Journal of Chromatography B (v.873, #1)

The QRAR model study of β-lactam antibiotics by capillary coated with cell membrane by Gengliang Yang; Weimin Cao; Tao Zhu; Ligai Bai; Yu Zhao (1-7).
With the accelerating development of new drugs, there is a great need for rapid and simple screening technologies. In this paper, a new in vitro method, capillary coated with cell membrane, was presented for drug screening based on the real biomembrane–drugs interaction, in which the cell membrane was applied to chromatography as pseudo-stationary phase directly. As the cell membrane was coated on the bare-fused capillary via sol–gel technology in our present work, it will be shown to be superior to other pseudo-stationary phases mimicking biological environment. Meanwhile, the quantitative retention–activity relationships (QRAR) model of β-lactam antibiotics was studied through investigating the effect of the membrane coating amount and pH of running buffer on the retention behaviors of the drugs and the logarithm of octanol–water partition coefficient (log  P) at pH 7.4 and pH 6.5 were also obtained for comparison. The results showed that the capillary coated with cell membrane could suit the study of QRAR model.
Keywords: Cell membrane; Capillary; β-Lactam antibiotics; QRAR;

Protein:protein aggregation induced by protein oxidation by Hamid Mirzaei; Fred Regnier (8-14).
When the level of reactive oxygen species (ROS) in cells exceeds a genetically coded defense capacity, the cells experience damage to vital components such as DNA, proteins and lipids that leads to non-specific interactions and the production of a series of high molecular weight protein aggregates. The dynamics of oxidative stress induced aggregation were studied here using model proteins and yeast. Model proteins were oxidized at increasing ROS concentrations and analyzed using size exclusion chromatography (SEC). Changes in the SEC elution profile showed that aggregation happens in stages and protein fragments produced as a result of oxidation also give rise to aggregates. Yeast cells were stressed with hydrogen peroxide to investigate in vivo aggregation. Equal amounts from control and oxidized lysates were chromatographed on a size exclusion column and proteins of molecular weight exceeding 700 kDa were collected from both samples which were then differentially labeled using light and heavy isotope coded N-acetoxysuccinamide and mixed in a 1:1 ratio. The coded mixture was analyzed using LC/MS and peptides that appeared as singlets representing the proteins that aggregated with higher molecular mass protein complexes were identified. Twenty-five proteins were identified to be of this type. Fifteen members in this group were found to have been carbonylated. These proteins are part of the proteome known as the aggresome. The protein content of the aggresome may provide vital information for mechanistic studies targeting disease and aging.
Keywords: Protein aggregation; Aggresome; Protein oxidation; Hydrophobic interaction; Protein carbonyls;

Capillary gas chromatographic (GC) determination of methylglyoxal (MGo) was developed on the basis of precolumn derivatization with 1,2-diaminopropane (DAP) at pH 3. The elution was carried out on an HP-5 (30 m × 0.32 mm i.d.) connected with FID. The linear calibration curve was obtained for MGo within 0.09–1.04 μg/ml with detection limit of 40 ng/ml. Dimethylglyoxal (DMGo) also formed derivative with DAP and eluted and separated from MGo at column temperature 100 °C for 1 min with heating rate 30 °C/min up to 200 °C with run time 4.6 min. The nitrogen flow rate was 1.5 ml/min with split ratio of 10:1, v/v. MGo was determined from serum and urine of diabetics and healthy volunteers. The amounts of MGo from serum and urine of diabetic patients were 0.180–0.260 μg/ml and 0.170–0.250 μg/ml with relative standard deviation (R.S.D.) within 1–4% and 1–3%, respectively. The amounts of MGo from serum of healthy volunteers were 0.032–0.054 μg/ml with an R.S.D. of 1.5–3%. DMGo was not detected from the biological fluids and was used as an internal standard.
Keywords: Methylglyoxal; 1,2-Diaminopropane; Capillary gas chromatography; Biological fluids;

A sensitive, rapid liquid chromatographic–electrospray ionization mass spectrometric method for the determination of xanthinol in human plasma was developed and validated. Xanthinol nicotinate in plasma (0.5 mL) was pretreated with 20% trichloroacetic acid for protein precipitation. The samples were separated using a Lichrospher silica (5 μm, 250 mm × 4.6 mm i.d.). A mobile phase of methanol–water containing 0.1% formic acid (50: 50, v/v) was used isocratically eluting at a flow rate of 1 mL/min. Xanthinol and its internal standard (IS), acyclovir, were measured by electrospray ion source in positive selected reaction monitoring mode. The method demonstrated that good linearity ranged from 10.27 to 1642.8 ng/mL with r  = 0.9956. The limit of quantification for xanthinol in plasma was 10.27 ng/mL with good accuracy and precision. The mean plasma extraction recovery of xanthinol was in the range of 90.9–100.2%. The intra- and inter-batch variability values were less than 4.8% and 7.9% (relative standard deviation, R.S.D.), respectively. The established method has been successfully applied to a bioequivalence study of two xanthinol nicotinate tablets for 20 healthy volunteers.
Keywords: Xanthinol nicotinate; Liquid chromatography/tandem mass spectrometry; Bioequivalence;

A sensitive and reproducible high-performance liquid chromatography (HPLC)–UV method for the determination of Z24, a tumorigenesis and angiogenesis inhibitor, has been developed and validated in mouse whole blood. Blood samples were extracted with ether, evaporated, and the residue was reconstituted in mobile phase. An aliquot was separated by isocratic reversed-phase HPLC on a Hypersil ODS-2 column and quantified using UV detection at 390 nm. The mobile phase was 50% (v/v) acetonitrile/water with a flow rate of 0.8 ml/min. A linear curve over the concentration range of 0.05–6 μg/ml (r 2  = 0.9976) was obtained. The coefficient of the variation for the intra- and inter-day precision ranged from 3.0 to 10.9% and 5.7 to 10.3%, respectively. The absolute recovery of Z24 was 89.2–108.5%. The method is simple, economical and sufficient for in vivo pharmacokinetic studies on Z24. Nonlinear pharmacokinetics was found in mice at doses from 20 to 80 mg/kg.
Keywords: Z24; HPLC; Pharmacokinetic; Mice; Tumorigenesis; Angiogenesis;

A solid phase extraction (SPE)-LC–MSMS method for the routine determination of oxalic acid (OX) in plasma, a diagnostic marker of primary hyperoxaluria (PH), was developed and validated. The normal range of OX was found to be 3–11 μmol/L (n  = 67), with no differences attributable to gender or age. The effect of pre-analytical factors on the in vitro production of OX was investigated, and plasma was found to be stable for 1–2 h at room temperature, less after ingestion of vitamin C; the process was not completely stopped by preservation at either −20 or −70 °C.
Keywords: Liquid chromatography–tandem mass spectrometry; Plasma; Oxalic acid; Laboratory diagnosis; Primary hyperoxaluria; Oxalogenesis;

Biochromatographic framework for analyzing magnesium chloride salt dependence on nor-NOHA binding to arginase enzyme by T. Bagnost; Y.C. Guillaume; M. Thomassin; A. Berthelot; C. Demougeot; C. André (37-40).
Our group demonstrated recently that arginase I inhibition reduces endothelial dysfunction and blood pressure rising in spontaneously hypertensive rats [C. Demougeot, A. Prigent-Tessier, C. Marie, A. Berthelot, J. Hypertens. 23 (2005) 971; C. Demougeot, A. Prigent-Tessier, T. Bagnost, C. Andre, Y. Guillaume, M. Bouhaddi, C. Marie, A. Berthelot, Life Sci. 80 (2007) 1128]. This discovery opens interesting perspectives in the development of new drugs against hypertension. As well, in a previous paper [T. Bagnost, Y.C. Guillaume, M. Thomassin, J.F. Robert, A. Berthelot, A. Xicluna, C. Andre, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 856 (2007) 113], a novel biochromatographic column was developed in our laboratory for studying the binding of N ω-hydroxy-nor-l-arginine (nor-NOHA), an arginase inhibitor, with this enzyme. In this manuscript, using this novel biochromatographic concept, the effect of magnesium chloride on the nor-NOHA/arginase binding was analyzed for the first time. This study demonstrated that the salt ions interacted with arginase and played a great role in the nor-NOHA/arginase association. For a salt concentration (x) in the medium less than 3 mM, the nor-NOHA/arginase binding decreased with x due to a decrease of the charge–charge interactions between nor-NOHA and its arginase binding site. Above 3 mM of salt in the medium, the affinity of nor-NOHA to arginase increased slightly with x because the net number of ions (n) (Mg2+ or Cl) released or bound upon complex formation is low. As well, it was clearly demonstrated, that above 3 mM the n value depend on the salt concentration in the bulk solvent and was approximately nil for x  = 12 mM. This dependence was due to a gradual and conformational change of the arginase enzyme which around 12 mM adopted a less flexible structure; its binding site was thus less accessible to nor-NOHA and nor-NOHA–arginase association decreased slightly.
Keywords: Arginase; Nor-NOHA; Magnesium;

The structural elucidation of metabolites of penehyclidine in rats, a novel anti-cholinergic drug, by the method of liquid chromatography–electrospray ionization mass spectrometry, gas chromatography–mass spectrometry with electron impact ion source and stable isotope ion cluster was described. Identification and elucidation of the phase I and phase II metabolites were performed by comparing the daughter ion pairs of stable isotope cluster, changes of the protonated molecular masses, full scan MS n spectra and retention times with those of the parent drug, penehyclidine and penehyclidine deuterium-labeled. Penehyclidine was easily biotransformed by the pathways of oxidative, hydroxylated, methoxylated and phase II conjugated reactions to form several metabolites that retained the some features of the parent molecules. Twelve metabolites (penehyclidine monoxide, hydroxypenehyclidine, penehyclidine dioxide, hydroxypenehyclidine monoxide, dihydroxypenehyclidine, dihydroxypenehyclidine (position isomer), dihydroxypenehyclidine monoxide, trihydroxypenehyclidine, methoxypenehyclidine dioxide, dimethoxypenehyclidine, trihydroxymethoxypenehyclidine and glucuronide conjugated hydroxypenehyclidine) were identified. The results from electrospray ion and electron impact ion data with the stable isotope cluster showed the qualitative differences in the mass spectral patterns, suggesting that these technologies should be used in parallel to ensure comprehensive metabolites detection and characterization. The described method has wide applicability to rapidly screen and provide structural information of metabolites.
Keywords: Structural elucidation; Penehyclidine; Metabolite; Liquid chromatography–mass spectrometry; Gas chromatography–mass spectrometry; Isopote cluster;

A traditional Chinese medicinal preparation (TCMP) named Guanxinning lyophilized powder for injection composed of Salvia miltiorrhiza Bge. (SMB) and Ligusticum chuanxiong Hort. (LCH) was studied. In order to learn the kinetic behaviors of the lyophilized powder and provide proofs for rational administration, we have developed a sensitive and reproducible method for determination and pharmacokinetic study of six main phenolic components {danshensu (DSS), protocatechuic acid (PAC), protocatechuic aldehyde (PAL), chlorogenic acid (CHA), caffeic acid (CAA) and salvianolic acid B (SAB)} of Guanxinning in rat plasma using liquid chromatography–mass spectrometric (LC–MS) method. Sample preparations were carried out by protein precipitation with the addition of methanol followed by liquid–liquid extraction with ethyl acetate–ethyl ether (3:1, v/v) after internal standard (IS, galic acid) spiked. After evaporation to dryness, the resultant residue was reconstituted in methanol and injected onto a Kromasil C18 column (150 mm × 4.6 mm i.d. with 5 μm particle size). The analytes were analyzed by using negative electrospray ionization (ESI) mass spectrometry in selected ion monitoring (SIM) mode. The method was with good linearity in the range 0.342–85.0 μg mL−1 for DSS, 0.0647–12.9 μg mL−1 for PAC, 0.0933–18.7 μg mL−1 for PAL, 0.0085–3.40 μg mL−1 for CHA, 0.0138–2.75 μg mL−1 for CAA and 0.0272–810 μg mL−1 for SAB (r  > 0.99). The average extract recoveries of the six analytes from rat plasma were all no less than 75%, the precision and accuracy determined were all within the required limits. This LC–MS method was successfully applied to pharmacokinetic study of the six phenolic components of Guanxinning lyophilized powder for injection in rats.
Keywords: LC–MS; Guanxinning lyophilized powder for injection; Phenolic components; Pharmacokinetic study;

Simultaneous determination of hydrochlorothiazide, quinapril and quinaprilat in human plasma by liquid chromatography-tandem mass spectrometry by Sagar A. Parekh; Ashutosh Pudage; Santosh S. Joshi; Vikas V. Vaidya; Noel A. Gomes; Sudhir S. Kamat (59-69).
A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous estimation of hydrochlorothiazide, quinapril and its metabolite quinaprilat in human plasma. After solid phase extraction (SPE), the analytes and IS were chromatographed on a hypurity C8 (100 mm × 2.1 mm i.d., 5 μm particle size) column using 2 μL injection volume with a run time of 2.8 min. An isocratic mobile phase consisting of 0.5% (v/v) formic acid:acetonitrile (25:75, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The proposed method was validated over the range of 5–500 ng/mL for hydrochlorothiazide method and 5–1500 ng/mL for quinapril and quinaprilat. Inter-batch and intra-batch precision (coefficient of variation – % CV) across five validation runs lower limit of quantitation (LLOQ), lower quality control (LQC), middle quality control (MQC), higher quality control (HQC) and upper limit of quantitation (ULOQ) was less than 15. The accuracy determined at these levels was within ±13% in terms of relative percentage error.
Keywords: Hydrochlorothiazide; Quinapril; Quinaprilat LC-MS/MS; Multiple reaction monitoring (MRM);

Plasma aromatic and sulfur containing amino acids are good indicators of protein anabolism/catabolism, while blood reduced and oxidized glutathione reflect oxidative status in an organism. Using a full factorial design for screening important variables (pH, concentration, temperature) we developed a capillary zone electrophoresis method permitting their measurements in the single run, without any derivatization procedures. The best separations were obtained within less than 30 min employing a 10 mmol/l phosphate buffer, pH 2.8, 18 °C, 15 kV voltage. Fairly good precision with a linear relationship between peak area and concentrations (r  = 0.995–0.999) were obtained. The method was used to analyze human capillary blood.
Keywords: Amino acids; Capillary electrophoresis; Factorial design; Glutathione;

Simultaneous determination of metoprolol succinate and amlodipine besylate in human plasma by liquid chromatography–tandem mass spectrometry method and its application in bioequivalence study by Amlan Kanti Sarkar; Debotri Ghosh; Ayan Das; P. Senthamil Selvan; K. Veeran Gowda; Uttam Mandal; Anirbandeep Bose; Sangeeta Agarwal; Uttam Bhaumik; Tapan Kumar Pal (77-85).
A simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of metoprolol succinate (MPS) and amlodipine besylate (AM) using hydrochlorothiazide (HCTZ) as IS in human plasma. Both the drugs were extracted by simple liquid–liquid extraction with chloroform. The chromatographic separation was performed on a reversed-phase peerless basic C18 column with a mobile phase of methanol–water containing 0.5% formic acid (8:2, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 1–100 ng/ml for MPS and 1–15 ng/ml AM in human plasma. The MRM transition of m/z 268.10–103.10, m/z 409.10–334.20 and m/z 296.00–205.10 were used to measure MPS, AM and HCTZ (IS), respectively. This method was successfully applied to the pharmacokinetic study of fixed dose combination (FDC) of MPS and AM formulation product after an oral administration to Indian healthy human volunteers.
Keywords: Metoprolol succinate; Amlodipine besylate; Hydrochlorothiazide; LC–MS/MS; Validation;

Chromatographic resolution, characterisation and quantification of VX enantiomers in hemolysed swine blood samples by Georg Reiter; John Mikler; Ira Hill; Kendal Weatherby; Horst Thiermann; Franz Worek (86-94).
The present study was initiated to develop a sensitive and highly selective method for the analysis of the enantiomers of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) in blood samples for toxicokinetic and therapeutic research. To achieve this goal, analytical and semi-preparative enantioseparation of VX were carried out with gas and liquid chromatography. The GC chiral stationary phase was HYDRODEX-β-TBDAc (beta cyclodextrin), on which VX was baseline-resolved. On the chiral HPLC phase CHIRALCEL OD-H the enantiomers of VX were isolated with enantiomeric excess >99.99%. They were characterised by specific optical rotation (±25.8 deg ml dm−1  g−1 at 20 °C and 589 nm) and by determination of cholinesterase inhibition rate constants. For the quantitative chiral detection of VX the enantioresolution was realized on the HPLC chiral phase CHIRAL AGP. A specific procedure was developed to isolate VX from swine blood samples thereby stabilising its enantiomers. The limit of detection was 200 fg per enantiomer on column. The absolute recovery of the overall sample preparation procedure was 75%. After an intravenous and percutaneous administration of a supralethal dose of VX in anesthetised swine (+)-VX and (−)-VX could be quantified up to 720 min.
Keywords: Liquid chromatography; Mass spectrometry; Gas chromatography–mass spectrometry; VX; (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate; Enantiomers; Cyclodextrin; Poisoning; Nerve agents; Organophosphates; Toxicokinetics; Swine; Acetylcholinesterase; Butyrylcholinesterase; Carboxylesterase; Enantiomers excess; Enantioresolution; Stereoselectivity; Inhibition; Elimination;

A simple, direct and accurate method for the determination of concentration and enrichment of free fatty acids (FFAs) in human plasma was developed. The validation and comparison to a conventional method are reported. Three amide derivatives, dimethyl, diethyl and pyrrolidide, were investigated in order to achieve optimal resolution of the individual fatty acids. This method involves the use of dimethylamine/Deoxo-Fluor to derivatize plasma free fatty acids to their dimethylamides. This derivatization method is very mild and efficient, and is selective only towards FFAs so that no separation from a total lipid extract is required. The direct method gave lower concentrations for palmitic acid and stearic acid and increased concentrations for oleic acid and linoleic acid in plasma as compared to methyl ester derivative after thin-layer chromatography. The [13C]palmitate isotope enrichment measured using direct method was significantly higher than that observed with the BF3/MeOH-TLC method. The present method provided accurate and precise measures of concentration as well as enrichment when analyzed with gas chromatography combustion-isotope ratio-mass spectrometry.
Keywords: Dimethylamide; Plasma free fatty acids; Mass spectrometry; Validation; GC–combustion isotope ratio; Enrichment;

Size-exclusion chromatography as a linear transfer system: Purification of human influenza virus as an example by Bernd Kalbfuss; Dietrich Flockerzi; Andreas Seidel-Morgenstern; Udo Reichl (102-112).
Preparative size-exclusion chromatography suffers from low selectivity and productivity. Empirical optimization of operating conditions constitutes a laborious task due to many parameters. Here, a modeling framework based on linear systems theory is presented for predicting the influence of volume overloading. Impulse-responses characterizing system behavior are derived from experimental data by maximum entropy deconvolution. Theoretical derivations are validated experimentally by study of a model system and chromatography of human influenza virus. By application of the theory it is demonstrated how group separation operations can be optimized with respect to yield, purity, productivity and dilution of the product.
Keywords: Size-exclusion chromatography; Group separation; Influenza virus; Linear time-invariant system; Maximum entropy deconvolution;

Separation/enrichment of active natural low content protein using protein imprinted polymer by Ruifang Han; Xiaocui Xing; Ying Wang; Yi Long; Yang Sun; Zhuo Zhao; Huaifeng Mi (113-118).
We describe a new type of protein-imprinted polymer for separation/enrichment of active natural protein present at a relatively low level in cell extracts, with a cloned bacterial protein as template. In this work, cloned pig cyclophilin 18 (pCyP18) was used as template. The template protein was selectively assembled with assistant recognition polymer chains (ARPCs) from their library, which consists of numerous limited length polymer chains with randomly distributed recognition and immobilizing sites. These assemblies of protein and ARPCs were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After removing the template, the synthesized imprinted polymer was used to adsorb authentic pCyP18 from cell extract, and its proportional content was enriched 200 times. The assay of peptidyl-prolyl cistrans-isomerase (PPIase) activity showed that natural pCyP18 is more active than cloned pCyP18 and, in particular, it is much more sensitive to the suppressant cyclosporine A (CsA).
Keywords: Protein-imprinted polymer; Assistant recognition polymer chains (ARPCs); Separation of natural protein; Cyclophilin 18; PPIase activity;

LC–ESI-MS determination and pharmacokinetics of adrafinil in rats by R. Nageswara Rao; Dhananjay D. Shinde; M.V.N. Kumar Talluri; Sachin B. Agawane (119-123).
A highly sensitive and specific liquid chromatography/tandem mass spectrometric (LC–MS/MS) method for investigating the pharmacokinetics of adrafinil in rats was developed. Rat serum pretreated by solid-phase extraction (SPE) was analyzed by LC–MS/MS with an electrospray ionization (ESI) interface. The mobile phase consisted of acetonitrile:water:acetic acid (35:65:0.1, v/v/v) in an isocratic elution mode pumped at 1.0 ml/min. The analytical column (250 mm × 4.6 mm i.d.) was packed with Kromasil C18 material (5.0 μm). The standard curve was linear from 16.5 to 5000 ng/ml. The assay was specific, accurate (R.S.D. < 2.6%), precise and reproducible (within- and between-day precisions R.S.D. <7.0% and <9.0%, respectively). Adrafinil in rat serum was stable over three freeze–thaw cycles at ambient temperature for 6 h. The method had a lower limit of quantitation of 16.5 ng/ml, which offered high sensitivity for the determination of adrafinil in serum. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.
Keywords: Adrafinil; Psychostimulants; Pharmacokinetics; Solid-phase extraction; LC–MS/MS;

A validated high performance liquid chromatographic assay for urinary catecholamines is presented. After addition of 3,4-dihydroxybenzylamine as internal standard (IS) to urine, norepinephrine (NE), epinephrine (E), dopamine (DA) are extracted by ion exchange chromatography and eluted with boric acid. After paired ion separation, quantitation is by electrochemical (coulometric) detection after correction of internal standard recovery. Novel interferences by anti-TB drugs on norepinephrine assay are discussed. A simple method for their removal using alumina is presented.
Keywords: Pheochromocytoma; Catecholamines; Norepinephrine; Rifampicin; Alumina;

Simple and validated HPLC–UV analysis of levetiracetam in deproteinized plasma of patients with epilepsy by Manuela Contin; Susan Mohamed; Fiorenzo Albani; Roberto Riva; Agostino Baruzzi (129-132).
We present a simple, fast and validated method for the determination of the new generation antiepileptic drug (AED) levetiracetam (LEV) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma sample (500 μL) pretreatment was based on simple deproteinization by methanol spiked with the internal standard (I.S.). HPLC analysis was carried out on a Synergi 4-μm Hydro-RP, 150 mm × 4 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5) and acetonitrile (94:6, v/v) at an isocratic flow rate of 1.5 mL/min. The UV detector was set at 205 nm. Calibration curves were linear (mean correlation coefficient = 0.999) over a range of 4–80 μg/mL. The quantitation limit was 2 μg/mL and the absolute recovery was >90% for LEV and the I.S. Both intra and interassay precision and accuracy were lower than 7.5%. The chromatographic run lasted 13 min. The present procedure omitting expensive solid phase or time-consuming liquid–liquid extraction and drying steps is cheaper, faster and simpler than mostly published analytical methods for levetiracetam. Applied to a large population of patients with epilepsy this assay proved very practical in our therapeutic drug monitoring setting (TDM).
Keywords: Levetiracetam; Antiepileptic drugs; High performance liquid chromatography; Deproteinization; UV detection;