Journal of Chromatography B (v.838, #2)

The molecular weight and size of recombinant Hepatitis B surface antigen (HBsAg) derived from Chinese hamster ovary (CHO) and the Hansenula polymorph have been characterized by high-performance size exclusion chromatography with multi-angle laser light scattering (HPSEC-MALLS). The average molecular weight of CHO-derived HBsAg particle (CHO-rHBsAg) (4921 kDa) was higher than that of H. polymorpha yeast strain (Hans-rHBsAg) (3010 kDa). The size of CHO-rHBsAg (22.1 nm) is nearly the same as that of native HBsAg compared to 18.1 nm for Hans-rHBsAg. The average monomer numbers were found to be 155 for CHO-rHBsAg and 86 for Hans-rHBsAg, respectively. The data obtained support the assumption that the higher immunogenicity of CHO-derived HBsAg is related to its more favorable macromolecular assembly structure.
Keywords: Hepatitis B surface antigen (HBsAg); Molecular weight; Molecular size; Distribution; HPSEC-MALLS;

The paper describes a method for the determination of selected lignans in plant foods. First, samples were submitted to methanolysis resulting in cleavage of ester bonds between lignan glycosides and organic acids. Glycosidic linkages were then broken by enzymatic hydrolysis using cellulase. The released aglycones were separated isocratically (acetonitrile/10 mM sodium acetate buffer, pH 4.8, 225:775, v:v) by reversed phase high performance liquid chromatography (RP-HPLC) and the compounds were detected coulometrically at four electrodes set on potentials between +260 and +330 mV against palladium reference electrodes. The selectivity and sensitivity of the method allowed quantitation of the lignans secoisolariciresinol, lariciresinol and isolariciresinol in various foodstuffs down to the upper ppb-range with recoveries between 44.7 and 97.0%. Unidentified peaks displaying similar current–voltage curves (CVCs) as the investigated lignans indicated the presence of further possible lignan representatives. In addition, investigation of various foodstuffs involving enzymatic hydrolysis with and without preceding methanolysis showed that the degree of esterification of lignans in plant foods is species dependent.
Keywords: Phytoestrogens; Lignans; Secoisolariciresinol; Lariciresinol; Isolariciresinol; HPLC–CEAD; Food;

ChanSu (toad venom) is a traditional Chinese medicine for the treatment of serious liver and gastric cancers. The major cytotoxic compounds in ChanSu are bufadienolides. In this paper, a strategy combining qualitative LC/MS analysis and quantitative HPLC determination of major bufadienolides was used for global quality control of ChanSu crude drug. Majority of the bufadienolides in methanol extract of ChanSu were unambiguously characterized by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), and by comparing with pure compounds. In addition, eight major bufadienolides were simultaneously determined in one single HPLC run within 30 min with photodiode array detection (DAD). All compounds showed good linearity in a wide concentration range, and their limits of detection (LOD) were around 1 ng. Thus, >95% of the bufadienolides in ChanSu could be characterized, and >90% of them were quantitated. The established method is rapid, simple and sensitive, and could be used for the routine analysis of ChanSu crude drug and its preparations. This research sets a good example for the comprehensive quality control of traditional medicine.
Keywords: Bufadienolide; ChanSu; Chinese medicine; Cytotoxicity; HPLC/DAD/APCI-MS; Quality control;

A method was developed for the determination of several phenylurea and triazine herbicides and their transformation products in oysters at the low μg/kg level. Pressurised liquid extraction (PLE) of lyophilisated samples had required successive SPE combined with a liquid/liquid extraction to provide relatively clean extracts for the determination in LC–MS/MS. This procedure was validated according to the 2002/657/EC analytical decision. Efficiency of the analytical method led to confirmatory CCα values ranging from 0.1 to 14 μg/kg with an R.S.D. value ranging from 14% to 66% and a recovery yield ranging from 32% to 46% for phenylureas and from 29% to 75% for triazines.
Keywords: Liquid chromatography; Electrospray ionization; Tandem mass spectrometry; Herbicides; Phenylurea; Triazine; Transformation products; Oysters; Validation;

HPLC determination of a novel aroylhydrazone iron chelator (o-108) in rabbit plasma and its application to a pilot pharmacokinetic study by Petra Kovaříková; Jiří Klimeš; Martin Štěrba; Olga Popelová; Vladimír Geršl; Přemysl Poňka (107-112).
A high performance liquid chromatographic method for the determination of a biocompatible iron chelator, pyridoxal 2-chlorobenzoyl hydrazone (o-108), in rabbit plasma was developed and validated. The separation was achieved on a C18 column with the mobile phase composed of a mixture of 0.01 M phosphate buffer (pH 6) with the addition of EDTA (2 mM), methanol and acetonitrile (42:24:14; v/v/v). The method was validated with respect to selectivity, linearity (0.8–150 μg/mL), intra- and inter-day variability and stability. This method was successfully applied to the analysis of the samples obtained from a pilot pharmacokinetic experiment, in which the chelator was administered intravenously to rabbits.
Keywords: Pyridoxal 2-chlorobenzoyl hydrazone; o-108; Iron chelator; HPLC; Pharmacokinetics;

Novel adsorptive polyamine coating for enhanced capillary electrophoresis of basic proteins and peptides by Angel Puerta; Jakob Axén; Lennart Söderberg; Jonas Bergquist (113-121).
In capillary electrophoresis (CE), the anionic and hydrophobic nature of the fused-silica capillary surface has long been known to present a problem in protein and peptide analysis. The use of capillary surface coating is one of the approaches to avoid the analyte–wall interactions. In this study, a new polymer, poly-LA 313, has been synthesized, physico-chemical characterized, and applied as polyamine coating for CE separations. The coating process is highly reproducible and provides fast separations of peptides and proteins in a few minutes and with high efficiency. The physically adsorbed polymer gives rise to a durable coating in the range of pH 2–10, in the presence of organic modifiers (acetonitrile and methanol) and with complex biological samples. The efficiency of the new cationic polymer was also tested performing protein and peptide separations with capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS).
Keywords: Capillary electrophoresis; Polyamine coating; Coated capillaries; Proteins; Peptides;

Recently, some research results showed that the circulating DNA in serum or plasma had potential for the molecular diagnosis and prognosis of certain cancers. Several methods have been employed for the quantification of circulating DNA. However, the circulating DNA levels obtained by various methods exhibited considerable differences. Additionally, these methods were labor-extensive and time-consuming, and not suitable for the quantification of circulating DNA in numerous samples due to the use of commercial DNA extraction kits for the purification of circulating DNA. We presented a new method for the quantification of circulating DNA in sera by capillary zone electrophoresis (CZE) with laser-induced fluorescence detection (LIF).In the present work, we want to make comparison between CZE-LIF assay and real time PCR for the quantification of circulating DNA levels. Linearity, intra and inter variability of two methods were evaluated.The intra and inter variability of circulating DNA quantification by real-time PCR were 7.3% and 14.92%, respectively. In CZE assay the intra and inter variability were 4.19% and 6.91%, respectively. The R.S.D. values of the same coated capillary and different coated capillaries were 5.14% and 9.02%, respectively. Our data showed that the circulating DNA levels obtained by two methods had a good correlation. Moreover, we further confirmed that blood samples collection, serum preparation and other treatment procedures had a significant impact on the DNA levels in sera.Our data further illustrated that CZE-LIF is a simple, rapid and sensitive method for the quantification of circulating DNA in human sera, and well suitable for the analysis of a large number of samples in clinical diagnosis.
Keywords: Human sera; Capillary zone electrophoresis; Circulating DNA; Laser induced fluorescence; Quantification; Real-time PCR;

Recently, an intravenous (i.v.) formulation of busulfan using the potentially hepatotoxic and neurotoxic N,N-dimethylacetamide (DMA) as a solvent was introduced. There is a need to assess the exposure of DMA in patients during the intravenous high dose therapy. A rapid and selective LC–MS method was developed to quantify relevant DMA concentration in plasma. After protein precipitation with trichloroacetic acid, the isocratic separation was achieved using a 150 mm × 2 mm C18 column and elution with a mobile phase containing 0.1% formic acid in water/acetonitrile (97:3). Detection of DMA was carried out with a ThermoFinnigan single-quadrupole mass spectrometer in selected-ion monitoring mode as H+-adduct at m/z 88.2. Deuterium-labelled DMA was used as the internal standard. The LC–MS method was accurate, precise and reproducible in the range from 0.25 to 150 mg/l and met the generally accepted criteria for bioanalytical methods. Two calibration ranges from 0.25 to 7.5 mg/l and 7.5 to 150 mg/l were used. The intra- (n  = 7) and interassay (n  = 7) accuracy and precision were both <7.7% and the limit of quantification is 0.25 mg/l. The method was successfully applied to investigate 203 plasma samples in children during the i.v.-busulfan therapy.
Keywords: N,N-Dimethylacetamide; LC–MS; Plasma; Busulfan;

Simple affinity chromatographic procedure to purify β-galactoside binding lectins by S.G. De-Simone; C.C. Netto; F.P. Silva (135-138).
Affinity chromatography based on the commercial resin Sepharose CL-6B was used to isolate new C1-β-type lectins from crude preparations of snake venoms (Bothrops jararaca, Bothrops jararacussu, Bothrops newiedi, Bothrops moojeni, Lachesis muta rhombeata). Most of the C-type lectins could be eluted with almost 100% recovery using the competitor isopropyl-β-d-thiogalactoside (IPTG) or through Ca2+ sequestration with EDTA. The lectin yield varied considerably among the different snake species, but B. newiedi venom was a particularly rich source of lectin, retaining 2.7 mg of lectin by milliliter of resin in saturating conditions. C1-α-lectins from Crotalus durisus terrificus venom, from the jack fruit (jacalin) and from bread fruit seeds extract (frutalin) had no affinity, either with or without Ca2+ added, for Sepharose CL-6B, showing that the resin is specific for C1-β-type lectins. Sepharose CL-6B used as galactose-affinity chromatography provides a simple and fast method for isolating C-type β-galactoside binding lectins from crude sample preparations.
Keywords: C-type β-galactoside binding lectins; Affinity chromatography; Lectins;

Scutellarin, a flavone glycoside, popularly applied for the treatment of cardiopathy, has been purified in two-step purification by high-speed counter-current chromatography (HSCCC) from Erigeron breviscapus (vant.) Hand. Mazz. (Deng-zhan-hua in Chinese), a well-known traditional Chinese medicinal plant for heart disease. Two solvent systems, n-hexane–ethyl acetate–methanol–acetic acid–water (1:6:1.5:1:4, v/v/v/v/v) and ethyl acetate–n-butanol–acetonitrile–0.1% HCl (5:2:5:10, v/v/v/v) were used for the two-step purification. The purity of the collected fraction of scutellarin was 95.6%. This study supplies a new alternative method for purification of scutellarin.
Keywords: Scutellarin; Erigeron breviscapus (vant.) Hand. Mazz.; Flavone glycoside; Polyphenol; Counter-current chromatography;