Journal of Chromatography B (v.877, #14-15)

Development of a sensitive and selective LC/MS/MS method for the simultaneous determination of intracellular 1-beta-d-arabinofuranosylcytosine triphosphate (araCTP), cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in a human follicular lymphoma cell line by Céline Crauste; Isabelle Lefebvre; Michael Hovaneissian; Jean Yves Puy; Béatrice Roy; Suzanne Peyrottes; Sabine Cohen; Jérôme Guitton; Charles Dumontet; Christian Perigaud (1417-1425).
A method was developed for the quantification of araCTP, CTP and dCTP in a human follicular lymphoma cell line. This method involves solid phase extraction (SPE) using a weak anion-exchanger (WAX) cartridge, a porous graphitic carbon high-performance liquid chromatography (HPLC) column separation, and tandem mass spectrometry (MS/MS) detection. By using a triple quadrupole mass spectrometer operating in negative ion multiple reaction monitoring (MRM) mode, the method was able to achieve a lower limit of quantification (LLOQ) of 0.1 μg mL−1 for araCTP and of 0.01 μg mL−1 for both CTP and dCTP. The method was validated and used to determine the amount of araCTP, CTP and dCTP formed after incubation of araC and an araCMP prodrug in the human follicular lymphoma cell line RL.
Keywords: araCTP; CTP; LC/MS/MS; WAX; Porous graphitic carbon;

Inositols and methylinositols in sea buckthorn (Hippophaë rhamnoides) berries by Heikki Kallio; Marika Lassila; Eila Järvenpää; Gudmundur G. Haraldsson; Sigridur Jonsdottir; Baoru Yang (1426-1432).
Sea buckthorn (Hippophaë rhamnoides L.) berries, especially of ssp. sinensis, contain significant quantities of an unknown, water-soluble compound, evidently a cyclitol derivative. The compound was isolated by HPLC and analyzed by GC–MS [trimethylsilyl (TMS) derivative, selected ion monitoring (SIM) and total ion chromatogram (TIC) analyses], by 1H and 13C NMR and by optical activity measurements. The results together with analyses of reference compound verified the unambiguous structure (−)-2-O-methyl-l-chiro-inositol (l-quebrachitol). In addition, chiro-inositol and myo-inositol existing in trace amounts were identified based on reference compounds, chromatographic data and mass spectra of the TMS derivatives. Methyl-myo-inositol was tentatively identified based on chromatography and mass spectrometry. Inositols and methyl inositols are bioactive compounds essential for regulating physiological processes of plants and humans. To our knowledge, this is the first report on the presence of chiro-inositol and myo-inositol in sea buckthorn and l-quebrachitol in edible berries. The identification of the inositols and l-quebrachitol in sea buckthorn may bring new insights into the sensory properties and also mechanisms behind the health effects of the berry.
Keywords: GC–MS; HPLC; 1H and 13C NMR; Methylinositol; Optical activity measurement; Sea buckthorn berry;

We compared sensitivity and selectivity of five dyes for detection of 2D PAGE-resolved proteins derived from Escherichia coli and MDCK cells. The sensitivity of these dyes was in the following order: SYPRO Ruby > Deep Purple > CBB-G250 > CBB-R250 > Colloidal Gold. Also, we report herein for the first time the application of Colloidal Gold (which is commonly used for staining proteins on blotted membranes) for in-gel staining of proteins. For E. coli, most of the dyes preferably detected proteins with pI range of 4.0–6.9, whereas Deep Purple preferably detected proteins with less acidic range (pI 5.0–7.9). For MDCK cells, while other dyes preferably stained proteins at pI range of 5.0–7.9, Colloidal Gold preferably stained more basic proteins (pI 7.0–9.9). This preferential staining property of Colloidal Gold to basic proteins was confirmed in SDS-PAGE-separated lysozyme (pI 9.4), compared to calmodulin (pI 4.0) and albumin (pI 6.0). These data provide useful information to select appropriate dyes for gel-based proteomic analysis of individual samples.
Keywords: CBB; Colloidal Gold; Deep Purple; Proteome; Proteomics; Staining; SYPRO Ruby;

Affinity ligands for immunoglobulins based on the multicomponent Ugi reaction by Jonathan M. Haigh; Abid Hussain; Michael L. Mimmack; Christopher R. Lowe (1440-1452).
This report describes a novel use of the four-component Ugi reaction to generate a solid-phase library suitable for the purification of immunoglobulins and their fragments by affinity chromatography. An aldehyde-functionalised Sepharose™ solid-support constituted one component in the four-component reaction, whereas the other three components (a carboxylic acid, a primary or secondary amine and an isonitrile) were varied in a combinatorial fashion to generate a tri-substituted peptoidal scaffold structure which provides a degree of rigidity and functionality suitable for rational investigation of immunoglobulin binding. The Ugi ligand library was initially screened chromatographically against whole human IgG and its fragments (Fc and Fab) to yield a Fab-specific lead ligand based on its ability to bind Fab differentially over Fc. Preparative chromatography of IgG from human serum showed 100% of IgG was adsorbed from the 20 mg/ml crude stock and subsequently eluted with a purity of 81.0% as determined by SDS-PAGE analysis under non-optimised conditions. High purity Fab and IgG isolation was achieved from both yeast and E. coli host cell proteins according to silver-stained SDS-PAGE lane densitometry. The ligand density and spacer-arm chemistry of the immobilised ligand was optimised to define an affinity adsorbent which binds 73.06 mg IgG/ml moist gel (dynamic binding capacity at 10% breakthrough) and a static binding capacity of 16.1 ± 0.25 mg Fab/ml moist resin displaying an affinity constant K d  = (2.6 ± 0.3) × 10−6  M. The lead candidate was modelled in silico and docked into a human Fab fragment (PDB: 1AQK) to suggest a putative binding interface to the constant CH1-CL Fab terminal through six defined hydrogen bond interactions together with putative hydrophobic interactions.
Keywords: Solid-phase combinatorial library; Immunoglobulin purification; Affinity ligand design; Multicomponent reaction; Ugi reaction;

In this study, we describe a bioanalytical method for quantification of NTBC in plasma of patients with hereditary tyrosinemia type 1 (HT-1) using high-performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). After protein precipitation with acetonitrile including Mesotrione as internal standard, separation of NTBC was achieved by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in selected reaction monitoring (SRM) mode. NTBC recovery in the developed method was found to be more than 90%. The lower limit of quantification was calculated to be 0.35 μM. The intra-day and inter-day precision of three different quality control samples (measured as RSD%) was less than 10% and 15%, respectively. The standard calibration curves showed good linearity within the range of 2.5–40 μM and the determined correlation coefficients were r 2  ≥ 0.995. This presented method is rapid, sensitive, specific and suitable for clinical practice and research.
Keywords: NTBC; Tyrosinemia type 1; HPLC–ESI-MS/MS; LTQ-Orbitrap;

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate aminoflavone (NSC 686288) in human plasma by Richard Wiegand; Jianmei Wu; Xianyi Sha; Patricia LoRusso; Elisabeth Heath; Jing Li (1460-1464).
A reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for determination of aminoflavone (AF) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 mL of plasma with 1.0 mL ethyl acetate containing 50 ng/mL of the internal standard zileuton. The analytes were separated on a Waters X-Terra™ MS C18 column using a mobile phase consisting of methanol/water containing 0.45% formic acid (70:30, v/v) and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the AF concentration range of 5–2000 ng/mL in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL for AF in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully applied to characterize AF plasma concentration-time profile in the cancer patients in a phase I trial.
Keywords: Aminoflavone; High-performance liquid chromatography; Mass spectrometry; LC–MS/MS; Pharmacokinetics;

A flexible and high throughput liquid chromatography–tandem mass spectrometric assay for the quantitation of telcagepant in human plasma by Lihong Du; Cynthia M. Miller-Stein; Robert J. Valesky; Ashley N. Martucci; Eric J. Woolf (1465-1471).
Telcagepant (MK-0974) is a novel oral calcitonin gene-related peptide (CGRP) receptor antagonist and is currently under clinical development. Results from phases II and III clinical trials have suggested that telcagepant is effective for migraine treatment. A reliable and high throughput protein precipitation (PPT) method for determination of telcagepant in human plasma using liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry has been developed. Clinical samples, internal standard (IS) and acetonitrile are transferred into 96-well plates using a robotic liquid handling system. An aliquot of 10 μL supernatant is directly injected into the LC–MS/MS system where separation is performed on a FluoPhase RP (150 × 2.1 mm, 5 μm) column with an isocratic mobile phase (60% acetonitrile with 0.1% formic acid and 40% water with 0.1% formic acid) at 0.2 mL/min. The interfering 3S-diastereomer of telcagepant, which is observed in clinical samples, is chromatographically resolved from telcagepant. The PPT procedure significantly reduces the time required for sample processing and the assay is sufficiently sensitive for detection using both API 4000 and API 3000 mass spectrometers. The linear calibration range is 5–5000 nM using 200 μL of plasma. Assay intraday validation was conducted using six calibration curves derived from six lots of human control plasma. Calibration standard accuracy did not deviate by more than 3% and 6% of nominal values, and precision did not exceed 4% coefficient of variation (CV) and 10% CV, respectively on the API 4000 and API 3000. Several clinical phases IIb and III studies have been successfully supported with this assay.
Keywords: Telcagepant (MK-0974); Protein precipitation; Human plasma; LC–MS/MS;

A capillary microtrap thermal desorption module is developed for near real-time analysis of volatile organic compounds (VOCs) at sub-ppbv levels in air samples. The device allows the direct injection of the thermally desorbed VOCs into a chromatographic column. It does not use a second cryotrap to focalize the adsorbed compounds before entering the separation column so reducing the formation of artifacts. The connection of the microtrap to a GC–MS allows the quantitative determination of VOCs in less than 40 min with detection limits of between 5 and 10 pptv (25 °C and 760 mmHg), which correspond to 19–43 ng m−3, using sampling volumes of 775 cm3. The microtrap is applied to the analysis of environmental air contamination in different laboratories of our faculty. The results obtained indicate that most volatile compounds are easily diffused through the air and that they also may contaminate the surrounding areas when the habitual safety precautions (e.g., working under fume hoods) are used during the manipulation of solvents. The application of the microtrap to the analysis of VOCs in breath samples suggest that 2,5-dimethylfuran may be a strong indicator of a person's smoking status.
Keywords: Thermal desorption; Breath; Smoking; Biomarker;

Crosslinked-poly(vinylbenzylchloride), poly(VBC), beads were prepared by suspension polymerization and poly(glycidylmethacrylate) was grafted by surface-initiated-atom radical polymerization (SI-ATRP) technique. Epoxy groups of the grafted poly(GMA) were reacted with hydrazine and ammonia to create an affinity binding sites. The hydrazine and amine functionalized poly(VBC-g-GMA) beads were used as an affinity support for adsorption of invertase from solution and yeast crude extract. The influence of pH, equilibrium time, ionic strength and initial invertase concentration on the adsorption capacities of both hydrazine and amine functionalized beads has been investigated. Maximum invertase adsorptions onto hydrazine and amine functionalized beads, were 86.7 and 30.4 mg/g at pH 4.0 and 5.5, respectively. The experimental equilibrium data fitted well to the Temkin isotherm model. Finally, the hydrazine functionalized poly(VBC-g-GMA) beads were used for the purification of invertase from crude yeast extract in a batch system and the purity of the eluted invertase from the hydrazine functionalized beads was determined as 92% by HPLC from single step purification protocol.
Keywords: SI-ATRP; Polymer brushes; Multi-modal ligand; Invertase; Adsorption; Purification;

Analysis of aminoglycoside residues in bovine milk by liquid chromatography electrospray ion trap mass spectrometry after derivatization with phenyl isocyanate by Sherri B. Turnipseed; Susan B. Clark; Christine M. Karbiwnyk; Wendy C. Andersen; Keith E. Miller; Mark R. Madson (1487-1493).
A derivatization procedure using phenyl isocyanate was adapted to liquid chromatography ion trap mass spectrometry (LC–MS n ) for confirmation and quantification of aminoglycoside residues in milk. Aminoglycoside residues were extracted from milk with acid and isolated from the matrix with a weak cation exchange solid-phase extraction cartridge. After isolating the compounds from the milk, derivatives of gentamicin, neomycin, and tobramycin were formed by reacting the drugs with phenyl isocyanate in the presence of triethylamine. The analytes were separated using a dilute formic acid/acetonitrile gradient on a reversed-phase LC column. The derivatized compounds were analyzed using positive ion electrospray LC–MS n with ion trap detection. Product ion spectra were generated from the derivatized protonated molecules. Specific ion transitions were evaluated for quantitative determination and qualitative confirmation of residues in milk. Using this procedure, residues were qualitatively confirmed in milk samples fortified with gentamicin and neomycin at levels ranging from 15 to 300 ng mL−1. Gentamicin has four major components that were successfully separated and confirmed independently; for quantitative determination the peak areas from the four analogs were summed. Tobramycin was added as an internal standard for quantitation to mitigate the effects of matrix ion suppression and variable recoveries. Overall recoveries for this method ranged from 80% to 120% with relative standard deviations of less than 25%. The method detection limits are 9.8 ng mL−1 for NEO and 12.8 ng mL−1 for total GEN residues.
Keywords: Aminoglycosides; Milk; Liquid chromatography–mass spectrometry; Derivatization; Phenyl isocyanate;

A rapid LC–MS/MS method has been developed and validated for the simultaneous identification, confirmation and quantitation of 10 nitroimidazoles in plasma. The method validated in accordance with Commission Decision (CD) 2002/657/EC is capable of analysing for metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ) and ternidazole (TRZ) which are rarely analysed by modern methods. MNZ, DMZ and RNZ have a recommended level (RL) of 3 ng mL−1 which this method is easily able to detect for all the nitroimidazole compounds. Plasma samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid–liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS). The decision limits (CCα) range from 0.5 to 1.6 ng mL−1 and the detection capabilities (CCβ), range from 0.8 to 2.6 ng mL−1. The results of the inter-assay study, which was performed by fortifying bovine plasma samples (n  = 18) on three separate days, show the accuracy calculated for the various analytes range between 101% and 108%. The precision of the method, expressed as CV% values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 ng mL−1), ranged between 4.9% and 15.2%. A day 4 analysis was carried out to examine species variances in animals such as avian, ovine, porcine and equine.
Keywords: Nitroimidazoles; Method validation; Liquid chromatography tandem mass spectrometry; Plasma;

d-Phenylalanine is capable of trapping reactive oxygen species (ROS) and reactive nitrogen species (RNS) by forming three major hydroxylation (o-, m-, p-tyrosine) and two major nitration products (nitrophenylalanine, nitrotyrosine). Here, we show how a method for the analysis of these phenylalanine derivatives was established using isocratic HPLC (Nucleosil120, C18 column) coupled with photodiode array detection and validated for cell-free in vitro and in vivo determination of radical formation. An ideal separation was achieved using a mobile phase consisting of 5% acetonitrile, 50 mM KH2PO4, pH 3.0, a column temperature of 35 °C and a flow rate of 1.0 mL/min. Limits of detection were in the range of 5–100 nM. Linearity was given within 5 nM–100 μM (correlation coefficient >0.999). Retention times as well as peak heights exhibited a high precision (RSD: ≤0.1% and <1.5%, respectively). The feasibility of d-phenylalanine for ROS/RNS measurement was demonstrated in a cell-free in vitro assay using peroxynitrite and by analysis of brain samples of mice treated with the dopaminergic neurotoxin 6-hydroxydopamine.
Keywords: Reactive nitrogen species; Reactive oxygen species; ROS; RNS; Hydroxyl radical; Peroxynitrite; 6-Hydroxydopamine; Phenylalanine; HPLC;

A quantitative HPLC–MS method for the simultaneous determination of testosterone, 11-ketotestosterone and 11-β hydroxyandrostenedione in fish serum by Martín Blasco; Pedro Carriquiriborde; Damián Marino; Alicia E. Ronco; Gustavo M. Somoza (1509-1515).
A simple and novel HPLC–MS method for the simultaneous quantification of testosterone, 11-ketotestosterone, and 11β-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water–acetonitrile mobile-phase with a cycle time of 12 min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2–50 ng/ml), limits of detection (0.1–0.2 ng/ml) and quantification (0.2–0.5 ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA).
Keywords: Androgens; Testosterone; 11-Ketotestosterone; 11β-Hydroxyandrostenedione; Fish serum; HPLC–MS;

The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95.
Keywords: Phage display; Monospecific antibodies; Echinococcus granulosus;

A sensitive and reliable method using gas chromatography-negative chemical ionization mass spectrometry (GC-NCI/MS) was developed for the simultaneous determination of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) at trace levels in muscle and liver. Before extraction with ethyl acetate, CAP-d 5 was added to tissue samples as internal standard. The organic extracts were frozen to remove lipid and further purified by liquid–liquid extraction (LLE) with hexane and solid-phase extraction (SPE) using Oasis HLB cartridges. The target compounds were derivatized with BSTFA + 1% TMCS prior to GC-NCI/MS determination in selected ion monitoring mode (SIM). The recovery values ranged from 78.5 to 105.5%, with relative standard deviations (RSD) <17%. The limits of detections (LODs) of 0.1 μg/kg for CAP and 0.5 μg/kg for TAP, FF, and FFA were obtain. Incurred sample and samples from local market were successfully analyzed using this method.
Keywords: Chloramphenicol; Thiamphenicol; Florfenicol; Florfenicol amine; GC-NCI/MS; Liver; Muscle;

A sensitive and specific electrospray ionization liquid chromatography–tandem mass spectrometry method was developed to detect diosgenin in the plasma of normal and hyperlipidemic rats. Diosgenin was extracted with n-hexane–ethyl acetate (9:1, v/v) using sarsasapogenin as an internal standard. With multiple reaction monitoring modes, linear calibration curves were obtained in the range 10–1500 ng/mL (r  ≥ 0.9979) and the limit of quantification was 10 ng/mL. Intra- and inter-assay variabilities were within 7.74%, and accuracies were between −5.33% and 1.50%. The assay was successfully applied to study pharmacokinetics in rats after oral administration of diosgenin. Significantly different pharmacokinetics between normal and hyperlipidemic rats were observed, which would be beneficial for the clinical use of diosgenin.
Keywords: Pharmacokinetics; Diosgenin; LC–MS/MS; Sarsasapogenin; Hyperlipidemic rat;

The O,N-deacylated derivative (deON) and polysaccharide part (PS) from the lipopolysaccharide (LPS) of Escherichia coli C strain were separated by strongly basic anion-exchange chromatography (SAX) based on the differences in the number of charged phosphate and ethanolamine substituents. They were also successfully separated and characterized by capillary zone electrophoresis and subsequent ESI-ion trap-MS (CZE/ESI-IT-MS). The O-deacylated LPS (deO) presented as a broad peak in CZE/ESI-IT-MS. However, more than twelve species could be discriminated by an extracted ion electropherogram (EIE) and monitoring the species which have different numbers of phosphate and ethanolamine substituents on polysaccharide backbone.
Keywords: Capillary zone electrophoresis; Strongly basic anion-exchange chromatography; Lipopolysaccharide; Escherichia coli C; Mass spectrometry; HPLC post-column fluorescence derivatization;

Evicting hitchhiker antigens from purified antibodies by Keith A. Luhrs; Debra A. Harris; Scott Summers; Missag H. Parseghian (1543-1552).
Antibodies that target common cellular structures may have a propensity to bind those very same antigens as they become exposed in dead or dying cells during production in a bioreactor. Those tendencies can be accentuated if the targeted epitope is highly conserved across species. While attention to contaminants such as endotoxin, viral particles, cellular DNA and even prions has grown coincident with the emergence of the monoclonal antibody industry, it is surprising how little attention has been focused on hitchhiker antigens that may co-elute while bound to the supposedly pure antibody. In this case study, we will focus on anti-histone antibodies and the measures we have taken to eliminate stowaways, such as histone–DNA complexes. These simple measures include the addition of a quartenary amine guard column to the protein A, adjusting the ionic strength of the cell culture supernatant to 400 mM sodium chloride, and establishing a mobile phase gradient from 400 mM to 2 M during protein A chromatography. Initially adjusting the cell culture to 600 mM can compromise the quartenary amine guard column. Also, we demonstrate the applicability of these techniques in both the R&D lab and the manufacturing plant, particularly in improving the apparent potency of antibodies destined for the clinic. Given the prominence of anti-histone antibodies in chromatin immunoprecipitation (ChIP), the implications of hitchhiker antigens interferring with the results of an experiment are far-reaching, indeed, we detect them in some popularly used antibodies. Moreover, a wide variety of monoclonals that may target antigens expressed by the producer cell line may face similar problems, resulting in a decreased production yield, as well as a diminished apparent binding potency.
Keywords: Antibody purification; Commercial-scale purification; Affinity chromatography; Host cell proteins; Hitchhiker antigens;

Fluorapacin, bis(4-fluorobenzyl)trisulfide, a small molecule natural product derivative of trisulfide, has revealed a broad spectrum of anti-proliferative activity and in vivo anti-tumor efficacy in human xenograft mice models with excellent safety profile. In the present study, two new metabolites, para-fluorohippuric acid (p-FHA) and para-fluorobenzoic acid (p-FBA), were identified by GC–MS and HPLC as the main metabolites in urine of rats after intravenous administration of fluorapacin. A simple HPLC-UV method for simultaneous determination of these two metabolites in urine has been developed and validated. The newly developed method demonstrated excellent specificity, accuracy, precision, and stability. This method was successfully employed to study the urinary excretion of fluorapacin in rats. The results indicated that p-FHA was the major metabolite in urine, and the total excretion recovery of p-FHA and p-FBA was 67.6 ± 4.9% (mean ± SE, n  = 6) of dosage after 48 h of administration.
Keywords: Trisulfide; Fluorapacin; Metabolites; Identification; Determination; Urinary excretion;

Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography by Fui Chin Chong; Wen Siang Tan; Dayang Radiah Awang Biak; Tau Chuan Ling; Beng Ti Tey (1561-1567).
Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap™ 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV onto and from Nickel Sepharose™ 6 Fast Flow were investigated. The optimal binding was achieved at pH 7.5, superficial velocity of 1.25 cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500 mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4 mg/ml in a scale-up immobilized metal affinity chromatography (IMAC) packed bed column of Nickel Sepharose™ 6 Fast Flow with the optimized condition obtained from the method scouting. The purification of histidine-tagged N protein using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94.
Keywords: Nucleocapsid protein; Nipah virus; Immobilized metal affinity chromatography; Escherichia coli;

Carbon nanotubes (CNTs) are a kind of new carbon-based nano-materials which have drawn great attention in many application fields. The potential single-walled carbon nanotubes (SWCNTs) as solid-phase microextraction (SPME) adsorbents for the preconcentration of environmental pollutants have been investigated in recent years. The goal of this work was to investigate the feasibility of SWCNTs used as adsorbents for solid-phase microextraction of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) in human urine. SWCNTs were attached onto a stainless steel wire through organic binder. Potential factors affecting the extraction efficiency were optimized, including extraction time, extraction temperature, desorption time, desorption temperature, and salinity. The developed method showed good performance according to the ICH performance criteria for bioanalytical methods. The calibration curves of the ethers were linear (r 2  ≥ 0.992) in the range from 10 to 5000 ng L−1. The limits of detection at a signal-to-noise (S/N) ratio of 3 were 10 ng L−1 for all the analytes. In addition, compared with the commercial carboxen/polydimethylsiloxane (CAR/PDMS) fiber, the SWCNT fiber showed better thermal stability (over 350 °C) and longer life span (over 150 times). The developed method was applied successfully to determine trace level of the ethers in urine of 10 healthy male volunteers.
Keywords: Fiber coating for SPME; Methyl tert-butyl ether; Ethyl tert-butyl ether; tert-Amyl methyl ether; Single-walled carbon nanotubes; Solid-phase microextraction; GC–MS;

The work described in this paper represents an improvement over a previously published method for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human urine by solid phase extraction on a molecularly imprinted polymer column coupled with HPLC and -MS/MS detection. The influence of ion suppression due to sample matrix effect was evaluated, and found to influence the response of NNAL. By changing the liquid chromatography conditions, the response for this method was enhanced approximately 25-fold through avoidance of ionization suppression that was found with a previously published method and sample throughput has been improved. The dynamic range of the assay extends from 20 to 2500 pg/mL with a mean r 2  > 0.998. The lower limit of quantitation for the assay was 20 pg/mL despite the use of an inherently lower sensitivity instrument. The method was validated according to current FDA guidelines for bioanalytical method validations.
Keywords: Tobacco specific nitrosamine; 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNAL; Molecularly imprinted polymer; Matrix effect; LC–MS/MS;

A sensitive and specific LC–MS/MS method for the quantification of the endocannabinoids and related structures anandamide, 2-arachidonoyl glycerol, 2-arachidonyl glycerol ether, O-arachidonoyl ethanolamide, dihomo-γ-linolenoyl ethanolamide, docosatetraenoyl ethanolamide, N-arachidonoyl dopamine, N-arachidonyl glycine, N-oleoyl dopamine, oleoyl ethanolamide, palmitoyl ethanolamide, and stearoyl ethanolamide in human plasma was developed and validated. Compounds were extracted using acetonitrile followed by solid-phase extraction. Separation was performed on a Xterra C8 column using gradient elution coupled to a triple-quadrupole MS. LLOQ levels ranged from 0.02 to 1.75 μg/mL, LODs ranged from 0.0002 to 0.1266 ng/mL, and accuracies were >80% (except stearoyl ethanolamide at lowest spike level) at all spike levels.
Keywords: Endocannabinoids; LC–MS/MS; Sample pre-treatment; Human plasma;

A new analytical method for puerarin using capillary electrophoretic (CE) separation and chemiluminescence (CL) detection has been developed. The detection was based on the enhanced CL intensity of the reaction between luminol and potassium ferricyanide by puerarin in alkaline solution. A laboratory-built CE–CL apparatus was deployed for the puerarin detection. Under the optimal conditions, a linear range from 5.0 × 10−8 to 2.5 × 10−6  M and a detection limit of 1.0 × 10−8  M (S/N = 3) for puerarin were achieved. The determination of puerarin was achieved in less than 5 min, and the proposed method was applied to the determination of puerarin in pharmaceutical, human urine and human plasma samples.
Keywords: Capillary electrophoresis; Chemiluminescence detection; Puerarin; Pharmaceutical; Biological samples;

Analysis of cyclosporin A in dried blood spots using liquid chromatography tandem mass spectrometry by A.J. Wilhelm; J.C.G. den Burger; R.M. Vos; A. Chahbouni; A. Sinjewel (1595-1598).
Dried blood spot sampling is a promising and patient friendly alternative for venous sampling. A liquid chromatography tandem mass spectrometer assay was developed for analyzing cyclosporin A in dried blood spots. Linearity ranged from 25 to 1440 μg/L. Within and between run accuracy and precision were within limits. The developed assay has a negligible matrix-effect and a recovery of 97%. The dried blood spots were stable during a period of at least 17 days in the refrigerator. The developed assay is suitable for analyzing cyclosporin A in dried blood spots.
Keywords: Cyclosporin; Dried blood spot; Fingerprick;

A molecular chromatographic approach to analyze the cell diffusion of arginase inhibitors by Teddy Bagnost; Claire André; Mireille Thomassin; Alain Berthelot; Céline Demougeot; Yves Claude Guillaume (1599-1602).
Our group demonstrated that arginase inhibition reduces endothelial dysfunction 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] which opens perspectives in the development of drugs against hypertension. In previous papers [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; T. Bagnost, Y.C. Guillaume, M. Thomassin, A. Berthelot, C. Demougeot, C. Andre, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 873 (2008) 37], we developed a biochromatographic column for studying the binding of an arginase inhibitor with this enzyme and the effect of magnesium on this binding. In this paper, the interaction of arginase inhibitors with an immobilized artificial membrane (IAM) has been studied using a biochromatographic approach. This IAM provided a biophysical model system to study the inhibitor passive transport across cells. It was demonstrated that more the inhibitor cross the cell membrane by passive diffusion more it is potent. As well, an analysis of the thermodynamics of the interaction of the arginase inhibitors with the IAM showed that van der Waals, hydrogen and ionic bonds were the main forces between the arginase inhibitors and the polar head groups of the IAM surface.
Keywords: Arginase; nor-NOHA; NOHA; BEC; HPLC; IAM;

Estimation of allele frequency in pooled DNA by using PCR–RFLP combined with microchip electrophoresis by Wei-peng Wang; Rui-hong Zhang; Ping Wu; Sen Wang; Rong Li (1603-1606).
Biallelic marker, most commonly single nucleotide polymorphism (SNP), is widely utilized in genetic association analysis, which can be speeded up by estimating allele frequency in pooled DNA instead of individual genotyping. Several methods have shown high accuracy and precision for allele frequency estimation in pools. Here, we explored PCR restriction fragment length polymorphism (PCR–RFLP) combined with microchip electrophoresis as a possible strategy for allele frequency estimation in DNA pools. We have used the commercial available Agilent 2100 microchip electrophoresis analysis system for quantifying the enzymatically digested DNA fragments and the fluorescence intensities to estimate the allele frequencies in the DNA pools. In this study, we have estimated the allele frequencies of five SNPs in a DNA pool composed of 141 previously genotyped health controls and a DNA pool composed of 96 previously genotyped gastric cancer patients with a frequency representation of 10–90% for the variant allele. Our studies show that accurate, quantitative data on allele frequencies, suitable for investigating the association of SNPs with complex disorders, can be estimated from pooled DNA samples by using this assay. This approach, being independent of the number of samples, promises to drastically reduce the labor and cost of genotyping in the initial association analysis.
Keywords: Allele frequency; Pool; SNP; PCR–RFLP; Microchip electrophoresis;

HPLC with pulsed amperometric detection for sorbitol as a biomarker for diabetic neuropathy by Hee-Jung Sim; Ji-Seon Jeong; Ha-Jeong Kwon; Tong Ho Kang; Hye Min Park; Yong-Moon Lee; Sun Yeou Kim; Seon-Pyo Hong (1607-1611).
This study describes the optimal analytical conditions for sorbitol analysis by a high-performance anion-exchange chromatography-pulsed amperometric detection method. Its clinical utility as a diagnostic tool was established by measuring sorbitol in the sciatic nerves or salivary glands of diabetes mellitus-induced mice. Sorbitol was completely separated from other monosaccharides on an anion-exchange column with 100 mM NaOH as eluent. The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were 0.03 ng (3 ng/g) and 0.10 ng (10 ng/g), respectively. The linear dynamic range was 0.01–50.0 μg/g (r 2  = 0.9997 and 0.9989 for sciatic nerves and salivary glands, respectively), and the mean recoveries for intra- or inter-day assays were in the range of 98.5–103.9%. This method easily identified diabetic and normal groups, making it a practical procedure for the rapid screening of diabetic neuropathy.
Keywords: Sorbitol; Diabetic neuropathy; Diabetes mellitus; HPAEC-PAD; Sciatic nerve; Salivary glands;