Journal of Chromatography B (v.925, #C)

► Fast HPLC–UV method for the determination of antiepileptic drugs in human plasma. ► Simultaneous quantification of six antiepileptic drugs and their main metabolites. ► Sample pre-treatment is a combination of deproteinization followed by SPE. ► The method was fully validated according to international recommendations. ► This method was applied to real plasma samples from epileptic patients.A sensitive and fast high-performance liquid chromatographic method coupled with ultraviolet detection is herein reported for the simultaneous determination of human plasma concentration of six antiepileptic drugs frequently used in clinical practice [phenobarbital (PB), primidone (PRM), phenytoin (PHT), carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC)] and some of their main metabolites, carbamazepine-10,11-epoxide (CBZ-E), 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine (trans-diol) and licarbazepine (Lic). Sample preparation consisted of a deproteinization step with methanol followed by a solid-phase extraction procedure. Chromatographic separation was achieved in approximately 15 min on a reversed-phase C18 column using a mobile phase composed by water–methanol–acetonitrile–triethylamine (68.7:25:6:0.3, v/v/v/v; pH 6.5) pumped isocratically at 1.0 mL/min. The detector was set at 237 nm. Calibration curves were linear with regression coefficients greater than 0.992 over the concentration ranges 0.25–100 μg/mL for PB, 0.4–50 μg/mL for PRM, 0.5–50 μg/mL for PHT, 0.1–50 μg/mL for CBZ, LTG and CBZ-E, 0.1–25 μg/mL for OXC, 0.25–10 μg/mL for trans-diol and 0.15–80 μg/mL for Lic. Inter- and intra-day imprecision did not exceed 12.15% and inaccuracy was within ±14.91%. Absolute mean recoveries ranged from 78.49 to 101.04% and no interferences were observed at the retention times of the analytes and internal standard (ketoprofen). This bioanalytical method was successfully applied to real plasma samples from epileptic patients and it seems to be a suitable tool for routine therapeutic drug monitoring and also to support other clinical pharmacokinetic-based studies.
Keywords: Antiepileptic drugs; High-performance liquid chromatography; Human plasma; Bioanalytical method validation; Therapeutic drug monitoring;

► Simultaneous determination of opiates and maintenance drugs by LC–MS/MS. ► The compound identification was fulfilled EU guideline. ► Drug monitoring ensures compliance and helps therapy.For monitoring compliance of methadone or buprenorphine maintenance patient, a method for the simultaneous determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), buprenorphine, norbuprenorphine, opiates (morphine, codeine, 6-monoacetylmorphine) in urine by superficially porous liquid chromatography tandem mass spectrometry was developed and validated. After enzyme digestion and liquid–liquid extraction, reverse-phase separation was achieved in 5.2 min and quantification was performed by multiple reaction monitoring. Chromatographic separation was performed at 40 °C on a reversed phase Poroshell column with gradient elution. The mobile phase consisted of water and methanol, each containing 0.1% formic acid, at a flow rate of 0.32 mL/min. Intra-day and inter-day precision were less than 12.1% and accuracy was between −9.8% and 13.7%. Extraction efficiencies were more than 68%. Although ion suppression was detected, deuterated internal standards compensated for these effects. Carryover was minimal, less than 0.20%. All analytes were stable at room temperature for 16 h, 4 °C for 72 h, and after three freeze–thaw cycles. The assay also fulfilled compound identification criteria in accordance with the European Commission Decision 2002/657/EC. We analyzed 62 urine samples from patients received maintenance therapy and found that 54.8% of the patient samples tested were detected for morphine, codeine, or 6-monoacetylmorphine. This method provides a reliable and simultaneous quantification of opiates, maintenance drugs, and their metabolites in urine samples. It facilitates the routine monitoring in individuals prescribed the drug to ensure compliance and help therapeutic process.
Keywords: Maintenance therapy: Methadone; Buprenorphine; Opiates; Superficially porous liquid chromatography; Tandem mass spectrometry;

Development and validation of a quantitative LC–tandem MS assay for hexadeca-4,7,10,13-tetraenoic acid in human and mouse plasma by Edwin C.A. Stigter; Sophia Letsiou; Niels J.F. vd Broek; Johan Gerrits; Kenji Ishihara; Emile E. Voest; Nanda M. Verhoeven-Duif; Arjan B. Brenkman (16-19).
► FA(16:4)n-3 is excreted by mesenchymal stem cells upon exposure to platinum analogs. ► FA(16:4)n-3 induces chemotherapy resistance. ► An LC–MS/MS quantification method for FA(16:4)n-3 was developed for use in human and mouse plasma. ► Validation parameters of aforementioned method were within limits of acceptance.Upon exposure to platinum analogs, mesenchymal stem cells were recently found to excrete minute amounts of specific lipid mediators that induce chemotherapy resistance. One of these lipids is hexadeca-4,7,10,13-tetraenoic acid (FA(16:4)n-3). Importantly, FA(16:4)n-3 is present in high concentrations in certain fish oils and algae and oral intake of these products also potently induced chemotherapy resistance. These findings suggested that certain foods could negatively affect clinical chemotherapy treatment. In order to allow further study of the relation between FA(16:4)n-3 and clinical chemotherapy resistance, a method for the detection and quantification of FA(16:4)n-3 in plasma is required. Therefore, a quantification method for FA(16:4)n-3 in human and mouse plasma was developed consisting of a liquid–liquid extraction, solid phase clean-up and LC–MS/MS (MRM) analysis. The method was fully validated over a period of three weeks according to the standard protocols and requirements. The linearity range of the method is 1–100 nmol/L (r 2  > 0.99) using deuterated FA(16:3)n-3 as an internal standard. The limits of quantification and detection are 1.0 nmol/L and 0.8 nmol/L, respectively. Recoveries for spiked concentrations range from 103 to 108%. The intra-day and inter-day mean precision amounts to 98–106% and 100–108%, respectively. No significant loss of FA(16:4)n-3 is observed upon storage at −80 °C. The developed assay for the detection and quantification of FA(16:4)n-3 in human plasma is robust and reproducible. The validation parameters are within limits of acceptance.
Keywords: Hexadeca-4,7,10,13-tetraenoic acid; Chemotherapy resistance; LC–MS/MS; Validation;

► An improved UPLC–MS/MS method for determination of aripiprazole in human plasma. ► Highly sensitive and rapid compared to all existing methods in biological matrices. ► Practically free from endogenous matrix interference. ► Successful bioequivalence study in healthy Indian subjects. ► Reproducibility of study data is demonstrated by incurred sample reanalysis.An improved and rugged UPLC–MS/MS method has been developed and validated for sensitive and rapid determination of aripiprazole in human plasma using aripiprazole-d8 as the internal standard (IS). The analyte and IS were extracted from 100 μL of human plasma by solid-phase extraction using Phenomenex Strata-X (30 mg, 1 cc) cartridges. Chromatography was achieved on an Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column using methanol: 10 mM ammonium formate (85:15, v/v) as the mobile phase with isocratic elution. Quantitation was done using multiple reaction monitoring in the positive ionization mode. The linearity of the method was established in the concentration range 0.05–80 ng/mL. The mean extraction recovery was greater than 96% across QC levels, while intra- and inter batch accuracy and precision (% CV) values ranged from 97.4 to 101.9% and from 1.20 to 3.72% respectively. The relative matrix effect in eight different lots of plasma samples, expressed as % CV for the calculated slopes of calibration curves was 1.08%. The stability of aripiprazole was studied under different storage conditions. The validated method was used to support a bioequivalence study of 10 mg aripiprazole formulation in 36 healthy Indian subjects.
Keywords: Aripiprazole; Aripiprazole-d8; UPLC–MS/MS; Solid-phase extraction; Human plasma; Bioequivalence;

► A combination of an aptamer sorbent with the ion mobility spectrometry is introduced. ► This method was used for the determination of tetracycline in urine and plasma samples. ► The proposed method has a low detection limit and a wide dynamic range.An extraction method based on aptamer sorbent followed by electrospray ionization-ion mobility spectrometry (ESI-IMS) has been developed for the analysis of tetracycline in human urine and plasma samples. The effect of extraction parameters on the extraction efficiency including washing (solvent type and volume) and elution (solvent type, volume and flow rate) were investigated. Under the optimized conditions, the linear dynamic ranges for tetracycline in urine and plasma samples were found to be 0.05–5.00 μg/mL and 0.10–5.00 μg/mL with detection limits of 0.019 and 0.037 μg/mL, respectively. The extraction efficiency was 86.5% for urine and it was 82.8% for plasma samples. The relative standard deviation was 5.9% and 6.3% for six replicate measurements of tetracycline at 1 and 2 μg/mL in urine and plasma samples, respectively.
Keywords: Tetracycline; Aptamer; Ion mobility spectrometry; Solid-phase extraction;

► Fast and reliable online LC/LC–MS/MS method for urinary N-acetyl-4-aminophenol (NAAP). ► NAAP is both the major metabolite of aniline and a common pain reliever (paracetamol). ► NAAP was quantifiable in all urine samples analyzed. ► Urinary concentrations ranged from 8.7 μg/L to 22100 μg/L. ► Also non-paracetamol users have a considerably body burden with NAAP. N-Acetyl-4-aminophenol (NAAP) is the major urinary metabolite of aniline. The general population is known to be ubiquitously exposed to aniline through various sources. Furthermore, NAAP, known under the trade name paracetamol (resp. acetaminophen), is one of the most commonly used over-the-counter analgesics. Recent studies suggest anti-androgenic properties of NAAP. Although NAAP has been used as a pain reliever over decades and its role in aniline metabolism is well known there is a lack of internal exposure data both in environmental and occupational settings.To determine the internal NAAP exposure of the general population, workers exposed to aniline and users of paracetamol we developed a fast on-line HPLC–MS/MS method with isotope dilution quantification of NAAP after enzymatic hydrolysis of its conjugates in urine. We achieved minimal sample pretreatment through on-line extraction and enrichment of the analyte by turbulent flow chromatography on a Waters Oasis HLB phase followed by back-flush transfer onto the analytical column. The limit of quantification (LOQ) was 0.75 μg/L.In a pilot study, urine samples of 21 volunteers, not occupationally exposed to aniline, were analyzed for NAAP. NAAP was detected in all samples in a wide concentration range between 8.7 μg/L and 22100 μg/L (median 85.7 μg/L). The highest concentration was measured in a volunteer who took paracetamol one day ago. Half of the volunteers quoted to either never have taken paracetamol or at least not during several weeks before the study. Therefore, other routes of exposure than direct use of paracetamol, like aniline or paracetamol contaminated foodstuff, leading to the NAAP excretions have to be taken into account.
Keywords: Paracetamol; Acetaminophen; Aniline; Human Biomonitoring; Urine; Turbulent flow chromatography;

► We validated a method for quantitation of hydrocodone and metabolites in plasma. ► Samples were extracted using solid phase extraction and analyzed by LC–MS–MS. ► Linearity, precision, LOQ, and other method validation parameters were evaluated. ► The method provided good precision, accuracy and sensitivity.A selective, sensitive and accurate high-performance liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the quantitation of hydrocodone, hydromorphone and norhydrocodone in human plasma was developed. The internal standard stock solution comprised of hydrocodone-d6, hydromorphone-d6 and norhydrocodone-d3 was added to 0.5 mL plasma samples. Samples were extracted using a copolymeric sorbent (mixed mode) solid phase extraction (SPE) column. Chromatographic separation was carried out using a reversed-phase C18 analytical column with a gradient mobile phase consisting of solvent A = 5% acetonitrile with 0.1% formic acid and solvent B = 100% acetonitrile. MS analysis was performed using positive electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. Linearity was established over the range 1–100 ng/mL with correlation coefficients ≥0.998 for all three analytes. The coefficient of variation (CV) of intra-day samples was ≤5.6% at 10 ng/mL. The precision of inter-day (6 days) samples resulted in CVs ≤8.1% at concentrations tested at 2.5, 10 and 25 ng/mL for all three analytes. The lower limit of quantification (LOQ) was 1.0 ng/mL with signal-to-noise (S/N) ratio >10, the limit of detection (LOD) was 0.25 ng/mL with S/N ratio >3 for the drug and its metabolites. Dilution effects, extraction recovery, stability, interference, carryover and ion suppression were also evaluated. This method was successfully applied to human subject plasma samples in support of a hydrocodone pharmacokinetic study.
Keywords: Hydrocodone; Hydromorphone; Norhydrocodone; LC–MS–MS; Plasma;

Combinatorial approach of LC–MS/MS and LC–TOF-MS for uncovering in vivo kinetics and biotransformation of ochratoxin A in rat by Zheng Han; Zhiyong Zhao; Jianxin Shi; Yucai Liao; Zhihui Zhao; Dabing Zhang; Yongning Wu; Sarah De Saeger; Aibo Wu (46-53).
► A combinatorial approach of LC–MS/MS and LC–TOF-MS was developed. ► The same conditions were used for the analysis in seven bio-matrices. ► The kinetics and distribution of OTA in rat were elucidated. ► Three typical metabolites of OTA were simultaneously identified.A combinatorial platform of liquid chromatography–tandem mass spectrometry (LC–MS/MS) and liquid chromatography coupled with time of flight mass spectrometry (LC–TOF-MS) has been developed to investigate the in vivo kinetics and biotransformation of ochratoxin A (OTA) in rats. The stable isotope dilution LC–MS/MS method was first validated by determining the linearity (R 2  ≥ 0.9990), sensitivity (lower limit of quantitation of 0.05 ng mL−1), accuracy (83.3–108.3), precision (RSD ≤ 15.6%) and stability (≥75.0%), and was approved for the determination OTA in plasma, heart, liver, spleen, lung, kidney and brain with a run time of 7.0 min. Simultaneously, an LC–TOF-MS method could unambiguously identify the metabolites of OTA in a total run time of 14 min. The subsequent studies on kinetics and distribution after oral administration of 0.2 mg/kg b.w. OTA in rat indicated that OTA could reach a maximum value of 1932.4 ± 124.9 ng mL−1 within 5 h due to its fast absorption, and then was slowly eliminated in plasma with a half-life time (t 1/2) of 75.6 ± 29.0 h. Results of tissue accumulation after a daily oral administration of 0.1 mg/kg b.w. OTA during 20 days showed that the highest concentration of OTA was observed in lung (95.9 ± 13.7 ng g−1), followed by liver (76.0 ± 9.7 ng g−1), heart (62.0 ± 4.2 ng g−1) and kidney (55.7 ± 4.7 ng g−1). Furthermore, three less toxic metabolites of OTA were clearly identified: Ochratoxin β (OTβ) and ochratoxin B (OTB) methyl ester were found in kidney and spleen, respectively, while phenylalanine was detected in heart and kidney. Thus, a possible metabolic pathway of OTA was proposed. The above achieved results justified that the application of combinatorial LC–MS/MS and LC–TOF-MS methods are valuable tools to uncover the kinetics and metabolism of OTA for the interpretation of toxicological findings in animals and extrapolation of the resulting data as reference to humans.
Keywords: Ochratoxin A; Liquid chromatography–tandem mass spectrometry; Liquid chromatography coupled with time of flight mass spectrometry; Kinetics; Biotransformation;

A highly sensitive and selective method for the determination of leukotriene B4 (LTB4) in ex vivo stimulated human plasma by ultra fast liquid chromatography–tandem mass spectrometry by Weisheng Lin; Mike-Qingtao Huang; Xiaohua Xue; Kirk Bertelsen; Guiyan Chen; Harry Zhao; Zhongping (John) Lin; Anne Fourie; Jan de Jong; Naidong Weng (54-62).
Leukotriene B4 (LTB4) is an important inflammatory component in a number of diseases and has been used as a pharmacodynamic (PD) biomarker. In this report, a highly sensitive and selective ultra fast liquid chromatography–tandem mass spectrometry (UFLC-MS/MS) method for the determination of LTB4 in plasma from ex vivo stimulated human blood, using leukotriene B4-d4 (LTB4-d4, contains four deuterium atoms at the 6, 7, 14, and 15 positions) as the internal standard (IS), was developed and validated. The chromatographic separation of LTB4 from its three isomers and an unknown interference peak from human plasma was crucial to achieve accurate determination of 0.2 ng/mL (LLOQ) of LTB4. LTB4 and the IS were extracted with methyl tertiary butyl ether (MTBE) from 200 μL human plasma. Reversed-phase HPLC separation was carried out with a Phenomenex Synergi Hydro-RP column (100 mm × 3 mm, 2.5 μm). MS/MS detection was set at mass transitions of 335.0 → 194.9  m/z for LTB4 and 339.0 → 196.9  m/z for LTB4-d4 in Turbo Ionization Spray (TIS) negative mode. The dynamic range of the method is 0.2–200 ng/mL. LTB4 was found to be stable in human plasma for at least three freeze (−20 °C)/thaw cycles, and on the benchtop (room temperature) for at least 6 h. The stock solution storage stability study demonstrated that the LTB4 stock solution, in 50:50 acetonitrile:water, was stable at 4 °C for at least 198 days. The processed samples were found to be stable for at least 72 h at room temperature. The long-term sample storage stability test demonstrated that LTB4 human plasma samples were stable at a storage temperature of −20 °C for at least 198 days. In addition, intraday and interday accuracy and precision, sensitivity, linearity, and recovery were evaluated. An additional partial validation was conducted to decrease the plasma sample volume from 200 to 100 μL. All the data reported in this study fulfilled the requirements and recommendations in the FDA guidance for bioanalytical method validation. Comparison of the validated UFLC-MS/MS method with an ELISA method using ex vivo stimulated samples indicated that although results from the two assays correlated relatively well, the UFLC-MS/MS method has been shown to be superior in selectivity and dynamic range to an ELISA method in our study. The validated UFLC-MS/MS method was successfully used to analyze samples generated from two clinical studies. The excellent assay performance and incurred sample reproducibility (ISR) results obtained from the study sample analysis demonstrated the assay is robust and reliable.
Keywords: Ultra fast liquid chromatography–tandem mass spectrometry (UFLC–MS/MS); Leukotriene B4 (LTB4); Bioanalysis; PD biomarker;

In matrix derivatization of trichloroethylene metabolites in human plasma with methyl chloroformate and their determination by solid-phase microextraction–gas chromatography-electron capture detector by Mohana Krishna Reddy Mudiam; Rajeev Jain; Meenu Varshney; Ratnasekhar Ch; Abhishek Chauhan; Sudhir Kumar Goyal; Haider A. Khan; R.C. Murthy (63-69).
► Rapid, sensitive and green analytical method for quantitative determination of TCE metabolites in human plasma samples. ► In matrix derivatization of TCE metabolites with MCF directly in plasma samples. ► Reduced derivatization time (30 s) and temperature (25 °C) in comparison to the existing methods. ► Solventless extraction of volatile derivatives by HS-SPME. ► Determination of TCE metabolites in real plasma samples was successfully performed.Trichloroethylene (TCE) is a common industrial chemical that has been widely used as metal degreaser and for many industrial purposes. In humans, TCE is metabolized into dichloroacetic acid (DCA), trichloroacetic acid (TCA) and trichloroethanol (TCOH). A simple and rapid method has been developed for the quantitative determination of TCE metabolites. The procedure involves the in situ derivatization of TCE metabolites with methyl chloroformate (MCF) directly in diluted plasma samples followed by extraction and analysis with solid-phase microextraction (SPME) coupled to gas chromatography-electron capture detector (GC-ECD). Factors which can influence the efficiency of derivatization such as amount of MCF and pyridine (PYR), ratio of water/methanol were optimized. The factors which can affect the extraction efficiencies of SPME were screened using 27−4 Placket–Burman Design (PBD). A central composite design (CCD) was then applied to further optimize the most significant factors for optimum SPME extraction. The optimum factors for the SPME extraction were found to be 562.5 mg of NaCl, pH at 1 and an extraction time of 22 min. Recoveries and detection limits of all three analytes in plasma were found to be in the range of 92.69–97.55% and 0.036–0.068 μg mL−1 of plasma, respectively. The correlation coefficients were found to be in the range of 0.990–0.995. The intra- and inter-day precisions for TCE metabolites were found to be in the range of 2.37–4.81% and 5.13–7.61%, respectively. The major advantage of this method is that MCF derivatization allows conversion of TCE metabolites into their methyl esters in very short time (≤30 s) at room temperature directly in the plasma samples, thus makes it a solventless analysis. The method developed was successfully applied to the plasma samples of humans exposed to TCE.
Keywords: TCE metabolites; Methyl chloroformate derivatization; SPME; Central composite design; GC-ECD;

► A novel RDX extraction method for small volume samples is proposed. ► Acceptable recoveries and % confidence were achieved in three different matrices. ► The proposed extraction method allowed detection of RDX degradation bi products by MS/MS. ► The method shows applicability in complex biological matrices.A simple, fast liquid–liquid extraction method was developed for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation using small sample volumes. The method was tested in vitro with anaerobic incubations of RDX with whole rumen fluid (WRF) and a commercial Sporanaerobacter acetigenes strain in methanogenic media for RDX. Additionally, validation experiments were conducted in deionized water in order to show applicability toward various aqueous matrices. Conditions for extraction were as follows: 300 μL of sample were mixed with an equal volume of a 0.34 M ammonium hydroxide solution to reach a basic pH, extracted with a hexane/ethyl acetate 1:1 (v/v) solution (1 mL) and shaken vigorously for 10 s. The resulting organic phase was transferred, then dried under a constant flow of N2 and reconstituted with acetonitrile (300 μL) for HPLC–UV and LC–MS/MS analysis. Percent recovery values were obtained (83–101%) in all matrices for RDX. In WRF (n  = 3 animals), RDX degradation was observed with almost 100% elimination of RDX after 4 h. The five nitroso and ring cleavage metabolites were observed by mass spectrometry. Liquid cultures of S. acetigenes did not show significant RDX biodegradation activity. RDX extractions from deionized water samples indicated acceptable recoveries with low variability, suggesting suitability of the method for aqueous matrices. Overall, the new method demonstrated acceptable efficiency and reproducibility across three matrices, providing an advantageous alternative for studies where complex matrices and small volume samples are in use.
Keywords: Biodegradation; Ovine ruminal microbiota; RDX; Organic extraction; HPLC–UV;

Analysis of anticoagulant rodenticide residues in Microtus arvalis tissues by liquid chromatography with diode array, fluorescence and mass spectrometry detection by Alma M. Hernández; José Bernal; José L. Bernal; María T. Martín; Constantino Caminero; María J. Nozal (76-85).
► Four rodenticides were determined in Microtus arvalis tissues for the first time. ► The proposed sample treatment was efficient, fast and simple. ► The chromatographic conditions were the same for the three detection modes used. ► Matrix (M. arvalis tissues) had an influence onto the ESI-MS signals. ► Rodenticide residues were found in few samples (8) at very low levels (<50 μg/kg).We describe here a fast and selective analytical method to determine the levels of four anticoagulant rodenticides (chlorophacinone, bromadiolone, brodifacoum and difenacoum) in animal tissues by liquid chromatography (LC) using different detection methods: fluorescence (FLD), diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS). Rodenticides were extracted from freeze-dried and homogenized tissue samples (liver, intestine and muscle) that had been obtained from the common vole (Microtus arvalis). These samples were diluted in 5 mL of methanol, the solution was shaken and centrifuged, and the supernatant was removed and evaporated to dryness. The residue was reconstituted in 1 mL of methanol (liver samples) or 1 mL of the mobile phase (muscle and intestine samples), and injected onto an LC–DAD–FLD–MS system coupled to electrospray ionization (ESI) in negative mode. After conducting an LC optimization study, we selected a Gemini 5 μm C18 column, a mobile phase composed of a mixture of 30 mM ammonium formate in water and methanol (26:74, v/v), and we used an isocratic elution mode. The method was fully validated and shown to be selective, precise, accurate, and linear in the range from ∼5 μg/kg (ESI-MS) or ∼50 μg/kg (DAD–FLD) to 10,000 μg/kg, depending on the compound analyzed. Recoveries ranged from 82% to 103%, while the limits of detection and quantification ranged from 9–89 μg/kg (FLD–DAD) and 0.6–4.6 μg/kg (ESI-MS). This method was successfully used to simultaneously measure the aforementioned compounds in M. arvalis tissues.
Keywords: Anticoagulant rodenticides; Vole; Microtus arvalis; Animal tissues; LC–DAD–FLD–MS;

A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules by Masaya Tsunehiro; Yuma Meki; Kanako Matsuoka; Emiko Kinoshita-Kikuta; Eiji Kinoshita; Tohru Koike (86-94).
A simple and efficient method based on magnetic-bead technology has been developed for the separation of phosphorylated and nonphosphorylated low-molecular-weight biomolecules, such as nucleotides, phosphorylated amino acids, or phosphopeptides. The phosphate-binding site on the bead is an alkoxide-bridged dinuclear zinc(II) complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag), which is linked to a hydrophilic cross-linked agarose coating on a magnetic core particle. All steps for the phosphate-affinity separation are conducted in buffers of neutral pH with 50 μL of the magnetic beads in a 1.5-mL microtube. The entire separation protocol for phosphomonoester-type compounds, from addition to elution, requires less than 12 min per sample if the buffers and the zinc(II)-bound Phos-tag magnetic beads have been prepared in advance. The phosphate-affinity magnetic beads are reusable at least 15 times without a decrease in their phosphate-binding ability and they are stable for three months in propan-2-ol.
Keywords: Magnetic bead; Nucleotide; Phosphate-affinity separation; Phosphopeptide; Phos-tag;

Metabolomic profiling of the response of susceptible and resistant soybean strains to foxglove aphid, Aulacorthum solani Kaltenbach by Dan Sato; Hiromichi Akashi; Masahiro Sugimoto; Masaru Tomita; Tomoyoshi Soga (95-103).
► We compared phenotypes of aphid-resistant and susceptible soybean leaves. ► We quantified their primary metabolites using CE–TOFMS. ► Citrate in resistant leaves was originally 34-fold higher than susceptible leaves. ► Metabolites to TCA cycle and flavonoids/alkaloids changed after aphid inoculation. ► Amino acids in aphids on the resistant leaves had similar profiles to starvation.Aphid infection reduces soybean (Glycine max [L.] Merr.) yield. Consequently, cultivation of aphid-resistant strains is a promising approach to pest control, and understanding the resistance mechanism is of importance. Here, we characterized the resistance of soybeans to foxglove aphid, Aulacorthum solani Kaltenbach, at the metabolite level. First, we evaluated aphid mortality and settlement rates on the leaves of two soybean strains, ‘Tohoku149’ and ‘Suzuyutaka’, and found that the former had strong resistance soon after introduction of the aphids. The metabolomic response to aphid introduction was analyzed using capillary electrophoresis–time-of-flight mass spectrometry. We found the following three features in the profiles: (1) concentrations of citrate, amino acids, and their intermediates were intrinsically higher for Tohoku149 than Suzuyutaka, (2) concentrations of several metabolites producing secondary metabolites, such as flavonoids and alkaloids, drastically changed 6 h after aphid introduction, and (3) concentrations of TCA cycle metabolites increased in Tohoku149 48 h after aphid introduction. We also profiled free amino acids in aphids reared on both soybean strains and under starvation, and found that the profile of the aphids on Tohoku149 was similar to that of the starved aphids, but different to that of aphids on Suzuyutaka. These tests confirmed that aphids suck phloem sap even from Tohoku149. This study demonstrates the metabolomic profiles of both soybean strains and aphids, which will contribute to the molecular level understanding of mechanisms of soybean resistance to aphids.
Keywords: Soybean; Aphid; Pest resistant; Metabolomics; Capillary electrophoresis time-of-flight mass spectrometry;

Isolation of α-arbutin from Xanthomonas CGMCC 1243 fermentation broth by macroporous resin adsorption chromatography by Chunqiao Liu; Peng Zhang; Luo Liu; Tao Xu; Tianwei Tan; Fang Wang; Li Deng (104-109).
► A simple and inexpensive method for isolation of α-arbutin from broth is developed. ► Macroporous resins were tested and S-8 was found best for α-arbutin purification. ► One-step column chromatography recovers 90.9% (w/w) of α-arbutin in a purity of 97.3% (w/w). ► The equilibrium adsorption data of S-8 were well-fitted to the Freundlich isotherm. ► We have scaled up S-8 column from laboratory test to large scale.α-Arbutin is a glycosylated hydroquinone which has inhibitory function against tyrosinase. In this work, a one-step isolation of α-arbutin from Xanthomonas CGMCC 1243 fermentation broth by macroporous resin adsorption chromatography was investigated. The research results indicated that S-8 resin offered the best adsorption and desorption capacities for α-arbutin than others and its equilibrium adsorption data were well-fitted to the Freundlich isotherm. In order to optimize the operating parameters for separating α-arbutin, dynamic adsorption and desorption tests on S-8 column chromatography were carried out. Under optimized conditions (adsorption volume of 7 bed volume (BV), mobile phase of 25% (v/v) ethanol solution and elution volume of 3 BV), the purity and recovery of α-arbutin were 97.3% (w/w) and 90.9% (w/w), respectively. The product was identified as α-arbutin by 13C NMR and 1H NMR analysis. Moreover, we scaled up S-8 column from laboratory test (10 cm × 2 cm ID) to large scale (500 cm × 100 cm ID) without diminishing α-arbutin yield. In conclusion, the results in this work provide a one-step and cost-effective method for large-scale production of α-arbutin.
Keywords: α-Arbutin; Isolation; Macroporous resin; Fermentation broth; Xanthomonas;

Obesity is becoming a health concern worldwide and metformin, a first line anti-diabetic drug, was associated with weight loss under different backgrounds. However, most researches focused on the anti-diabetic mechanism and less attention has been paid on the mechanism of weight loss of metformin. Therefore, we established a metabonomic method to evaluate metformin action in preventing obesity in a high fat diet-induced-obesity (DIO) mice model. 36 male C57BL/6 mice (8-week old) were randomly divided into control group (n  = 12, normal chow), model group (n  = 12, high fat chow) and metformin group (n  = 12, high fat chow and dosed with metformin) over 16 weeks. A urinary metabonomic study using UPLC-TOF/MS was performed in combination with multivariate statistical analysis. In addition, indices of body weight and food intake as well as fasting blood glucose, fed blood glucose, oral glucose tolerance test (OGTT) and plasma insulin were collected. Significant weight loss in metformin-treated mice was achieved and 21 potential biomarkers were identified. Decreased glucose, myristic acid, stearidonic acid, lysoPC (16:0), lysoPC (18:0), l-glutamic acid, l-methionine, l-threonine, l-phenylalanine, l-histidine, l-carnitine, l-malic acid and pantothenic acid in urine indicated that metformin may have exerted effects on energy metabolism. Further, based on the biomarkers, we cautiously propose that tricarboxylic acid cycle (TCA) may have been compromised by metformin and might contribute to the activation of adenosine monophosphate kinase (AMPK), then AMPK activation led to more β-oxidation of certain fatty acids and augmented lipolysis and thus induced weight loss. Related cellular and molecular studies are being considered to further investigate the underlying mechanism.
Keywords: Metformin; Metabonomics; Obesity; Prevention treatment;

► Combined assay of cabazitaxel and docetaxel in human plasma. ► Semi-quantitative determination of the metabolites RPR112698 and RPR123142. ► Lower limit of quantification for cabazitaxel and docetaxel is 1.0 and 0.1 ng/mL, respectively. ► Method can be applied to support clinical pharmacokinetic studies.We present a sensitive validated LC–MS/MS assay for the simultaneous determination of cabazitaxel and docetaxel in human plasma, with calibration ranges of 1.0–150 ng/mL for cabazitaxel and 0.1–15 ng/mL for docetaxel. Sample pretreatment consisted of liquid–liquid extraction with tert-butyl methyl ether. Chromatographic separation was achieved on a Zorbax Extend C18 column using a gradient mixture of 10 mM ammonium hydroxide and methanol. Mass detection was carried out by turbo ion spray ionization in positive ion multiple reaction monitoring mode. All inter-day accuracies and precisions were within ±15% of the nominal value and within ±20% at the lower limit of quantitation. Demethylations of cabazitaxel yielding the metabolites RPR112698 and RPR123142 were monitored semi-quantitatively and quantified as ng docetaxel equivalents. Plasma samples of a prostate cancer patient treated with cabazitaxel were analyzed to demonstrate the usefulness of the presented assay for clinical drug monitoring. In conclusion, this method can be applied to support clinical pharmacokinetic studies with the novel anticancer drug cabazitaxel.
Keywords: Cabazitaxel; Docetaxel; LC–MS/MS; Validation; Human plasma; Taxane;

Liquid chromatography–tandem mass spectrometric assay for the mutated BRAF inhibitor dabrafenib in mouse plasma by Rolf W. Sparidans; Selvi Durmus; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (124-128).
► The first validated bioanalytical assay for dabrafenib has been reported. ► The assay has successfully been validated in the 2–2000 ng/ml range. ► The drug is sufficiently stable under all conditions relevant for the assay. ► First pharmacokinetics in male wild type FVB mice have reported.A quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for the mutated BRAF inhibitor dabrafenib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing PLX4720 as internal standard. The extract was directly injected into the reversed-phase chromatographic system after dilution with water. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 2–2000 ng/ml calibration range with r 2  = 0.993 ± 0.002 for linear regression with quadratic weighting (n  = 5). Within day precisions (n  = 6) were 3.3–5.2%, between day (3 days; n  = 18) precisions 4.7–8.2%. Accuracies were between 95–104% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully used to determine drug pharmacokinetics in mice.
Keywords: Dabrafenib; Mutated BRAF inhibitor; LC–MS/MS; plasma;