Journal of Chromatography B (v.1033-1034, #C)

New on-line separation workflow of microbial metabolites via hyphenation of analytical and preparative comprehensive two-dimensional liquid chromatography by Xia Yan; Li-Juan Wang; Zhen Wu; Yun-Long Wu; Xiu-Xiu Liu; Fang-Rong Chang; Mei-Juan Fang; Ying-Kun Qiu (1-8).
Microbial metabolites represent an important source of bioactive natural products, but always exhibit diverse of chemical structures or complicated chemical composition with low active ingredients content. Traditional separation methods rely mainly on off-line combination of open-column chromatography and preparative high performance liquid chromatography (HPLC). However, the multi-step and prolonged separation procedure might lead to exposure to oxygen and structural transformation of metabolites. In the present work, a new two-dimensional separation workflow for fast isolation and analysis of microbial metabolites from Chaetomium globosum SNSHI-5, a cytotoxic fungus derived from extreme environment. The advantage of this analytical comprehensive two-dimensional liquid chromatography (2D-LC) lies on its ability to analyze the composition of the metabolites, and to optimize the separation conditions for the preparative 2D-LC. Furthermore, gram scale preparative 2D-LC separation of the crude fungus extract could be performed on a medium-pressure liquid chromatograph ÿ preparative high-performance liquid chromatography system, under the optimized condition. Interestingly, 12 cytochalasan derivatives, including two new compounds named cytoglobosin Ab (3) and isochaetoglobosin Db (8), were successfully obtained with high purity in a short period of time. The structures of the isolated metabolites were comprehensively characterized by HR ESI⿿MS and NMR. To be highlighted, this is the first report on the combination of analytical and preparative 2D-LC for the separation of microbial metabolites. The new workflow exhibited apparent advantages in separation efficiency and sample treatment capacity compared with conventional methods.
Keywords: Comprehensive two dimensional liquid chromatography; Medium-pressure liquid chromatograph ÿ preparative high-performance liquid chromatography; Chaetomium globosum SNSHI-5; Cytochalasan derivatives;

A liquid chromatography⿿tandem mass spectrometry assay for the detection and quantification of trehalose in biological samples by Philip M. Kretschmer; Austin M. Bannister; Molly K. O⿿Brien; Laura A. MacManus-Spencer; Margot G. Paulick (9-16).
Trehalose is an important disaccharide that is used as a cellular protectant by many different organisms, helping these organisms better survive extreme conditions, such as dehydration, oxidative stress, and freezing temperatures. Methods to detect and accurately measure trehalose from different organisms will help us gain a better understanding of the mechanisms behind trehalose⿿s ability to act as a cellular protectant. A liquid chromatography⿿tandem mass spectrometry (LC⿿MS/MS) assay using selected reaction monitoring mode for the detection and quantification of trehalose using maltose as an internal standard has been developed. This assay uses a commercially available LC column for trehalose separation and a standard triple quadrupole mass spectrometer, thus allowing many scientists to take advantage of this simple assay. The calibration curve from 3 to 100 μM trehalose was fit best by a single polynomial. This LC⿿MS/MS assay directly detects and accurately quantifies trehalose, with an instrument limit of detection (LOD) that is 2⿿1000 times more sensitive than the most commonly-used assays for trehalose detection and quantification. Furthermore, this assay was used to detect and quantify endogenous trehalose produced by Escherichia coli (E. coli) cells, which were found to have an intracellular concentration of 8.5 ± 0.9 mM trehalose. This method thus shows promise for the reliable detection and quantification of trehalose from different biological sources.
Keywords: Trehalose; LC⿿MS/MS; Biological samples; Cellular protectant; Quantification;

A novel approach for the simultaneous quantification of 18 small molecule kinase inhibitors in human plasma: A platform for optimised KI dosing by Madelé van Dyk; John O Miners; Ganessan Kichenadasse; Ross A McKinnon; Andrew Rowland (17-26).
Small molecule kinase inhibitors (KIs) are a rapidly expanding class of narrow therapeutic index antineoplastic drugs that exhibit substantial inter-individual variability in exposure. This manuscript describes a novel approach for the quantification of 18 KIs in plasma, providing a platform that is unparalleled in terms of scope for the assessment of KI therapeutic drug monitoring (TDM) and facilitating pharmacokinetic studies with KIs. Following the addition of a panel of four deuterated internal standards, plasma samples were prepared by solvent precipitation with acidified methanol. Analytes were separated on a Waters ACQUITY⿢ T3 HSS C18 analytical column (150 ÿ 2.1 mm, 1.8 μm particle size) by linear gradient elution, with subsequent detection by time-of-flight mass spectrometry. Time-of-flight data were collected in wide pass mode, with selected ion (pseudo-MRM) spectra extracted at the precursor m/z of analytes in ESI+ mode. The analytical performance of this approach in terms of specificity, linearity, accuracy, precision range, quantification limit and detection limit meet all criteria for an analytical platform for the quantification of drugs. This approach was developed, validated and reported in accordance with the 2015 version of the FDA guidance for industry on ⿿analytical procedures are methods validation for drugs and biologics⿿ facilitating direct application as a clinical trials platform.
Keywords: Small molecule kinase inhibitors; Ultraperformance liquid chromatography; Time-of-flight mass spectrometry; Therapeutic drug monitoring; Pharmacokinetics;

Molecularly imprinted pipette-tip solid phase extraction for selective determination of fluoroquinolones in human urine using HPLC-DAD by Hanna Leijoto de Oliveira; Sara da Silva Anacleto; Anny Talita Maria da Silva; Arnaldo César Pereira; Warley de Souza Borges; Eduardo Costa Figueiredo; Keyller Bastos Borges (27-39).
A simple method using HPLC-DAD was developed for the determination of fluoroquinolones in human urine including ciprofloxacin (CIPRO), enrofloxacino (ENRO), marbofloxacino (MARBO) and norfloxacin (NOR). In addition, it was studied the extraction of fluoroquinolones in human urine samples using pipette tip-based molecularly imprinted polymers solid phase extraction (PT-MIPs-SPE). With the goal of finding the best procedure for extraction of four fluoroquinolones in human urine, several parameters that are likely to affect the efficiency of extraction during sample preparation, including the washing solvent, type and volume of eluent, amount of material, the volume of the sample, pH and the ionic strength were systematically optimized. Chromatographic separations of fluoroquinolones were hit within 10 min using a Synergi® C18 (250 × 4.6 mm, 4 μm) column and mobile phase consisting of water (10 mM of phosphoric acid, the pH adjusted at 3.29 with triethylamine) : acetonitrile (85.7: 14.3, v/v) at a flow rate of 1.5 mL min−1. Detection was performed at 290 nm. The average extraction recoveries/standard deviation relative to ENRO, CIPRO, NOR and MARBO were 96.40 ± 5.51%, 42.47 ± 4.81%, 41.82 ± 7.99% and 87.49 ± 4.70, respectively. The method was liner from 39 to 1260 ng mL−1 for each fluoroquinolone with correlation coefficient of 0.9904, 0.9910, 0.9914 and 0.9919, to ENRO, CIPRO, NOR and MARBO, respectively. The assays of within-day and between-day precision and accuracy for all analytes were studied at three concentration levels and were lower than 15%. The method was successfully employed in a preliminary cumulative urinary excretion study after administration of CIPRO to a healthy volunteer.
Keywords: Fluoroquinolones; Molecularly imprinted polymer; PT-MIPs-SPE; Human urine;

In this study, green and efficient deep eutectic solvent-based negative pressure cavitation-assisted extraction (DES-NPCE) followed by macroporous resin column chromatography was developed to extract and separate four main isoflavonoids, i.e. prunetin, tectorigenin, genistein and biochanin A from Dalbergia odorifera T. Chen leaves. The extraction procedure was optimized systematically by single-factor experiments and a Box-Behnken experimental design combined with response surface methodology. The maximum extraction yields of prunetin, tectorigenin, genistein and biochanin A reached 1.204, 1.057, 0.911 and 2.448 mg/g dry weight, respectively. Moreover, the direct enrichment and separation of four isoflavonoids in DES extraction solution was successfully achieved by macroporous resin AB-8 with recovery yields of more than 80%. The present study provides a convenient and efficient method for the green extraction and preparative separation of active compounds from plants.
Keywords: Deep eutectic solvent; Negative pressure cavitation-assisted extraction; Dalbergia odorifera T. Chen; Isoflavonoids; Macroporous resin column chromatography;

MP 3950 is an active metabolite of mosapride which exhibits high 5-HT4 receptor agonistic effect. The present paper describes an enantioselective chiral HPLC method for separation and quantification of MP 3950 enantiomers in rat plasma. The plasma samples were prepared by liquid–liquid extraction with ethyl acetate and the baseline chromatographic separation was achieved on a Chiralcel OJ-H column with a mobile phase consisting n-hexane/ethanol/methanol/diethylamine (85:5:10:0.4, v/v/v/v) at the flow rate of 1.0 mL/min. The ultraviolet detection was performed at 276 nm. The calibration curve was linear in the range from 0.100 to 5.00 μg/mL with the lower limit of quantification of 0.100 μg/mL for each enantiomer. The intra- and inter-day precisions were not more than 14.7%. The accuracy was from −6.4% to 14.0%. The validated method was successfully applied to chiral inversion and stereoselective pharmacokinetic study in rats after oral administration of MP 3950 racemate. The results indicated that no stereochemical inversion was occurred in rats. And the plasma concentrations and area under plasma concentration-time curve of (S)-MP 3950 were all significantly higher than those of (R)-MP 3950 in both male and female rats, which indicated that the disposition of MP 3950 in rats might be stereoselective.
Keywords: MP 3950; Enantiomer separation; Chiral HPLC; Chiral inversion; Stereoselective pharmacokinetics;

Simultaneous determination of major phytocannabinoids (THC, CBD, CBN), their main metabolites (11-OH-THC, THC-COOH, THC-COOH-glucuronide) and common synthetic cannabinoids (HU-210, JWH-018, JWH-073, JWH-250) remains an issue in forensic toxicology. The present study has developed a liquid chromatography⿿tandem mass spectrometry (LC⿿MS/MS) method to simultaneously detect the above 10 analytes in human urine samples. The chromatographic separation was performed on an ACQUITY UPLC®BEH Phenyl 1.7 μm (2.1 ÿ 100 mm) column, using a mobile phase consisting of 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min in gradient elution mode. The limit of detection (LOD) and limit of quantification (LOQ) of all analytes were 0.01⿿0.5 ng/mL and 0.05⿿1 ng/mL, respectively. The assay was linear from LOQ to 100 ng/mL for phytocannabinoids, their main metabolites and HU-210, and from 0.05 to 50 ng/mL for JWH-250, JWH-018 and JWH-073. The extraction recoveries were over 50% and the matrix effects were between 59.4% and 100.1%. The accuracy and precision were <10.4% of bias and <10.5% of relative standard deviation (RSD), respectively. The developed method was applied to 5 urine samples from real caseworks, and the results that THC metabolites together with synthetic cannabinoids were detected demonstrated the effectiveness of our method.
Keywords: Phytocannabinoids; Metabolites; Synthetic cannabinoids; LC⿿MS/MS;

Hair cortisol levels are increasingly applied as a measure for stress in humans and mammals. Cortisol is an endogenous compound and is always present within the hair matrix. Therefore, “cortisol-free hair matrix” is a critical point for any analytical method to accurately quantify especially low cortisol levels. The aim of this project was to modify current methods used for hair cortisol analysis to more accurately determine low endogenous cortisol concentrations in hair. For that purpose, 13C3-labeled cortisol, which is not naturally present in hair (above 13C natural abundance levels), was used for calibration and comparative validation applying cortisol versus 13C3-labeled cortisol. Cortisol was extracted from 20 mg hair (standard sample amount) applying an optimized single step extraction protocol. An LC–MS/MS method was developed for the quantitative analysis of cortisol using either cortisol or 13C3-cortisol as calibrators and D7-cortisone as internal standard (IS). The two methods (cortisol/13C3-labeled cortisol) were validated in a concentration range up to 500 pg/mg and showed good linearity for both analytes (cortisol: R2  = 0.9995; 13C3-cortisol R2  = 0.9992). Slight differences were observed for limit of detection (LOD) (0.2 pg/mg/0.1 pg/mg) and limit of quantification (LOQ) (1 pg/mg/0.5 pg/mg). Precision was good with a maximum deviation of 8.8% and 10% for cortisol and 13C3-cortisol respectively. Accuracy and matrix effects were good for both analytes except for the quality control (QC) low cortisol. QC low (2.5 pg/mg) showed matrix effects (126.5%, RSD 35.5%) and accuracy showed a deviation of 26% when using cortisol to spike. These effects are likely to be caused by the unknown amount of endogenous cortisol in the different hair samples used to determine validation parameters like matrix effect, LOQ and accuracy. No matrix effects were observed for the high QC (400 pg/mg) samples. Recovery was good with 92.7%/87.3% (RSD 9.9%/6.2%) for QC low and 102.3%/82.1% (RSD 5.8%/11.4%) for QC high. After successful validation the applicability of the method could be proven. The study shows that the method is especially useful for determining low endogenous cortisol concentrations as they occur in cow hair for example.
Keywords: Hair; Stress; Endogenous cortisol; 13C3-labeled cortisol; LC–MS/MS;

Qualitative and Quantitative Drug residue analyses: Florfenicol in white-tailed deer (Odocoileus virginianus) and supermarket meat by liquid chromatography tandem-mass spectrometry by Shanoy C. Anderson; Seenivasan Subbiah; Angella Gentles; Galen Austin; Paul Stonum; Tiffanie A. Brooks; Chance Brooks; Ernest E. Smith (73-79).
A method for confirmation and detection of Florfenicol amine residues in white-tailed deer tissues was developed and validated in our laboratory. Tissue samples were extracted with ethyl acetate and cleaned up on sorbent (Chem-elut) cartridges. Liguid chromatography (LC) separation was achieved on a Zorbax Eclipse plus C18 column with gradient elution using a mobile phase composed of ammonium acetate in water and methanol at a flow rate of 300 μL/min. Qualitative and quantitative analyses were carried out using liquid chromatography − heated electrospray ionization(HESI) and atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry in the multiple reaction monitoring (MRM) interface. The limits of detection (LODs) for HESI and APCI probe were 1.8 ng/g and 1.4 ng/g respectively. Limits of quantitation (LOQs) for HESI and APCI probe were 5.8 ng/g and 3.4 ng/g respectively. Mean recovery values ranged from 79% to 111% for APCI and 30% to 60% for HESI. The validated method was used to determine white-tailed deer florfenicol tissue residue concentration 10-days after exposure. Florfenicol tissue residues concentration ranged from 0.4 to 0.6 μg/g for liver and 0.02-0.05 μg/g for muscle and a trace in blood samples. The concentration found in the tested edible tissues were lower than the maximum residual limit (MRL) values established by the federal drug administration (FDA) for bovine tissues. In summary, the resulting optimization procedures using the sensitivity of HESI and APCI probes in the determination of florfenicol in white-tailed deer tissue are the most compelling conclusions in this study, to the extent that we have applied this method in the evaluation of supermarket samples drug residue levels as a proof of principle.
Keywords: Chromatography; Florfenicol; White-tailed deer; LC–MS/MS; Drug residue florfenicol amine;

The urinary and plasma metabonomics method based on a Agilent-1200 LC system coupled to an Agilent-6410 mass spectrometry (HPLC⿿MS/MS) had been established to investigate the anti-inflammatory activity of Yi-Guan-Jian (YGJ) decoction and explore its potential anti-inflammatory mechanism. Rat acute inflammation was induced by subcutaneous injection of carrageenan in hind paws. Multivariate statistical approaches, such as principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA) and XCMS online software were used to distinguish normal control group (NG), model group (MG), aspirin-treated group (AG) and Yi-Guan-Jian decoction group (YGJ), aimed at finding out the potential biomarkers. There was a clear separation among the four groups in PCA model. Twenty-five potential biomarkers had been identified using PCA, PLS-DA and XCMS online software. Lastly, we had an enrichment for the related metabolic pathways and screened out the pathways that influence the organism a lot in MetPA, then five mainly metabolism: tryptophan metabolism, lipid metabolism, oxidative stress, glyoxylate and dicarboxylate metabolism, taurine and hypotaurine metabolism were found. In this study, YGJ showed good anti-inflammatory effects and it could suppress the changes of pathologic inflammatory cytokines of carrageenan-induced rat paw edema (CIE). There might be a correlation between these results and the regulation of the disturbed metabolites in urine and plasma. This study demonstrates that metabonomics is a powerful methodology to gain insight into the mechanisms of traditional Chinese medicine (TCM) formula in therapy.
Keywords: Metabonomics; HPLC⿿MS/MS; Carrageenin-induced rat paw edema; Yi-Guan-Jian decoction; Traditional Chinese medicine;

Separation of pegylated recombinant proteins and isoforms on CIM ion exchangers by Jernej Gašperšič; Aleš Podgornik; Petra Kramberger; Marko Jarc; Janez Jančar; Mirjan Žorž; Nika Lendero Krajnc (91-96).
Protein pegylation is a process of covalent attachment of a polyethylene glycol (PEG) group to the protein tertiary structure that can “mask” the agent from the immune system and also increases the hydrodynamic size of the agent. Usually the pegylation prolongs the protein stability in the organism due to reduced renal clearance and provides superior water solubility to hydrophobic molecules. The mono-pegylated form of protein is usually prefered for medical applications. Different conditions with different PEG reagents have to be tested to find optimal pegylation procedure with specific protein.The goal of this study was to prepare screening method for separation of random mono-pegylated protein. Cytochrome C and beta lactoglobulin were pegylated with four reagents and a complete screening of several chromatographic monoliths in ion exchange mode with different buffers was performed to optimaly separate each mono-pegylated protein.The screening method was developed that produces optimal separation of target pegylated protein on CIM monoliths. Because of short chromatographic run time, CIM monoliths are perfect candidates to test alot of parameters.The results obtained show that each protein has its own unique separation parameters (pH, ionexchange ligand, buffer type). Two biopharmaceuticals were isolated using protocol: super human leptin antagonist (SHLA) was purified from inclusion bodies and mono-pegylated super mouse leptin antagonist (SMLA) from pegylated mixture. During study it was observed that the convective interaction media (CIM) monoliths additionally discriminate between protein isoforms pegylated on different sites in 3D structure of the protein.
Keywords: Monolith; Ion exchange; Pegylating; Purification;

Screening of nerve agent markers with hollow fiber-chemosorption of phosphonic acids by Karin Höjer Holmgren; Tomas Gustafsson; Anders Östin (97-105).
This report describes a method developed for extracting nerve gas markers such as phosphonic acids from urine and other aqueous samples. It involves single-step microextraction with chemosorption to hollow fibers that have been pre-soaked in a solution containing a derivatization reagent (3,5 triflouro methyl benzene diazomethane). The derivatives it forms with phosphonic acids can be sensitively detected by mass spectrometric detectors operating in negative chemical ionization (NCI) mode. Limits of quantification obtained in analyses of water and urine extracts by GC/MS in negative chemical ionization and selected ion monitoring mode were 0.1–10 and 0.5–10 ng/mL, respectively. Pentaflourophenyl diazomethane can also be used as a derivatization reagent, and the micro-extracts (which generate low background signals) can be sensitively analyzed by GC–MS/MS in NCI selected reaction monitoring (SRM) mode, using two specific transitions for both reagents. Thus, this sensitive approach can be flexibly modified to obtain confirmatory information, or address potential problems caused by interferences in some samples.
Keywords: GC/MS; Derivatization; Microextraction; Hollow fiber; Phosphonic acids; Nerve gas markers; Urine;

Oncrasins are a class of RNA polymerase II inhibitors. Oncrasin-72 is an indole-3 carbinol analog that has shown to inhibit growth and induce the cell death of various human cancer cell lines. Oncrasin-266, a prodrug of oncrasin-72, has been shown to have improved pharmacokinetic properties and safety than Oncrasin-72. With respect to the potential therapeutic advantages of this class of compounds, there is a need for further preclinical assessment for future clinical trials. The development of and validation of an analytical method is essential for the quantification of oncrasins in biological fluids for pharmacokinetic studies. This study focuses on the HPLC–MS/MS method development and validation of oncrasin-266, oncrasin-72 and its aldehyde metabolite in rat plasma. Blank rat plasma, coupled with 1-(3-chlorobenzyl)-1H-indole, as internal standard, was used for generating standard curves ranging from 1 to 250 ng/mL for oncrasin-266 and oncrasin-72; and 0.5–125 ng/mL for the aldehyde metabolite. The chromatographic separation was achieved by a Zorbax 300SB-C18 HPLC column at 50°C with a flow rate of 1.1 mL/min under gradient elution. Mass detection was performed under positive ionization electrospray. Intra- and inter-day accuracy and precision of the assay were less than 10%. We report a simple, specific and reproducible HPLC–MS/MS method for the quantification of oncrasins in rat plasma. This study was successfully used for the quantification of oncrasins in rat plasma for pharmacokinetic studies in three dose groups of 10, 25, and 50 mg/kg via intravenous administration.
Keywords: LC–MS/MS; Pharmacokinetics; Oncrasin; Metabolite; Prodrug;

This paper presents the trouble-shooting for a very unusual stability case. Tetracaine was found unstable in neat solutions only at high concentrations, but not at low concentrations. Moreover, its stable-isotope labeled internal standard did not show similar behavior. A series of trouble-shooting experiments were conducted to uncover the root cause. Some generally applicable precautions/insights can be drawn from this investigation to avoid potential stability issues during bioanalytical method development and validation.
Keywords: Tetracaine; Stability; Stock solution; LC–MS; Bioanalysis;

Metabolic characterization of pyrotinib in humans by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry by Yunting Zhu; Liang Li; Ge Zhang; Hong Wan; Changyong Yang; Xingxing Diao; Xiaoyan Chen; Lianshan Zhang; Dafang Zhong (117-127).
Pyrotinib is a novel irreversible tyrosine kinase inhibitor developed for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. The results of phase I clinical trial demonstrated that pyrotinib was well tolerated and exhibited potent antitumor activity. As a promising therapeutic agent for HER2-positive breast cancer, it is of great importance to investigate the biotransformation of pyrotinib in humans and identify the major enzymes involved in its metabolism during its early stage of development for safety consideration. For this purpose, a robust analytical method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was established to characterize the metabolites of pyrotinib in human plasma, feces, and urine, and identify the primary enzymes responsible for its metabolism. As a result, a total of 24 metabolites were identified, including 16 phase I metabolites resulting from dealkylation, oxidation, dehydrogenation, and carbonylation, and 8 phase II metabolites originating from cysteine and N-acetylcysteine conjugation. Pyrotinib was absorbed into blood by 1 h, reached its peak level at 4 h, and afterwards underwent slow elimination. The principal metabolites detected in humans (M1, M2, and M5) were products resulting from O-depicoline and pyrrolidine lactam formation, whose structures have been confirmed by the synthetic references. In addition, fecal clearance was the major route of excretion for pyrotinib. Further phenotyping experiment proved that CYP3A4 was the most active enzyme responsible for the biotransformation of pyrotinib, implying the vital necessity of the assessment of the potential CYP3A-mediated drug–drug interactions in humans. Taken together, this study provided valuable metabolic data to explicate the dynamic process of pyrotinib in humans, and important reference basis for its safety evaluation and rational clinical application. The results will also benefit the assessment of the contributions to the overall activity or toxicity from the key metabolites.
Keywords: Pyrotinib; UPLC/Q-TOF MS; Metabolism; Human; CYP3A4; Drug–drug interaction;

Display OmittedIn this work the habitual behaviour of low pH in environmental organic trace analysis is challenged by investigating the full potential of building a multi-component UHPLC-ESI–MS/MS method adapted to cover common emerging contaminants of many different polarities, minimizing the elements of compromise in the performance of the final analytical separation and detection. Contributes have been made by taking advantage of common commercially available technology in understanding the impact from solvent components and the ionization of analytes which can facilitate future development of robust, sensitive and precise UHPLC–MS/MS methods. All contaminants were evaluated and optimized without prejudices regarding historical residence in terms of chromatographic conditions and ESI mode; increasing multi-method's flexibility that can be implemented in routine analysis in response to new requests as well as to emerging contaminants yet to be discovered. Our data strongly supports the questioning of the assumption that equilibrium concentrations of ions in solution reflect those produced during the electrospray process. ESI responses of [M+H]+ and limits of detection were comparable, or often better at high pH compared to acidic eluents. Presence of nitrogen basic groups such as tertiary and secondary amines in a compound increased the intensity of the ESI+ signal, and was even further elevated in basic eluent. The proton affinity probably changes for many nitrogen-containing compounds during the ionization process, making the gas-phase processes very important in generation of these ions by ESI+. There were also an unexpected large number of compounds showing their highest response at pH 7 and weak ionic strength. A flow optimized, buffert free, neutral UHPLC–MS/MS method enhanced the sensitivity for the environmental important synthetic hormone ethinyl estradiol significantly.

The repair of DNA mediated by O6-alkylguanine-DNA alkyltransferase (AGT) provides protection against DNA damage from endogenous or exogenous alkylation of the O6 position of guanine. However, this repair acts as a double-edged sword in cancer treatment, as it not only protects normal cells from chemotherapy-associated toxicities, but also results in cancer cell resistance to guanine O6-alkylating antitumour agents. Thus, AGT plays an important role in predicting the individual susceptibility to guanine O6-alkylating carcinogens and chemotherapies. Accordingly, it is necessary to establish a quantitative method for determining AGT activity with high accuracy, sensitivity and practicality. Here, we describe a novel nonradioactive method for measuring AGT activity using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI⿿MS/MS). This method is based on the irreversibility of the removal of the O6-alkyl group from guanine by AGT and on the high affinity of O6-benzylguanine (O6-BG) as an AGT substrate. HPLC-ESI⿿MS/MS was used to measure the AGT activities in cell protein extracts from eight tumour lines, demonstrating that AGT activity was quite variable among different cell lines, ranging from nondetectable to 1021 fmol/mg protein. The experiments performed in intact tumour cells yielded similar results but exhibited slightly higher activities than those observed in cell protein extracts. The accuracy of this method was confirmed by an examination of AGT expression levels using western blotting analysis. To our knowledge, this method is the first mass spectrometry-based AGT activity assay, and will likely provide assistance in the screening of cancer risk or the application of chemotherapies.
Keywords: O6-alkylguanine-DNA alkyltransferase; AGT activity; O6-benzylguanine; Tumour cells; HPLC-ESI⿿MS/MS;

Profiling and identification of metabolites of isorhynchophylline in rats by ultra high performance liquid chromatography and linear ion trap Orbitrap mass spectrometry by Jianwei Wang; Peng Qi; Jinjun Hou; Yao Shen; Bingpeng Yan; Qirui Bi; RuiHong Feng; Xiaojian Shi; Min Yang; Wanying Wu; De-an Guo (147-156).
The searching of potentially bioactive metabolites in the biological body is an interesting and meaningful work for the drug study. However, the structural clarification of possible metabolites is one of the most challenging tasks in drug metabolism studies because of the variety of metabolic reactions and complexity of metabolites in vivo. Here, an ultra high performance liquid chromatography/linear ion trap-Orbitrap mass spectrometry (U-HPLC/LTQ-Orbitrap-MS) with combination of data post-processing techniques, including extracted ion chromatogram (EIC) and multiple mass defect filters (MMDF), was established for profiling and identification of metabolites of isorhynchophylline (IR) in vivo and in vitro, and the possible metabolic pathways were subsequently proposed after the oral dose of 20 mg/kg; A total of 47 metabolites of IR were tentatively identified, including 47, 21, 18, and 25 metabolites in rat urine, plasma, liver and rat liver microsomes (RLM) samples, respectively. To our knowledge, most of them were reported for the first time. Seven metabolic pathways, including dehydrogenation, oxidation, hydrolysis, reduction, demethylation, hydroxylation and glucuronide conjugation were involved in the metabolism. Among them, dehydrogenation, hydrolysis, hydroxylation and oxidation were considered as the main metabolic pathway of metabolism according to metabolic profile of in vivo and in vitro. The relative percentage of each metabolite and main metabolite types were also determined to better understand the metabolic behavior of IR in rats. The newly discovered IR metabolites significantly expanded our understanding and were going to be greatly helpful for the further pharmacokinetic study of IR in vivo.
Keywords: Isorhynchophyllin; UHPLC/LTQ-Orbitrap-MS; Metabolic profile; Metabolites;

A Previous metabolomics study has demonstrated that tyrosine metabolism might be disrupted by treating with Semen Strychni on the cell nephrotoxicity model. To investigate the relationship between Semen Strychni alkaloids (SAs) and endogenous tyrosine, tyramine under the nephrotoxicity condition, an HILIC-ESI-MS/MS based analytical strategy was applied in this study. Based on the established Semen Strychni nephrotoxicity cell model, strychnine and brucine were identified and screened as the main SAs by an HPLC-Q Exactive hybrid quadrupole Orbitrap mass system. Then, a sensitive HILIC-ESI-MS/MS method was developed to simultaneously monitor strychnine, brucine, tyrosine and tyramine in cell lysate. The analytes were separated by a Shiseido CAPCELL CORE PC (150 mm ÿ 2.1 mm, 2.7 μm) HILIC column in an acetonitrile/0.1% formic acid gradient system. All the calibration curves were linear with regression coefficients above 0.9924. The absolute recoveries were more than 80.5% and the matrix effects were between 91.6%⿿107.0%. With the developed method, analytes were successfully determined in cell lysates. Decreased levels of tyrosine and tyramine were observed only in combination with increased levels of SAs, indicating that the disturbance of tyrosine metabolism might be induced by the accumulation of SAs in kidney cell after exposure of Semen Strychni. The HILIC-ESI-MS/MS based analytical strategy is a useful tool to reveal the relationships between the toxic herb components and the endogenous metabolite profiling in the toxicity investigation of herb medicines.
Keywords: HILIC-ESI-MS/MS; Nephrotoxicity; Semen Strychni alkaloids; Tyrosine;

A rapid and sensitive UHPLC–MS/MS assay for the determination of trelagliptin in rat plasma and its application to a pharmacokinetic study by Xiao-xia Hu; Tian Lan; Zhe Chen; Cheng-cheng Yang; Peng-fei Tang; Ling-jing Yuan; Guo-Xin Hu; Jian-Ping Cai (166-171).
This study aims to develop and validate a simple, rapid and sensitive ultra-performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method for exploring pharmacokinetic characteristics of trelagliptin. Protein precipitation by acetonitrile was used to prepare plasma sample. A RRHD Eclipse Plus C18 (2.1 × 50 mm, 1.8 μ) column with gradient mobile phase (containing acetonitrile and 0.1% formic acid) help to achieve the separation of trelagliptin and carbamazepine (IS) with high selectivity. Detection of target fragment ions m/z 358.2 → 133.9 for trelagliptin, and m/z 237.1 → 194.0 for IS was performed in positive-ion electrospray ionization mass spectrometry by multiple reaction monitoring. Linear calibration plots were achieved in the range of 5–4000 ng/mL for trelagliptin (R2  = 0.999) in rat plasma. The recovery of trelagliptin ranged from 87.8% to 93.7%. The method was showed to be accurate, precise and stable. No obvious matrix effect was found. It has been fully validated and successfully applied to pharmacokinetic study of trelagliptin.
Keywords: Trelagliptin; UHPLC–MS/MS; Pharmacokinetics; Rat plasma;

A simple and fast multiresidue extraction and purification method was developed for the determination of 61 veterinary drugs, belonging to seven classes, in milk and milk powder. The extraction depends on the acetonitrile solvent, followed by a single step to remove lipids with fatty acid chains using a new reversed phase SPE without traditional pre-equilibration and washing steps before eluting SPE. The purifying lipid effect of the present preparation method was evaluated by comparing the response changes of ion peak areas of the milk endogenous metabolites before and after SPE treatment using ultra-fast LC coupled to tandem quadrupole and TOF MS. Subsequently, UPLC coupled to tandem quadrupole MS was performed for the quantitative analysis of milk and milk powder samples spiked with 61 veterinary drugs, including β-lactam, macrolide, amide alcohol, forest amine, sulfanilamide, tetracyclines, and quinolones antibiotics. This method is very simple, fast, sensitive, and selective, and allows the good recoveries of all compounds, with a recovery range of 61.5⿿118.6%, and coefficients of variation of less than 11.6%. The 61 compounds behave in the dynamic range 0.01⿿200 μg kg⿿1, with correlation coefficient >0.99. The limits of quantification for the analytes are in the range 0.01⿿5.18 μg kg⿿1. Finally, this method has been successfully applied to the screening of veterinary drugs in 50 commercial bovine milk and milk powder samples, and ceftiofur and ciprofloxacin were detected in some brand samples.
Keywords: Veterinary drugs; Multiresidue extraction method; Milk and milk powder; UPLC⿿MS/MS;

Fructus Gradeniae, the fruit of Gardenia jasminoides Ellis, was used alone or in combination with other herb medicines in the treatment of type 2 diabetes mellitus in China for a long time. In present investigation, the HPLC method for the determination of geniposide in rat plasma was developed and validated, and the pharmacokinetics of geniposide in type 2 diabetic rats after oral administration of Fructus Gradeniae extract or pure was studied. The results showed that the pharmacokinetic profile (especially the area under the plasma concentration-time curve, AUC) of geniposide in type 2 diabetic rats after orally administered with Fructus Gradeniae extract or pure geniposide was remarkably different from that in normal rats. The results indicated that the increased AUC of geniposide in type 2 diabetic rats did not result from the effects of other components contained in Fructus Gradeniae. It could be speculated that the increased AUC of geniposide might result from the pathological state of type 2 diabetes mellitus which resulted in the pharmacokinetic alterations of geniposide.
Keywords: Geniposide; Pharmacokinetics; Type 2 diabetes; Fructus Gradeniae (Gardenia jasminoides Ellis);

Development of a UPLC–MS/MS method for the determination of lomefloxacin in rabbit aqueous humor and its application to a pharmacokinetic study by Shiwen Song; Dongyang Zhao; Jing Sun; Qiuyan Miao; Xiaojie Liu; Yanling Wang; Lu Zhong; Meng Xu; Peng Zhang (187-192).
Lomefloxacin is a kind of synthetic fluoroquinolone antibiotic, which is used for the treatment of infectious diseases. In this study, a rapid and efficient liquid chromatography–tandem mass spectrometric assay was developed to determine the concentration of lomefloxacin in rabbit aqueous humor quantitatively. Aqueous humor samples were extracted by protein precipitation. Ofloxacin was chosen as internal standard. The chromatographic separation was achieved on a Kinetex C18 (50 mm × 2.10 mm, 2.6 μm, Phenomenex Corp, USA) column, with a gradient of methanol (0.1% formic acid) and water (0.1% formic acid). Multiple reaction monitoring (MRM) with positive ionization mode was used for the mass analysis.The validation of this method was based on the European Medicines Agency (2011) [1] and US FDA Guidelines (2001) [2]. The calibration range of aqueous humor samples was 5–1200 ng/mL with r  = 0.9990 (n  = 6). For all QC samples, Inter-and intra-run precisions were less than 15% and accuracies were between 80%–120%. In conclusion, the assay was rapid, sensitive and able to determinate the lomefloxacin in rabbit aqueous humor accurately. At the same time, this method was successfully applied to study the pharmacokinetics of lomefloxacin hydrochloride eye drops and lomefloxacin hydrochloride ophthalmic gel in rabbit aqueous humor.
Keywords: Lomefloxacin; Eye drops; Rabbit aqueous humor; UPLC–MS/MS; Pharmacokinetics;

Cytochrome P450 3A (CYP) enzymes are involved in the elimination of many drugs and are known to be regulated by several environmental factors. Thus, it was the aim of this study to develop and validate an analytical method allowing estimation of the hepatic CYP3A enzyme activity using the 4-hydroxycholesterol to cholesterol ratio as an endogenous biomarker in serum. Both compounds were isolated from the biological matrix by liquid–liquid extraction using n-hexane after saponification with ethanolic sodium methoxide solution (2 M) to cleave the steroids from their esterified forms without any kind of further derivatization. Chromatographic separation was achieved on a reversed-phase column (SupelcoAcsentis®, C8) within 7 min using an isocratic elution with ammonium acetate 5 mM (pH = 3.8, 10%) and acetonitrile (90%) at a flow rate of 300 μl/min. d6-cholesterol and d7-4β-hydroxycholesterol were used as internal standards. Detection was done on a triple quadrupole mass spectrometer using the following mass transitions: 369.3/161.5, 369.3/147.1 and 369.3/95.2 for cholesterol; 385.2/367.4, 385.2/109.1 for 4-hydroxycholesterol; 374.4/152.7 and 392.2/108.9 for d6-cholesterol and d7-4-hydroxycholesterol, respectively as the internal standards.The method was validated according to current bioanalytical guidelines considering selectivity, linearity, accuracy, precision, recovery, stability. The analytical range was 5–250 and 50–1000 ng/ml, for 4-hydroxycholesterol and cholesterol, respectively.The method was shown to be selective for both compounds with good linearity over the selected range (r > 0.99) as well as good within- and between day accuracy (error: −1.2–3.7% for 4-hydroxycholesterol and −7.7–9.5% for cholesterol) and within- and between day precision (2.1–14.6% for 4-hydroxycholesterol and 1.1–14.9% for cholesterol). Recovery was found to be over 80% for both analytes while significant stability issues could not be observed. Finally, the validated assay was applied to measure 4-hydroxycholesterol and cholesterol in serum samples of clinical studies in humans and foals that could verify induction of hepatic CYP3A4 (human) and CYP3A89 (foals) after premedication with the known enzyme inducer rifampicin.
Keywords: 4β-Hydroxycholesterol; Cholesterol; LC–MS/MS; CYP3A;

Validation of a SPE-LC–MS/MS method for the determination of ketamine and norketamine in micropulverized hair after a single IV dose by Adriana S. Barreto; Vinnicius F. Brant; Eliani Spinelli; Silvana V. Rodrigues (200-209).
A SPE-LC–MS/MS method for the determination of ketamine (KET) and norketamine (NKET) was developed and validated. Extensive pulverization (25 min at 25 Hz) of previously cooled samples (5 min in liquid nitrogen) allowed for extraction in a phosphate buffer (pH 6) solution after 10 min vortex agitation at room temperature, simplifying the coupling of the extraction to an effective mixed-mode SPE (solid phase extraction) clean-up procedure. The extraction optimization was performed with samples fortified by drug incorporation according to a published procedure involving incubation of blank matrices for 16 days. The method was validated for selectivity, matrix effect, linearity, LLQ (lower limit of quantification), precision, accuracy, recovery, carryover and stability after preparation and has proven to be accurate and reliable within a range of 0.02–10 ng/mg for KET and 0.04–4 ng/mg for NKET, meeting proposed KET cutoffs for discrimination from chronic use. In addition, the method was sensitive enough to detect the drugs after unique small (1 mg/kg) intravenous doses received by patients submitted to general anesthesia before surgical procedures. Ketamine levels varied from 0.060 to 0.111 ng/mg and norketamine was positive (<LLQ) in all samples. The high sensitivity achieved with conventional 3D-ion-trap MS/MS detection is probably due to the simple but very effective proposed procedure.
Keywords: LC–MS/MS; Hair analysis; Ketamine; Norketamine; Extraction;

Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste by Renata Cristina da Penha França; Caio Rodrigo Dias Assis; Juliana Ferreira Santos; Ricardo José Soares Torquato; Aparecida Sadae Tanaka; Izaura Yoshico Hirata; Diego Magno Assis; Maria Aparecida Juliano; Ronaldo Olivera Cavalli; Luiz Bezerra de Carvalho Jr; Ranilson Souza Bezerra (210-217).
A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50 °C and 8.5, respectively. The enzyme was thermostable until 55 °C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent k m value of the purified trypsin was 0.38 mM, k cat value was 3.14 s−1, and k cat /k m was 8.26 s−1  mM−1. The catalytic proficiency of the purified enzyme was 2.75 × 1012  M−1 showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93 kcal mol−1 while the resulting rate enhancement of this reaction was found to be approximately in a range from 109 to 1010-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes.
Keywords: Rachycentron canadum; Digestive peptidase; Pyloric caecum; Fish; Trypsin;

Preparative isolation of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from Cordyceps militaris by macroporous resin and purification by recycling high-speed counter-current chromatography by Zhong Zhang; Tuernisan Tudi; Yanfang Liu; Shuai Zhou; Na Feng; Yan Yang; Chuanhong Tang; Qingjiu Tang; Jingsong Zhang (218-225).
In this study, cordycepin, N6-(2-hydroxyethyl)-adenosine (HEA) and adenosine from the fruiting bodies of Cordyceps militaris were separated by using macroporous resin NKA-II adsorption. The parameters of static adsorption were tested and the optimized conditions were as follow: the total adsorption time was 12 h, 50% ethanol was used for desorption and the desorption time was 9 h. The crude sample that was prepared by macroporous resin NKA-II contained 3.4% cordycepin, 3.7% HEA and 4.9% adenosine. Then the crude sample was further purified by recycling high-speed counter-current chromatography (HSCCC) with ethyl acetate, n-butanol, 1.5% aqueous ammonium hydroxide (1:4:5, v/v/v) as the optimized two-phase solvent system. Three nucleosides including 15.6 mg of cordycepin, 16.9 mg of HEA and 23.2 mg of adenosine were obtained from 500 mg of crude sample in one-step separation. The purities of three compounds were 98.5, 98.3 and 98.0%, respectively, as determined by high performance liquid chromatography.
Keywords: Cordyceps militaris; Macroporous resin; High-speed counter-current chromatography; Cordycepin; N6-(2-hydroxyethyl)-adenosine; Adenosine;

Ambinine (AM), one of the major hexahydrobenzo[c]phenanthridine alkaloids from Corydalis ambigua Cham. et Schlecht. var. amurensis Maxim, is considered to be an important compound because of its special content and activity. However, there are few published studies on AM metabolism. In this research, the metabolism of AM in vivo was comprehensively studied for the first time. A total 44 metabolites (including 13 phase I and 31 phase II metabolites) as well as its parent drug in plasma, bile, urine and feces of rats were identified and 41 of them were reported for the first time. The results obtained indicated that demethylation, sulfation and glucuronidation were the major metabolic pathways of AM in vivo. Furthermore, this study provides valuable and new information about the metabolism of AM, which will be very helpful for understanding the safety and efficacy of AM, as well as its analogues.
Keywords: Ambinine; Metabolites; UPLC-Q/TOFMS;

Development and validation of a LC–MS/MS method for the quantification of tenofovir and emtricitabine in seminal plasma by S.M. Illamola; E. Valade; D. Hirt; E. Dulioust; Y. Zheng; J.P. Wolf; J.M. Tréluyer (234-241).
Accurate and sensitive liquid-chromatography tandem mass spectrometry method for the quantification of tenofovir and emtricitabine in seminal plasma has been developed and full validated. Molecules were separated by high-performance liquid chromatography on an Atlantis T3 C18 column using a gradient of deionized water and methanol, including 0.05% formic acid (250 μl/min) and detected by electrospray ionisation/tandem mass spectrometry in positive ion mode. The method was validated over a clinical range of 3.13–1000 ng/mL for tenofovir and 6.25–2000 ng/mL for emtricitabine. Inter and intra-assay precisions were <9.37% for tenofovir and<10.88% for emtricitabine, and accuracies were between 0.48% and 8.43% for tenofovir, and between 0.64% and 13.87% for emtricitabine. The developed method was successfully applied for analysing tenofovir and emtricitabine concentrations in seminal plasma samples from a clinical study. The use of tandem mass spectrometry can be a suitable method for the analysis of this kind of matrices, providing high sensitivity and specificity to the analysis.
Keywords: Tenofovir; Emtricitabine; Seminal plasma; LC–MS/MS;

A rapid, sensitive and selective ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC⿿MS/MS) method was developed and validated for simultaneous determination of ten Aconitum alkaloids in rat tissues. The tissue samples were prepared by a simple procedure protein precipitation with acetonitrile containing 0.1% acetic acid and separated on an Agilent XDB C18 column (4.6 mm ÿ 50 mm, 1.8 μm) using gradient elution with a mobile phase consisting of water and acetonitrile (both containing 0.1% formic acid) at a flow rate of 0.3 mL/min. The quantitive determination was performed on an electrospray ionization (ESI) triple quadrupole tandem mass spectrometer using selective reaction monitoring (SRM) under positive ionization mode. The established method was fully validated according to the USA Food and Drug Administration (FDA) bioanalytical method validation guidance and the results demonstrated that the method was sensitive and selective with the lowest limits of quantification (LLOQ) at 0.025 ng/mL in rat tissue homogenates. Meanwhile, the linearity, precision, accuracy, extraction recovery, matrix effect and stability were all within the required limits of biological sample analysis. After method validation, the validated method was successfully applied to the tissue distribution study on the compatibility of Heishunpian (HSP, the processed product of Aconitum carmichaelii Debx) and Fritillariae thunbergii Bulbus (Zhebeimu, ZBM). The results indicated that the distribution feature of monoester diterpenoid aconitines (MDAs), diester diterpenoid aconitines (DDAs) and non-ester alkaloids (NEAs) were inconsistency, and the compatibility of HSP and ZBM resulted in the distribution amount of DDAs increased in tissues. What⿿s more, the results could provide the reliable basis for systematic research on the substance foundation of the compatibility of the herbal pair.
Keywords: Heishunpian; Aconitum alkaloids; UHPLC⿿MS/MS; Tissue distribution;

Identification of in vitro and in vivo metabolites of alantolactone by UPLC-TOF-MS/MS by Donggui Yao; Zhe Li; Changhong Huo; Yufang Wang; Yibing Wu; Manli Zhang; Ligeng Li; Qingwen Shi; Hiromasa Kiyota; Xiaowei Shi (250-260).
Alantolactone (AL), an active sesquiterpene originating from Inula helenium, is a potential anticancer and anti-inflammatory agent. However so far, studies on AL metabolism have not been reported. In the present study, we have investigated for the first time the in vivo and in vitro metabolites of AL using ultra performance liquid chromatography combined with time of flight mass spectrometry (UPLC-TOF-MS/MS). A unique on-line information-dependent acquisition (IDA) method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was applied to trace all of the probable metabolites of AL. Five MMDF templates were set according to the core structure of AL and the general metabolite biotransformation patterns, and other five sulfur-containing dimer filter templates were first established on the basis of structural elucidation of AL metabolites obtained from rat intestinal bacteria biotransformation. As a result, 44 metabolites were characterized: 41 metabolites from rat urine, bile and feces after oral administration of AL, and 13 metabolites from AL biotransformation by rat intestinal bacteria. Particularly, 26 metabolites were identified as novel sulfur-containing products. The results indicated that addition of double bond at ο(11,13) and oxidization were the main metabolic reactions of AL. A new metabolism pathway to produce addition products of H2S to AL and further generate a series of sulfur-containing dimers of AL was revealed. This study significantly enriched our knowledge about AL metabolism, which will lead to a better understanding of the safety and efficacy of AL. At the same time, the established methodology can be widely applied for the structural determination of the metabolites of other sesquiterpene containing α-methylene-γ-lactone moiety.
Keywords: Alantolactone; Intestinal bacteria biotransformation; UPLC-TOF-MS/MS; Multiple mass defect filter; Metabolites;

A simple, rapid and sensitive RP-HPLC-UV method for the simultaneous determination of sorafenib & paclitaxel in plasma and pharmaceutical dosage forms: Application to pharmacokinetic study by Ismail Khan; Zafar Iqbal; Abad Khan; Muhammad Hassan; Fazle Nasir; Abida Raza; Lateef Ahmad; Amjad Khan; Muhammad Akhlaq Mughal (261-270).
A simple, economical, fast, and sensitive RP-HPLC-UV method has been developed for the simultaneous quantification of Sorafenib and paclitaxel in biological samples and formulations using piroxicam as an internal standard. The experimental conditions were optimized and method was validated according to the standard guidelines. The separation of both the analytes and internal standard was achieved on Discovery HS C18 column (250 mm ÿ 4.6 mm, 5 μm) using Acetonitrile and TFA (0.025%) in the ratio of (65:35 V/V) as the mobile phase in isocratic mode at a flow rate of 1 ml/min, with a wavelength of 245 nm and at a column oven temperature of 25 °Cin a short run time of 12 min. The limits of detection (LLOD) were 5 and 10 ng/ml while the limits of quantification (LLOQ) were 10 and 15 ng/ml for sorafenib and paclitaxel, respectively. Sorafenib, paclitaxel and piroxicam (IS) were extracted from biological samples by applying acetonitrile as a precipitating and extraction solvent. The method is linear in the range of 15⿿20,000 ng/ml for paclitaxel and 10⿿5000 ng/ml for sorafenib, respectively. The method is sensitive and reliable by considering both of its intra-day and inter-day co-efficient of variance. The method was successfully applied for the quantification of the above mentioned drugs in plasma. The developed method will be applied towards sorafenib and paclitaxel pharmacokinetics studies in animal models.
Keywords: RP-HPLC; Sorafenib; Paclitaxel; Biological samples; Pharmacokinetics;

A novel microflow LC⿿MS method for the quantitation of endocannabinoids in serum by Jay S. Kirkwood; Corey D. Broeckling; Seth Donahue; Jessica E. Prenni (271-277).
Endocannabinoids (ECs) represent a class of endogenous, small molecules that bind and activate the G-protein coupled EC receptors. They are involved in a variety of fundamental biological processes and are associated with many disease states. Endocannabinoids are often present in complex matrices and at low concentrations, complicating their measurement. Here we describe a highly sensitive method for the quantitation of the following ECs in serum: N-arachidonoylethanolamine (anandamide), N-oleoylethanolamine, N-palmitoylethanolamine, 2-arachidonoylglycerol, and its inactive isomer 1-arachidonoylglycerol. On-line sample trapping coupled with separation via microflow liquid chromatography and detection by tandem quadrupole mass spectrometry results in the necessary sensitivity for accurate quantitation of ECs in less than 50 μL of serum, without the need for off-line solid phase extraction. Limits of quantitation between 1.2 and 13.4 pg/mL were achieved, representing a significant increase in sensitivity compared to previous methods using analytical flow rates. An additional benefit of microflow chromatography is the reduction of solvent consumption by more than two orders of magnitude. The experimental utility of the assay is demonstrated through the analysis of serum from hibernating bears to assess seasonal changes in circulating EC concentrations.
Keywords: Endocannabinoids; Microflow; Chromatography; SRM; Quantitation; LC⿿MS; Serum; Sensitivity;

Apolipoprotein F (APO-F) is a novel low abundance liver fibrosis biomarker and its concentration decreases in human serum and plasma across liver fibrosis stages. Current antibody based assays for APO-F suffer from limitations such as unspecific binding, antibody availability and undetectable target if the protein is degraded; and so an antibody-free assay has the potential to be a valuable diagnostic tool. We report an antibody-free, rapid, sensitive, selective and robust LC⿿MS/MS (MRM and MRM3) method for the detection and quantitation of APO-F in healthy human plasma. With further analysis of clinical samples, this LC⿿MS based method could be established as the first ever antibody-free biomarker assay for liver fibrosis. We explain the use of Skyline software for peptide selection and the creation of a reference library to aid in true peak identification of endogenous APO-F peptides in digests of human plasma without protein or peptide enrichment. Detection of a glycopeptide using MRM-EPI mode and reduction of interferences using MRM3 are explained. The amount of APO-F in human plasma from a healthy volunteer was determined to be 445.2 ng/mL, the coefficient of variation (CV) of precision for 20 injections was <12% and the percentage error of each point along the calibration curve was calculated to be <8%, which is in line with the assay requirements for clinical samples.
Keywords: Apolipoprotein F; Multiple reaction monitoring; Multiple reaction monitoring cubed; Liquid chromatography⿿mass spectrometry; Skyline; Liver fibrosis;

Electrospinning technique was used to convert polydimethyl siloxane (PDMS) sol-gel solution to a new nanostructure on a stainless steel wire. The surface morphology of the fiber was observed by scanning electron microscopy (SEM). It showed a diameter range of 30⿿60 nm for PDMS nanoparticles with a homogeneous and porous surface structure. The applicability of this coating was assessed for the headspace SPME (HS-SPME) of benzene, toluene, ethylbenzene and xylenes (BTEX) from water samples followed by gas chromatography⿿mass spectrometry. The important parameters affecting extraction efficiency such as extraction time and temperature, desorption conditions, agitation rate and ionic strength were investigated and optimized.Under the optimized conditions, LODs and LOQs of 0.3⿿5 μgL⿿1 and 1⿿10 μgL⿿1 were obtained, respectively. The method showed linearity in the broad range of 1⿿5000 μgL⿿1 with correlation coefficient of >0.99. Inter-day and intra-day precisions of the developed method ranged from 2.43% to 6.54% and from 5.24% to 13.73%, respectively. The thermal stability of the fiber was investigated on stainless steel wire. It was found to be durable at 260 °C for more than 360 min. Furthermore, the proposed method was successfully applied for quantification of BTEX in real water samples.
Keywords: Polydimethylsiloxane; Electrospray; Solid phase microextraction; BTEX; Gas chromatography⿿mass spectrometry;

Tentative identification of in vitro metabolites of 5-APDB, a synthetic benzofuran, by LC-Q/TOF-MS by Jong Suk Park; Shaheed Ur Rehman; In Sook Kim; Min Sun Choi; Jae Sin Lee; Sanghwan In; Hye Hyun Yoo (296-300).
5-(2-Aminopropyl)-2,3-dihydrobenzofuran (5-APDB) is a designer drug of phenethylamine and amphetamine class. In this study, the in vitro metabolism of 5-APDB was investigated in rat and human liver microsomes and human hepatocytes to characterize its metabolites. 5-APDB was incubated with microsomes or hepatocytes, and the reaction mixture was analyzed using liquid chromatography-quadrupole time-of-flight with tandem mass spectrometry (LC-Q/TOF-MS). 5-APDB was metabolized to yield three metabolites (M1, M2 and M3). These metabolites were structurally characterized on the basis of accurate mass analysis and MS/MS fragmentation patterns. Metabolite M1 and M2 were identified as hydroxylated metabolites in the benzofuran moiety; M3 was a reduced metabolite which may be generated from M1 or M2 via dehydration. These results provide evidence for the in vivo 5-APDB metabolism, and would be forensically useful for the detection of 5-APDB and its metabolites in biological samples.
Keywords: 5-APDB; Designer drug; Metabolite; LC-Q/TOF-MS;

Simultaneous determination of curcumin diethyl disuccinate and its active metabolite curcumin in rat plasma by LC–MS/MS: Application of esterase inhibitors in the stabilization of an ester-containing prodrug by Pahweenvaj Ratnatilaka Na Bhuket; Nuansri Niwattisaiwong; Patanachai Limpikirati; Phisit Khemawoot; Pasarapa Towiwat; Boonsri Ongpipattanakul; Pornchai Rojsitthisak (301-310).
Four esterase inhibitors, ethylenediamine tetraacetic acid disodium (Na2EDTA), sodium fluoride (NaF), bis(4-nitrophenyl) phosphate (BNPP) and phenylmethanesulfonyl fluoride (PMSF), were evaluated for their inhibitory effects on enzymatic hydrolysis of labile phenolate esters in curcumin diethyl disuccinate (CDD), a prodrug of curcumin (CUR), in rat plasma. BNPP and PMSF at 10 mM exhibited stabilization by preventing degradation of CDD. BNPP at a final concentration of 10 mM was subsequently selected to prevent ex vivo metabolism of CDD throughout LC–MS/MS analysis of CDD and CUR in rat plasma. A simple protein precipitation technique using acetonitrile as a precipitating agent was used to extract CDD, CUR and dimethylcurcumin (DMC), an internal standard, from rat plasma. Chromatographic separation was performed on a Halo C8 column (4.6 × 50 mm, 2.7 μm) using an isocratic mobile phase containing acetonitrile-0.2% formic acid in water (73:27 v/v) with a flow rate of 0.4 mL min−1. An AB SCIEX QTRAP® 6500 mass spectrometer was operated using a positive ion electrospray mode for ionization and detection of analytes and internal standard. Calibration curves for CDD and CUR were established using 50 μL of rat plasma over the concentration range of 1–500 ng mL−1. The developed method was fully validated according to US Food and Drug Administration (FDA) guidelines for selectivity, sensitivity, linearity, accuracy, precision, dilution integrity, recovery, matrix effect, and stability. The validated method was applied to evaluate the pharmacokinetics of CDD and CUR in rats after a single intravenous dose of 40 mg kg−1. The method using BNPP as an esterase inhibitor was successful in determining the remaining CDD in rat plasma. The pharmacokinetic results indicate that CDD in rats is converted instantaneously to CUR after intravenous administration and a higher CUR plasma concentration at 5 min is achieved in comparison with direct intravenous injection of CUR.
Keywords: Esterase inhibitors; Curcumin diethyl disuccinate; Curcumin; LC–MS/MS; Validation; Rat plasma;

Determination and pharmacokinetics of chinensinaphthol methyl ether in rat urine by a sensitive and specific UFLC-ESI-MS/MS method by Jia’an Qin; Jiaoyang Luo; Hongzheng Zhao; Shanshan Zhang; Xingguo Zhang; Meihua Yang (311-316).
A rapid, stable, and sensitive method based on ultra-fast liquid chromatography combined with electrospray ionization tandem mass spectrometry (UFLC-ESI-MS/MS) was established and optimized for quantification and pharmacokinetics analysis of chinensinaphthol methyl ether (CME) in rat urine. Samples were prepared by liquid phase extraction with ethyl acetate, and chromatographic separation was performed on an ACQUITY UPLC® BEH Phenyl column (2.1 × 50 mm, 1.7 μm). For gradient elution, we used a mobile phase consisting of water containing 0.1% formic acid and 5 mmol/L ammonium formate and methanol with 0.1% formic acid. The quantification was executed under multiple reaction monitoring (MRM) in positive mode. The precursor/product transition (m/z) in the positive ion mode was [M+H]+ m/z  = 395.1 → 346.1. This method was validated by evaluating specificity, linearity, matrix effects, recovery, accuracy, precision, and stability, which were all shown to be reasonable and reliable. The lower limit of quantification (LOQ) was 0.5 ng/mL, and the linear range was 0.5–100 ng/mL. The method was successfully applied to quantify and analyze the pharmacokinetics of CME in rat urine. After oral administration of a single dose of CME (5.0 mg/kg), the accumulated amount of CME excreted in urine was 162.3 ± 54.1 ng, and the terminal elimination half-life was 53.4 ± 5.3 h, indicating low CME excretion in urine and significant CME metabolism in vivo.
Keywords: Chinensinaphthol methyl ether; UFLC-ESI-MS/MS; Determination; Urine pharmacokinetics;

The serum concentration of 7α-hydroxy-4-cholesten-3-one (C4), a marker of cholesterol 7α-hydroxylase activity, has recently become an attractive diagnostic tool for researchers interested in cholesterol and bile acid metabolism. The rapidly increasing demand of C4 measurement led to the development of various fast, mostly mass spectrometry-based analytical methods. Our aim was to compare four simple (i.e., not requiring solid phase extraction) extraction procedures (two ⿿one-phase⿿, and two ⿿two-phase⿿) in terms of basic analytical performance and their labouriousness. All methods exhibited comparable extraction recoveries (ranging from 88 to 97%) and intra-assay precision (variation coefficients below 10%), and failed in the removal of phospholipids. Although marked differences were observed in desalting and deproteination, all methods can be considered satisfactory. Simple acetonitrile precipitation can be recommended if a fast extraction and minimal hands-on time is preferred; while two-phase ammonium sulphate:acetonitrile extraction should be chosen when maximal deproteination is required.
Keywords: 7α-Hydroxy-4-cholesten-3-one; Bile acids; Cholesterol 7α-hydroxylase; Mass spectrometry;

We established and validated a specific and sensitive analytical method for the determination of 1-vinyl-2-pyrrolidone (VP) as 1-vinyl-2-pyrrolidone-mercapturic acid (VPMA) in urine using an electrospray liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS) column switching method. An online solid phase extraction (SPE) for sample cleanup was performed by column switching to a restricted access material and back-flushing to the analytical column. A Phenomenex Luna C8 column was used for sample separation (150 mm; ID 4,6 mm; 3 μm). D4-VPMA served as an isotope labeled internal standard and was detected in negative multiple-reaction monitoring (MRM) mode.The Limit of quantification (LOQ) for VPMA was 1.5 μg/L, the intra-day precision of three concentrations (2 μg/L, 75 μg/L and 400 μg/L) of spiked urine samples ranged from 2.7 to 7.3%, the inter-day precision from 3.4 to 14.4%. The accuracy ranged from 6.2 to 9.0%, for the intra-day experiments and from 0.3 to 6.9% for the inter-day experiments. The method was applied to urines of Sprague-Dawley rats exposed to VP as a proof of principle of VPMA as a potential biomarker.
Keywords: 1-Vinyl-2-pyrroldione; Mercapturic acid; Biomarker; Urine; LC/MS/MS;

Development and validation of a sensitive UHPLC–MS/MS method for quantitation of prucalopride in rat plasma and its application to pharmacokinetics study by Zhi Sun; Lihua Zuo; Jian Kang; Lin Zhou; Mengmeng Jia; Zeyun Li; Zhiheng Yang; Xiaojian Zhang; Zhenfeng Zhu (328-333).
A rapid, sensitive, selective and accurate ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for the quantitation of prucalopride in rat plasma using carbamazepine as an internal standard (IS). Separation was achieved on a Waters ACQUITY UHPLC® HSS C18 column (2.1 mm × 50 mm, 1.8 μm) column with a gradient mobile phase consisting of acetonitrile–water (containing 0.1% formic acid) as mobile phase at a flow rate of 0.2 mL/min. Prucalopride and IS were monitored using positive electrospray triple quadrupole mass spectrometer (Waters Xevo TQD) via multiple reaction monitoring (MRM) mode. The monitored transitions were set at m/z 367.99 → 195.89 and m/z 236.97 → 194.04 for prucalopride and IS, respectively. The achieved lower limit of quantitation was 0.1 ng/mL. The validated method had an excellent linearity in the range of 0.1–100 ng/mL (r > 0.996). The intra- and inter-day precisions were both ≤ 7.8% for prucalopride and IS, and the average intra- and inter-day accuracies ranged from −3.0% to 8.5%. Extraction recoveries at three levels QC concentrations were in the range of 90.0–110.0% for prucalopride and 99.6% for IS. Matrix effects were found to be acceptable. The validated assay was successfully applied to a pharmacokinetic study of prucalopride following oral administration of 0.25, 0.5, 1.0 mg/kg to female and male rats respectively.
Keywords: Prucalopride; Pharmacokinetics; Rat plasma; UHPLC–MS/MS;

Extraction of polyphenols in Himanthalia elongata and determination by high performance liquid chromatography with diode array detector prior to its potential use against oxidative stress by Mariola Belda; Desiré Sanchez; Elena Bover; Beatriz Prieto; Carolina Padrón; Dolores Cejalvo; Jose Miguel Lloris (334-341).
Several studies have demonstrated the antioxidant capacity of seaweeds, which can be used for the development of biopharmaceuticals with extensive medical application. Antioxidant therapies appear to attenuate the organic deterioration originated by an excessive oxidative stress, which could prevent the harmful effects of various injuries such as ischemia-reperfusion (I/R) among others. Marine brown seaweeds play a significant role, as they are the only organisms on earth producing phlorotannins, which are polyphenols that exhibit important biological activity. To ensure obtaining an extract with the greatest antioxidant activity, some variables that affect the extraction of polyphenols are optimized, including seaweed amount, type of solvent, and time and temperature of extraction. Subsequently, the total phenolic content (TPC) and the antioxidant activity have been determined. The optimized condition was obtained for 6 g of seaweed, ethanol: water proportion of 60:40 and 2 h/60 °C, achieving 548.33 mg AG/100 g seaweed and 76% of antioxidant activity. The characterization of the extracted polyphenols was made by HPLC/DAD. 11 polyphenols were identified in the extract: Phloroglucinol, Gallic Acid, Catechin, Rutin, Gentisic Acid, Chlorogenic Acid, Caffeic Acid, Coumaric, Ferulic, Myricetin and Quercetin.
Keywords: Himanthalia elongata; Total phenolic content (TPC); Polyphenols; Reverse phase high performance liquid chromatography (RP-HPLC); 2,2-Diphenyl-1-picrylhydrazyl (DPPH); Antioxidant activity;

Applying mini-bore HPAEC-MS/MS for the characterization and quantification of Fc N-glycans from heterogeneously glycosylated IgGs by Maria Maier; Dietmar Reusch; Cees Bruggink; Patrick Bulau; Manfred Wuhrer; Michael Mølhøj (342-352).
High-performance anion-exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) is a highly sensitive method for the analysis of oligosaccharides without the need for prior derivatization. However, the method suffers from the lack of chemical information with peak assignments based on the retention times of authentic standards or known peaks of reference materials. Here we applied HPAEC coupled on-line with electrospray ion trap mass spectrometry (HPAEC-MS) using a prototype mini-bore (1 mm I.D.) CarboPac PA200 column and challenged the analytical separation based method for the structural assignment of heterogeneous mixtures of N-glycans derived from immunoglobulin G from human plasma, glyco-engineered CHO cells, and Sp2/0 mouse myeloma cells. Compared to an analytical scale 3 mm I.D. column, the mini-bore column demonstrated a superior performance with up to 8-fold improved limit of detection for specific N-glycans determined by PAD. Quantitative evaluation by extracted ion current chromatograms revealed detection limits in the 50⿿100 femtomole range using ion trap MS operated in positive ionization mode. In our hands HPAEC-MS/MS allowed the detection and quantification of even low abundant glycan species including biantennary complex-type, high mannose, hybrid and hybrid bisected structures. In comparison to the detection of N-glycans as lithiated or sodiated adducts, we obtained a 65-fold improved signal-to-noise ratio with protonated ions only. Relative quantitative evaluation by single ion current chromatograms was successfully applied and demonstrated an excellent performance with respect to selectivity in the relative quantification of heterogeneous samples of N-glycans compared to HPAEC-PAD and HILIC-UPLC of 2-AB labelled N-glycans.
Keywords: HPAEC-MS/MS; Mass spectrometry; Pulsed amperometric detection; N-Glycan; IgG; Oligosaccharides; Sialyltransferase;

Icariin (ICA), the major constituent of Epimedium brevicornu Maxim, is recognized as an effective agent for the treatment of osteoporosis. In our previous report, the relatively short half-life (74 min) and poor bioavailability (approximately 0.1%) of ICA in rats suggested that not only ICA itself but also its metabolites could be responsible for the observed osteoporosis treatment effect. Therefore, the present study aimed at identifying the metabolites of ICA in rat plasma, bile, urine, and feces after the administration of a single oral dose of ICA (150 mg/kg). In this work, ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS) method was established to identify the metabolic profiles following intragastric administration of pure ICA in rats. The blood, bile, urine, and feces samples of the rats were investigated to explore the complete metabolic pathway of ICA in vivo. A total of 14 metabolites were detected in the bile, revealing that the bile is the main excretion pathway for ICA and its metabolites. The conjugated metabolites as observed in vivo, was further confirmed by the in vitro enzymatic study. Five metabolites of ICA, including demethylicariin, icariside I-3-O-glucuronide, demethylicariside II, demethylicariside II-7-O-glucuronide, and dehydroxyicaritin-glucuronide, were reported for the first time in the literature. In addition, the results revealed that the principal metabolism pathways of ICA in rat were deglycosylation and glucuronidation after deglycosylation.
Keywords: Icariin; Metabolite characterization; UHPLC/QTOF-MS; Rats; Enzymatic study;

A multiresidue approach for the simultaneous quantification of antibiotics in macroalgae by ultra-high performance liquid chromatography–tandem mass spectrometry by Sara Leston; Andreia Freitas; João Rosa; Jorge Barbosa; Marco F.L. Lemos; Miguel Ângelo Pardal; Fernando Ramos (361-367).
Together with fish, algae reared in aquaculture systems have gained importance in the last years, for many purposes. Besides their use as biofilters of effluents, macroalgae’s rich nutritional profiles have increased their inclusion in human diets but also in animal feeds as sources of fatty acids, especially important for the fish industry. Nonetheless, algae are continuously exposed to environmental contaminants including antibiotics and possess the ability for bioaccumulation of such compounds. Therefore, the present paper describes the development and validation of an ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous quantification of antibiotics in the green macroalgae Ulva lactuca. This multi-residue method enables the determination of 38 compounds distributed between seven classes and was fully validated according to EU Decision 2002/657/EC.
Keywords: Antibiotics; Macroalgae; Aquaculture; Quantification; UHPLC-MS/MS;

Stabilization and detection of hydrophylloquinone as di-O-methyl derivative by Rodrigo A.C. Sussmann; Marcilio M. de Moraes; Gerardo Cebrián-Torrejón; Exequiel O. Porta; Antonio Doménech-Carbó; Lydia F. Yamaguchi; Alejandro M. Katzin; Massuo J. Kato (368-371).
Phylloquinone is a redox active naphthoquinone involved in electron transport in plants. The function of this reduced form remains unclear due to its instability, which has precluded detection. Herein, a simple method that permits the stabilization of the reduced form of phylloquinone by di-O-methylation and HPLC detection is described.
Keywords: Phylloquinone; Hydrophylloquinone; Redox; Di-O-methyl derivative; HPLC; Electrochemistry;

Multivariate optimization of the hollow fibre liquid phase microextraction of muscimol in human urine samples by Somandla Ncube; Anna Poliwoda; Hlanganani Tutu; Piotr Wieczorek; Luke Chimuka (372-381).
A liquid phase microextraction based on hollow fibre followed by liquid chromatographic determination was developed for the extraction and quantitation of the hallucinogenic muscimol from urine samples. Method applicability on polar hallucinogens was also tested on two alkaloids, a psychedelic hallucinogen, tryptamine and a polar amino acid, tryptophan which exists in its charged state in the entire pH range. A multivariate design of experiments was used in which a half fractional factorial approach was applied to screen six factors (donor phase pH, acceptor phase HCl concentration, carrier composition, stirring rate, extraction time and salt content) for their extent of vitality in carrier mediated liquid microextractions. Four factors were deemed essential for the effective extraction of each analyte. The vital factors were further optimized for the extraction of single-spiked analyte solutions using a central composite design. When the simultaneous extraction of analytes was performed under universal factor conditions biased towards maximizing the enrichment of muscimol, a good composite desirability value of 0.687 was obtained. The method was finally applied on spiked urine samples with acceptable enrichments of 4.1, 19.7 and 24.1 obtained for muscimol, tryptophan and tryptamine respectively. Matrix-based calibration curves were used to address matrix effects. The r2 values of the matrix-based linear regression prediction models ranged from 0.9933 to 0.9986. The linearity of the regression line of the matrix-based calibration curves for each analyte was directly linked to the analyte enrichment repeatability which ranged from an RSD value of 8.3–13.1%. Limits of detection for the developed method were 5.12, 3.10 and 0.21 ng mL−1 for muscimol, tryptophan and tryptamine respectively. The developed method has proven to offer a viable alternative for the quantitation of muscimol in human urine samples.
Keywords: Hallucinogen; Muscimol; Hollow fibre liquid phase microextraction; Central composite design; Urine sample;

The signatures of natural stable nitrogen isotopic composition (δ15N) of individual amino acid (AA) have been confirmed to be a potentially effective tool for elucidating nitrogen cycling and trophic position of various organisms in food webs. In the present study, a two-stage derivatisation approach of esterification followed by acylation was evaluated. The biological samples underwent acid hydrolysis and the released individual AA was derivatived into corresponding N-pivaloyl-isopropyl (NPIP) esters for nitrogen isotopic analysis in gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Usually, 13 individual AA derivatives were separated with fine baseline resolution based on a nonpolar gas chromatography column (DB-5 ms). The minimum sample amount required under the presented conditions is larger than 20 ngN on column in order to accurately determine the δ15N values. The δ15N values determined by GC-C-IRMS with a precision of better than 1‰, were within 1‰ after empirical correction compared to the corresponding measured by element analysis (EA)-IRMS. Bland-Altman plot showed highly consistency of the δ15N values determined by the two measurement techniques. Cation-exchange chromatography was applied to remove interfering fraction from the extracts of plant and animal samples and without nitrogen isotope fractionation during the treatment procedure. Moreover, this approach was carried out to estimate the trophic level of various natural organisms in a natural lake environment. Results highly proved that the trophic level estimated via the presented AA method well reflected the actual food web structure in natural environments.
Keywords: GC-C-IRMS; Amino acid; Trophic level; NPIP ester;

A quantitative bioanalytical liquid chromatography⿿tandem mass spectrometric (LC⿿MS/MS) assay for tyrosine kinase inhibitors afatinib, erlotinib, gefitinib and encorafenib and the O⿿desmethyl metabolites of erlotinib and gefitinib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing stable isotopically labeled internal standard of afatinib, erlotinib, gefitinib and OSI-420. After dilution, the extract was directly analyzed by a reversed-phase liquid chromatography⿿tandem mass spectrometric system. Both O⿿desmethyl-erlotinib isomers, OSI⿿420 and OSI⿿413 could be separated, OSI-413 was only determined semi-quantitatively.The assay was completely validated for human plasma in a 10⿿10,000 ng/ml range for encorafenib, 5⿿5000 ng/ml for erlotinib, 2⿿2000 ng/ml for gefitinib and 1⿿1000 ng/ml for the other compounds. Calibration models were only linear when the corresponding labeled internal standard was used for quantification. Within-run precisions (n  = 18) and between-run precisions (3 runs; n  = 18) all were ⿤12.3% and accuracies were between 93 and 108% for each analyte over the tested concentration ranges. All compounds were sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine drug levels in plasma from patients treated with a kinase inhibitor.
Keywords: Afatinib; Erlotinib; Gefitinib; Encorafenib; LC⿿MS/MS; Plasma;

Large size virion is unable to diffuse into pores of conventional porous chromatography particles. Therefore, separation of virion by conventional column-packing materials is not quite efficient. To solve this problem, a monolithic column with large convective pores and quaternary amine groups was prepared and was applied to separate Enterovirus 71 (EV71, ≈5700–6000 kDa). Cross-section, pore structure, hydrodynamic performance, adsorption property and dynamic binding capacity of prepared monolithic column were determined. Double-pore structures, macropore at 2472 nm and mesopore at 5–60 nm, were formed. The porosity was up to 63.3%, which enable higher permeability and lower back pressure of the monolithic column than commercial UNO™ Q1 column. Based on the breakthrough curves, the loading capacity of bovine serum albumin was calculated to be 42.0 mg per column. In addition, prepared quaternary amine monolithic column was proved to be suitable for the separation of protein mixture by strong anion-exchange chromatography. As a practical application, prepared monolith column presents excellent performance to the separation of EV71 from virus-proteins mixture.
Keywords: Monolithic column; Strong anion-exchange chromatography; Enterovirus 71; Quaternary amine;

Development of supermacroporous monolithic adsorbents for purifying lectins by affinity with sugars by Gabriel Ramos Ferreira Gonçalves; Olga Reinert Ramos Gandolfi; Carilan Moreira Souza Santos; Renata Cristina Ferreira Bonomo; Cristiane Martins Veloso; Rafael da Costa Ilhéu Fontan (406-412).
Affinity techniques are frequently used to purify biocompounds, because of specific interactions observed in many cases. One example are the lectins, proteins connected in a reversible manner and specific to carbohydrates or sugar-containing molecules. Four different methods were investigated (epoxy, Schiff base, glutaraldehyde and ethylenediamine) to immobilize the carbohydrate N-acetyl-d-glucosamine (d-GlcNAc) on the surface of supermacroporous cryogels made for lectin purification. The glutaraldehyde method presented the highest immobilization capacity of d-GlcNAc (147.77 mg/g), while the ethylenediamine method presented the lowest capacity (32.47 mg/g). FTIR spectra analysis confirmed the presence of the immobilized carbohydrate. The cryogels containing d-GlcNAc immobilized by the different methods were characterized in terms of swelling capacity, degree of expansion, porosity and constituent fractions. Results showed that the activation methods did not affect the macroporous structure. Images obtained from scanning electron microscopy evidenced the presence of interconnected macropores in the structure of the cryogels produced. The cryogels presented even lower flow resistance in the permeability analysis. Finally, the cryogel modified by the glutaraldehyde method was used in the Concanavalin A lectin adsorption process, presenting an adsorptive capacity of 44.49 mg/g and high stability after five cycles of use.
Keywords: Cryogels; Acrylamide; Functionalization; Characterization; Affinity;

Determination of thyroid hormones in mouse tissues by isotope-dilution microflow liquid chromatography–mass spectrometry method by Meri De Angelis; Florian Giesert; Brian Finan; Christoffer Clemmensen; Timo D. Müller; Daniela Vogt-Weisenhorn; Matthias H. Tschöp; Karl-Werner Schramm (413-420).
Thyroid hormones (THs) play a critical role in the regulation of many biological processes such as growth, metabolism and development both in humans and wildlife. In general, TH levels are measured by immunoassay (IA) methods but the specificity of the antibodies used in these assays limits selectivity. In the last decade, several analytical methods using liquid chromatography-mass spectrometry (LC–MS) and tandem mass spectrometry (LC–MS/MS) have been developed to measure THs. These new techniques proved to be more accurate than the IA analysis and they were widely used for the determination of TH level in different human and animal tissues. A large part of LC–MS/MS methods described in literature employed between 200 and 500 mg of sample, however this quantity can be considered too high especially when preclinical studies are conducted using mice as test subjects. Thus an analytical method that reduces the amount of tissue is essential. In this study, we developed a procedure for the analysis of six THs; L-thyroxine (T4), 3,3′,5-triiodo-l-thyronine (T3), 3,3′,5′-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (rT2), 3,3′-diiodo-l-thyronine (T2), 3-iodo-l-thyronine (T1) using isotope (13C6-T4, 13C6-T3, 13C6-rT3, 13C6-T2) dilution liquid chromatography–mass spectrometry. The major difference with previously described methods lies in the utilization of a nano-UPLC (Ultra Performance Liquid Chromatography) system in micro configuration. This approach leads to a reduction compared to the published methods, of column internal diameter, flow rate, and injected volume. The result of all these improvements is a decrease in the amount of sample necessary for the analysis. The method was tested on six different mouse tissues: liver, heart, kidney, muscle, lung and brown adipose tissue (BAT). The nano-UPLC system was interfaced with a quadrupole time-of-flight mass spectrometer (Q-TOF2-MS) using the positive ion mode electrospray ionization. In our analytical method the instrumental calibration curves were constructed from 0 to 100 pg μL−1 and all of them showed good linearity (r2  > 0.99). The limit of quantification was from 2.5 to 5 pg injected into the column. The method recoveries calculated using spiked mouse liver and spiked mouse muscle were between 83% and 118% (except T1 and rT2 at high concentration) with a coefficient of variation (CV) of <10% for all derivatives. The new methodology allows us to measure T4 and T3 concentrations in a range from 21 to about 100 mg and give a more extensive insight on thyroid hormone concentration in different mouse tissue.
Keywords: Thyroid hormones; Microflow liquid chromatography; Mouse tissues; Mass spectrometry;

Antioxidant and possible chemopreventive properties of betacyanins, natural plant pigments, contribute to a growing interest in their chemistry and separation. Mixtures of betacyanins from fresh red Gomphrena globosa L. cultivar flowers were separated in three highly polar solvent systems by high-speed counter-current chromatography (HSCCC) for a direct comparison of their separation effectiveness. Three samples of crude extract (600 mg) were run on semi-preparative scale in solvent system (NH4)2SO4soln – EtOH (2.0:1.0, v/v) (system I) and the modified systems: EtOH – ACN – 1-PrOH – (NH4)2SO4satd.soln – H2O (0.5:0.5:0.5:1.2:1.0, v/v/v/v/v) (system II) and EtOH – ACN – (NH4)2SO4satd.soln – H2O (1.0:0.5:1.2:1.0, v/v/v/v) (system III). The systems were used in the head-to-tail (system I) or tail-to-head (systems II and III) mode. The flow rate of the mobile phase was 2.0 ml/min and the column rotation speed was 860 rpm. The retention of the stationary phase was 52.0% (system I), 80.2% (systems II) and 82.0% (system III). The betacyanins in the crude extract as well as HSCCC fractions were analyzed by LC–MS/MS. System I was applied for the first time in HSCCC for the separation of betacyanins and was quite effective in separation of amaranthine and 17-decarboxy-amaranthine (αI  = 1.19) and very effective for 17-decarboxy-amaranthine and betanin (αI  = 2.20). Modification of system I with acetonitrile (system III) as well as acetonitrile and propanol (system II) increased their separation effectiveness. Systems II–III enable complete separation of 17-decarboxy-amaranthine (K D(II)  = 2.94,  K D(III)  = 2.42) and betanin (K D(II)  = 2.46,  K D(III)  = 1.10) as well as betanin and gomphrenin I (K D(II)  = 1.62, K D(III)  = 0.74). In addition, separation of amaranthine and 17-decarboxy-amaranthine is the most effective in system II, therefore, this system proved to be the most suitable for the separation of all polar betacyanins.
Keywords: Betacyanins; Amaranthine; Betanin; HSCCC; Gomphrena globosa L.;

Display OmittedCyclophosphamide (CY) is a commonly-used nitrogen mustard alkylating agent, but its clinical application is severely limited by its cardiotoxicity. Since the development of metabolomics, the change of metabolite profiles caused by cyclophosphamide has been studied by metabolomics and has gained much attention. In this study, we analyzed rat plasma samples collected one, three and five days after cyclophosphamide administration using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Multiple statistical analyses, such as principal component analysis (PCA) and partial least squares ⿿ discriminant analysis (PLS-DA), were used to examine metabolite profile changes in plasma samples in order to screen for potential cardiotoxicity biomarkers and metabolic pathways. Levels of a dozen of metabolites changed significantly in plasma from the CY-treated group after one, three, and five days compared with the control group treated with normal saline (NS). Receiver operator characteristic (ROC) curve analysis suggested that the total 16 metabolites play important roles in different times of CY-induced cardiotoxicity respectively. Our results suggest that these metabolites in linoleic acid metabolism and glycerol phospholipid metabolism may be related to CY-induced cardiotoxicity. These metabolites could act as sensitive biomarkers for CY-induced cardiotoxicity and be useful for investigating toxic mechanisms. They may also lay a foundation for clinical use of cyclophosphamide.
Keywords: Cyclophosphamide; Cardiotoxicity; Metabolomics; Multivariate data analysis; Receiver operating characteristic curve;