Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (J Chrom B )

Publisher: Elsevier


The Journal of Chromatography B. addresses advancements in and applications of analytical methodologies related to drugs, other pharmacologically active compounds, metabolites, as well as to bio-polymers such as proteins and nucleic acids. The areas considered include: clinical analysis, therapeutic drug monitoring, pharmaceutical analysis and novel approaches to sample preparation in bioanalysis; the qualitative and quantitative analysis of proteins, peptides and their post-translational modifications as well as nucleic acids; the screening and profiling of body fluids and tissues related to monitoring the level of active substances, including metabolites, biomarkers and toxicants; the comparative analysis of biological systems and proteomics and genomics including novel ways of data handling and interpretation; analytical techniques covered include the various facets of chromatography, electrophoresis and related methods, including mass spectrometry and affinity-based methodologies.

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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Enzymatic protein digestion in polyacrylamide gel has been used for sample pretreatment in mass spectrometry-based proteomics due to its effectiveness in removing contaminants that interfere with sample ionization. However, the difficulty of recovering the digested peptides from the solid gel matrix has been a drawback of this method. Here we have developed a novel in-gel digestion method to enhance peptide recovery using a dissolvable, bis-acrylylcystamine (BAC)-crosslinked polyacrylamide gel. After enzymatic protein digestion in BAC gel, we completely dissolved the gel by reductive treatment with tris-(2-carboxyethyl) phosphine to release the digested peptides from the gel. Our analysis revealed that the reductive dissolution of the BAC gel enhances the peptide recovery, which has a significantly higher protein identification capability than the conventional method, using an insoluble polyacrylamide gel. In addition, protein samples trapped in dehydrated BAC gel were stable at room temperature and reproducible sample recovery was obtained after storage for one week. These results indicate that the proposed method could be an effective tool for conducting sample pretreatment for mass spectrometry-based protein analysis.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 967C:36-40.
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    ABSTRACT: 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and formoterol are newly revised prohibited threshold substances (150ng/mL for THC-COOH and 40ng/mL for formoterol) by the World Anti-Doping Agency (WADA). In continuation of our direct quantitation work of the prohibited threshold substances, direct LC-MS/MS methods combined with a simple sample preparation procedure have been developed and validated for the measurement of these two threshold substances in urine samples. After the enzymatic hydrolysis of urine samples, the resulting samples were diluted with acetonitrile and centrifuged. The supernatant was directly analyzed by LC-MS/MS using the selected reaction monitoring mode. The calibration curve range of the assay was ranged over 50-200% of the threshold value according to WADA guidelines. The limit of detection and limit of quantification were 6.1 and 18.4ng/mL for THC-COOH and 2.0 and 6.2ng/mL for formoterol, respectively. Intra- and inter-day precisions were between 2.08% and 7.28% and the accuracies ranged from 95.16% to 104.49%. The present methods were successfully applied to the analysis of the proficiency test samples.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 967C:8-12.
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    ABSTRACT: Monitoring of the plasmatic levels of levodopa (LEV) and carbidopa (CAR) is necessary to adjust the dose of these drugs according to the individual needs of Parkinson's disease patients. To support drug therapeutic monitoring, a method using HILIC mode and LC-MS/MS was developed for the simultaneous determination of carbidopa, levodopa, and its metabolites (3-o-methyldopa (3-OMD) and dopamine (DOPA)) in human plasma. A triple quadrupole mass spectrometry was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. After straightforward sample preparation via protein precipitation, an Atlantis HILIC (150×2.1mm, 3μm, Waters, USA) column were used for separation under the isocratic condition of acetonitrile/water (79:21, v/v) containing 0.05% formic acid and 3mmol/L ammonium formate and the total run time was 7min. Deuterated LEV was used as internal standard for quantification. The developed method was validated in human plasma with a lower limit of quantitation of 75ng/mL for LEV, 65ng/mL for CAR and 3-OMD, and 20ng/mL for DOPA. The calibration curve was linear within the concentration range of 75-800ng/mL for LEV, 65-800ng/mL for CAR and 3-OMD, and 20-400ng/mL for DOPA (r>0.99). The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±13.44% of nominal and inter-assay and intra-assay precision≤13.99%. All results were within the acceptance criteria of the US FDA and ANVISA guidelines for method validation. LEV, CAR, 3-OMD and DOPA were stable in the battery of stability studies, long-term, bench-top, autosampler, and freeze/thaw cycles. Samples from patients undergoing treatment were analyzed, and the results indicated that this new method is suitable for therapeutic drug monitoring in Parkinson's disease patients.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 967C:41-49.
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    ABSTRACT: Docetaxel lipid microsphere (DT-LM) is a novel formulation of docetaxel without Tween-80. A sensitive, robust and reproducible ultrafiltration (UF) followed by UPLC-MS/MS method was developed and validated for the quantification of unbound docetaxel in human plasma using paclitaxel as IS. Ultrafiltrate samples were chromatographed on Acquity UPLC BEH C18 column (50mm×2.1mm, 1.7μm). The mobile phase was a mixture of 10mM ammonium formate in water containing 0.2% formic acid (A) and acetonitrile containing 0.2% formic acid (B). The volume of plasma utilized was only 450μL. The calibration curve was linear over the range of 0.2-200ng/mL, with LLOQ of 0.2ng/mL. The method was shown to be reliable and reproducible with intra- and inter-day precision and accuracy <±15%, and extraction recovery of 98.1-104.8%. Docetaxel was stable during stability studies, e.g., short term, post-preparation and freeze-thaw cycles. The validated method was utilized to support the pharmacological study of DT-LM in patients with advanced cancer.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 967C:28-35.
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    ABSTRACT: A novel large-volume immobilized enzyme reactor (IMER) on small column was prepared with organic-inorganic hybrid silica particles and applied for fast (10min) and oriented digestion of protein. At first, a thin enzyme support layer was formed in the bottom of the small column by polymerization with α-methacrylic acid and dimethacrylate. After that, amino SiO2 particles was prepared by the sol-gel method with tetraethoxysilane and 3-aminopropyltriethoxysilane. Subsequently, the amino SiO2 particles were activated by glutaraldehyde for covalent immobilization of trypsin. Digestive capability of large-volume IMER for proteins was investigated by using bovine serum albumin (BSA), cytochrome c (Cyt-c) as model proteins. Results showed that although the sequence coverage of the BSA (20%) and Cyt-c (19%) was low, the large-volume IMER could produce peptides with stable specific sequence at 101-105, 156-160, 205-209, 212-218, 229-232, 257-263 and 473-451 of the amino sequence of BSA when digesting 1mg/mL BSA. Eight of common peptides were observed during each of the ten runs of large-volume IMER. Besides, the IMER could be easily regenerated by reactivating with GA and cross-linking with trypsin after breaking the -CN- bond by 0.01M HCl. The sequence coverage of BSA from regenerated IMER increased to 25% comparing the non-regenerated IMER (17%). 14 common peptides. accounting for 87.5% of first use of IMER, were produced both with IMER and regenerated IMER. When the IMER was applied for ginkgo albumin digestion, the sequence coverage of two main proteins of ginkgo, ginnacin and legumin, was 56% and 55%, respectively. (Reviewer 2) Above all, the fast and selective digestion property of the large-volume IMER indicated that the regenerative IMER could be tentatively used for the production of potential bioactive peptides and the study of oriented protein digestion.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 967C:13-20.
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    ABSTRACT: This work reports that Cu(II) complexes with l-proline were used as chiral additives for the enantioseparations and determination of three underivatized amino acids by ligand-exchange micellar electrokinetic chromatography (LE-MEKC). Sodium dodecylsulfate (SDS) was shown to be necessary for simultaneous separation of the enantiomeric amino acids. Separation parameters such as SDS concentrations, the Cu(II)-l-proline ratio, the concentration of the copper(II) complex at a specific Cu(II)-l-proline ratio, pH and separation voltage were investigated for the enantioseparation in order to achieve the maximum possible resolution. A good separation was achieved in the BGE composing of 10mM ammonium acetate, 10mM Cu(II) and 20mM l-proline and 30mM SDS at pH 5.0, and an applied voltage of 15kV performed. Under above-mentioned optimum conditions, linearity was achieved within concentration ranges of up to two orders of magnitudes for the investigated amino acids with the correlation coefficients ranging from 0.9917 to 0.9984. The proposed method has been successfully applied to the determination of amino acid enantiomers in human urine, compound amino acids injection, and amino acid oral liquid.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 965C:254-259.
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    ABSTRACT: The purification and enrichment of most natural products with potential pharmaceutical applications has been performed mainly employing conventional batch-mode chromatographic processes. There is a growing interest in use of simulated moving bed (SMB) chromatography for natural product enrichment as this method enables conservation of mobile phase, while increasing productivity of chromatography medium. SMB increases yield while decreasing cost. Cyclolinopeptides C ([1-9-NaC],[1-MetO]-CLB, 3) and E ([1-8-NaC],[1-MetO]-CLE, 8) were extracted as a mixture from flaxseed oil and then enriched using a three-zone simulated moving bed. The current research extends the SMB technology to enrichment of cyclolinopeptides (CLs), a group of biologically active hydrophobic cyclic peptides that occur in flaxseed oil. Of interest are [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) that provide synthetic scaffolds for modified CLs. The influence of flow rate (feed, desorbent, and extract) on the separation of [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) was investigated.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 965C:231-237.
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    ABSTRACT: Lopinavir is an HIV protease inhibitor with high protein binding (98-99%) in human plasma. This study was designed to develop an ultrafiltration method to measure the unbound concentrations of lopinavir overcoming the non-specific binding issue. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of total concentrations of lopinavir in plasma was developed and validated, and an adaptation was also optimized and validated for the determination of unbound concentrations. The chromatographic separation was performed with a C18 column (100mm×2.1mm i.d., 5μm particle size) using a mobile phase containing deionized water with formic acid, and acetonitrile, with gradient elution at a flow-rate of 350μLmin(-1). Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionization in positive ion mode. The method was validated over a clinical range of 0.01-1μg/mL for human plasma ultrafiltrate and 0.1-15μg/mL in human plasma. The inter and intra-assay accuracies and precisions were between 0.23% and 11.37% for total lopinavir concentrations, and between 3.50% and 13.30% for plasma ultrafiltrate (unbound concentration). The ultrafiltration method described allows an accurate separation of the unbound fraction of lopinavir, circumscribing the loss of drug by nonspecific binding (NSB), and the validated LC-MS/MS methodology proposed is suitable for the determination of total and unbound concentrations of lopinavir in clinical practice.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 965C:216-223.
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    ABSTRACT: A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method using multi-walled carbon nanotubes (MWCNTs) as a reversed-dispersive solid phase extraction (r-dSPE) material combined with ultra-high liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was developed for the simultaneous determination of amantadine, rimantadine and memantine in chicken muscle. The satisfactory separation of isomers (rimantadine and memantine) was obtained on an Acquity BEH C18 column (2.1mm×100mm, 1.7μm) after optimization of mobile phase composition, column temperature and flow rate. The method involved an acetonitrile-based sample preparation and a dSPE clean-up procedure with MWCNTs material. Variations in the type and amount of MWCNTs, the pH value of the extract, the extraction time for MWCNTs, and the type of eluent were used to determine the optimal parameters for increasing the sample throughput and the sensitivity. The samples were quantified using amantadine-D15, rimantadine-D4 and memantine-D6 as the internal standards. Under the optimized conditions, recoveries of 96.8-104.6% and the values of coefficient of variation (CV) of 3.8-6.4% were obtained for the three drugs in chicken muscle at three spiked levels (0.5, 1.0 and 1.5μg/kg), and the decision limits (CCα) and detection capabilities (CCβ) were 0.15-0.20μg/kg and 0.20-0.25μg/kg, respectively. Positive results were obtained from local supermarket using this method, and the concentrations obtained from the newly developed method compared well to the previously reported method.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 965C:197-205.
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    ABSTRACT: Novel magnetic molecularly imprinted polymers based on multiwalled carbon nanotubes (MWNTs@MMIPs) with specific selectivity toward bisphenol A were synthesized using bisphenol A as the template molecule, methacrylic acid, and β-cyclodextrin as binary functional monomers and ethylene glycol dimethacrylate as the cross-linker. The MWNTs@MMIPs were characterized by Fourier transform infrared, vibrating sample magnetometer, and transmission electron microscopy. Batch mode adsorption experiment was carried out to investigate the specific adsorption equilibrium and kinetics of the MWNTs@MMIPs. The MWNTs@MMIPs exhibited good affinity with a maximum adsorption capacity of 49.26μmolg(-1) and excellent selectivity toward bisphenol A. Combined with high-performance liquid chromatography analysis, the MWNTs@MMIPs were employed to extract bisphenol A in tap water, rain water, and lake water successfully with the recoveries of 89.8-95.4, 89.9-93.4, and 87.3-94.1%, respectively.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 965C:190-196.
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    ABSTRACT: Secnidazole is a long-lasting nitroimidazole antimicrobial agent that is used as racemic mixture in clinical settings. We developed and validated an enantioselective high-performance liquid chromatography method to determine secnidazole enantiomers in rat plasma. Secnidazole enantiomers and S-(-)-ornidazole (internal standard) were extracted from 50μL of rat plasma using diethyl ether-dichloromethane (3:2, v/v). Baseline resolution (Rs=2.45) was achieved within 7.0min on a Chiral-AGP column (150mm×4.0mm, 5μm) at 20°C. The mobile phase consisted of 10mM ammonium acetate-methanol (96:4, v/v) and was delivered at a flow rate of 0.5mL/min with ultraviolet detection at 318nm. The method was linear over the concentration range 0.500-100μg/mL for both enantiomers. The lower limit of quantification was 0.500μg/mL for both enantiomers. The relative standard deviation values for intra- and inter-day precision were 0.8-8.6 and 1.8-8.2% for S-(+)-secnidazole and R-(-)-secnidazole, respectively. The relative error values of accuracy ranged from -7.8 to 1.1% for S-(+)-secnidazole and from -7.3 to -0.1% for R-(-)-secnidazole. The method was successfully used to determine the pharmacokinetic properties of secnidazole enantiomers in rats after administration of the racemate and individual enantiomers. The pharmacokinetic results indicate that the disposition of secnidazole enantiomers is not stereoselective and chiral inversion and enantiomer-enantiomer interaction do not occur in rats.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 965C:224-230.
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    ABSTRACT: A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed, using testosterone-d3 as a surrogate analyte, for the simultaneous quantification of goserelin and testosterone in rat plasma. According to this method, the pharmacokinetic and pharmacodynamic data were obtained from a single plasma sample aliquot. The method involved the addition of alarelin as an internal standard (IS) for goserelin and testosterone-(13)C3 for testosterone or testosterone-d3. The conditions for the separation of these two compounds were achieved on a ZORBAX Eclipse Plus C18 column (Agilent, 2.1×50mm, 1.8μm, Stockport, UK) in a single chromatographic run at a flow rate of 400μL/min. In order to minimize interferences of complex matrix, the extraction of plasma consisted of a protein precipitation step using methanol, followed by purification using an Oasis(®) HLB solid-phase extraction column. The method was validated in the concentration range of 0.01-30.0ng/mL for goserelin and 0.05-30.0ng/mL for testosterone-d3, respectively. The within- and between-run precisions were 1.7-9.2% and 2.1-6.9%, respectively. The within- and between-run accuracies were -1.8 to 5.3% and -4.9 to 4.0%, respectively. This accurate and highly specific assay provides a useful method to evaluate the pharmacokinetics and pharmacodynamics of goserelin in rats.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2014; 965C:183-189.
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    ABSTRACT: Diaveridine (DVD) is a popular antibacterial synergist that is widely used in combination with sulfonamide. It has been reported to be genotoxic to mammalian cells, but more studies are required to clarify this. Moreover, there is very little information on its pharmacokinetics, metabolic elimination and mechanism of toxicity. Therefore, in order to gain a better understanding of the metabolism of DVD, we performed high-performance liquid chromatography linear ion trapped orbitrap mass spectrometer (LC-LTQ-Orbitrap). With this approach, we identified 15 metabolites of DVD in chicken after a single oral administration of DVD; 10 of these metabolites have been identified in vivo for the first time. Nine phase I and five phase II metabolites were detected in the plasma, and eight phase I and six phase II metabolites were found in feces. The major phase I metabolites were formed via the O-demethylation and N-oxidation pathways, and the major phase II metabolites were glucuronide conjugates. These results are essential for understanding this compound more clearly and lay the basis for further studies about the metabolism of DVD. Therefore, using this approach, we were able to identify and characterize metabolites of DVD with high sensitivity and resolution. We were able to detect a broad range of metabolites, even some trace ones and some so far unknown metabolites.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 06/2014; 965C:91-99.
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    ABSTRACT: Tolvaptan is a competitive vasopressin V2-receptor antagonist that inhibits water reabsorption in the renal collecting ducts. A selective high-performance liquid chromatography method for determining tolvaptan enantiomers in rat and dog sera was developed and validated. Benzyl salicylate was used as an internal standard. Sample preparation involved liquid-liquid extraction with an n-hexane:diethyl ether (1:1, v/v) solution, followed by solid-phase extraction using a silica-gel cartridge. Chromatographic separation was performed on a cellulose-based chiral stationary phase in the reversed phase mode. The analytes were monitored with UV detection. The calibration curve showed linearity over the concentration range from 0.025 to 2.5μg/mL for each analyte. Precision as the percentage coefficient of variation did not exceed 14.8%, and accuracy as relative error was within ±14.6% for the analytes. The validated method was successfully applied to evaluate the pharmacokinetics of oral tolvaptan enantiomers in rats and dogs, indicating gender and species differences in the systemic exposure to tolvaptan enantiomers.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 06/2014; 965C:112-118.
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    ABSTRACT: Kushen-Gancao Decoction (KGD) is a classic traditional Chinese herb combination in treating viral hepatitis and chronic liver diseases. This study aims to investigate the pharmacokinetic (PK) study of matrine (MT), oxymatrine (OMT), glycyrrhizic acid (GL) and glycyrrhetinic acid (GA) following oral administration of KGD in rats. A rapid, sensitive and reliable HPLC-MS/MS method was successfully developed for the simultaneous determination of MT, OMT, GL and GA in rat plasma. A Inertsil C18 analytical column was used with a gradient mobile phase system of methanol-ammonium acetate (5mM) with a flow rate of 0.5mL/min. The analysis was performed on a positive and negative ionization electrospray mass spectrometer via multi reaction monitoring (MRM). Linear calibration curves were obtained for the following concentration range: 10-5000ng/mL for MT, OMT and GL, 50-15,000ng/mL for GA in rat plasma (R(2)>0.99). The lower limit of quantification (LLOQ) was 5ng/mL (MT, OMT and GL) and 20ng/mL (GA). The intra- and inter-day accuracies ranged from -7.91 to 9.10% and precisions (RSD) were within 15%. The analytes were found to be stable under short-term temperature conditions, post-preparative temperature conditions, and after three freeze-thaw cycles conditions. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of KGD.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 06/2014; 965C:19-26.
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    ABSTRACT: A rapid and sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of microcystin-RR (MC-RR) and its glutathione and cysteine conjugates (MC-RR-GSH and MC-RR-Cys, respectively) in fish plasma and bile. The analytes were extracted using methanol, followed by an Oasis mixed-mode cation-exchange polymeric sorbent. The separation was performed on a reversed-phase Waters XBridge C18 column with the gradient mobile phase, consisting of water and acetonitrile (both acidified with 0.5‰ formic acid). Mean recoveries of MC-RR, MC-RR-GSH and MC-RR-Cys ranged from 80.7 to 93.7%, 81.1 to 93.1% and 80.3 to 93.2%, respectively, at three concentrations (0.2, 1.0 and 5.0μgmL(-1)). Limits of detection (LODs) for MC-RR, MC-RR-GSH and MC-RR-Cys were 6, 12 and 9ngmL(-1), respectively. Limits of quantification (LOQs) were 15, 30 and 22.5ngmL(-1) for MC-RR, MC-RR-GSH and MC-RR-Cys, respectively. This method makes it feasible for the identification and quantification of MC-RR, MC-RR-GSH and MC-RR-Cys in limited and complex biological fluid samples (such as plasma and bile, typically 50μL), which were previously excluded or difficult to study due to the relatively large sample volumes.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 06/2014; 963C:113-118.
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    ABSTRACT: Vitamin K, comprising phylloquinone (PK) and menaquinones (MKn), is a family of vitamers found in multiple biological and environmental matrices. Advancing emerging evidence for novel and distinct physiologic roles of these vitamers in human health and disease necessitates sensitive and selective methods for quantifying PK and MKn in these matrices. We developed a novel method employing high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS) for simultaneous quantification of 11 vitamin K vitamers that can be applied in feces, serum and food. Minimal detectable concentrations of vitamin K vitamers ranged from 1pmol/g to 30pmol/g. Limits of quantification ranged from 5pmol/g to 90pmol/g. Inter-assay and intra-assay variations were <17% and <8%, respectively, in food, and <12% and <8%, respectively, in feces. Recovery exceeded 80% for all vitamers in both food and feces. The method successfully quantified PK and MKn concentrations in rat chow, feces and serum. In summary, this LC-APCI-MS method provides a sensitive and selective tool for quantifying vitamin K vitamers in feces, serum and food. This method can be applied in human and animal studies examining the role of vitamin K vitamers derived from the diet and gut bacteria synthesis in health and disease.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 06/2014; 963C:128-133.
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    ABSTRACT: We developed and validated an ultra-performance liquid chromatographic (UPLC) method coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry for simultaneous determination of levornidazole and its first-pass metabolites, l-chloro-3-(2-hydroxymethyl-5-nitro-l-imidazolyl)-2-propanol (Ml), 2-methyl-5-nitroimidazole (M2) and 3-(2-methyl-5-nitro-1-imidazolyl)-1,2-propanediol (M4), in human plasma and urine. The biological samples were pretreated by protein precipitation and liquid-liquid extraction and analyzed using an ACQUITY UPLC CSH C18 column (2.1×50mm, 1.7μm) and a QTRAP mass spectrometer in multiple reaction monitoring mode via APCI. Acetonitrile and 0.1% formic acid in water was used as the mobile phase in gradient elution at a flow rate of 0.6mL/min. The lower limit of quantification of this method was 0.0100, 0.00500, 0.0200 and 0.00250μg/mL for levornidazole, M1, M2 and M4, respectively. The linear calibration curves were obtained for levornidazole, M1, M2, and M4 over the range of 0.0100-5.00, 0.00500-2.50, 0.0200-10.0 and 0.00250-1.25μg/mL, respectively. The intra- and inter-batch precision was less than 12.2% in plasma and less than 10.8% in urine. The intra- and inter-batch accuracy was 87.8-105.7% in plasma and 92.8-109.2% in urine. The mean recovery of levornidazole, M1, M2 and M4 was 91.1-105.1%, 95.8-103.8%, 87.8-96.8%, 96.8-100.6% from plasma and 96.0-100.9%, 96.9-107.9%, 95.1-102.7%, 103.7-105.9% from urine respectively. This method was validated under various conditions, including room temperature, freeze-thaw cycles, long-term storage at -40±5°C, after pretreatment in the autosampler (at 10°C), and 10- and 100-fold dilution. This newly established analytical method was successfully applied in a pharmacokinetic study following single intravenous infusion of levornidazole in 24 healthy Chinese subjects.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 06/2014; 963C:119-127.

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