Journal of Chromatography B (v.911, #C)

► DPC was synthesized and used to develop a highly sensitive CL detection system. ► A CE-CL method was established for highly sensitive assay of EP in biological sample. ► Using this CE-CL method, smoking was found to stimulate the release of EP.Epinephrine (EP) is one of the most important neurotransmitters and hormones. Some previous literatures show that there is a close relation between its release and smoking. To compare the levels of EP in urines of smokers and nonsmokers, a sensitive chemiluminescence (CL) system, luminol-diperiodatocuprate (III) (K5[Cu(HIO6)2], DPC), has been developed and validated for the determination of EP after CE separation. The DPC-luminol-EP CL reaction showed very intensive emission and fast kinetic characteristics, thus led to a high sensitivity in the flow-through detection mode for capillary electrophoresis. With the peak height as a quantitative parameter, the relative CL intensity was linear with the EP concentration in the range of 2.0–400 ng/mL, with a limit of detection of 0.82 ng/mL (S/N = 3). The reproducibility was assessed by intra- and inter-day relative standard deviations (RSDs) for 11 replicate determinations of EP standard samples at low, medium and high concentrations. The intra- and inter-day RSDs for CL signals were 5.5%–6.6% and 6.1%–7.5%, respectively, and those for migration times were 3.4%–5.8% and 4.3%–6.3%, respectively. The presented method was successfully applied to the determination of EP in EP injection and urine samples of smokers and nonsmokers. The recovery test results for urine samples ranged from 86.5 to 112.0%, which demonstrated the reliability of this method. The results for urine sample detection indicate that the average level of EP in the urines of the smoker group is obviously higher than that in the urines of the nonsmoker group, which may demonstrate that smoking can stimulate the release of EP in human body.
Keywords: Epinephrine; Smoker; Diperiodatocuprate; Capillary electrophoresis; Chemiluminescence;

► Developed a simple SDS-assisted sample preparation method for shotgun analysis of membrane proteomes. ► Utilized the strong solubilization ability of SDS for the extraction and solubilization of membrane proteins. ► Optimized KDS precipitation for high efficiency of SDS-removal and peptide recovery. ► The developed method obviously improves the identification of membrane proteins particularly IMPs. ► The developed method is easy to operate at low cost.Analysis of the membrane proteins, particularly the integral membrane proteins, is limited by the inherent membrane hydrophobicity. Sodium dodecyl sulfate (SDS) is one of the most efficient reagents used for the extraction of membrane proteins, but its presence in samples interferes with LC–MS-based proteomic analyses because it affects RP-LC separations and electrospray ionization. In this paper, we present an improved sample preparation strategy based on SDS-assisted digestion and peptide-level SDS-removal using an optimized potassium dodecyl sulfate (KDS) precipitation method (SSDP method) for shotgun analysis of the membrane proteome. This method utilizes a high concentration of SDS (1.0%) to lyse the membranes and to solubilize the hydrophobic membrane proteins, resulting in a more complete protein digestion in the diluted SDS buffer (0.1% SDS), and a high efficiency of SDS removal and peptide recovery by the optimized KDS precipitation for protein identification. The SSDP method provides evidence that proteins can be efficiently digested, and the SDS can be decreased to <0.01% allowing >95% peptide recovery. Compared to other sample preparation methods commonly used in shotgun membrane proteomics, the newly developed method not only increased the identified number of the total proteins, membrane proteins and integral membrane proteins by an average of 33.1%, 37.2% and 40.5%, respectively, but also leading to the identification of highest number of matching peptides. All the results showed that the method yielded better recovery and reliability in the identification of the proteins especially the highly hydrophobic integral membrane proteins, and thus providing a promising tool for the shotgun analysis of membrane proteome.
Keywords: Membrane proteome; Shotgun analysis; SDS-assisted digestion; SDS-removal; Peptide recovery; LC–MS/MS;

► Rapid and simultaneous determination of four total aminothiols levels in plasma. ► RP-HPLC isocratic elution within 6 min of all analytes, including the IS. ► Validated method exhibits an excellent precision and recovery for all aminothiols. ► Method appropriated for high-throughput routine clinical analysis.Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53 mm × 7 mm I.D., 3 μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r 2) ≥0.99. The LOD for the four plasma thiols was lower than 0.10 μmol/L, while LOQ varied from 0.5 to 15 μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6 min. By reducing the total run time, a larger number of analysis can be performed daily.
Keywords: Plasma aminothiols; Homocysteine; Cysteine; Glutathione; Cysteinylglycine; HPLC;

► Rapid and straightforward saliva sample preparation and LC–ESI-MS/MS detection. ► Untargeted analysis detected physiological proteolysis events. ► Extreme proteolytic diversity of salivary alpha-amylase isoforms detected. ► Implications for biomarker discovery, validation and clinical application.Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC–ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva.
Keywords: Saliva; Alpha-amylase; Liquid chromatography; Mass spectrometry; Proteolysis; Biomarker;

► This is the first LC–MS/MS method to determine DHBE regioisomers in human plasma. ► Five DHBE isomers were separated and determined by this method within 16.5 min. ► This method is simple and sensitive and easily reproducible. ► It was applied to a pharmacokinetic study of DHBE regioisomers in humans.Dihydroxybutylether (DHBE), a strong choleretic drug, is a mixture of three regioisomers: 4-(3-hydroxybutoxy)-2-butanol (I), 3-(4-hydroxy-2-butoxy)-1-butanol (II) and 3-(3-hydroxylbutoxy)-1-butanol (III). A liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of dihydroxybutylether (DHBE) regioisomers in human plasma. After plasma samples were deproteinized with 10% perchloric acid, the post-treatment samples were analyzed on a Capcell Pak C18 MGII column interfaced with a triple quadrupole tandem mass spectrometer in positive electrospray ionization mode. Methanol and water was used as the mobile phase with a gradient elution at a flow rate of 1 mL/min. Acetaminophen was used as an internal standard (IS). Multiple selected reaction monitoring was performed using the transitions m/z 163 → 55 and m/z 152 → 110 to quantify DHBE regioisomers and IS, respectively. Five DHBE isomers (a, b, c, d and e) were separated under the present chromatographic condition. The assay was linear over the concentration range of 5.0–200 ng/mL for DHBE isomers a, b and c, and 10.0–400 ng/mL for DHBE isomers d and e. The intra- and inter-day precision was within 13.6% in terms of relative standard deviation (RSD%) and the accuracy within 7.3% in terms of relative error. This simple and sensitive and easily reproducible LC–MS/MS method was successfully applied to the pharmacokinetic study of DHBE regioisomers in healthy male Chinese volunteers after an oral dose of 1.0 g DHBE.
Keywords: Dihydroxybutylether; LC–MS/MS; Plasma; Pharmacokinetics;

► Identified a new metabolite of taxifolin in Caco-2 cells and in rat plasma. ► Prepared the metabolite using Caco-2 cells. ► Evaluated the pharmacokinetic studies of 3′-O-methyltaxifolin in rats.A new metabolite of taxifolin: 3′-O-methyltaxifolin (3′-O-MTAX) in Caco-2 cells and in rat plasma was identified. The chemical structure of 3′-O-MTAX was determined by MS and 1H NMR. A rapid, sensitive and specific UPLC–MS method to determine 3′-O-MTAX in rat plasma was also developed. Following ethyl acetate extraction, 3′-O-MTAX in plasma was separated on a Sunfire™ (2.1 mm × 50 mm, 3.5 μm) column and analyzed in the selected ion recording with a negative electrospray ionization mode using puerarin as the internal standard. The lower limit of quantification (LLOQ) was 2.75 ng/mL. Intra- and inter-day precisions (% RSD) were all within 7.2% and accuracy (% deviation) ranged from −5.0 to 4.7%. The overall recoveries at four concentrations were all >72.0%. This validated method was successfully applied to measure 3′-O-MTAX in rat plasma after oral administration of taxifolin.
Keywords: 3′-O-methyltaxifolin; Taxifolin; UPLC–MS; Pharmacokinetics;

► A novel chimeric protein was expressed in E. coli inclusion bodies. ► A process to purify and refold the protein was developed. ► The protein is effective in blocking the infcetion of PRRSV. ► The protein can be a promising vaccine candidate against PRRSV.Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease currently affecting the swine industry worldwide. In the US alone, it causes economic losses of more than 560 million dollars every year. Although killed-virus and modified-live PRRS vaccines are commercially available, the unsatisfactory efficacy and safety of current vaccines drives the impetus of developing novel PRRSV vaccines. To fulfill this purpose, we designed a chimeric protein consisting of the ectodomains of viral GP5 and M protein, the two most widely studied subunit vaccine targets, and expressed it in E. coli. An optimized purification/refolding process composed of immobilized metal ion affinity chromatography, dialysis refolding and anion exchange chromatography was developed to purify the chimeric protein from the inclusion bodies. This process could recover approximately 12 mg protein/l E. coli broth with near 100% purity and very low endotoxin level. In addition, the purified protein is antigenic, can bind to a cellular receptor for the virus (heparan sulfate), and can block virus infection of susceptible cells. Therefore, the chimeric protein is a promising subunit vaccine candidate against PRRSV.
Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV); Vaccine; Heparan sulfate; Protein refolding; Protein purification;

► We prepared molecularly imprinted monolithic column for the assay of tramadol. ► The column showed highly specific recognition for the template. ► The work was applied for automated analysis of tramadol in urine and plasma.The applicability of an on-line solid phase extraction method using molecularly imprinted monolithic column was developed for the assay of tramadol (TRD) in urine and plasma samples. The monolithic column was prepared by using TRD as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and chloroform as the porogen with in situ molecular imprinting polymerization technique. Various parameters affecting the extraction efficiency of the monolithic column were evaluated. Chromatographic analysis of TRD after on-line clean-up of samples was performed by reversed-phase HPLC on an ACE column with ultraviolet detection at 218 nm. The present work was successfully applied for automated simple analysis of TRD in urine and plasma samples with high recoveries between 90.5–93.1% and 93.3–96.0%, respectively. The results revealed that in concentration up to 500 ng/mL of dextromethorphan (DEX), timolol (TMO) and O-desmethyltramadol (M1), the recoveries were not reduced more than 4.3% and 4.0% for plasma and urine samples, respectively. The limit of detection (S/N = 3) and limit of quantification (S/N = 10) for TRD in urine samples were 0.03 ng/mL and 0.10 ng/mL, and in plasma samples were 0.3 and 1.0 ng/mL, respectively. Inter-column precision of the assays (n  = 3) for urine and plasma samples at the 100 ng/mL TRD level were 4.0% and 4.2%, respectively.
Keywords: Molecularly imprinted polymer; Tramadol; Human Plasma; Monolithic column; Sample clean-up;

► Intermediates of iron-catalyzed dopamine oxidation were identified by IPC–ESI-MS. ► The short-lived intermediate, 6-hydroxydopamine, was detected and quantified. ► Regenerable anion exchange column is used for on-line ion-pairing reagent removal. ► Mechanism and kinetics of complex reactions can be studied with IPC–ESI-MS.Reversed-phase ion-pairing chromatography (RP-IPC) is coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) through an interface comprising a four-way switch valve and an anion exchange column. Regeneration of the anion exchange column can be accomplished on-line by switching the four-way switch valve to interconnect the column to a regeneration solution. Positioning the anion exchange column between the RP-IPC and ESI-MS instruments allows the ion-pairing reagent (IPR) sodium octane sulfonate to be removed. The IPC–ESI-MS method enabled us to separate and detect four intermediates of the Fe(III)-catalyzed dopamine oxidation. In particular, 6-hydroxydopamine, which is short-lived and highly neurotoxic, was detected and quantified. Together with the separation of other intermediates, gaining insight into the mechanism and kinetics of the Fe(III)-catalyzed dopamine oxidation becomes possible.
Keywords: Reversed-phase ion-pairing chromatography; Electrospray ionization-mass spectrometry; Dopamine oxidation; 6-Hydroxydopamine; Short-lived intermediates;

Determination of tolperisone in human plasma by liquid chromatography/tandem mass spectrometry for clinical application by Chang-Ik Choi; Jung-In Park; Hye-In Lee; Yun-Jeong Lee; Choon-Gon Jang; Jung-Woo Bae; Seok-Yong Lee (59-63).
► A simple, rapid, and sensitive LC–MS/MS method for human plasma tolperisone was developed. ► The lower limit of quantification using 200 μL of human plasma was 0.5 ng/mL. ► This method allows for minimal sample processing. ► The developed and validated method has been successfully applied to a pharmacokinetic study in humans.We have developed and validated a simple, rapid, and sensitive liquid chromatography analytical method employing tandem mass spectrometry (LC–MS/MS) for the determination of tolperisone, a centrally acting muscle relaxant, in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of tolperisone was performed using a reversed-phase Luna C18 column (2.0 mm × 50 mm, 5 μm particles) with a mobile phase of 10 mM ammonium formate buffer (pH 3.5) – methanol (12:88, v/v) and quantified by tandem mass detection in ESI positive ion mode. The flow rate of the mobile phase was 250 μL/min and the retention times of tolperisone and the internal standard (IS, dibucaine) were both 0.6 min. The calibration curves were linear over a range of 0.5–300 ng/mL (r  > 0.999). The lower limit of quantification, using 200 μL human plasma, was 0.5 ng/mL. The mean accuracy and precision for intra- and inter-day validation of tolperisone were within acceptable limits. The LC–MS/MS method reported here showed improved sensitivity for quantification of tolperisone in human plasma compared with previously described analytical methods. Lastly, the validated method was successfully applied to a pharmacokinetic study in humans.
Keywords: Tolperisone; LC–MS/MS; Human plasma; Pharmacokinetics;

Automated multi-step purification protocol for Angiotensin-I-Converting-Enzyme (ACE) by Thomas Eisele; Timo Stressler; Bertolt Kranz; Lutz Fischer (64-70).
► Detergent free solubilisation of Angiotensin-I-Converting-Enzyme (ACE). ► Development of an automated multi-step purification protocol for ACE. ► ACE purification about 300-fold to a specific activity of 37 U mg−1 in less than 8 h. ► Development of a novel activity staining protocol for ACE. ► The automated protein purification protocol can be easily applied to other proteins.Highly purified proteins are essential for the investigation of the functional and biochemical properties of proteins. The purification of a protein requires several steps, which are often time-consuming. In our study, the Angiotensin-I-Converting-Enzyme (ACE; EC 3.4.15.1) was solubilised from pig lung without additional detergents, which are commonly used, under mild alkaline conditions in a Tris–HCl buffer (50 mM, pH 9.0) for 48 h. An automation of the ACE purification was performed using a multi-step protocol in less than 8 h, resulting in a purified protein with a specific activity of 37 U mg−1 (purification factor 308) and a yield of 23.6%. The automated ACE purification used an ordinary fast-protein-liquid-chromatography (FPLC) system equipped with two additional switching valves. These switching valves were needed for the buffer stream inversion and for the connection of the Superloop™ used for the protein parking. Automated ACE purification was performed using four combined chromatography steps, including two desalting procedures. The purification methods contained two hydrophobic interaction chromatography steps, a Cibacron 3FG-A chromatography step and a strong anion exchange chromatography step. The purified ACE was characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE. The estimated monomer size of the purified glycosylated ACE was determined to be ∼175 kDa by SDS-PAGE, with the dimeric form at ∼330 kDa as characterised by a native PAGE using a novel activity staining protocol. For the activity staining, the tripeptide l-Phe-Gly-Gly was used as the substrate. The ACE cleaved the dipeptide Gly-Gly, releasing the l-Phe to be oxidised with l-amino acid oxidase. Combined with peroxidase and o-dianisidine, the generated H2O2 stained a brown coloured band. This automated purification protocol can be easily adapted to be used with other protein purification tasks.
Keywords: Automated multi-step purification; Angiotensin-I-Converting-Enzyme (ACE); Solubilisation of ACE; Activity staining of ACE;

The experimental study of Astragalus membranaceus on meridian tropsim: The distribution study of astragaloside IV in rat tissues by Yan-xu chang; Yu-gang Sun; Jin Li; Qiu-Hong Zhang; Xin-Rong Guo; Bo-li Zhang; Hua Jin; Xiu-mei gao (71-75).
► A LC-ESI-MS/MS method was developed and validated for astragaloside IV in heart, liver, spleen, lung and kidney. ► The method was sensitive enough for distribution study of astragaloside IV in rat tissues. ► Liver, kidney, lung, heart and spleen were detected to contain astragaloside IV. ► Astragaloside IV was one of the material bases of the meridian tropism of Huangqi.According to Traditional Chinese Medicine (TCM) theories, TCM with different meridian tropism have different therapeutic effects. In view of the meridian tropism of Astragalus membranaceus (Huangqi), astragaloside IV, one of the effective phytochemicals of Huangqi, was appointed and observed its distribution in rat tissues following a single intravenous (i.v.) dose. A simple and accurate LC-ESI-MS/MS method was developed and validated for astragaloside IV quantification in heart, liver, spleen, lung and kidney using warfarin as an internal standard (IS). Chromatographic separation was performed on a Eclipse plus C18 (4.6 mm × 100 mm, 1.8 μm) when the flow rate was set at 0.300 mL min−1 and ammonium acetate aqueous solution - acetonitrile was used as mobile phase. The intra- and inter-day precisions of the quality control samples were within 15% and accuracies were within 90.0–110%. The recoveries were more than 90.0% at high, medium and low concentrations, respectively. This method was successfully applied for distribution of astragaloside IV after intravenous (i.v.) dose of 4 mg kg−1 astragaloside IV in rats. Astragaloside IV concentration was highest in liver and kidney and remained much higher than that in other tissues over the experiment course. Lung, heart and spleen were also detected to contain astragaloside IV. The results clearly demonstrated that astragaloside IV was one of the material bases of the meridian tropism of Huangqi.
Keywords: LC–MS/MS; Astragaloside IV; Meridian tropism; Astragalus membranaceus;

Development and validation of a HPLC method for the assay of dapivirine in cell-based and tissue permeability experiments by José das Neves; Bruno Sarmento; Mansoor Amiji; Maria Fernanda Bahia (76-83).
► A HPLC-UV method was developed for assaying dapivirine in biological matrices. ► Box-Behnken experimental design was successfully used to optimize the method. ► Validation according to FDA and EMA guidelines on bioanalytical assays was performed. ► Method was applied to cell-based/mucosal tissues uptake and permeability assays.Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is being currently used for the development of potential anti-HIV microbicide formulations and delivery systems. A new high-performance liquid chromatography (HPLC) method with UV detection was developed for the assay of this drug in different biological matrices, namely cell lysates, receptor media from permeability experiments and homogenates of mucosal tissues. The method used a reversed-phase C18 column with a mobile phase composed of trifluoroacetic acid solution (0.1%, v/v) and acetonitrile in a gradient mode. Injection volume was 50 μL and the flow rate 1 mL/min. The total run time was 12 min and UV detection was performed at 290 nm for dapivirine and the internal standard (IS) diphenylamine. A Box-Behnken experimental design was used to study different experimental variables of the method, namely the ratio of the mobile phase components and the gradient time, and their influence in responses such as the retention factor, tailing factor, and theoretical plates for dapivirine and the IS, as well as the peak resolution between both compounds. The optimized method was further validated and its usefulness assessed for in vitro and ex vivo experiments using dapivirine or dapivirine-loaded nanoparticles. The method showed to be selective, linear, accurate and precise in the range of 0.02–1.5 μg/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02 μg/mL), limit of detection (0.006 μg/mL), recovery (equal or higher than 90.7%), and sample stability at different storage conditions, were also determined and found adequate for the intended purposes. The method was successfully used for cell uptake assays and permeability studies across cell monolayers and pig genital mucosal tissues. Overall, the proposed method provides a simple, versatile and reliable way for studying the behavior of dapivirine in different biological matrices and assessing its potential as an anti-HIV microbicide drug.
Keywords: Microbicides; Liquid chromatography; Box-Behnken design; Method validation; Polymeric nanoparticles; Diphenylamine;

Ion mobility spectrometry for detection of skin volatiles by Veronika Ruzsanyi; Pawel Mochalski; Alex Schmid; Helmut Wiesenhofer; Martin Klieber; Hartmann Hinterhuber; Anton Amann (84-92).
► Fast and direct method is proposed for monitoring of trace VOCs emitted by human skin.Volatile organic compounds (VOCs) released by humans through their skin were investigated in near real time using ion mobility spectrometry after gas chromatographic separation with a short multi-capillary column. VOCs typically found in a small nitrogen flow covering the skin are 3-methyl-2-butenal, 6-methylhept-5-en-2-one, sec-butyl acetate, benzaldehyde, octanal, 2-ethylhexanol, nonanal and decanal at volume fractions in the low part per billion-(ppb) range. The technique presented here may contribute to elucidating some physiological processes occurring in the human skin.
Keywords: Volatile organic compounds (VOC); Ion mobility spectrometry (IMS); Multi-capillary column (MCC); Gas chromatography–mass spectrometry (GC–MS); Skin emissions;

► Two LC approaches for analysis of therapeutic monoclonal antibodies are compared. ► ZIC-HILIC enables identification of major glycan species using fluorescence or ESI-MS detection. ► Porous graphitized carbon improves sensitivity, providing information on minor species. ► Fluorescence-based approaches offer simpler data analysis for routine glycan profiling. ► MS-based approaches provide detailed glycosylation analysis of complex glycoprotein samples.Two LC approaches for analysis of therapeutic monoclonal antibodies (MAbs) are presented and compared. In the first approach, zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) of 2-aminobenzamide-labelled glycans was coupled with fluorescence or electrospray ionisation mass spectrometric (ESI-MS) detection. The ZIC-HILIC method enabled relative quantification and identification of major glycan species. The sensitivity of fluorescence detection was higher compared to ESI-MS; however, MS detection enabled identification of co-eluted peaks. The new ZIC-HILIC approach was compared with porous graphitized carbon (PGC) separation of reduced glycans coupled with ESI-MS. Using PGC higher sensitivity was achieved compared to ZIC-HILIC due to the lower chemical background originating from the mobile phase and the derivatisation step, providing detailed information on minor glycan species. Furthermore, PGC exhibited excellent capability for separation of isobaric glycans with various degrees of mannosylation and galactosylation. The structures of glycans from MAbs used in this study were confirmed by exoglycosidase digestions. The two methods were applied to two monoclonal antibodies expressed in Chinese Hamster ovary cell lines and a monoclonal antibody expressed in a murine NS0 cell line. While the fluorescence-based approach is more suitable for routine glycan profiling due to the simplicity of data analysis, MS-based approaches were shown to provide detailed glycosylation analysis of complex glycoprotein samples.
Keywords: Monoclonal antibodies; Glycans; 2-Aminobenzamide; Porous graphitized carbon; ZIC-HILIC; ESI-MS; Fluorescence;

HPLC–MS/MS method for the simultaneous determination of clopidogrel, its carboxylic acid metabolite and derivatized isomers of thiol metabolite in clinical samples by Marta Karaźniewicz-Łada; Dorota Danielak; Artur Teżyk; Czesław Żaba; Gilles Tuffal; Franciszek Główka (105-112).
► The method allowed to determine clopidogrel and its metabolites in one analytical run. ► The method fulfilled the validation criteria designed for bioanalytical methods. ► Plasma levels of clopidogrel and its metabolites were determined in patients. ► Pharmacokinetic parameters were calculated after a dose of 75 mg clopidogrel.A fast and reproducible HPLC–MS/MS method was developed for the simultaneous determination of clopidogrel (CLP), its carboxylic acid derivative (CLPM), derivatized thiol metabolite isomers MP-H3 and the active MP-H4 in incurred human plasma. CLP, CLPM, MP-H3 and MP-H4 isomers together with the internal standard piroxicam were extracted from plasma samples using a simple protein precipitation with acetonitrile. The analytes were separated on HPLC Zorbax Plus C18 column via gradient elution with water and acetonitrile, both containing 0.1% (v/v) formic acid. Detection of the analytes were performed on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. Calibration curves of the analytes prepared in 250 μL plasma were found to be linear in ranges: 0.25–5.00 ng/mL for CLP, 0.25–50.00 ng/mL for MP-H3 and MP-H4 isomers and 50–10,000 ng/mL for CLPM. The lower limit of quantitation was 0.25 ng/mL for CLP, MP-H3, MP-H4 and 50.00 ng/mL for CLPM. Intra- and inter-assay precision, expressed as relative standard deviation, was ≤18.1% for CLP, ≤15.2% for CLPM, ≤10.1% for MP-H3 and ≤19.9% for MP-H4. Intra- and inter-day accuracy of the method, expressed as relative error, was ≤16%. The analytes were stable in samples stored for 6 h in autosampler, in plasma samples for 24 h at room temperature and for 3 months at −25 °C. Resolution of CLP, CLPM and MP-H3 and MP-H4 isomers of thiol metabolite during one analytical run was reported in patient plasma. The HPLC–MS/MS method was applied for pharmacokinetic studies of CLP and its metabolites in patients treated with daily dose of 75 mg CLP.
Keywords: Clopidogrel; Thiol metabolite active isomer; HPLC–MS/MS resolution; Pharmacokinetics; Validation;

LC–MS/MS for the simultaneous analysis of arachidonic acid and 32 related metabolites in human plasma: Basal plasma concentrations and aspirin-induced changes of eicosanoids by Dhananjay D. Shinde; Kwon-Bok Kim; Kyung-Suk Oh; Nagi Abdalla; Kwang-Hyeon Liu; Soo Kyung Bae; Ji-Hong Shon; Ho-Sook Kim; Dong-Hyun Kim; Jae Gook Shin (113-121).
► We developed simultaneous quantitation tool of 32 eicosanoids in human plasma. ► High recovery of all analytes was achieved by solid phase extraction. ► LC/MS/MS method afforded high sensitivity to detect pg level of eicosanoids. ► This method is suitable for eicosanoid profiling in human plasma.Eicosanoids play an important role in various biological responses and can be used as biomarkers for specific diseases. Therefore, we developed a highly selective, sensitive, and robust liquid chromatography–tandem mass spectrometric method to measure arachidonic acid and its 32 metabolites in human plasma. Sample preparation involved solid phase extraction, which efficiently removed sources of interference present in human plasma. Chromatographic separation was performed using a Luna C8-column with 0.5 mM ammonium formate buffer and acetonitrile as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in negative ion mode. The matrix did not affect the reproducibility and reliability of the assay. All analytes showed good linearity over the investigated concentration range (r  > 0.997). The validated lower limit of quantitation for the analytes ranged from 10 to 400 pg/mL. Intra- and inter-day precision (RDS%) over the concentration ranges for all eicosanoids were within 16.8%, and accuracy ranged between 88.1 and 108.2%. This assay was suitable for the determination of basal plasma levels of eicosanoids and the evaluation of effect of aspirin on eicosanoid plasma levels in healthy subjects.
Keywords: Arachidonic acid; Eicosanoids; Aspirin; Liquid chromatography–mass spectrometry;

► A fast method was developed to identify the metabolites of benzbromarone in rats. ► Seventeen metabolites were obtained. ► Two phase I metabolites and four phase II metabolites were rarely reported before. ► The work gave us a new insight into the metabolic fate of benzbromarone in vivo. ► More information about the cleavage rules of compounds by ESI-QTOF was collected.A high performance liquid chromatography–quadrupole time of flight mass spectrometry (HPLC–QTOF-MS) method was employed in investigation of benzbromarone metabolites in rat plasma, urine, feces and bile samples. Meanwhile, the metabolic pathways of benzbromarone in rats were discussed. The identification was achieved on a reversed-phase C18 column with mobile phase gradient method. The QTOF-MS was operated under full scan of MS or MS/MS in negative mode. The fragments were acquired by raising collision induced dissociation (CID) energy for speculating the structures of parent ions. According to the information from the chromatograms and mass spectra, 17 metabolites were obtained. Among them, the deoxidized phase I metabolites and an array of phase II metabolites—sulfate conjugates detected in the biological samples made the work more significant.
Keywords: Benzbromarone; Metabolite; HPLC–QTOF-MS;

► Simple and low-cost method for cefazolin in serum suitable for labs with basic HPLC. ► Satisfactorily validated fulfilling new EMA guidelines (precision, accuracy, range). ► Stability (freeze–thaw, short- and long-term) tests proved repeatability and performance. ► The procedure was found resistant to potential human errors during clinical usage.The paper presents an HPLC method for cefazolin determination in human serum. The preparation step was based on serum protein precipitation with acetonitrile followed by supernatant evaporation and sample reconstitution in water before injection. The separation of cefazolin and internal standard cefamandole was performed at ambient temperature under isocratic conditions on LiChrosorb RP8-5 column (250 mm × 4.6 mm) using the mixture: CH3CN:H2O:0.5 M KH2PO4 (100:894:6, v/v) as a mobile phase with a flow rate of 1.5 mL/min. UV detection was performed at 272 nm with LLOQ of 0.2 μg/mL. The precision was satisfactory in the whole range tested with RSD of 2.3–12.5% (accuracy: from −2.3% to +3.6%) and of 1.7–7.1% (accuracy: from −3.5% to +1.1%) for intra- and inter-assay, respectively. The method stability was confirmed in a series of experiments including: freeze–thaw and short- and long-term stability testing. Finally, the procedure described was found resistant to potential human errors.
Keywords: HPLC; Cefazolin; Validation; Stability;

► Drug-facilitated crime (DFC) is a significant problem in China; the majority of these cases are robberies. ► Recently, UPLC–MS/MS has been shown to be one of the most promising developments in the area of fast chromatographic separations. ► This method allowed for the rapid and accurate determination of zolpidem and its metabolites in human blood and urine and may be applied to forensic toxicological analyses involving zolpidem.Zolpidem (ZPD) is an imidazopyridine derivative used as a new type of hypnotic and is commonly used in drug-facilitated crimes. A rapid, sensitive, and specific ultra-performance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) assay method for the simultaneous determination of zolpidem and its main metabolites zolpidem 6-carboxylic acid (ZCA) and zolpidem phenyl-4-carboxylic acid (ZPCA) in biological fluids was developed and validated. Aliquots of 0.1 mL blood or urine specimens were used for the analysis, and zolpidem and its metabolites were extracted in a single step using acetonitrile (containing 0.1% formic acid) precipitation. The supernatant was then dried, and 100 μL methanol was added. The separation was performed on an Acquity™ UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm) analytical column by API 4000Q ultra-performance liquid chromatography–tandem mass spectrometry. Positive ionisation tandem MS detection in the multiple reaction monitoring (MRM) mode was used. The mobile phases consisted of either acetonitrile or water containing 20 mmol/L ammonium acetate and 0.1% formic acid, and the flow rate was 0.5 mL/min. The chromatographic separation time was 4 min, and calibration curves for human blood were generated over the range of 0.1–300 ng/mL for ZPD, 0.1–500 ng/mL for ZPCA and 0.1–200 ng/mL for ZCA. For urine, the linear range was 0.1–600 ng/mL for ZPD and ZPCA, and 0.1–300 ng/mL for ZCA. The limit of detection was 0.05 ng/mL and the limit of quantitation was 0.1 ng/mL for ZPD, ZCA and ZPCA. The linear correlation coefficients were greater than 0.9995. Both the inter-day and intra-day precisions were less than 15%, the recoveries were in the range of 70.0–98.3%, the matrix effects were approximately 79.5–99.0%, and the process efficiency was between 60.7% and 94.4%. This method allowed for the determination of zolpidem and its metabolites in human blood and urine and may be applied to forensic toxicological analyses.
Keywords: Zolpidem; Zolpidem 6-carboxylic acid; Zolpidem phenyl-4-carboxylic acid; Precipitation; UPLC–MS/MS;

► LC–MS/MS method for measurement of stable isotope enrichment of l-[2H5]-phenylalanine. ► Method is suited for investigations of nutritive effects on tissue protein synthesis. ► No need for derivatization.Stable isotope labeled amino acids are frequently used to examine nutritive effects on protein synthesis. This technique is characterized by tracing the incorporation of the label into newly synthesized proteins. In the present investigation, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the determination of very low enrichment of protein-bound l-[2H5]-phenylalanine ([2H5]-phe) in chicken liver. The LC–MS/MS measurements were carried out in positive atmospheric pressure chemical ionization (APCI) mode. Two mass transitions each for [2H5]-phe (171.1/125.1 and 171.1/106.1) and l-phenylalanine (phe) (166.1/91.1 and 166.1/93.1) were chosen for quantification and qualification. Due to the high excesses of phe, less sensitive transitions were chosen in the case of phe. The separation was carried out on a phenyl-hexyl column using 0.1% formic acid as eluent A and methanol as eluent B. The method was calibrated with calibration standard solutions in the range of 0.01–0.5 mole percent excess (MPE). Linear regression analysis led to coefficients of determination (r 2) greater than 0.9995. The method was applied on liver samples from experiments investigating nutritive effects on tissue protein synthesis in broiler chickens. These samples were analyzed with a gas chromatography–mass spectrometry (GC–MS) method and reanalyzed with the developed LC–MS/MS method one year later. Compared to GC–MS, the main advantages of the LC–MS/MS method are its higher selectivity as well as the elimination of the need to convert and derivatize the samples prior to measuring.
Keywords: Stable isotope labeled phenylalanine; Phenylalanine; LC–MS/MS; Mole percent excess; Liver;

► Nitrite and nitrate anions are easily determined using an HPLC-UV/VIS method. ► Nitrite is determined by pre-column derivatization using Griess reaction. ► Nitrate is simultaneously determined in UV domain. ► Limits of quantification are of ppb level for nitrite and hundreds of ppb for nitrate. ► New findings suggest that Griess reaction is not absolutely specific to nitrite.Measurements of nitrite and nitrate are used in biomedical research to estimate the endogenous formation of nitric oxide (an important biomolecule). These anions are also toxins and their concentration is regulated in certain foodstuffs. There are many published methods for detecting nitrite and nitrate but most of them fail to detect nitrite in biological samples. A new HPLC-UV/VIS method was developed which easily detects low concentrations of nitrite and nitrate present in mammal blood, urine and in vegetal samples. The method is based on a pre-column derivatization of nitrite anion using the Griess reaction and direct determination of nitrate using its UV absorbance. A chromatographic process with detection at two wavelengths allows the determination of both anions in one run (23 min with column reequilibration included). The limits of quantification in mammal blood are 2 ng/ml and 200 ng/ml for nitrite and nitrate, respectively. As regards vegetables, due to the need of sample dilution in the preparation steps, these limits are 3 times higher. Concentrations measured in rabbit blood samples ranged from 1.09 to 42.65 μg/ml for nitrate and 15.8 to 384.6 ng/ml for nitrite. Concentrations in vegetables ranged from below the limit of detection to 4 g/kg for nitrate and from below the limit of detection to 369.2 μg/kg for nitrite. The specificity of Griess reaction toward nitrite is under discussion since substances able to mimic this reaction were found, leading to compounds with spectral properties in visible domain indistinguishable from that of nitrite related azo dye.
Keywords: Nitrite; Nitrate; HPLC; Griess reaction; Blood; Vegetables;

► A sensitive nucleotide pyrophosphatase (NPP) assay was developed. ► p-Nitrophenyl 5′-thymidine monophosphate was used as an artificial substrate. ► A CE-VIS method coupled with dynamically polybrene-coated capillaries was used. ► A combination of phosphate–HEPES buffer notably increased the method sensitivity. ► The obtained LOD and LOQ for p-nitrophenolate are in the nanomolar range.A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20 cm effective length × 75.5 μm (id)), electrokinetic injection for 60 s, 70 mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of −80 μA, constant capillary temperature of 15 °C and detection at 400 nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415 nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (K m values for NPP1 and NPP3, K i values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.
Keywords: AOPCP; Benzoylbenzoyl-ATP; Capillary electrophoresis; Dial-ADP; p-Nitrophenyl 5′-thymidine monophosphate; NPP inhibitors; NPP substrates; Nucleotide pyrophosphatase; Polybrene;

Validation of an LC–MS/MS method for the quantification of choline-related compounds and phospholipids in foods and tissues by Yeping Xiong; Yuan-Yuan Zhao; Sue Goruk; Kirsten Oilund; Catherine J. Field; René L. Jacobs; Jonathan M. Curtis (170-179).
► Quantification of 14 choline-related compounds or phospholipid classes is achieved. ► The validated method uses a single hydrophilic interaction (HILIC) LC–MS/MS run. ► Choline, betaine and phospholipid classes are extracted by a single phase solvent. ► Phospholipid quantification is consistent with 31P NMR data. ► Total choline measurements in foods consistent with USDA choline database.A hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC LC–MS/MS) method was developed and validated to simultaneously quantify six aqueous choline-related compounds and eight major phospholipids classes in a single run. HILIC chromatography was coupled to positive ion electrospray mass spectrometry. A combination of multiple scan modes including precursor ion scan, neutral loss scan and multiple reaction monitoring was optimized for the determination of each compound or class in a single LC/MS run. This work developed a simplified extraction scheme in which both free choline and related compounds along with phospholipids were extracted into a homogenized phase using chloroform/methanol/water (1:2:0.8) and diluted into methanol for the analysis of target compounds in a variety of sample matrices. The analyte recoveries were evaluated by spiking tissues and food samples with two isotope-labeled internal standards, PC-d3 and Cho-d3. Recoveries of between 90% and 115% were obtained by spiking a range of sample matrices with authentic standards containing all 14 of the target analytes. The precision of the analysis ranged from 1.6% to 13%. Accuracy and precision was comparable to that obtained by quantification of selected phospholipid classes using 31P NMR. A variety of sample matrices including egg yolks, human diets and animal tissues were analyzed using the validated method. The measurements of total choline in selected foods were found to be in good agreement with values obtained from the USDA choline database.
Keywords: Hydrophilic interaction chromatography (HILIC); LC–MS/MS; Method validation; Choline; Phospholipids; Egg yolk;

► Using of a high-sensitivity cell/ACE for the estimation of precise high binding constants. ► Investigating simultaneously of retinol and retinoic acid in nanomolars with HSA and BSA. ► Decreasing the adsorption of protein by reducing the required additives concentrations. ► Using of mobility ratio based on nonlinear regression to deduce high binding constants. ► Studying the competitive binding between retinol and retinoic acid on HSA and BSA.Retinol and retinoic acid are Vitamin A components that are critical for many biological processes. Both of them are strongly complexing with serum albumins giving constants of the order of 105  L mol−1 or higher. With respect to this fact, affinity capillary electrophoresis (ACE) is not applicable in its commonly used form. Therefore, for the first time, the hyphenated ACE with a high-sensitivity cell was developed and employed to investigate the binding of retinol and retinoic acid in nanomolars with human serum albumin (HSA) and bovine serum albumin (BSA) under physiological conditions. ACE/high-sensitivity coupled cell had contributed to fast the association and dissociation rates of the complexes in nanomolar scale of analytes ensuring the establishment of a dynamic equilibrium within a short electrophoresis time. In addition, this hyphenation led to reduce the concentrations of serum albumins as additives in background electrolyte making a sense beside the proper rinsing protocol for the negligible possibility of their adsorption. The mobility ratio based on nonlinear regression analysis was used to deduce precise binding constants of analytes with serum albumins. The binding constants (K, L mol−1) of retinol were 1.28 × 105 and 5.25 × 106 and retinoic acid were 3.29 × 105 and 2.27 × 106 with HSA and BSA, respectively. The displacement and reciprocal competitive binding of analytes were investigated and indicated that retinoic acid was able to replace retinol from HSA and vice versa in the case of BSA.
Keywords: ACE; High sensitivity cell; Retinol; Retinoic acid; HSA; BSA;

A liquid chromatography mass spectrometry method for the measurement of cystathionine β-synthase activity in cell extracts by Desirée E.C. Smith; Marisa I.S. Mendes; Leo A.J. Kluijtmans; Mirian C.H. Janssen; Yvo M. Smulders; Henk J. Blom (186-191).
► A straightforward LC–MS/MS method for the determination of CBS enzyme activity. ► The method can be applied to different cell types. ► The method can be used for diagnosis of cystathionine β-synthase deficiency. ► The method can be used to study effects of treatment of homocystinuria. ► The method can be used to study effects of diet on mild hyperhomocystenemia. Background: In order to correctly assess the efficacy of therapy or diet in intervention studies on the activity of cystathionine β-synthase (CBS) a sensitive analytical method is necessary. Methods: An electrospray LC–MS/MS method preceded by a solid phase extraction step was developed for the measurement of CBS activity in cell extracts. Nonafluoropentanoic acid was used as an ionpair to provide the underivatized cystathionine the desired retention on a C18 column. Results: A detection limit of 50 pmol cystathionine/h/mg protein was achieved. In fibroblasts, intra- and inter-assay CVs for the CBS activity were 5.2% and 14.7%, respectively. A K m value of 8 μmol/L for homocysteine, and 2.5 μmol/L for serine was calculated. In fibroblasts wildtype, heterozygous, and homozygous CBS activity ranges measured were 8.5–27.0, 4.2–13.4, 0.0–0.7 nmol/h × mg protein, respectively. The method was applied to a study where rats were fed 2 diets. Increase of dietary methionine (7.7 versus 3.8 mg/kg methionine) significantly increased the CBS activity in rat liver lysates from a median of 58.0 to a median of 71.5 (P  = 0.037) nmol/h × mg protein. In a lymphoblasts cell culture experiment, the addition of Hcy to the culture media increased the activity of CBS 3 fold. Conclusion: This LC–MS/MS is able to diagnose CBS deficiency at the enzyme level, and can accurately measure the effect diets or therapy might have on the CBS activity in a variety of cell types.
Keywords: Cystathionine β-synthase; Homocystinuria; Liquid chromatography; Tandem mass spectrometry;

Comparison of different linear calibration approaches for LC–MS bioanalysis by Aimin Tan; Kayode Awaiye; Besy Jose; Paresh Joshi; Fethi Trabelsi (192-202).
► Two-concentration linear calibration is better than multi-concentration one. ► One curve may have more failed runs and higher variation at low end than two curves. ► A new in-between approach is proposed to take advantage of the two common ones. ► Maximum tolerable quadratic behaviour in linear calibration has been determined. ► Two-concentration linear calibration has potential use in regulated bioanalysis.Many different calibration approaches are used for linear calibration in LC–MS bioanalysis, such as different numbers of concentration levels and replicates. However, direct comparison of these approaches is rare, particularly using experimental results. The purpose of this research is to compare different linear calibration approaches (existing and new ones) through simulations and experiments. Both simulation and experimental results demonstrate that linear calibration using two concentrations (two true concentrations, not forced through zero) is as good as or even better than that using multiple concentrations (e.g. 8 or 10) in terms of accuracy. Additionally, two-concentration calibration not only significantly saves time and cost, but is also more robust. Furthermore, it has been demonstrated that the extrapolation of a linear curve at the high concentration end to a linearity-known region is acceptable. When multi-concentration calibration is used, the difference between the two commonly used approaches, i.e. singlet (one curve) or duplicate (two curves) standards per concentration level is small when a method is very precise. Otherwise, one curve approach can result in larger variation at the low concentration end and higher batch failure rate. To reduce the variation and unnecessary reassays due to batch failure or possible rejection of the lowest and/or highest calibration standards, a partially duplicate-standard approach is proposed, which has duplicate-standard-like performance but still saves time and cost as singlet-standard approach does. Finally, the maximum allowable degrees of quadratic (non-linear) response in linear calibration are determined for different scenarios. Because of its multiple advantages and potential application in regulated bioanalysis, recommendations as how to implement two-concentration linear calibration in practice are given and some typical “concerns” regarding linear calibration using only two concentrations are addressed, e.g. how does one know if the response is truly linear over a given range when only two concentrations are used?.
Keywords: Linear calibration; Linear regression; Calibration standard; Bioanalysis; Quantitation; LC–MS;