Journal of Chromatography B (v.906, #C)

Liquid chromatography–tandem mass spectrometry method for simultaneous determination of seven commonly used anticancer drugs in human plasma by Jingya Zhou; Shouhong Gao; Feng Zhang; Bo Jiang; Qin Zhan; Fei Cai; Jingxian Li; Wansheng Chen (1-8).
► We developed a novel, sensitive, reproducible, and accurate LC–MS/MS method. ► This method could simultaneous determination of seven anticancer drugs in human plasma. ► This new method was successfully applied to clinical samples from cancer patients.This paper describes the development and validation of a novel, general liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of cyclophosphamide, ifosfamide, irinotecan, etoposide, gemcitabine, carboplatin and pemetrexed concentrations in human plasma. Samples were prepared by two kinds of extraction method and analyzed using a gradient separation over an Atlantis T3-C18 column (2.1 mm × 100 mm, 3 μm, Waters). Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (0.1% formic acid and 10 mM ammonium acetate) at a flow rate of 0.25 mL/min. Linear coefficients of correlation were >0.992 for all analytes. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 9.2%, while the accuracy was within ±10.5%. The mean recovery of all the analytes ranged from 50.0 to 81.0%. This method was successfully applied to clinical samples from cancer patients.
Keywords: Anticancer drug; Liquid chromatography–tandem mass spectroscopy; Human plasma;

Multi-detection of preservatives in cheeses by liquid chromatography–tandem mass spectrometry by Fabio Fuselli; Chiara Guarino; Alessandro La Mantia; Lucia Longo; Angelo Faberi; Rosa Maria Marianella (9-18).
► An RP-HPLC–ESI-MS/MS based method was developed for the multi-determination of seven preservatives in three typologies of cheeses. ► The analytical strategy was evaluated in terms of accuracy, precision, limit of detection, and limit of quantification. ► Our method was applied to commercial cheese samples with good results.The incorrect use of preservatives in cheeses may compromise food safety and damage consumers. According to the law, more than one preservative may be contemporarily used in cheeses. So a method for their contemporary detection may be useful for both manufacturers and control agencies quality control. In this research a liquid chromatography–tandem mass spectrometric with electrospray ionization method for the multi-determination of seven preservatives (benzoic acid, citric acid, hexamethylenetetramine, lysozyme, natamycin, nisin and sorbic acid) in cheese was developed. The preservatives were contemporarily extracted from cheese by a single procedure, and analyzed by RP-LC/ESI-MS/MS (Ion Trap) in positive ionization mode, with single reaction monitoring (SRM) acquisition. Three sample types (hard, pasta filata and fresh cheese) were used for method evaluation. Recoveries were mostly higher than 90%; MDLs ranged from 0.02 to 0.26 mg kg−1, and MQLs were included between 0.07 and 0.88 mg kg−1. Due to matrix effect, quantitation was performed by referring to a matrix matched calibration curve, for each cheese typology. This method was also applied to commercial cheese samples, with good results. It appears fast, reliable and suitable for both screening and confirmation of the presence and quantitation of the preservatives in a single, multi-detection analysis.
Keywords: Cheese; Preservatives; HPLC; Mass spectrometry;

Novel affinity purification of xanthine oxidase from Arthrobacter M3 by Yuran Zhang; Yu Xin; Hailin Yang; Ling Zhang; Xiaole Xia; Yanjun Tong; Yi Chen; Li Ma; Wu Wang (19-24).
► A novel affinity protocol for the purification of xanthine oxidase is developed. ► Only three steps successfully purified xanthine oxidase. ► The most important step of this protocol is affinity chromatography. ► The mechanism relies on the affinity interaction between ligand and the enzyme. ► This method has advantage of fewer steps, better recoveries, and higher purity.An affinity protocol for purification of xanthine oxidase (XOD) from Arthrobacter M3 was developed. The isolation procedure consisted of only three steps, ammonium sulfate precipitation, affinity extraction to exclude the major impurities, and the final refining procedure with DEAE ion-exchange chromatography for removal of minor contaminants. In this affinity preparation, guanine, an analogue of xanthine, was chosen as the affinity ligand, and was coupled with Sepharose 4B through spacers composed of epichlorohydrin and ethylenediamine. Crude protein has been run through ammonium sulfate precipitation and the affinity column, 99.1% of proteins were removed. After DEAE ion-exchange chromatography, the purity of the refined XOD was 97.5% by Native-PAGE analysis. The activity recovery of purified XOD (36.1%) was almost higher than that of other methods reported. Reducing SDS-PAGE analysis showed that the purified XOD (one band in Native-PAGE analysis) showed two polypeptides with the molecular weights ∼35 kDa and ∼100 kDa, respectively. The desorption constant K d and the theoretical maximum absorption Q max on the affinity medium were 3.0 μg/ml and 2.2 mg/g medium in absorption analysis.
Keywords: Xanthine oxidase; Guanine; Affinity chromatography; DEAE ion-exchange chromatography; Arthrobacter M3;

Development of a sensitive HPLC method to measure in vitro permeability of E- and Z-isomeric forms of thiosemicarbazones in Caco-2 monolayers by Zufan Debebe; Sergei Nekhai; Meseret Ashenafi; David B. Lovejoy; Danuta S. Kalinowski; Victor R. Gordeuk; W. Malcolm Byrnes; Des R. Richardson; Pradeep K. Karla (25-32).
► We developed a sensitive HPLC method for assessment of BpT-E/Z isomers. ► We demonstrated effective iron chelation of BpT-iron chelators that inhibit HIV-1. ► We estimated the permeability profile of BpT-E/Z isomers in Caco2 monolayers. ► We anticipate that BpT chelators could hold promise as orally effective agents.In the current study, we developed a HPLC method to quantitatively measure the permeability of the BpT-based chelators, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma (Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak area was linear with respect to chelator concentration. Acceptable within-day and between-day precision (<22%) and accuracy (85–115% of true values) were obtained over a range of 1.0–100 μM for Bp4eT and 1.5–300 μM for Bp4aT. Limits of detection were 0.3 μM and 1 μM for Bp4eT and Bp4aT, respectively, while corresponding limits of quantification were 1 μM and 5 μM. Both chelators showed significant ability to chelate iron in THP-1 cells using a calcein-based assay and no apparent cytotoxicity was observed within 24 h. Ratios of the apical to basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at 100 μM and 300 μM respectively, indicating equal bi-directional movement of the compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This study demonstrates that Bp4eT and Bp4aT can be efficiently transported through Caco-2 cells and can potentially be formulated for oral delivery.
Keywords: HIV-1 iron chelator; HPLC method development; Inter-convertible Z and E isomers; Caco2 drug permeability;

► After intravenous administration of GN A in rats, urinary metabolites of GN A were identified. ► Metabolite identification was performed using UPLC–QTOP-MS analysis in conjunction with data processing programs. ► Reduction, nitro-reduction and hydration were the primary metabolic processes affecting GN A in vivo. ► Demethylation, oxidative deamination to alcohol and conjugations were also included in GN A metabolism.Glionitrin A (GN A) is a new diketopiperazine disulfide with an aromatic nitro group, which is isolated from the coculture of an Aspergillus fumigatus fungal strain and a Sphingomonas bacterial strain. After intravenous administration of GN A in rats, 13 urinary metabolites of GN A were identified using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectroscopy (UPLC–QTOP-MS) analysis in conjunction with data processing programs such as MetaboLynx™ and MassFragnent™. Reduction, nitro-reduction and hydration were the primary metabolic processes affecting GN A in vivo, followed by demethylation or oxidative deamination to alcohol, as well as cysteine, glycine, glucuronide or sulfate conjugation. The metabolite resulting from reduction was found to be a molecule with a dithiol group, and the metabolite made by nitro reduction was found to be an aromatic amine corresponding to GN A. Both of these products may have pharmacological or toxicological activity, which is valuable information in terms of using GN A as a lead compound. In addition, this work showed that UPLC–QTOP-MS analysis coupled with efficient data processing programs is useful for rapid and reliable characterization of GN A metabolites in vivo.
Keywords: Gliotrin A; Urinary metabolites; UPLC–QTOP-MS; Diketopiperazine disulfide;

► On-line sample concentration and analysis of some cationic alkaloids in human plasma. ► Micelle to solvent stacking in capillary zone electrophoresis. ► High sensitivity and good recovery.A sensitive method for the determination of three cationic alkaloids (berberine, palmatine and jatrorrhizine) from human plasma samples was developed by micelle to solvent stacking (MSS) in capillary zone electrophoresis (CZE). In MSS, the sample preconcentration mainly relies on the reversal in the effective electrophoretic mobility of the analytes at the boundary zone between the sample and CZE background solution (BGS). Under the optimized conditions, the sensitivity enhancement factors achieved in terms of corrected peak area were in the range from 47 to 53 for the alkaloids. The limits of detection (LODs) (S/N = 3) for berberine, palmatine and jatrorrhizine were 0.01, 0.01 and 0.02 μg/mL, respectively. The intraday (n  = 6) and interday repeatabilities (n  = 12) expressed as the relative standard deviations (RSDs) were less than 6.9% in terms of peak height and less than 7.3% in terms of corrected peak area, respectively. The recoveries of the method for the three alkaloids were in the range of 95.9–101.5% with peak height as the quantitative signal, and 92.6–103.6% with corrected peak area as the quantitative signal, respectively. The MSS-CZE method proved to be suitable for the analysis of the alkaloids in human plasma samples.
Keywords: On-line sample preconcentration; Micelle to solvent stacking; Capillary zone electrophoresis; Cationic alkaloids; Human plasma;

Generic and rapid determination of veterinary drug residues and other contaminants in raw milk by ultra performance liquid chromatography–tandem mass spectrometry by Jia Zhan; Xue-jun Yu; Ying-ying Zhong; Zai-ting Zhang; Xiao-mei Cui; Jin-feng Peng; Rui Feng; Xiao-tao Liu; Yan Zhu (48-57).
► First, the proposed method was generic for a wide range polarity compounds. ► Second, the method, with only two steps, was rapid and straightforward. ► Third, the way to remove water in milk for concentration was novelty. ► Finally, this kind of method is urgently needed in dairy plants.A generic, rapid and simple analytical method able to identify 255 veterinary drug residues and other contaminants in raw milk had been developed. The method was based on two-step simple precipitation and ultra performance liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (UPLC–ESI–MS/MS) operating both in positive and negative multiple reaction mode (MRM). For most of the target analytes, the optimized pretreatment processes led to no significant interference on analysis from complicated sample matrix. For quantification, matrix-fortified calibration curves were performed to compensate for the matrix effect and loss in sample preparation. Competent linearity was found for over 90% of target compounds with linear regression coefficients (R) higher than 0.99. Detection limits ranged from 0.05 to 10 μg/kg. Average recoveries spiked into raw milk were in the range from 63% to 141% with associated RSD values from 1% to 29% under the selected conditions. The method had been validated for its extraction sensitivity, linearity, recoveries and precision. The results clearly demonstrated the feasibility of the approach proposed. Application of this method, which improved efficiency and coverage of residues, would imply a drastic reduction of both effort and time in routine monitoring programs.
Keywords: Generic; Multi-class; Veterinary drug; Contaminant; Ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS); Raw milk;

► Aqueous normal phase chromatography was studied for the separation of zanamivir in human sera. ► Five different columns were compared for the capability to retain zanamivir. ► Effects of mobile phase constituents on the retention time were examined. ► The Diamond Hydride column was the most effective to retain zanamivir and avoid matrix effects.An aqueous normal phase (ANP) liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometry (ANP-LC-micrOTOFQ) method was used for the determination of zanamivir in human serum. Zanamivir was extracted with methanol from protein-precipitated human serum samples and further purified with SCX solid-phase extraction cartridges. Scherzo SM-C18, Agilent Zorbax SB-Aq, Cogent Diamond Hydride, Cogent Bidentate and Luna HILIC columns were compared and optimized for the retention and separation of zanamivir and the Luna HILIC and Diamond Hydride columns exhibited the best retention of zanamivir. The former provided a shorter retention time, a sharper peak and relatively high sensitivity, whereas the latter exhibited a longer retention time and less matrix interference. The analytical range of the calibration curve was between 5 and 1000 ng/mL.
Keywords: Aqueous normal-phase; Zanamivir; Human serum;

► We prepared a simple CZE running buffer. ► We developed and optimized a CZE method in dynamically coated capillary. ► We performed fast separation in less than 5 min with high resolution.This work aimed to develop a rapid capillary zone electrophoresis (CZE) method to provide abundant purity and identity information of monoclonal antibodies. The CZE running buffer system was optimized to be 20 mM acetate–acetic acid (pH 6.0) together with the co-addition of 0.3% polyethylene oxide (PEO) and 2 mM triethylenetetramine (TETA), which was further tested with advantages on the peak resolution improvements. The conditioning period was scheduled to 1 min for both 0.1 M HCl and CZE running buffer to reduce total separation time. Additionally, the applied voltage and effective separation length were optimized at 30 kV and 20 cm separately. Compared with the method reported by Yan [1], this newly developed method showed a higher resolution in separating the two unknown basic peaks by testing monoclonal antibody sample (mAb1). The further validation results showed that for all five of charge isoform peaks of test mAb1, repeatability, intraday and interday precision had a RSD less than 0.58% for migration time and less than 3.18% for corrected area percent. The correlation coefficients of more than 0.98 for all peaks also demonstrated the good linearity for the method. In addition to the application of distinguishing intact antibody from C-terminal Lys variants, the method also has advantage in separating the Fab, Fc and intact antibody-relevant substances quickly, which facilitated the rough evaluation of papain induced digestion.
Keywords: Capillary zone electrophoresis; Therapeutic monoclonal antibody; Optimization; Validation; Application;

► LC–MS/MS method for simultaneous quantitation of azelastine and desmethylazelastine. ► Low LOQ of 10 pg/mL for both compounds were achieved. ► An abnormal signal loss issue for desmethylazelastine was investigated and resolved.A selective and sensitive high performance liquid chromatography–tandem mass spectrometric method was developed for the analysis of azelastine and its major metabolite, desmethylazelastine, in human plasma. Azelastine-13C, d3 was used as internal standard. Azelastine, desmethylazelastine and the internal standard were extracted by a liquid–liquid extraction method and separation was performed under isocratic chromatographic condition. An abnormal signal loss issue for desmethylazelastine during method development was investigated and resolved. The developed method was precise and reproducible as shown by good intraday assay and interday assay precision (CV% ≤ 12.8%). The calibration curve was linear over a range of 10.0/10.0–1000/200 pg/mL for azelastine/desmethylazelastine. The method was successfully applied to a pilot bioequivalence study subsequently.
Keywords: Azelastine; Desmethylazelastine; LC–MS/MS; Human plasma; Quantification;

Simultaneous determination of eight corticosteroids in bovine tissues using liquid chromatography–tandem mass spectrometry by Ádám Tölgyesi; Virender K. Sharma; Szabolcs Fekete; Dóra Lukonics; Jenő Fekete (75-84).
► A new LC–MS/MS method is developed for corticosteroids in tissue samples. Acidic pH control and mixed-mode SPE cartridges are essential for sample clean-up. ► Corticosteroid epimers can be separated simultaneously using Kinetex HPLC column. ► The enhanced clean-up and LC–MS/MS separation improved the analytical limits. ► The method is successful in analyzing dexamethasone in incurred samples.This paper describes a newly developed method for the simultaneous determination of eight corticosteroid residues in bovine muscle, liver and kidney samples using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The determination of methylprednisone, the main metabolite of methylprednisolone, in bovine tissues using LC–MS/MS is carried out for the first time. The method development demonstrates that the pH is important in optimizing the sample preparation. Tests performed using different solid-phase extraction (SPE) cartridges were enabled to produce conditions for reducing the matrix effects (ion suppression and enhancement) of analysis. Acidic condition and mixed-mode cation exchange SPE columns resulted in the most suitable clean-up for muscle and liver, and also yielded acceptable results for kidney. The enhanced sample clean-up resulted in excellent clear baselines of ion transitions, and therefore, a higher delta electron multiplier voltage (ΔEMV) could be set in the MS/MS detector. The application of 500 V of ΔEMV improved the signal responses, however, the noise level did not change, and consequently, the overall sensitivity and analytical limits (limit of detection, limit of quantification) could be enhanced. In the HPLC separation, the recently introduced Kinetex phenyl-hexyl core–shell type column was used that enabled baseline separation for dexamethasone and its β-epimer, betamethasone. Dexamethasone and betamethasone were eluted within 12 min and such reduced retention, obtained with core–shell HPLC type column, further enhanced the sensitivity. The method was validated according to the European Union (EU) 2002/657/EC Decision; the studied parameters met the EU standards. The decision limits and limit of detections were calculated in each matrix for all corticosteroids and varied from 0.01 to 13.3 μg/kg and from 0.01 to 0.1 μg/kg, respectively.
Keywords: Corticosteroids; LC–MS/MS; Food analysis; Kinetex phenyl-hexyl HPLC column; Strata solid-phase extraction; Matrix effect;

► A chiral LC–MS/MS method was validated to quantify pantoprazole enantiomers. ► Separation was performed on an ovomucoid protein column using MS compatible mobile phases. ► Baseline resolution within 10 min leads to a reduction in the overall analysis time.Pantoprazole (PAN), a selective proton pump inhibitor, is used clinically as a racemic mixture for the treatment of acid-related gastrointestinal disorders. To investigate its stereoselective pharmacokinetics, a chiral liquid chromatography–tandem mass spectrometry method was developed and validated to determine the pantoprazole enantiomers in dog plasma. After liquid–liquid extraction, a baseline resolution of enantiomers was achieved on an ovomucoid column using the mobile phase of methanol:acetonitrile:10 mM ammonium formate (pH 7) (10.4:2.6:87, v/v/v) at 30 °C within 10 min. Stable isotopically labeled (+)-d3-pantoprazole and (−)-d3-pantoprazole were used as internal standards. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode via positive atmospheric pressure chemical ionization. The method was linear in the concentration range of 20.0–10,000 ng/mL for each enantiomer using 25 μL of dog plasma. The lower limit of quantification (LLOQ) for each enantiomer was 20.0 ng/mL. Intra- and inter-day precision ranged from 3.2% to 10.3% for (+)-pantoprazole and 3.7–10.0% for (−)-pantoprazole. Accuracy varied from −1.4% to −0.2% for (+)-pantoprazole and −1.6% to 0.8% for (−)-pantoprazole. The validated method was applied successfully for stereoselective pharmacokinetic studies of racemic pantoprazole.
Keywords: Chiral LC–MS/MS; Pantoprazole; Stereoselective pharmacokinetics;

Rapid resolution liquid chromatography (RRLC) analysis of amino acids using pre-column derivatization by Xiaoli Zhang; Tong Zhao; Ting Cheng; Xiaoyan Liu; Haixia Zhang (91-95).
► The RRLC method was the first time used for amino acids detection. ► Compared with common HPLC and some UPLC, the RRLC was superior performance. ► The reduced solvent consumption was friendly to environment protection. ► The RRLC based on UV detection was economic and available in common laboratories.A rapid resolution liquid chromatography (RRLC) method was developed for the simultaneous determination of 23 amino acids in rat serum after pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The amino acid derivatives were separated on an Agilent Zorbax Eclipse Plus C18 (4.6 mm × 50 mm, 1.8 μm) column at 45 °C. Ultraviolet (UV) detection was set at 360 nm. Good separation of 23 amino acids was achieved within 10 min with a ternary gradient elution of mobile phase at a flow rate of 1.5 mL min−1. Calibration curves were linear over the range from 1 to 500 μmol L−1 with coefficients 0.9962 or better for each amino acid. The lower limits of quantification (LLOQ) of all 23 amino acids were 1 μmol L−1 with signal-to-noise (S/N) ratio ≥4. Intra- and Inter-day precisions, expressed as relative standard deviation (RSD) percentages, were ranged from 0.32% to 3.09% and 0.67% to 5.82%, respectively. Finally, it was successfully applied to the determination of amino acids in rat serum with recoveries ranged from 90.8% to 106.0% and RSD percentages ranged from 1.78% to 4.68%, respectively. The results showed that the proposed method provided a shorter elution time, better resolution and sharper peak shapes for all amino acids. Compared with the conventional high performance liquid chromatography (HPLC) methods, even some ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), the established RRLC method was superior performance.
Keywords: Rapid resolution liquid chromatography; Amino acids; Pre-column derivatization; 2,4-Dinitrofluorobenzene;