Journal of Chromatography B (v.877, #16-17)

Analysis of human antibody IgG2 domains by reversed-phase liquid chromatography and mass spectrometry by Boxu Yan; Tamer Eris; Zac Yates; Robert W. Hong; Sean Steen; Gerd Kleemann; Weichun Wang; Jennifer L. Liu (1613-1620).
It has been well documented that papain cleaves an IgG1 molecule to release Fab and Fc domains; however, papain was found unable to release such domains from an IgG2. Here we present a new combinatory strategy to analyze the heterogeneity of the light chain (LC), single chain Fc (sFc), and Fab portion of the heavy chain (Fd) of an IgG2 molecule released by papain cleavage under mild reducing conditions. These domains were well separated on reversed-phase high performance liquid chromatography (RP-HPLC) and analyzed by in-line liquid chromatography time-of-flight mass spectrometry (LC–TOF/MS). In addition, some modifications of these domains were revealed by in-line mass spectrometry, and confirmed by the peptide mapping on LC–MS/MS analysis. This same strategy was proven suitable for IgG1 molecules as well. This procedure provides a simplified approach for the characterization of antibody biomolecules by facilitating the detection of low-level modifications in a domain. In addition, the technique offers a new strategy as an identification assay to distinguish IgG2 molecules on RP-HPLC, by which highly conserved Fc domains remain at a constant retention time (RT) unique to its subisotype, while varying RTs of the light chain and the Fd distinguish the monoclonal antibody from other molecules of the same isotype based on the underlying characteristics of each antibody.
Keywords: Antibody IgG2; Hinge; Papain; RP-HPLC; Mass spectrometry; Modification;

Determination of 2,3-dimercaptosuccinic acid in mice blood and tissues by HPLC with fluorescence detection by Xue Hai Ju; Ying Shi; Na Liu; Rui Chen Guo; Ben Jie Wang; Xi Cui (1621-1626).
2,3-Dimercaptosuccinic acid (DMSA) is an orally effective chelating agent for the treatment of heavy metal poisoning. The increasing therapeutic use of DMSA has stimulated the need for sensitive and selective methods for its determination in biological samples, as well as study on pharmacokinetics and tissue distribution. According to the previously reported method, an improved method was established for the determination of DMSA in mice blood and tissues, in which oxidized DMSA was reduced by the disulfide-reducing agent, dithiothreitol (DTT), and DMSA was converted to a highly fluorescent and stable derivative by reaction with monobromobimane (mBBr) in alkaline solution. Acetonitrile was used for deproteinization and dichloromethane was used for condensation and purification, which significantly shortened the amount of time used to process the sample. Meanwhile isocratical elution was performed and excellent separation of the DMSA derivative was obtained, this enabled a run finish within 20 min. The limits of quantitation were 0.025 μg/ml in brain and 0.1 μg/ml in blood, lung, heart, intestine, liver, spleen and kidney, respectively. The calibration curves were linear in all samples (r 2  > 0.992) with a range of 0.025–1.6 μg/ml for brain homogenate and 0.1–6.4 μg/ml for blood and homogenates of lung, heart, intestine, liver, spleen and kidney, respectively. Therefore, the method is simple, rapid and sensitive, and it could be applicable to the studies in an animal model to evaluate the distribution of DMSA in blood and tissues.
Keywords: 2,3-Dimercaptosuccinic acid; Monobromobimane; HPLC; Fluorescence detection;

A sensitive and selective HPLC method using a diode array detector (DAD) and an electrochemical detector (ECD) in series has been developed and validated for the quantitative measurement of p-phenylenediamine and its acetylated metabolites N-acetyl-p-phenylenediamine (MAPPD) and N,N′-diacetyl-p-phenylenediamine (DAPPD) in biological samples. The separation was carried out on a hydrophilic modified AQUA C18 column and the mobile phase was composed of acetonitrile: ammonium acetate solution (5:95, 25 mM, v/v). Spectrophotometric detection was performed at 240 or 255 nm and amperometric detection was carried out using a positive oxidation potential of 400 mV. The quantification of the three analytes was validated in the range of 0.05–50 μM and the established limits of determination were 0.5 μM for PPD and MAPPD and 1 μM for DAPPD. The standard deviations (N  = 9) were lower than 7.5% at a concentration of 1 μM. The samples were stabilised with ascorbic acid to prevent PPD from oxidizing. Pretreatment of samples or analyte enrichment before sample injection is not required. The method proved to be accurate, sensitive and sufficiently specific. It was applied to the ecotoxicological study of the kinetics of the PPD N-acetylation in cell lysates in two different media.
Keywords: Paraphenylenediamine (PPD); N-acetylation; MAPPD; DAPPD; Amperometric detection;

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5–500 nM and 5–5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C18 column (50 mm × 0.5 mm, 50 μm), followed by HPLC separation on a FluoPhase RP column (100 mm × 2.1 mm, 5 μm) and MS/MS detection. The off-line SPE assay used Waters Oasis®HLB μElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm × 2.1 mm, 5 μm) for LC–MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of <2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.
Keywords: Telcagepant; MK-0974; CGRP receptor antagonist; Diastereomer; On-line extraction; High turbulent-flow liquid chromatography (HTLC); Off-line solid-phase extraction (SPE); Human plasma;

Expression and purification of soluble E-Syt2: Low protein stability impedes tag removal by Gerhard J. Groer; Martin Haslbeck; André Gessner (1643-1650).
Affinity tags are valuable tools for high-throughput protein isolation in automated screenings or downstream processing approaches and are also widely used in laboratory applications for quick and easy access to many proteins. Here, we describe the preparative purification of soluble extended synaptotagmin 2 (rE-Syt2) at bench scale for basic structural and functional studies. Due to the low protein stability, a classical purification procedure without affinity tag was more powerful than isolation of His(6)-tagged rE-Syt2 and subsequent proteolytic tag-removal. Furthermore, expression analysis of truncated rE-Syt2 variants suggested a concept of interdependent-domain organization in proteins containing multiple C2 domains.
Keywords: E-Syts; C2 domain; Purification; Chromatography;

Open-loop simulated moving bed (SMB) has been used for immobilized metal affinity chromatographic (IMAC) purification of his-tagged β-glucosidase expressed in E. coli. A simplified approach based on an optimized single column protocol is used to design the open-loop SMB. A set of columns in the SMB represent one step in the chromatographic cycle i.e. there will be one set each of columns for load, wash, elution etc within the SMB. Only the wash and elution are operated with columns in sequence. The β-glucosidase was purified to almost single band purity with a purification factor of 15 and a recovery of 91%. SMB-performance showed reduced buffer consumption, higher purification fold, a better yield and higher productivity.
Keywords: IMAC; His tag; SMB;

In this study, a label-free relative quantification strategy was developed for quantifying low-abundance glycoproteins in human serum. It included three steps: (1) immunodepletion of 12 high-abundance proteins, (2) enrichment of low-abundance glycoproteins by multi-lectin column, (3) relative quantification of them between different samples by micrOTOF-Q. We also evaluated the specificity and efficiency of immunodepletion, the accuracy of protein quantification and the possible influence of immunodepletion, glycoprotein enrichment, trypsin digestion and peptide ionization on quantification. In conclusion, the relative quantification method can be effectively applied to the screening of low-abundance biomarkers.
Keywords: Relative quantification; Glycoprotein; Serum; Depletion; Immunoaffinity; Multi-lectin column chromatography; micrOTOF-Q; Mass spectrometry; Proteomics;

On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows by Medhat A. Al-Ghobashy; Martin A.K. Williams; Brigid Brophy; Götz Laible; David R.K. Harding (1667-1677).
Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads™ was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.
Keywords: Transgenic; Milk; Myelin basic protein; Biopharmaceuticals; Casein micelles;

A simple, fast, sensitive and specific high-performance liquid chromatography (HPLC) method is developed for simultaneous determination of kynurenine (Kyn) and tryptophan (Trp) with ultraviolet (UV) detection setting programmed wavelength. The separation was carried out on an Agilent Hypersil ODS column (125 mm × 4.0 mm, 5 μm) in less than 6 min and the eluate was monitored by the programmed wavelength detection setting at 360 nm from 0 min to 4 min for Kyn, and at 278 nm from 4 min to 6 min for Trp in a single run with UV detector. The linearities of the method were from 0.20 μmol/L to 21.2 μmol/L for Kyn and 2.25–678.0 μmol/L for Trp, and the detection limits were 0.028 μmol/L for Kyn and 0.053 μmol/L for Trp, respectively. Satisfactory precisions and recoveries were obtained by this method. The assay was employed to analyze plasma samples of children patients with Kawasaki disease (KD). The result showed great difference between Kawasaki disease and control group.
Keywords: Tryptophan; Kynurenine; Indoleamine 2,3-dioxygenase; HPLC; Kawasaki disease;

Ion-pairing separation of bioactive peptides using an aqueous/octan-1-ol micro-extraction system from bovine haemoglobin complex hydrolysates by Mathieu Vanhoute; Renato Froidevaux; Aurelien Vanvlassenbroeck; Didier Lecouturier; Pascal Dhulster; Didier Guillochon (1683-1688).
The ion-pair concept was applied on complex haemoglobin hydrolysates to extract two opioid peptides, LVV-haemorphin-7 and VV-haemorphin-7, in an aqueous/octan-1-ol micro-extraction system in the presence of alkyl-sulfonic acid as a surfactant agent and in relation to the haemorphin physico-chemical properties (charge, hydrophobicity). The effect of combined alkyl chain length/aqueous phase pH and the haem behaviour during the extraction, on the haemorphin recovery yield and enrichment has been determined. It has proved that transport over the organic phase is mediated by the alkyl-sulfonic acids, whatever be the aqueous phase pH. However, increasing both the alkyl chain length and the pH in the aqueous phase shows an haemorphin enrichment ratio increase but a recovery decrease of the extracted opioid peptides in the organic phase. Therefore, the best conditions to extract LVVh-7 and VVh-7 are the use of the octane-sulfonic acid at aqueous phase pH of 5 or 7 and the octane or the heptane-sulfonic acid with an aqueous phase pH of 5 or 7 respectively. In these conditions, a partition coefficient of 1.64 and 1.60 respectively for LVVh-7 and VVh-7 are obtained and represent about 40 times that acquired without agent.
Keywords: Ion-pair micro-extraction; Liquid/liquid biphasic system; Haemoglobin; Peptides; Haemorphins;

Simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma by HPLC coupled with tandem mass spectrometry by Maha F. Tutunji; Husam M. Ibrahim; Manal H. Khabbas; Lara F. Tutunji (1689-1697).
A sensitive, specific and selective method has been developed for the simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma. The method employed a state of the art LC–MS/MS operated in the positive and negative ionization switching modes. A simple sample preparation step involving protein precipitation with acetonitrile has been optimized; the analytes and the internal standard moxifloxacin were separated on a Purosphere® STAR C8 column (125 mm × 4 mm, 5 μm). The mobile phase was an ammonium acetate solution (1 mM) with formic acid (0.2%): methanol and acetonitrile (65:17.5:17.5, v/v/v (%)), the flow rate was set at 0.65 mL/min. Bisoprolol and hydrochlorothiazide were ionized using ESI source prior to detection by Multiple Reaction Monitoring (MRM) mode while monitoring at the following transitions: positive m/z 326 → 116 for bisoprolol, negative m/z 296 → 269 and m/z 296 → 205 for hydrochlorothiazide. Linearity was demonstrated over the concentration range 0.10–30.0 (ng/mL) for bisoprolol and 1.00–80.00 ng/mL for hydrochlorothiazide. The limits of detection were 0.100 (ng/mL) for bisoprolol and 1.00 (ng/mL) for hydrochlorothiazide. The validated method was successfully applied to a pharmacokinetic study of 5 mg bisoprolol fumarate with 12.5 mg hydrochlorothiazide tablet in healthy volunteers.
Keywords: Bisoprolol; Hydrochlorothiazide; LC–MS/MS; Pharmacokinetic;

Ethambutol and pyrazinamide are two first-line anti-tuberculosis drugs. Though they are normally combined for the treatment, their highly different polarity complicates simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of these two drugs in human plasma with decent peak shape and retention. Here we report a rapid and robust LC/MS/MS method for the simultaneous determination of these two drugs in human plasma. Human plasma samples, together with the isotopically labeled internal standards were extracted using protein precipitation, and then separated on a Chromolith SpeedROD RP-18e column and detected with mass spectrometry. The mobile phase is 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in methanol. Addition of trifluoroacetic acid in the mobile phases was found to be able to improve peak shape as well as to increase the retention of ethambutol, thus being able to analyze these two drugs at the same time with both drugs having decent peak shape and enough retention on a C18 column. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The standard curve range was 10.0–5000 ng/mL for ethambutol and 50.0–25,000 ng/mL for pyrazinamide using a plasma sample volume of 50.0 μL. This method has a very short run time of 3.8 min. The method has been fully validated, and <15% relative standard deviation was obtained for both analytes.
Keywords: Ethambutol; Pyrazinamide; Human plasma; Liquid chromatography; Mass spectrometry;

Detection of landiolol using high-performance liquid chromatography/fluorescence: A blood esterase-sensitive ultra-short-acting β1-receptor antagonist by Manabu Suno; Takayuki Kunisawa; Akio Yamagishi; Takashi Ono; Joe Yamamoto; Takehiro Yamada; Yoshikazu Tasaki; Keiko Shimizu; Hiroshi Iwasaki; Kazuo Matsubara (1705-1708).
Landiolol hydrochloride, a new adrenergic β1-selective antagonist having an ultra-short half-life, is used to prevent tachyarrhythmia during surgery. Since landiolol is thought to be rapidly hydrolyzed to an inactivate metabolite by esterases, quantification of the drug concentration in the blood is impractical. The landiolol concentration in blood was halved within 5 min after blood sampling. This degradation was effectively prevented by pre-treatment with neostigmine (100 μg) in the sampling tube, but not by EDTA pre-treatment, indicating that landiolol could be metabolized by pseudocholinesterase in plasma. After the one-step solid-phase extraction, fluorescence detection of landiolol reduced chromatographic background signals and then improved assay sensitivity to the lower limit of 10 ng/ml in blood; this reproducible approach yielded coefficient variation of less than 6%. The blood concentration-time profile of landiolol hydrochloride in patients of the present investigation afforded more practical assessment than previously reported studies, thus improving accuracy and facilitating detailed pharmacokinetic study in relation to the pharmacological action of drug.
Keywords: Landiolol; Ultra-short-acting β1-receptor antagonist; HPLC/fluorescence; Pseudocholinesterase; One-step solid-phase extraction;