Journal of Chromatography B (v.807, #2)

News Section (N1-N2).

OFC: Update (OFC).

An analytical method for the simultaneous quantitation of arseneous acid (As(III)), arsenic acid (As(V)), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and trimethylarsine oxide (TMAO) in human urine by coupling of high-performance liquid chromatography with hydride generation atomic absorption spectrometry (HPLC/HG-AAS) via a flow-injection interface is presented. After arsenic species separation by anion-exchange displacement chromatography the compounds are on-line reduced to their corresponding hydrides and detected by atomic absorption spectrometry. Detection limits range from 1.1 (TMAO) to 2.6 μg/L (As(V)). The method has been applied to determine arsenic species in the urine of a volunteer before and after consumption of seafood as well as to analyse certified reference urine samples for their arsenic species content.
Keywords: Arseneous acid; Arsenic acid; Monomethylarsonic acid; Dimethylarsonic acid; Trimethylarsine oxide;

A highly selective and sensitive column liquid chromatographic method for fluorescence determination of serotonin (5-HT), dopamine (DA), noradrenaline (NA) and their related metabolites 5-hydroxyindole-3-acetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) following derivatization with benzylamine and 1,2-diphenylethylenediamine (DPE) is described. The monoamines and the metabolites (20 μl samples) were derivatized in a two-step reaction, initiated with 20 μl of 0.3 M benzylamine in 0.3 M 3-cyclohexylaminopropanesulfonic acid (CAPS) buffer (pH 10.0), (for 5-HT, 5-HIAA, 2 min, 24 °C) and followed by 20 μl of 0.1 M DPE in 0.3 M glycine buffer (pH 10.0), (for DA, NA, DOPAC, 20 min, 50 °C). Both reagents contained 0.02 M potassium hexacyanoferrate(III) and 50% (v/v) methanol. The resulting highly fluorescent and stable benzoxazole derivatives were isocratically separated on a reversed-phase column (150  mm×1.5  mm i.d., packed with C18 silica, 5 μm) within 45 min. Using fluorescence detection at ex. and em. wavelengths of 345 and 480 nm, respectively, the detection limit (signal-to-noise ratio of 3) for 5-HT, DA, NA, 5-HIAA, L-DOPA and DOPAC ranged between 0.08 and 5.65 fmol per 20-μl injection (12–847.5 pM in standard solution). The concentrations of monoamines (expressed in μg/g wet weight, mean±S.E.M., n=5) in tissue extracts from the rat striatum were: 0.45±0.05 (5-HT), 4.27±0.08 (DA), 0.27±0.04 (NA), 0.55±0.06 (5-HIAA), 1.26±0.16 (L-DOPA) and 1.62±0.11 (DOPAC). Microdialysis samples were collected in 20 min intervals from the probes implanted in the striatum of awake rats. The basal monoamine levels (in fmol/20 μl, mean±S.E.M., n=5) in the dialysates were: 4.1±0.7 (5-HT), 78.4±9.1 (DA), 6.4±0.8 (NA), 785.5±64.5 (5-HIAA) and 5504.5±136.5 (DOPAC). It is concluded that the new fluorescence derivatization protocol provides an excellent means for simultaneous determination of all three monoamines both in the complex samples (e.g. brain homogenates) and also at trace levels, such as those found in the microdialysis samples.
Keywords: Derivatization, LC; Serotonin; Noradrenaline; Dopamine; 5-Hydroxyindole-3-acetic acid; 3,4-Dihydroxyphenylacetic acid;

This study describes a capillary gas chromatography–mass spectrometry (GC–MS) method for the simultaneous determination of endogenous thyroid hormone (thyroxine, T4) and its 13 C -labelled analogue ( 13 C 6 -thyroxine) in plasma. 13 C 9 -thyroxine was used as analytical internal standard. A double derivatization (CH3OH/HCl and HFBA) inducing good GC mobility was used for the GC–MS analysis of the thyroid hormones. Quantification was carried out by selected ion monitoring (SIM) of specific ions of the fragment ions (m/z 970/976/979). The detection limit of the present GC–MS–SIM method was found to be 100 pg per injection for thyroxine (S/N=3.0). A first implementation in in vivo tests of 13 C 6 -T4 like metabolic tracer was carried out under veterinary control on one cat and one rabbit. The thyroxine follow-up was done by GC–MS and based on double isotopic dilution with two different regio-selective 13 C -labelled molecules of the same hormone. The present paper discusses the possibilities and limitations of this methodology. The in vivo experiment demonstrated that the use of stable isotopes and mass spectrometry provide a reliable methodology for hormonal monitoring.
Keywords: Thyroid hormones;

Potential of biopartitioning micellar chromatography as an in vitro technique for predicting drug penetration across the blood–brain barrier by L Escuder-Gilabert; M Molero-Monfort; R.M Villanueva-Camañas; S Sagrado; M.J Medina-Hernández (193-201).
The blood–brain barrier (BBB) is considered to be the main barrier to drug transport into the central nervous system (CNS). The BBB restricts the passive diffusion of many drugs from blood to brain. The ease with which any particular drug diffuses across the BBB is determined largely by the molecular features of drugs, and it is therefore possible to predict the BBB permeability of a drug from its molecular structure. Biopartitioning micellar chromatography (BMC), a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 in adequate experimental conditions, can be useful in mimicking the drug partitioning process into biological systems. Retention in BMC depends on the hydrophobicity, electronic and steric properties of drugs. In this paper, the usefulness of BMC for predicting the BBB penetration ability of drugs expressed as the brain/blood distribution coefficient (BB) is demonstrated. A multiple linear regression (MLR) model that relates the BB distribution coefficients data with BMC retention data and total molar charge is proposed. The model is obtained using 44 heterogeneous drugs including, neutral, anionic, and cationic compounds. A comparison with other reported methodologies to predict the BBB permeability is also presented.
Keywords: Drug penetration; Blood–brain barrier; Biopartitioning micellar chromatography;

Fumitremorgin C (FTC) was recently discovered to be a potent and selective inhibitor of the breast cancer resistance protein (BCRP/ABCG2). FTC was shown to reverse multidrug resistance mediated by BCRP and to increase the cytotoxicity of several anticancer agents in vitro. To support in vivo studies a reverse phase HPLC method with ultraviolet detection was developed to quantitate FTC in mouse plasma and tissues. Further, assay method validation was performed for the determination of FTC in mouse plasma. Plasma standard curves ranged from 0.03 to 30 μg/ml, while the various tissue assay ranges differed to some extent. The sample preparation consisted of acetonitrile precipitation with separation accomplished with a C18 Novapak column and a C18 pre-column utilizing an isocratic mobile phase of ammonium acetate and acetonitrile. UV detection was set at 225 nm for FTC and at 312 nm for roquefortine, the internal standard. The retention times were approximately 9.5 min for FTC and 13.0 min for roquefortine. The recoveries for FTC and roquefortine from plasma were 90.8±5.8% and 111.6±13.6%, respectively. The reported assay can be used for future study of BCRP resistance in vivo in different biological matrices. Further, we found that a more potent analogue of FTC, Ko143, was able to be extracted and detected, with a maximal UV absorbance at 320 nm under the conditions reported.
Keywords: Fumitremorgin; Breast cancer resistance protein;

Improving automatic peptide mass fingerprint protein identification by combining many peak sets by Thorsteinn Rögnvaldsson; Jari Häkkinen; Claes Lindberg; György Marko-Varga; Frank Potthast; Jim Samuelsson (209-215).
An automated peak picking strategy is presented where several peak sets with different signal-to-noise levels are combined to form a more reliable statement on the protein identity. The strategy is compared against both manual peak picking and industry standard automated peak picking on a set of mass spectra obtained after tryptic in gel digestion of 2D-gel samples from human fetal fibroblasts. The set of spectra contain samples ranging from strong to weak spectra, and the proposed multiple-scale method is shown to be much better on weak spectra than the industry standard method and a human operator, and equal in performance to these on strong and medium strong spectra. It is also demonstrated that peak sets selected by a human operator display a considerable variability and that it is impossible to speak of a single “true” peak set for a given spectrum. The described multiple-scale strategy both avoids time-consuming parameter tuning and exceeds the human operator in protein identification efficiency. The strategy therefore promises reliable automated user-independent protein identification using peptide mass fingerprints.
Keywords: Peak set combining; Peptide mass fingerprinting; Protein identification;

Determination of rofecoxib in human plasma and breast milk by high-performance liquid chromatographic assay by Mei Zhang; Grant A Moore; Sharon J Gardiner; Evan J Begg (217-221).
A rapid and simple HPLC assay was developed for the determination of rofecoxib in human plasma and breast milk. After solid-phase extraction, rofecoxib was resolved on a C18 column and detected by UV detection at 272 nm. Standard curves were linear over the concentration range 10–2000 μg/L (r 2>0.99). Intra- and inter-day coefficients of variation for both matrices were <10% and the limit of quantification was around 10 μg/L.
Keywords: Rofecoxib;

The successful separation of β-lactoglobulin from other bovine whey proteins was performed by ceramic hydroxyapatite chromatography with a fluoride ion gradient in phosphate buffer as displacement agent. The method was applied to acid whey originating from milk of healthy as well as of mastitic cows. β-Lactoglobulin was completely eluted in one peak at a fluoride concentration of about 0.6 mol/l. The purity of β-lactoglobulin in this fraction was at least 96% if whey from healthy milk was processed. Co-eluted contaminants are traces of immunoglobulin G, serum albumin and lactoferrin. In case of mastitic whey the proportion of β-lactoglobulin is diminished as the amounts of immunglobulin G, serum albumin and lactoferrin are increased within this fraction. Size exclusion chromatography on Superdex 75 pg effectively removed contaminants resulting in a purity for β-lactoglobulin from normal whey of approximately 99%. The yield of β-lactoglobulin from physiological whey was 50–55% referring to the fraction highly enriched with β-lactoglobulin by hydroxyapatite chromatography. In case of mastitic milk the higher amounts of contaminants were also removed successfully by size exclusion chromatography.
Keywords: Ceramic hydroxyapatite chromatography; β-Lactoglobulin; Bovine whey proteins;

A new HPLC assay for plasma arginine–vasotocin (AVT) and isotocin (IT) determination based on fluorescence detection preceded by combination of solid-phase extraction (SPE) and fluorescence derivatization is presented. Plasma samples retained on solid support were purified and then derivatized by the fluorescent compound 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The peptide derivatives were eluted from cartridges, pre-concentrated and analyzed by HPLC system with fluorescent detection. The separation was carried out on a reversed-phase column with solvent gradient system. The assay was linear in the range 15–220 pmol ml−1 for AVT (r 2=0.998) and 10–220 pmol ml−1 for IT (r 2=0.996). The detection limits for AVT and IT were 0.8 and 0.5 pmol ml−1 (3:1, signal-to-noise), respectively. The recoveries of derivatized hormones were in the range 89–93%. Both of the inter- and intra-day assay precision were below 5.5 and 9% for AVT and IT, respectively. The assay should be also applicable to plasma and tissue samples from other animals with only minor modification.
Keywords: Derivatization, LC; Arginine–vasotocin; Isotocin;

A rapid precolumn high-performance liquid chromatography method based on fluorescence detection has been developed for the measurement of multiple amino acids from both ex vivo and in vivo biological samples using monolithic C18 columns. A mixture of 18 primary amino acids were derivatised with napthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide. The resulting isoindole derivatives were resolved within 10 min using a linear binary gradient elution profile with Rs values in the range 1.2–9.0. The limit of detection (LOD) was found to be between 6.0 and 60 fmol for 5 μl injection with a signal to noise ratio of 3:1. The NDA derivatives were found to be stable for 9 h at 4 °C. This assay has been employed for the rapid analysis of amino acids from brain tissue and microdialysis samples. Examples of application of the method are given.
Keywords: Microdialysis; Derivatisation, LC; Monolithic columns; Amino acids; Neurotransmitters;

Methods based on high-performance liquid chromatography (HPLC) with atmospheric-pressure chemical ionization (APCI) mass spectrometric (MS) detection using either single (MS) or triple (MS/MS) quadrupole mass spectrometric detection for the determination of (2R)-[1(R)-(3,5-bis-trifluoromethylphenyl)ethoxy]-3(S)-(4-fluoro-phenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-[1,2,4]triazol)methyl morpholine (Aprepitant, Fig. 1) in human plasma has been developed. Aprepitant (I) and internal standard (II, Fig. 1) were isolated from the plasma matrix buffered to pH 9.8 using a liquid–liquid extraction with methyl-t-butyl ether (MTBE). The analytes were separated on a Keystone Scientific’s Javelin BDS C-8 2  mm×4.6  mm 3 μm guard column coupled to BDS C-8 50  mm×4.6  mm 3 μm analytical column, utilizing a mobile phase of 50% acetonitrile and 50% water containing 0.1% formic acid and 10 mM ammonium acetate delivered at a flow rate of 1 ml/min. The single quadrupole instrument was operated in a single ion monitoring (SIM) mode analyzing the protonated molecules of Aprepitant and II at m/z 535 and 503, respectively. The triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode (MRM) monitoring the precursor→product ion combinations of m/z 535→277 and 503→259 for Aprepitant and II, respectively. The linear calibration range for both single and triple quadrupole detectors was from 10 to 5000 ng/ml of plasma with coefficients of variation less than 8% at all concentrations. Both single and triple quadrupole instruments yielded similar precision and accuracy results. Matrix effect experiments performed on both instruments demonstrated the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. Both instruments were used successfully to support numerous clinical trials of Aprepitant.
Keywords: Substance P inhibitor;

N-(2-Mercaptopropionyl)-glycine (MPG) is a synthetic aminothiol antioxidant that is used in the treatment of cystinuria, rheumatoid arthritis, liver and skin disorders. Recent studies have shown that MPG can function as a chelating, cardioprotecting and a radioprotecting agent. Several other studies have shown that it may also act as a free radical scavenger because of its thiol group. Thiol-containing compounds have been detected in biological samples by various analytical methods such as spectrophotometric and colorimetric methods. However, these methods require several milliliters of a sample, time-consuming procedures and complicated derivatization steps, as well as having high detection limits. The present study describes a rapid, sensitive and relatively simple method for detecting MPG in biological tissues by using reverse-phase HPLC. With ThioGlo™ 3 [3H-Naphto[2,1-b] pyran, 9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) phenyl-3-oxo-)] as the reagent, highly fluorescent derivatives of thiols can be obtained that are suitable for HPLC. MPG is derivatized with ThioGlo™ 3 and is then detected flourimetrically by reverse phase HPLC using a C18 column as the stationary phase. Acetonitrile: Water (75:25) with acetic acid and phosphoric acid (1 mL/L) is used as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). The calibration curve for MPG is linear over a range of 10–2500 nM (r=0.999) and the coefficients of the variation of within-run and between-run precision were found to be 0.3 and 2.1%, respectively. The detection limit was 5.07 nM per 20 μL injection volume. Quantitative relative recovery of MPG in the biological samples (plasma, lung, liver, kidney and brain) ranged from 90.5±5.3 to 106.7±9.3%. Based on these results, we have concluded that this method is suitable for determining MPG in biological samples.
Keywords: Derivatization, LC; N-(2-Mercaptopropionyl)-glycine;

Modification with homocysteine (Hcy)-thiolactone leads to the formation of Nε-Hcy-Lys-protein. Although Nε-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nω-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-Nε-Hcy-Lys-protein antibodies. Nω-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of ω-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with Nε-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-Nε-Hcy-Lys-protein IgG was adsorbed on Nω-homocysteinyl-aminohexyl-Agarose column at pH 8.6 and eluted with a pH 2.3 buffer. Enzyme-linked immunosorbent assays demonstrate that the antibody recognizes specifically N-homocysteinylated variants of hemoglobin, albumin, transferrin, and antitrypsin.
Keywords: Homocysteine-thiolactone; Proteins; Immune tolerance;

A rapid, sensitive and specific method was developed for the simultaneous assay of testosterone, androstenedione and 6β-hydroxytestosterone (6β-OHT) in the TC199 tissue culture medium used in intestinal drug metabolism studies with the rat everted gut sac model. An electrospray LC–MS method was validated in the concentration range of 0.025–9.5 μM (7.2 ng–2.7 μg/mL) for testosterone and androstenedione and 0.01–4 μM (3 ng–1.2 μg/mL) for 6β-hydroxytestosterone. The limits of quantification (LOQ) with an injection volume of 10 μL were 0.0005 μM (4.9 fmol, 1.4 pg injected), 0.004 μM (0.04 pmol, 11.4 pg injected) and 0.03 μM (0.3 pmol, 91 pg injected), respectively. The method also detected the other testosterone metabolites, the 16α-, 16β-, 2β- and 2α-hydroxytestosterones and was then used to study the metabolism of testosterone during its absorption by rat intestine in vitro, using everted gut sacs.
Keywords: Testosterone; Cytochrome P450;

A simple and sensitive high-performance liquid chromatographic (HPLC) method with spectrophotometric detection was developed for the determination of moclobemide in human plasma. Plasma samples were extracted under basic conditions with dichloromethane followed by back-extraction into diluted phosphoric acid. Isocratic separation was employed on an ODS column (250  mm×4.6  mm, 5 μm) at room temperature. The mobile phase consisted of 5 mM NaH2PO4–acetonitrile–triethylamine (1000:350:10 (v/v/v), pH 3.4). Analyses were run at a flow-rate of 1.0 ml/min and ultraviolet (UV) detection was carried out at 240 nm. The method was specific and sensitive with a quantification limit of 15.6 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 98.2%, while the intra- and inter-day coefficient of variation and percent error values of the assay method were all at acceptable levels. Linearity was assessed in the range of 15.6–2000 ng/ml in plasma with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailibility studies.
Keywords: Moclobemide;

Glutamate oxidase (GOX, EC from Streptomyces catalyses the oxidation of l-glutamate to α-ketoglutarate. Its kinetic constants for l-glutamate were measured equal to 2 mM for K m and 85.8 s−1 for k cat. BLAST search and amino acid sequence alignments revealed low homology to other l-amino acid oxidases (18–38%). Threading methodology, homology modeling and CASTp analysis resulted in certain conclusions concerning the structure of catalytic α-subunit and led to the prediction of a binding pocket that provides favorable conditions of accommodating negatively charged aromatic ligands, such as sulphonated triazine dyes. Eleven commercial textile dyes and four biomimetic dyes or minodyes, bearing a ketocarboxylated-structure as their terminal biomimetic moiety, immobilized on cross-linked agarose gel. The resulted mini-library of affinity adsorbents was screened for binding and eluting l-glutamate oxidase activity. All but Cibacron® Blue 3GA (CB3GA) affinity adsorbents were able to bind GOX at pH 5.6. One immobilized minodye–ligand, bearing as its terminal biomimetic moiety p-aminobenzyloxanylic acid (BM1), displayed the higher affinity for GOX. Kinetic inhibition studies showed that BM1 inhibits GOX in a non-competitive manner with a K i of 10.5 μM, indicating that the dye–enzyme interaction does not involve the substrate-binding site. Adsorption equilibrium data, obtained from a batch system with BM1 adsorbent, corresponded well to the Freundlich isotherm with a rate constant k of 2.7 mg1/2  ml1/2/g and Freundlich isotherm exponent n of 1. The interaction of GOX with the BM1 adsorbent was further studied with regards to adsorption and elution conditions. The results obtained were exploited in the development of a facile purification protocol for GOX, which led to 335-fold purification in a single step with high enzyme recovery (95%). The present purification procedure is the most efficient reported so far for l-glutamate oxidase.
Keywords: l-Glutamate oxidase; Enzymes; Triazine dyes;

A new liquid chromatographic–mass spectrometric (LC–MS) method for determining trace concentrations of γ-hydroxybutyric acid (GHB) in biological samples has been developed. This method utilizes solid-phase extraction for separation, deuterated GHB as an internal standard (IS) and multiple reaction monitoring (MRM) in the negative ion mode to detect the parent and product ions (103 and 57 for GHB, and 109 and 61 for D6-GHB, respectively). The assay produces excellent linearity and reproducibility, with a limit of quantification (LOQ) of about 0.1 μg/ml. The method has been applied for the determination of endogenous GHB in various rat brain regions.
Keywords: γ-Hydroxybutyrate;

A capillary electrophoresis (CE) method has been developed and validated for separating the tetrapeptide H-Tyr-(d)Arg-Phe-Phe-NH2 and nine related substances. The method was developed using experimental design in a four-step procedure, in which eight variables were investigated in a total of 47 experiments. The preferred background electrolyte (BGE) consisted of 0.1 M malonic acid at pH 2.5 with 7 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin (2,6-DM-β-CD). The separation of H-Tyr-(d)Arg-Phe-Phe-NH2 and the related substances was accomplished within 15 min, with a resolution greater than 1.5 between all peaks. The method was then investigated with respect to its selectivity, linearity, precision, detection limit (LOD) and quantitation limit (LOQ). In addition, a system suitability test was performed and response factors were determined, essentially following International Conference of Harmonization guidelines for the validation of analytical methods. LOD and LOQ for the related substance H-Arg-Phe-NH2 were found to be 0.3 and 0.8 μg/ml, respectively, at a target H-Tyr-(d)Arg-Phe-Phe-NH2 concentration of 1 mg/ml. The method performed well with respect to all of the validation parameters.
Keywords: H-Tyr-(d)Arg-Phe-Phe-NH2; Tetrapeptides;

Trimethoprim is an anti-infective agent used in the treatment of urinary and respiratory tract infections and mild to moderate pneumocystis carinii pneumonia. Trimethoprim is also a selective in vitro inhibitor of cytochrome P450 2C8 and may have utility as an in vivo inhibitor of this enzyme. A simplified high performance liquid chromatography (HPLC) method was developed to determine trimethoprim in human plasma. Samples are processed by protein precipitation with perchloric acid and chromatographic separation is achieved on a Synergi Polar-RP column (4 micron, 150  mm×4.6  mm) using a mobile phase consisting of 50 mM ammonium formate-acetonitrile-methanol (pH=3.0; 90:6:4 (v/v/v)). Detection is monitored at 280 nm. Intra- and inter-day precision ranged from 1.1 to 1.9 and 0.9 to 4.1%, respectively. The assay is simple, economical, precise, and is directly applicable to human studies involving steady state trimethoprim pharmacokinetics.
Keywords: Trimethoprim;

Interest in the analysis of low abundance neuropeptides particularly using matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI–TOF–MS) is increasing because these neuropeptides are essential to the mechanism of transportation and the metabolism. This article describes an immunoprecipitation procedure that is suitable for MALDI–MS analysis of substance P (SP), a neuropeptide, in rat brain tissues. Substance P was precipitated from brain tissue extracts by immunoprecipitation with antibodies directed against SP, and are analyzed by MALDI–TOF–MS. Mass spectrometric analysis showed a singly charged [M+H]+ ion peak that corresponded to the SP molecular mass and was observed with a detection error of 1.6%. The average mass errors between the observed and theoretical molecular mass were within the 0.11 Da range. Capillary zone electrophoresis analysis was subsequently performed, and the effects of the different separation parameters were examined. Beginning with milligram quantities of brain tissue, picomole quantities of SP could be detected using this method.
Keywords: Substance P;

In this study, an affinity membrane containing l-histidine as an amino acid ligand was used in separation and purification of human immunoglobulin G (HIgG) from solution and human serum. The polarities and the surface free energies of the affinity membranes were determined by contact angle measurements. HIgG adsorption and purification onto the affinity membranes from aqueous solution and human serum were investigated in a batch and a continuous system. Effect of different system parameters such as ligand density, adsorbent dosage, pH, temperature, ionic strength and HIgG initial concentration on HIgG adsorption were investigated. The maximum adsorption capacity of p(HEMA-MAAH-4) membranes for HIgG was 13.06 mg ml−1. The reversible HIgG adsorption on the affinity membrane obeyed both the Langmuir and Freundlich isotherm models. The adsorption data was analysed using the first- and second-order kinetic model and the experimental data was well described by the first-order equations. In the continuous system, the purity of the eluted HIgG, as determined by HPLC, was 93% with recovery 58% for p(HEMA-MAAH-4) membrane. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.
Keywords: Poly(hydroxyethylmethacrylate-co-methacrylolyamido-histidine); Immunoglobulin G;

Novel methylcellulose-immobilized cation-exchange precolumn for on-line enrichment of cationic drugs in plasma by Eiichi Yamamoto; Takahisa Sakaguchi; Takashi Kajima; Nariyasu Mano; Naoki Asakawa (327-334).
We developed a novel methylcellulose-immobilized strong cation-exchange (MC-SCX) precolumn for direct analysis of drugs in plasma. MC-SCX consists of silica gel with a methylcellulose outer-surface and a 2-(4-sulfophenyl) ethyl phase inner-surface. The MC-SCX precolumn was evaluated by direct analysis using pyridoxine, atenolol and sulpiride spiked in plasma, using a column-switching HPLC system. Each drug was retained and enriched on MC-SCX using an acidic mobile phase, which resulted in good linearity, sufficient reproducibility, intra- and inter day precision, and accuracy in analytical ion-pair LC with trifluoroacetic acid. The analytical methods for model drugs were applied to pharmacokinetics of atenolol and sulpiride in rats.
Keywords: Pharmacokinetics; Pyridoxine; Atenolol; Sulpiride;

Development and validation of a gas chromatography–mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study by Stephane Pirnay; Stephane Bouchonnet; Françoise Hervé; Danielle Libong; Nathalie Milan; Philippe d’Athis; Frédéric Baud; Ivan Ricordel (335-342).
Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography–mass spectrometry (GC–MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid–liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/μl plasma for BUP, 0.125 and 12.5 ng/μl for NBUP and N-DMFNZ, 0.125 and 5 ng/μl for FNZ, and between 0.025 and 50 ng/μl for 7-AFNZ. The limit of quantification was 0.025 ng/μl plasma for 7-AFNZ and 0.125 ng/μl for the four other compounds. A good reproducibility (intra-assay CV=0.32–11.69%; inter-assay CV=0.63–9.55%) and accuracy (intra-assay error=2.58–12.73%; inter-assay error=0.83–11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC–MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.
Keywords: Derivatization, GC; Pharmacokinetics; Buprenorphine; Flunitrazepam;

Chloramphenicol (CAP) is subjected to monitoring in food products, with a minimum required performance level set at 0.3 ng/g. CAP was isolated from chicken meat and seafood by very simple solvent extraction procedure. For honey, a fast SPE procedure was applied. CAP-D5 was used as internal standard. HPLC separation was done on RP18 123  mm×3  mm column in acetonitrile–ammonium formate 10 mM, pH 3.0 (40:60) at flow rate of 0.3 ml/min. A TSQ Quantum instrument with ESI source has been used in negative ionization mode. A MRM procedure has been applied and following transitions were monitored: m/z 321>152 (quantifier), 321>194, 321>257 (qualifiers), 326>157 (IS). CAP peak was eluted at around 5 min; the total run time was 7 min. LOD was around 0.1 ng/g meat or 0.05 ng/g honey. Matrix effects were studied for all materials used, involving injection of blank extracts with post-column infusion of CAP, as well as checking the influence of the co-injected blank extracts on the signal intensity of CAP. No influence of matrix on the results of CAP determination were observed. The method allows analyzing up to 30 duplicate samples per day, including all calibration standards. Additionally, the method for determination of CAP glucuronide (CAP-G) was established, using urine from rats that were given this drug as a source of the metabolite. Full validation of the metabolite was not possible, due to the unavailability of reference standard.
Keywords: Chloramphenicol; Chloramphenicol glucuronide;