Journal of Chromatography B (v.840, #2)

In this study, hexa-histidine tagged VP3 protein of infectious bursal disease virus (IBDV) was purified using immobilized metal ion affinity technique from the fermentation of Escherichia coli BL21 (DE3) containing a recombinant plasmid with a VP3 gene. The purification efficiencies of VP3 protein (TVP3 and ΔTVP3) using Ni2+-NTA commercial agarose gels and Ni2+-IDA regenerated cellulose-based membranes at 4 °C were compared. A good washing condition for removing most impurity proteins was found as 20 mM NaH2PO4, 500 mM NaCl, 40 mM imidazole, pH 7.8, whereas an efficient elution condition was 20 mM NaH2PO4, 500 mM NaCl, 500 or 750 mM imidazole, pH 7.8. By applying these conditions to the flow experiments, similar recovery (86–88%) and purity (98–99%) for VP3 were obtained in both gel column (1 ml gel) and membrane cartridge (four membrane disks) under the flow rate of 1.7 ml/min for protein loading and 2.7 ml/min for protein elution. Regarding that the membrane process exhibited some advantages such as shorter residence time and lower cost, a better process efficiency in a large-scale system could be expected for the Ni2+-IDA membranes.
Keywords: Infectious bursal disease virus; Immobilized metal ion affinity chromatography; Immobilized metal ion affinity membrane; VP3 protein;

Hydrophobic interaction adsorption of hen egg white proteins albumin, conalbumin, and lysozyme by Edwin E. Garcia Rojas; Jane S. dos Reis Coimbra; Luis A. Minim; Sérgio H. Saraiva; César A. Sodré da Silva (85-93).
Hydrophobic adsorption equilibrium data of the hen egg white proteins albumin, conalbumin, and lysozyme were obtained in batch systems, at 25 °C, using the Streamline Phenyl® resin as adsorbent. The influence of three types of salt, NaCl, Na2SO4, or (NH4)2SO4, and their concentration on the equilibrium data were evaluated. The salt Na2SO4 showed the higher interaction with the studied proteins, thus favoring the adsorption of proteins by the adsorbent, even though each type of salt interacted in a distinct manner with each protein. The isotherm models of Langmuir, Langmuir exponential, and Chen and Sun were well fitted to the equilibrium data, with no significant difference being observed at the 5% level of significance. The mass transfer model applied simulated correctly adsorption kinetics of the proteins under the studied conditions.
Keywords: Hydrophobic adsorption; Egg white proteins; Modeling;

In this paper the determination of Ochratoxin A (OTA) in low volumes of human blood serum by enzyme-linked immunosorbent assay (ELISA) is compared with an appropriate capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) method. In order to use ELISA for high-throughput analysis in epidemiological studies no sample cleanup was performed. Both methods showed a limit of detection (LOD) of 0.5 ng/mL. Comparing the precisions of both methods, the data show that the quantified concentrations in ELISA are higher than the corresponding concentrations in the CE-LIF method. Using a matrix calibration curve instead of a standard calibration curve the reproducibilities of both methods are comparable. No additional matrix effect could be observed by adding phenylalanine as probable matrix compound to the serum.
Keywords: Capillary electrophoresis; CE-LIF human blood serum; Low sample volume; Ochratoxin A; ELISA;

Simultaneous determination of sulforaphane and its major metabolites from biological matrices with liquid chromatography–tandem mass spectroscopy by Shruti Agrawal; Bozena Winnik; Brian Buckley; Lixin Mi; Fung-Lung Chung; Thomas J. Cook (99-107).
A simple, sensitive and specific LC–MS/MS method for the simultaneous determination of sulforaphane (SFN) and its major metabolites, the glutathione (SFN–GSH) and N-acetyl cysteine conjugates (SFN–NAC) from biological matrices was developed and validated. The assay procedure involved solid-phase extraction of all three analytes from rat intestinal perfusate using C2 extraction cartridges, whereas from rat plasma, metabolites were extracted by solid-phase extraction and SFN was extracted by liquid–liquid extraction with ethyl acetate. Chromatographic separation of SFN, SFN–GSH and SFN–NAC was achieved on a C8 reverse phase column with a mobile phase gradient (Mobile Phase A: 10 mM ammonium acetate buffer, pH: 4.5 and Mobile Phase B: acetonitrile with 0.1% formic acid) at a flow rate of 0.3 mL/min. The Finnigan LCQ LC–MS/MS was operated under the selective reaction monitoring mode using the electrospray ionization technique in positive mode. The nominal retention times for SFN–GSH, SFN–NAC and SFN were 8.4, 11.0 and 28.2 min, respectively. The method was linear for SFN and its metabolites with correlation coefficients >0.998 for all analytes. The limit of quantification was 0.01–0.1 μM depending on analyte and matrix, whereas the mean recoveries from spiked plasma and perfusate samples were approximately 90%. The method was further validated according to U.S. Food and Drug Administration guidance in terms of accuracy and precision. Stability of compounds was established in a battery of stability studies, i.e., bench-top, auto-sampler and long-term storage stability as well as freeze/thaw cycles. The utility of the assay was confirmed by the analysis of intestinal perfusate and plasma samples from single-pass intestinal perfusion studies with mesenteric vein cannulation in rats.
Keywords: Sulforaphane; LC–MS/MS; Validation; Cancer chemo-prevention;

Vorinostat (suberoylanilide hydroxamic acid, SAHA) is undergoing evaluation as an antineoplastic agent. We developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for quantitating vorinostat and its major metabolites, vorinostat glucuronide and 4-anilino-4-oxobutanoic acid, in human serum. The assay uses: deuterated internal standards; acetonitrile protein precipitation; a BDS Hypersil C18 (3 μm, 100 mm × 3 mm) column; a gradient mobile phase of 0.5% acetic acid in acetonitrile and water; and electrospray positive-mode ionization with selected reaction monitoring (SRM) detection. The lower limit of quantitation was 3.0 ng/ml for each analyte. The assay is being employed in at least 12 clinical studies of vorinostat-containing regimens.
Keywords: Vorinostat; Suberoylanilide hydroxamic acid; SAHA; Histone deacetylase inhibitor; LC–MS/MS;

A novel method for simultaneous determination of cortisol and prednisolone in body fluids has been developed in this paper. Three-way data recorded by high-performance liquid chromatography with a diode array detector (HPLC–DAD) have been analyzed by second-order calibration based on the alternating trilinear decomposition (ATLD) algorithm. The chemometric methodology selected exploits the second-order advantage of the three-way data arrays, which allows one to obtain concentrations of individual calibrated analytes even in the presence of interferences not present in the calibration samples (e.g. background in urine or plasma). It was applied to simultaneous determination of cortisol and prednisolone in both plasma and urine samples. Though the chromatographic and spectral peaks of the analytes were heavily overlapped and interferents coeluted with the compounds studied, good recoveries of the analytes could be obtained with HPLC–DAD coupled with second-order calibration based on ATLD. Sample preparation was based on solvent extraction (SE), and quantification can be carried out with simple mobile phase. The time required for the quantification process is shorter than other methods.
Keywords: Second-order calibration; HPLC–DAD; Alternating trilinear decomposition; Cortisol; Prednisolone; Plasma; Urine;

An analytical method was developed for the determination of enantiomers of dencichine in plasma. Sample extraction from plasma was achieved by a solid-phase extraction (SPE) procedure using a C18 cartridge, with carbocisteine as the internal standard. Plasma was deproteinized using inorganic acid and derivatizated before the SPE. Chiral separation of dencichine enantiomers was achieved by pre-column derivatization using o-phthaldialdehyde (OPA) and the chiral thiol N-isobutanoyl-l-cysteine (NIBC) to form diastereoisomeric isoindole derivatives that were separable by ODS column using a gradient solvent programme. The column eluent was monitored using mass spectrometry (MS). The conditions of MS detection were optimized, and selected ion monitoring was used to selectively detect d-dencichine and its arrangement isomer. High sensitivity and selectivity were obtained using this method. The limit of detection was determined to be 10 ng/ml for d-dencichine and 8 ng/ml for l-dencichine in plasma. The linearity was demonstrated over a wide range of concentrations, from 0.5 to 50 μg/ml for both enatiomers. The intra- and inter-day precision (C.V.), studied at four concentrations, was less than 7.0%. No interferences from endogenous amino acids and isomers of dencichine were found. The method was suitable for pharmacokinetic studies of dencichine enantiomers.
Keywords: Dencichine; Eanatiomer separation; LC-ESI-MS;

A liquid chromatographic (LC) method for determining 14 sulfonamide (SA) (sulfanilamide, sulfadiazine (SDZ), sulfathiazole, sulfapyridine, sulfamerazine (SMR), sulfamethazine (SMZ), sulfamethizole, sulfamethoxypyridazine, sulfachloropyridazine (SCP), sulfamonomethoxine, sulfadoxine, sulfamethoxazole, sulfadimethoxine (SDM), and sulfaquinoxaline (SQX)) residues in edible catfish, shrimp and salmon tissues was developed and validated at 5, 10 or 20 ng g−1. The method was then used to determine residues in tissues of catfish, shrimp and salmon dosed with six selected sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfachloropyridazine, sulfadimethoxine and sulfaquinoxaline). All assays were within U.S. Food and Drug Administration guidelines for recovery and intra-assay variability. The method was developed to determine possible sulfonamide residues in aquacultured catfish, shrimp and salmon produced for food.
Keywords: Sulfonamides; Catfish; Shrimp; Salmon; Liquid chromatography;

A new method was developed for the rapid extraction and unequivocal determination of barbital, amobarbital and phenobarbital residues in pork. The isolation of the analytes from pork samples was accomplished by utilizing an accelerated solvent extractor ASE 300. The procedure was automatically carried out in series for fat removing and extraction, respectively with n-hexane and acetonitrile pressurized constantly at 10.3 MPa for 30 min. After evaporation, the extracts were cleaned up on a C18 solid phase extraction (SPE) cartridge and the barbiturates were eluted with hexane–ethyl acetate (7:3), evaporated on a rotary evaporator and derivatized with CH3I. The methylated barbiturates were separated on a HP-5MS capillary column and detected with a mass detector. Electron impact ion source (EI) operating in time program-selected ion monitoring mode (SIM) was used for identification and external standard method was used for quantification. Good linearity was obtained in the range from 0.5 μg/kg to 25 μg/kg. Average recoveries of the three barbiturates spiked in pork ranged from 84.0% to 103.0%, with relative standard deviations from 1.6% to 12%. The limit of detection (LOD) was 0.5 μg/kg for the three barbiturates (S/N ≥ 3). The quantification limit (LOQ) was 1 μg/kg for the three barbiturates (S/N ≥ 10).
Keywords: Barbiturates; Accelerated solvent extraction (ASE); Solid phase extraction; Methylation; Gas chromatography–mass spectrometry; Pork;