Journal of Chromatography B (v.774, #1)

News Section (N1-N2).

Rational combinatorial chemistry-based selection, synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII by Paul R. Morrill; Geeta Gupta; Kenny Sproule; Don Winzor; Jesper Christensen; Inger Mollerup; Christopher R. Lowe (1-15).
The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca2+-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (∼1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability.
Keywords: Recombinant human clotting factor VII;

A simple, sensitive and accurate method for the estimation of free and total (free plus protein-bound) melatonin (MLT) in human plasma and cerebrospinal fluid (CSF) is described. Via Chem-Elut cartridges, free and total MLT (the latter obtained after a deproteinization step) were quantified in dichloromethane-extracted samples and analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with fluorimetric detection. The column used was an Extrasil ODS-2 (3 μm, 150×4.6 mm I.D.), while the mobile phase consisted of 75 mM sodium acetate–acetonitrile (72:28, v/v) (pH 5.0). Repeatability and reproducibility of the method were 3.24 and 9.4%, respectively. The recovery of melatonin from plasma and CSF was 99.9±4.0% for non-deproteinized samples and 93.2±4.8% for deproteinized samples. The detection limit of the assay was 0.5 pg/ml. In human plasma, the mean±SD concentrations in the darkness period were 23.18±7.44 pg/ml for free melatonin and 82.5±36.48 pg/ml for total melatonin, while the lowest concentrations detected during daytime were 2.23±2.22 and 7.40±5.68 pg/ml, respectively. Detection of MLT in CSF was 5.01±2.31 and 28.55±6.95 pg/ml for the free and total fraction, respectively.
Keywords: Melatonin;

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles by Bohuslav Rittich; Alena Španová; Yuriy Ohlashennyy; Jiřı́ Lenfeld; Ivo Rudolf; Daniel Horák; Milan J Beneš (25-31).
Magnetic bead cellulose particles and magnetic poly(HEMA–co–EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA–co–EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg2+, Ca2+, Mn2+ and Co2+) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co2+ ions.
Keywords: Deoxyribonuclease I;

We have developed a sensitive and specific HPLC-fluorescence assay for the determination of estradiol-3-glucuronide and estradiol-17-glucuronide in human and rat liver microsomal incubations. The method utilizes a mobile phase comprised of acetonitrile and 50 mM ammonium phosphate buffer (35:65, v/v) that is pumped though a phenyl column at 1 ml/min; the run time is less than 15 min. Calibration curves for both metabolites were linear over the range 20–4000 pmol. The intra- and inter-day coefficients of variation were <6%. In both rat and human liver microsomes, the formation of estradiol-3-glucuronide displayed atypical kinetics (consistent with activation), while estradiol-17-glucuronide formation was consistent with classical Michaelis–Menten kinetics. Overall, the assay described is a sensitive and reproducible method for the determination of estradiol glucuronides in liver microsomal preparations.
Keywords: Estradiol glucuronide;

Two complementary methods for identifying and measuring sulfonamide residues in eggs were developed for use in surveying eggs for potential drug residues. The first method uses liquid chromatography–tandem mass spectrometry (LC–MS–MS) to confirm the presence of sulfonamide residues in eggs. During its validation the limit of confirmation was estimated to be 5–10 ng/g (ppb) depending on the drug. Also, a method for measuring residue level by liquid chromatography with ultraviolet detection (LC–UV) was validated using the same extraction procedure as the confirmatory method. The determinative method was validated over the 50–200 ppb range. Samples were prepared by homogenizing whole egg, extracting with acetonitrile, and cleaning up with a C18 solid-phase extraction cartridge. For confirmation, analytes were separated by gradient LC on a C18 column, ionized by electrospray ionization (ESI), and detected by MS–MS with an ion trap mass spectrometer. For determination, analytes were separated by a different gradient LC procedure and detected by UV at 287 nm. Fifteen drugs were dosed individually in laying hens, and residues of parent drug and/or metabolites were found in eggs for all the drugs. Validation was based on repetitive analyses of control samples, control samples fortified at 100 ppb sulfonamides, and samples of blended incurred eggs.
Keywords: Sulfonamides;

Three-dimensional protein map according to pI, hydrophobicity and molecular mass by Daniel B. Wall; Stephen J. Parus; David M. Lubman (53-58).
A three-dimensional method has been developed to map the protein content of cells according to pI, M w and hydrophobicity. The separation of complex protein mixtures from cells is performed using isoelectric focusing (IEF) in the liquid phase in the first dimension, non-porous silica (NPS) RP-HPLC in the second dimension and on-line electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) detection in the third dimension. The experimentally determined pI, M w and hydrophobicity can then be used to produce a three-dimensional map of the protein expression of a cell, where now each protein can be tagged by three independent parameters. The ESI-TOF-MS provides an accurate M w for the intact protein while the hydrophobicity dimension results from the RP-HPLC component of the separation. The elution time, or percent acetonitrile at time of elution, of the protein is related to the hyrophobicity, which is an inherent property of the protein. 3D protein maps can thus be generated showing pI, M w and % acetonitrile at time of elution as well as pI, M w and hydrophobicity. The potential of the 3D plot for effective mapping of proteins from cells compared to current 2D methods is discussed.
Keywords: Proteins;

Beta-agonist compounds are widely used in human therapeutics because of bronchodilator or heart tonic properties; they are also used as growth promoters in food-producing animals. Ractopamine is a forbidden molecule in the EU, but is registered as an additive in other countries such as in the USA for pigs. Consequently, efficient analytical methods were developed to survey residues in edible tissue and urine samples. This paper describes a protocol based on a powerful extraction and purification process and a liquid chromatography–positive electrospray mass spectrometry identification method. A validation was performed according to the “DG SANCO 1805/2000” European decision. The obtained decision limit (CCα) and detection capability (CCβ) were as low as 10 and 30 ng/kg (ppt), respectively. This method appeared very efficient on incurred samples, including porcine edible tissues (meat, liver, kidney), tissues enriched in β-agonist receptors (lung, retina), and finally bovine urine samples.
Keywords: Ractopamine;

A simple, rapid and specific high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) has been developed and validated for the determination of ketoconazole in human plasma. The method used diethyl ether to extract the ketoconazole and the internal standard (I.S.) R51012 from alkalinized plasma sample. The LC separation was on a C18 column (50×3 mm, 5 μm) using acetonitrile–water–formic acid (75:25:1, v/v/v) mobile phase. The retention times were approximately 1.8 min for both ketoconazole and the I.S. The MS–MS detection was by monitoring 531.2→82.1 (m/z) for ketoconazole, and 733.5→460.2 (m/z) for the I.S. The dynamic range was from 20.0 to 10 000 ng/ml based on 0.1 ml plasma, with linear correlation coefficient of ≥0.9985. The run time was 2.5 min/injection. The recoveries of ketoconazole and the I.S. were 102 and 106%, respectively. The precision and accuracy of the control samples were with the relative standard deviations (RSDs) of ≤4.4% (n=6) and the relative errors (REs) from −0.6 to 1.4% for intra-day assay, and ≤8.6% RSD (n=18) and −1.4 to 0.9% RE for inter-day assay. The partial volume tests demonstrated good dilution integrity. Three freeze–thaw cycles, keeping plasma samples at ambient for 24 h, storing extracted samples at ambient for 24 h, and storing frozen plasma samples at approximately −20 °C for up to 2 months did not show substantial effects.
Keywords: Ketoconazole;

A simple reversed-phase high-performance liquid chromatography assay for the simultaneous quantitative determination of three HIV non-nucleoside reverse transcriptase inhibitors (nevirapine, delavirdine, and efavirenz) in human blood plasma is described. The method was validated over the range of 10 ng/ml to 50 μg/ml for nevirapine, 25 ng/ml to 25 μg/ml for delavirdine, and 10 ng/ml to 10 μg/ml for efavirenz. The method is accurate (average accuracies over eight concentrations ranging from 87.3 to 113%), and precise (within-day and between-day precision measures ranging from 0.12 to 7.9% and 0.26 to 5.9%, respectively). All three non-nucleoside reverse transcriptase inhibitors proved to be stable under various conditions. Due to its simplicity, this assay can readily be used for investigational or clinical monitoring of plasma concentrations.
Keywords: Nevirapine; Delavirdine; Efavirenz;

Chromatographic approach for determining the relative membrane permeability of drugs by Qing C. Meng; Jonas S. Johansson; Roderic G. Eckenhoff (89-95).
With the aid of the experimental dependence of the theoretical plate height (H) on the flow-rate (U), values of diffusion coefficients as the permeation rate, of the compounds in a polymeric stationary phase were calculated from solute mass transfer. This approach is proposed for modeling the relative diffusion rate of a drug within the membrane. After the successful separation of opioid compounds using a C18 derivatized polystyrene–divinylbenzene polymer HPLC column, the slopes of HU plots increase quantitatively in the order of meperidine<alfentanil<fentanyl<sufentanil, indicating that the large mass transfer resistance slows down the penetration of molecules. A constant intercept for the experimental plate height supports the proposal interpretation. A good correlation between the diffusion coefficients and hydrophobicity (log P oct) from the traditional shake-flask method was obtained for the opioid compounds, demonstrating that the more lipophilic molecules penetrate deeper into the stationary phase leading to a lower migration rate under the specified conditions. Plot of the diffusion coefficients versus potency ratio for different opioids after intravenous administration reflect the values of the dynamic process in drug studies. The work herein differs from existing studies by measuring the permeability of drugs into the stationary phase rather than providing membrane partition coefficients for a series of analogues. Thus, the study of drug permeability combined with other physico-chemical properties, such as hydrophobicity, may provide additional information on drug–membrane interactions.
Keywords: Meperidine; Alfentanil; Fentanyl; Sufentanil;

Nitrites and nitrates are widely used reporters of endogenous activity of nitric oxide synthases (NOS), an important group of enzymes producing the gaseous signal molecule nitric oxide (NO). However, due to the great chemical heterogeneity of neuronal tissues, standard analytical protocols for evaluation of neuronal nitrite/nitrate concentrations are inefficient. We optimized a high-performance capillary zone electrophoresis (CZE) technique to analyze nitrite/nitrate concentrations in submicroliter samples from mammalian neuronal tissues. The measurements were made using a PrinCE 476 computerized capillary electrophoresis system with a Crystal 1000 contact conductivity detector. Isotachophoretic stacking injection of 1000- to 10 000-fold diluted samples, which had been pretreated with a custom-designed solid-phase microextraction (SPME) cartridge, was employed to assay micromolar and nanomolar nitrite and nitrate levels in the presence of the high millimolar chloride concentrations characteristic of many biological samples. In the presented technique, a 10-μl volume of diluted ganglionic sample was used for chloride removal and sample cleanup. The method yields high analytical performance, including good reproducibility, resolution, and accuracy. The limits of detection relative to undiluted sample matrix were 8.9 nM (0.41 ppb) and 3.54 nM (0.22 ppb) for nitrite and nitrate, respectively. In addition, this technique resolves other anions that are present in neuronal tissues at sub-nanomolar concentrations and can be broadly applied for high-throughput anionic profiling. In rat dorsal root ganglia, endogenous levels of nitrate (231±29 μM; n=6) and nitrite (24–96 μM) were found. These concentrations exceeded those previously found in neuronal tissue homogenates using different techniques.
Keywords: Nitrite; Nitrate;

High-performance liquid chromatographic assay for amiodarone N-deethylation activity in human liver microsomes using solid-phase extraction by Nobumitsu Hanioka; Yoshiro Saito; Akiko Soyama; Masanori Ando; Shogo Ozawa; Jun-ichi Sawada (105-113).
A selective and sensitive assay for amiodarone N-deethylation activity in human liver microsomes by high-performance liquid chromatography (HPLC) with UV detection is reported. The extraction of desethylamiodarone from incubation samples was performed by means of an original solid-phase extraction (SPE) procedure using a polymeric reversed-phase sorbent (Oasis HLB). The method was validated for the determination of desethylamiodarone with respect to specificity, linearity, precision, accuracy, recovery, limit of quantitation and stability. Amiodarone N-deethylation activity from low to high substrate concentrations using human liver microsomes was precisely determined without a concentration step. This method is applicable to the study in vitro of the metabolism of amiodarone.
Keywords: Amiodarone; Desethylamiodarone; Cytochrome P450;

A reversed-phase HPLC method compatible with evaporative light scattering (ELS) and electrospray mass spectrometric (ES-MS) detection was developed for separation of phosphatidylserine (PS) molecular species. The method was optimised for separation of three disaturated synthetic species: dipalmitoyl glycerophosphoserine, palmitoyl-stearoyl glycerophosphoserine and distearoyl glycerophosphoserine using isocratic elution with a mixture of 2-propanol, tetrahydrofuran and ammonium formate. Baseline separation was obtained on three different columns: one polystyrene/divinylbenzene (PS/DVB) column and two silica based C18 and C30 columns. The best chromatographic resolution was achieved with the C30 column. The limit of detection for DPPS was 5 μg/ml (S/N=3) with ELS detection and 0.1 μg/ml (S/N=3) with negative ion ES-MS in the single ion monitoring mode. Baseline separation of the five main species in a biological PS sample, bovine brain PS, was obtained with the PS/DVB column. Species identification was done by using the retention times of the intact PS species and their corresponding carboxylate anion fragments obtained by in-source fragmentation. Data have shown that individual PS species can be identified by their retention times using direct ELS detection in a mixture of disaturated PS species. However, for the bovine brain PS electrospray-MS detection was necessary for species identification due to the many possible fatty acid combinations in biological PS.
Keywords: Phosphatidylserines;

Capillary zone electrophoresis was employed for the determination of lactate using end-column amperometric detection at a carbon fiber bundle microdisk electrode. The optimum conditions of separation and detection are 3.6×10−3 mol/l Na2HPO4–1.4×10−3 mol/l NaH2PO (pH 7.2) for the buffer solution, 18 kV for the separation voltage and 1.60 V versus the saturated calomel electrode for the detection potential. The limit of detection is 7.6×10−7 mol/l or 1.7 fmol (S/N=3) and the linear range is 1.7×10−6–8.2×10−4 mol/l for the injection voltage of 6 kV and injection time of 5 s. The RSD is 1.8% for the migration time and 3.3% for the electrophoretic peak current. The method was applied to the determination of lactate in human saliva. The recovery of the method is between 95 and 109%.
Keywords: Lactate;