB, 0.8% (v/v) TEA and 1.0 × 10−4  M ammonium acetate, pH 3.65) at a flow rate of 1.0 ml/min. For the four QNs, the detection limits at a signal-to-noise of 3 ranged from 0.36 to 2.4 ng/ml. The relative standard deviations for the determination of QNs ranged from 1.6 to 4.5% within a day (n  = 11) and from 3.7 to 6.2% in three days (n  = 15), respectively. The method was successfully applied to the determination of QNs in prawn samples. The possible mechanism of the CL reaction was also discussed briefly.
Keywords: Quinolones; HPLC; Chemiluminescence; Prawn; Ce(IV); Ru(bpy)3 2+;

A high-performance liquid chromatography (HPLC) method for determining sophoridine in rat plasma was developed for application in the pharmacokinetic studies. The plasma was deproteinized with acetonitrile that contained an internal standard (ephedrine) and was separated from the aqueous layer by adding sodium chloride and sodium carbonate. The HPLC assay was carried out using a YMC-ODS column. The mobile phase was methanol–ethanol–0.01 mol l−1 ammonium acetate buffer–triethylamine (10:0.5:89.5:0.03, v/v/v/v) (pH 6.80). The flow rate was 0.8 ml min−1. The detection wavelength was set at 210 nm. The method was used to determine the concentration–time profiles of sophoridine in the plasma following oral administration or injection of sophoridine aqueous solution. The fractions of sophoridine reaching the systemic circulation were estimated for the first time by a deconvolution method.
Keywords: Sophoridine; HPLC–DAD; Rat plasma;

Diagnosing AICA-ribosiduria by capillary electrophoresis by Petr Hornik; Petra Vyskočilová; David Friedecký; Tomáš Adam (15-19).
AICA-ribosiduria is a recently discovered inherited metabolic disease caused by a defect in final steps of purine de novo biosynthesis—5-amino-4-imidazolecarboxamide ribotide (AICAR)-transformylase/inosinemonophosphate (IMP)-cyclohydrolase (ATIC). A rapid and selective capillary electrophoretic method for screening of patients with AICA-ribosiduria is described. The method is based on direct ultraviolet detection of 5-amino-4-imidazolecarboxamide (AICA) and 5-amino-4-imidazolecarboxamide riboside (AICAr) in untreated urine. Background electrolyte consists of 100 mM malonic acid adjusted with γ-aminobutyric acid (pH 2.7). Under the given separation conditions both compounds of interest are well separated from other substances with separation efficiency of 1 020 000 and 130 000 theoretical plates/m for AICA and AICAr, respectively. Total analysis time is 3 min with the limits of detection of 3.6 μM and 4.5 μM for AICA and AICAr, respectively. The usefulness of the presented method for screening of patients with ATIC deficiency is demonstrated on samples of Chinese hamster ovary cell line defective in ATIC activity, spiked urine samples and urine samples from patients treated with high-dose MTX which do not excrete increased amounts of AICA and AICAr compared to untreated controls (p  < 0.05). The described method is fast and effective enough for diagnostic applications.
Keywords: Inborn errors; Purines; Metabolism; Screening;

A simple and sensitive method was developed for the quantification of amikacin in human plasma and urine samples. The method involves centrifugation of body fluid plasma after dilution with an ethanol/sodium carbonate mixture, and then an aliquot of the supernatant is directly injected into the chromatograph. After separation on a reversed-phase C18 column (runtime 20 min), aminoglycoside is detected on the basis of its complex formation reaction with Cu(II), the catalyst of the luminol/hydrogen peroxide chemiluminescence system. Using a volume of 500 μl biological sample, linearity is established over the concentration range 0.15–2.0 μg/ml and the limit of detection (LOD) is ca. 50 μg/l in plasma or urine. The intra-day and inter-day precision (measured by relative standard deviation, R.S.D.%) are always less than 9%, and relative recoveries are found to be over 92%.
Keywords: Amikacin; Chemiluminescence; Reversed-phase chromatography; Human plasma and urine samples;

Capillary liquid chromatography (cLC) coupled with matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) was used to compare small proteins and peptides extracted from Bacillus subtilis spores grown on four different media. A single, efficient protein separation, compatible with MALDI–MS analysis, was employed to reduce competitive ionization between proteins, and thus interrogate more proteins than possible using direct MALDI–MS. The MALDI–MS data files for each fraction are assembled as two-dimensional data sets of retention time and mass information. This method of visualizing small protein data required careful attention to background correction as well as mass and retention time variability. The resulting data sets were used to create comparative displays of differences in protein profiles between different spore preparations. Protein differences were found between two different solid media in both phase bright and phase dark spore phenotype. The protein differences between two different liquid media were also examined. As an extension of this method, we have demonstrated that candidate protein biomarkers can be trypsin digested to provide identifying peptide fragment information following the cLC–MALDI experiment. We have demonstrated this method on two markers and utilized acid breakdown information to identify one additional marker for this organism. The resulting method can be used to identify discriminating proteins as potential biomarkers of growth media, which might ultimately be used for source attribution.
Keywords: Capillary liquid chromatography; Bacillus; Spores; MALDI mass spectrometry; Differential display; Small proteins;

Organophosphorus (OP) pesticides are among the most widely used pesticides in the United States. Human exposure to these pesticides may occur from their use on crops in agriculture and for pest control in residential settings. Most of the OP pesticides used in the United States are metabolized to up to three of six common urinary dialkyl phosphate metabolites. Quantification of these metabolites provides information on cumulative exposure to most OP pesticides. To accurately quantify OP pesticide metabolites in human urine, we developed a simple, highly sensitive, analytic method involving automated solid-phase extraction (SPE) of human urine, followed by post-extraction derivatization of the organophosphorus metabolites with 1-chloro-3-iodopropane, and analysis by isotope dilution gas–chromatography–tandem mass spectrometry. The styrene-divinyl benzene polymer-based SPE cartridges yielded good SPE recoveries of the metabolites because of their enhanced non-polar interactions. This method is less labor-intensive, more time-efficient, and reproducible than previously reported methods. Automation of the SPE allowed unattended extraction of urine samples, and hence, increased the sample throughput and reduced the inter- and intra-day variations. The method limits of detection were excellent for all analytes ranging from 50 pg/ml to 170 pg/ml. Relative standard deviations ranged from 2% to 12%.
Keywords: Organophosphorous pesticides; Dialkyl phosphate metabolites;

Hydrolysis of retinyl esters and phospholipids is important for visual functions of the animal retina. This study aimed to examine hydrolytic activity of an enzyme with native substrates such as retinyl esters and phospholipids responsible for this function in porcine retina. After cytosolic proteins were extracted from porcine retina, the proteins were separated using non-denaturing two-dimensional electrophoresis (2DE). Some major proteins and phospholipase Cα were identified by matrix-assisted laser desorption ionisation–time of flight–mass spectrometry (MALDI–TOF–MS) or electrospray ionisation–tandem mass spectrometry (ESI–MS/MS). The phospholipase Cα showed hydrolytic activities with not only α-naphtyl acetate but also with retinyl palmitate and phosphatidylcholine when effects of different substrates were investigated using enzyme activity staining on 2DE or MALDI–TOF–MS. Results indicated that hydrolytic activity of the enzyme with non-native and native substrates could be examined using a combination of non-denaturing 2DE and MALDI–TOF–MS.
Keywords: Retinyl palmitate; Phosphatidylcholine; Eserine; Phospholipase Cδ; Phospholipase Cα;

A high-throughput ultrafiltration method with a direct injection assay has been developed to determine unbound concentrations of a high-protein binding compound, an αvβ3 bone integrin antagonist (I), in human plasma for a clinical pharmacokinetic study. The 96-well MultiScreen® filter plate with Ultracel-PPB membrane was evaluated for the separation of unbound from protein-bound compound I by ultrafiltration. The sample preparation was automated using a Packard MultiPROBE II EX liquid handling system to transfer the plasma samples to the 96-well PPB plate for centrifugation and to prepare ultrafiltrate samples for analysis. Using on-line extraction with a column-switching setup for sample clean-up and separation, the ultrafiltrate samples were directly injected onto a reversed-phase HPLC system and analyzed using a mass spectrometer interfaced with an electrospray ionization (ESI) source in the positive ionization mode (LC/ESI-MS/MS). The performance of the ultrafiltration using Ultracel-PPB 96-well plate for unbound I analysis was evaluated and optimized with respect to sample volume, centrifugation temperature, speed and time, and the relationship of the well positions of the PPB plate versus filtrate volumes and concentrations. The assay intraday accuracy and precision were between 93.9 and 104.8 and <7.3% (CV), respectively. The linear range of the calibration curve for the assay was 0.1–500 ng/mL on a Finnigan TSQ Quantum LC/ESI-MS/MS system. Evaluation and validation of the unbound plasma assay demonstrated it to be rapid, sensitive and reproducible.
Keywords: Unbound drug; Ultrafiltration; Ultracel-PPB 96-well plate; Packard liquid handling system; Human plasma; LC–MS/MS;

2-Mercaptoethylamine (cysteamine) is an aminothiol compound used as a drug for the treatment of cystinosis, an autosomal recessive lysosomal storage disorder. Because of cysteamine's important role in clinical settings, its analysis by sensitive techniques has become pivotal. Unfortunately, the available methods are either complex or labor intensive. Therefore, we have developed a new rapid, sensitive, and simple method for determining cysteamine in biological samples (brain, kidney, liver, and plasma), using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent and reversed-phase high performance liquid chromatography (HPLC) with a fluorescence detection method (λ ex  = 330 nm, λ em  = 376 nm). The mobile phase was acetonitrile and water (70:30) with acetic acid and o-phosphoric acid (1 mL/L). The calibration curve for cysteamine in serine borate buffer (SBB) was found to be linear over a range of 0–1200 nM (r 2  = 0.9993), and in plasma and liver matrix, the r 2 values were 0.9968 and 0.9965, respectively. The coefficients of the variation for the within-run and between-run precisions ranged from 0.68 to 9.90% and 0.63 to 4.17%, respectively. The percentage of relative recovery ranged from 94.1 to 98.6%.
Keywords: CSH; Cysteamine; NPM; Thiols; Cystinosis; Oxidative stress; HPLC; 2-Mercaptoethylamine;

To study the flow of shaped particles in porous media, elution of spherical and rod-like micro-organisms was performed through beds of spherical glass beads. A 0.04 cm/s constant flow rate was used with 5 μm yeast suspensions, 1 μm latex micro-spheres and rod-like bacilli Lactobacillus bulgaricus 6 μm long and 0.5 μm in diameter. Yeast cells’ diameter is close to the bacilli length and micro-spheres have the same diameter as bacilli. All particle types have similar density. To make the different packing beds, 1.125 mm coarse beads and 0.1115 mm fine beads were used. Experiments were carried out using a column loaded with the binary packing (volume fraction of coarse particles in the mixture 0.7) or a monosize packing with the same amount of coarse or fine particles as used in the binary packing. Analysis of experimental results was based on two models: pure exclusion effect and hydrodynamic separation model [hydrodynamic chromatography (HDC)]. Results for spheres show that the classic HDC model fits to the experimental data whenever the ratio of particle size to the pathway bend scale is high (∼1/100, micro-spheres). However, if this ratio increases and becomes ∼1/20, the HDC model needs to be corrected due to the effect of channel wall curvature on exclusion. This led to a modified HDC equation of the form R  =  B/(1 + 2λ  − 2.8λ 2), where R is the retention, λ is the aspect ratio and constant B  ≥ 1. Bacillus separation follows an exclusion mechanism, since pore topology is important in the separation of shaped particles when the aspect ratio approaches λ  = 0.1. In the case of a binary packing bed, rod-like particles display a different behaviour than the one exhibited by the spherical particles of the same scale as bacilli, either in length or in diameter. This may be explained by the interaction between rod-like bacilli and the bed's pore topology. A generalised exclusion model for particles was proposed to be R  =  A/(1 −  λ) z , where A is the coefficient proportional to the tortuosity and the parameter z  = 1, 2 or 3 depends mainly on pore shape. Controlled pore topology opens interesting applications for bio-separation (in porous micro-fluidic devices, deep bed filtration) and might be especially important for macromolecules and micro-organisms separation with different shapes.
Keywords: Porous media; Flow; Particle shape; Separation; Models; Pore topology;

Enantioselective and highly sensitive determination of carvedilol in human plasma and whole blood after administration of the racemate using normal-phase high-performance liquid chromatography by Masako Saito; Junichi Kawana; Tetsuro Ohno; Masahiro Kaneko; Kiyoshi Mihara; Kazuhiko Hanada; Risa Sugita; Natsuki Okada; Sachiko Oosato; Masatoshi Nagayama; Tetsuya Sumiyoshi; Hiroyasu Ogata (73-77).
A highly sensitive HPLC method for enantioselective determination of carvedilol in human whole blood and plasma was developed. Carvedilol and S-carazolol as an internal standard extracted from whole blood or plasma were separated using an enantioselective separation column (Chiralpak AD column; 2.0 Ø × 250 mm) without any chiral derivatizations. The mobile phase was hexane:isopropanol:diethylamine (78:22:1, v/v). The excitation and emission wavelengths were set at 284 and 343 nm, respectively. The limits of quantification for the S(−)- and R(+)-carvedilol enantiomers in plasma and blood were both 0.5 ng/ml. Intra- and inter-day variations were less than 5.9%. As an application of the assay, concentrations of carvedilol enantiomer in plasma and blood samples from 15 patients treated with carvedilol for congestive heart failure were determined.
Keywords: Carvedilol enantiomers; HPLC; Human whole blood; Human plasma;

Sensitive HPLC–APCI–MS method for the determination of cyclovirobuxine D in human plasma by Li Ding; Jingjing Hu; Meng Jiang; Ningning Xiong (78-83).
A sensitive high performance liquid chromatography–atmospheric pressure chemical ionisation–mass spectrometry (HPLC–APCI–MS) assay for determination of cyclovirobuxine D (CVB-D) in human plasma using mirtazapine as internal standard (I.S.) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of 30 mM ammonium acetate buffer solution containing 1% formic acid–methanol (48:52, v/v). CVB-D was determined with atmospheric pressure chemical ionisation–mass spectrometry (APCI–MS). HPLC–APCI–MS was performed in the selected-ion monitoring (SIM) mode using target ions at [M + H]+ m/z 403.4 for CVB-D and [M + H]+ m/z 266.2 for I.S. Calibration curves were linear over the range 10.11–4044 pg/ml. The lower limit of quantification was 10.11 pg/ml. The intra- and inter-run variability values were less than 9.5 and 12.4%, respectively. The mean plasma extraction recovery of CVB-D was in the range of 85.3–92.8%. The method was successfully applied to determine the plasma concentrations of CVB-D in Chinese volunteers.
Keywords: Cyclovirobuxine D; HPLC–APCI–MS;

Stability studies of amphetamine and ephedrine derivatives in urine by C. Jiménez; R. de la Torre; M. Ventura; J. Segura; R. Ventura (84-93).
Knowledge of the stability of drugs in biological specimens is a critical consideration for the interpretation of analytical results. Identification of proper storage conditions has been a matter of concern for most toxicology laboratories (both clinical and forensic), and the stability of drugs of abuse has been extensively studied. This concern should be extended to other areas of analytical chemistry like antidoping control. In this work, the stability of ephedrine derivatives (ephedrine, norephedrine, methylephedrine, pseudoephedrine, and norpseudoephedrine), and amphetamine derivatives (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA)) in urine has been studied. Spiked urine samples were prepared for stability testing. Urine samples were quantified by GC/NPD or GC/MS. The homogeneity of each batch of sample was verified before starting the stability study. The stability of analytes was evaluated in sterilized and non-sterilized urine samples at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 °C and −20 °C was determined at different time intervals for 24 months for sterile urine samples, and for 6 months for non-sterile samples. For short-term stability testing, analyte concentration was evaluated in liquid urine stored at 37 °C for 7 days. The effect of repeated freezing (at −20 °C) and thawing (at room temperature) was also studied in sterile urine for up to three cycles. No significant loss of the analytes under study was observed at any of the investigated conditions. These results show the feasibility of preparing reference materials containing ephedrine and amphetamine derivatives to be used for quality control purposes.
Keywords: Ephedrine; Norephedrine; Methylephedrine; Pseudoephedrine; Norpseudoephedrine; Amphetamine; Methamphetamine; 3,4-Methylenedioxyamphetamine (MDA); 3,4-Methylenedioxymethamphetamine (MDMA); Stability; Urine; Doping control;

Solid phase extraction (SPE) was coupled at line to capillary electrophoresis (CE) for the determination of three basic and neutral diabetic drugs (metformin, phenformin and glyburide) in human plasma. The SPE procedure employed a C18 cartridge to remove most of the water and proteins from the plasma sample. Analyte detectability was increased due to trace enrichment during the SPE process. Elution of metformin, phenformin and glyburide was achieved with methanol + 3% acetic acid. CE analysis was performed using a non-aqueous buffer, acetonitrile + 5 mM ammonium acetate + 5% acetic acid, which afforded rapid separation of metformin from phenformin within 3 min. Glyburide, with a migration time longer than 6 min, did not cause any interference. The present SPE–CE method, with an electrokinetic injection time of 6 s and UV detection at 240 nm, was useful for monitoring down to 1 μg/mL of metformin and phenformin in human plasma. When the electrokinetic injection time was increased to 36 s, the detection limits were improved to 12 ng/mL for metformin and 6 ng/mL for phenformin.
Keywords: Metformin; Phenformin; Glyburide; Human plasma; Solid phase extraction; Capillary electrophoresis; Non-aqueous buffer;

This method describes the simultaneous determination of amisulpride, amitriptyline, aripiprazole, benperidol, chlorpromazine, chlorprothixene, citalopram, clomipramine, clozapine, desipramine, doxepin, fluoxetine, flupentixol, fluphenazine, fluvoxamine, haloperidol, hydroxyrisperidone, imipramine, levomepromazine, maprotiline, mianserine, mirtazapine, moclobemide, norclomipramine, nordoxepin, norfluoxetine, nortriptyline, O-desmethylvenlafaxine, olanzapine, opipramol, paroxetine, perazine, perphenazine, pimozide, pipamperone, quetiapine, reboxetine, risperidone, sertraline, sulpiride, thioridazine, trazodone, trimipramine, venlafaxine, viloxazine, ziprasidone, zotepine and zuclopenthixol with a single sample/triple injection approach. Drugs were assigned to subgroups covering low, medium and high concentrations (overall range of therapeutic levels to be considered: 0.5–2000 ng/mL) by further dilution of the supernatant obtained after the first protein precipitation. Chromatographic separation was necessary for isobaric mass fragments and performed on a monolithic C18 column (50 mm × 4.6 mm) with methanol gradient and 5 mM acetate buffer at pH 3.9. The injection interval was 8 min. A set of three internal standards was used for quantification of drugs with widely varying hydrophobicity. After electrospray ionization positive ion fragments were detected in the multiple reaction monitoring (MRM) mode with an API 4000 tandem mass spectrometer. Regression parameters of calibration curves and limits of quantification showed good covering of therapeutic and subtherapeutic ranges with an average correlation coefficient of 0.9988. Imprecision and inaccuracy measures were prepared for intra- and inter-assay comparisons at three concentration ranges in all subgroups. Average coefficients of variation were 6.1% for intra-assay and 7.4% for inter-assay comparisons, while average deviations from spiked concentrations were 4.8% for intra-assay and 4.2% for inter-assay comparisons, respectively. Recovery rates, measured as the percent recoveries of spiked serum samples against standard solutions without serum matrix, varied between 92 and 111%, with an average of 101%. As the only exception, the olanzapine response was much higher (185%) in serum matrix than in matrix-free controls.
Keywords: Therapeutic drug monitoring; Antidepressants; Antipsychotics; LC–MS/MS;

Structural features in carrageenan that interact with a heparin-binding hematopoietic growth factor and modulate its biological activity by Aiye Liang; Xiaomian Zhou; Qiuyan Wang; Xin Liu; Xiaojun Liu; Yuguang Du; Keyi Wang; Bingcheng Lin (114-119).
The effects of carrageenans’ structural features on its interaction with granulocyte colony-stimulating factor (G-CSF) and on the growth and differentiation of a G-CSF dependent leukemia cell line (NFS-60) were studied. λ, ι, and κ carrageenans, with decreasing contents of sulfation, bound to G-CSF with binding constants of (6.2 ± 0.6) × 105  M−1, (7.4 ± 0.5) × 105  M−1 and (6.0 ± 0.4) × 105  M−1, and with 27.7 ± 0.2, 17.4 ± 0.1 and 8.4 ± 0.1 binding sites, respectively. However, κ carrageenan oligosaccharide had no affinity for G-CSF. The three carrageenans significantly inhibited G-CSF-induced growth of NFS-60 cells. The high sulfate content λ carrageenan could also induce the maturation of the cells, but relatively low sulfate content ι and κ carrageenans could not. The results suggested that G-CSF–carrageenan bindings were dependent on carrageenans’ sulfate contents and chain lengths, which could also affect the growth and differentiation of NFS-60 cells.
Keywords: Carrageenan; Granulocyte colony-stimulating factor; Interaction; Capillary zone electrophoresis; Leukemia cells;

Faecal sterols determination in wastewater and surface water by Giorgio Gilli; Renato Rovere; Deborah Traversi; Tiziana Schilirò; Cristina Pignata (120-124).
A simplified method to detect faecal sterols, as an alternative assessment of environmental faecal pollution is proposed. The aim of this study is the development of a method to determine sterols in water samples avoiding sample filtration through glass fibre filter. The method is based on a liquid–liquid extraction and a final GC–FID determination. The quantified sterols are coprostanol and 24-ethylcoprostanol, while 5α-cholestane is used as internal standard. The recovery of coprostanol and 24-ethylcoprostanol in wastewater ranges from 90 to 100% and the detection limit is 1–2 μg l−1. Moreover the method proved to be useful for the sterols determination in surface water too.
Keywords: Sterols; Faecal pollution; Coprostanol; 24-Ethylcoprostanol; Wastewater; Surface water;

Efficient purification and preconcentration of erythropoietin in human urine by reusable immunoaffinity column by Jiebo Mi; Shan Wang; Xiaojie Ding; Zhenquan Guo; Meiping Zhao; Wenbao Chang (125-130).
In this paper, an efficient method is proposed for purification and preconcentration of erythropoietin (EPO) in human urine samples. The EPO-specific immunoaffinity column (IAC) was generated by covalent immobilization of anti-EPO polyclonal antibodies on Sepharose 4B support. The EPO-binding capacity of the IAC was found to be about 2.0 μg (6.6 IU) per 1.5 mL of gel and the activity recoveries of EPO at low concentrations of 7.8, 10 and 120 mIU/mL by the IAC were between 78 and 86%. Substantial cleanup effect was observed when the IAC was applied to human urine samples.
Keywords: Erythropoietin (EPO); Immunoaffinity column (IAC); Purification; Preconcentration;