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  • Monitoring of selected skin- and breath-borne volatile organic compounds emitted from the human body using gas chromatography ion mobility spectrometry (GC-IMS)
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-13
    Paweł Mochalski, Helmut Wiesenhofer, Maria Allers, Stefan Zimmermann, Andreas T. Güntner, Nicolay J. Pineau, Wolfgang Lederer, Agapios Agapiou, Christopher A. Mayhew, Veronika Ruzsanyi

    Human smuggling and associated cross-border crimes have evolved as a major challenge for the European Union in recent years. Of particular concern is the increasing trend of smuggling migrants hidden inside shipping containers or trucks. Therefore, there is a growing demand for portable security devices for the non-intrusive and rapid monitoring of containers to detect people hiding inside. In this context, chemical analysis of volatiles organic compounds (VOCs) emitted from the human body is proposed as a locating tool. In the present study, an in-house made ion mobility spectrometer coupled with gas chromatography (GC-IMS) was used to monitor the volatile moieties released from the human body under conditions that mimic entrapment. A total of 17 omnipresent volatile compounds were identified and quantified from 35 ion mobility peaks corresponding to human presence. These are 7 aldehydes (acrolein, 2-methylpropanal, 3-methylbutanal, 2-ethacrolein, n-hexanal, n-heptanal, benzaldehyde), 3 ketones (acetone, 2-pentanone, 4-methyl-2-pentanone), 5 esters (ethyl formate, ethyl propionate, vinyl butyrate, butyl acetate, ethyl isovalerate), one alcohol (2-methyl-1-propanol) and one organic acid (acetic acid). The limits of detection (0.05–7.2 ppb) and relative standard deviations (0.6–11%) should be sufficient for detecting these markers of human presence in field conditions. This study shows that GC-IMS can be used as a portable field detector of hidden or entrapped people.

    更新日期:2018-01-13
  • Quantitative determination of cyclic phosphatidic acid and its carba analog in mouse organs and plasma using LC–MS/MS
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-08
    Yoshibumi Shimizu, Masaki Ishikawa, Mari Gotoh, Keiko Fukasawa, Shinji Yamamoto, Kensuke Iwasa, Keisuke Yoshikawa, Kimiko Murakami-Murofushi

    Cyclic phosphatidic acid (cPA), an analog of lysophosphatidic acid, is involved in the regulation of many cellular processes. A sensitive and specific method to quantify the molecular species of cPA is important for studying the physiological and pathophysiological roles of cPA. Here, we developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based quantification method for the simultaneous detection of cPA species having various fatty acids (16:0, 18:0, 18:1, and 18:2) as well as 2-carba-cPA, a chemically synthesized analog of cPA. Chromatography was performed using a reversed-phase C18 column. cPA species were detected using a triple quadrupole mass spectrometer. cPA 17:0 was used as an internal standard. Intra- and interday precision values (CV%) were within 10%. The linear range of detection for each cPA species was 0.01 μg/mL to 5 μg/mL, with correlation coefficients of 0.998 or higher. The developed method was applied to the quantification of cPA species in mouse plasma and organs. The concentrations of cPA 16:0, 18:0, and 18:1 were revealed to be significantly reduced in the brains of cuprizone-treated mice, a model of multiple sclerosis, compared with control mice. These findings could be important for understanding the roles of cPA in the neurodegenerative processes associated with multiple sclerosis.

    更新日期:2018-01-08
  • Hydrophilic interaction liquid chromatography in the speciation analysis of selenium
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-08
    Aleksandra Sentkowska, Krystyna Pyrzynska

    The hydrophilic interaction liquid chromatography (HILIC) coupled to mass spectrometry was employed to study retention behavior of selected selenium compounds using two different HILIC stationary phases: silica and zwitterionic. Two organic solvents – acetonitrile and methanol – were compared as a component of mobile phase. Separation parameters such as a content of organic modifier, the eluent pH and inorganic buffer concentration were investigated. Based on all observations, methanol seems to be beneficial for the separation of studied compounds. The optimal HILIC separation method involved silica column and eluent composed of 85% MeOH and CH3COONH4 (8 mM, pH 7) was compared to RP method in terms of time of the single run, the separation efficiency and limit of detection.

    更新日期:2018-01-08
  • Capillary electrophoresis-mass spectrometry for targeted and untargeted analysis of the sub-5 kDa urine metabolome of patients with prostate or bladder cancer: A feasibility study
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-07
    Matthew S. MacLennan, Miranda G.M. Kok, Laiel Soliman, Alan So, Antonio Hurtado-Coll, David D.Y. Chen

    Targeted and untargeted analyses of the sub-5 kDa urine metabolome of genitourinary cancer patients (prostate and/or bladder) were performed without chemical derivatization using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). For targeted analysis, endogenous levels of sarcosine and 5 other amino acid metabolites implicated in the progression of prostate cancer were quantified in four patients and in a pooled urine sample from healthy volunteers. An untargeted analysis (m/z 50 to 850) of patient urine was performed using the same CE-ESI-MS system identifying over 400 distinct molecular features per patient. All patient urine samples were collected at prostatectomy/cystectomy via catheter. Patient urine samples were filtered by centrifugation, with endogenous sarcosine enriched by solid-phase extraction, and the processed samples loaded onto CE-ESI-MS for analysis. Diagnostic information, digital pathological slides, and tissue samples were collected and stored in a comprehensive biobanking database. The introduction of urine sample collection into the surgery workflow was facile and is a promising strategy for addressing the translational research challenge of moving smoothly from “chromatogram to nomogram”.

    更新日期:2018-01-07
  • Simultaneous detection of 5-fluorocytosine and 5-fluorouracil in human cells carrying CD/5-FC suicide gene system by using capillary zone electrophoresis
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-07
    Yajing Liu, Pan Zhu, Zhiwei Huang, Li Zhou, Ping Shi

    A well-known suicide gene therapy approach, cytosine deaminase (CD) in combination with prodrug 5-flurocytosine (5-FC), has become an effective strategy of tumor treatment. However, there are short of simple and convenient detection methods to evaluate the efficiency of 5-FC conversion to 5-fluorouracil (5-FU) in human cells carrying various CD/5-FC systems. In this study, we developed an effective capillary zone electrophoresis (CZE) method to simultaneously measure 5-FC and 5-FU in cells carrying CD/5-FC suicide gene system. Under the condition of 60 mM borate buffer (pH 9.5) and 25 kV separation voltage with 0.5 psi × 15 s injection in 210 nm, the separation of 5-FC and 5-FU could be completely achieved within 15 min. The linearity of the calibration curve of standard 5-FC and 5-FU was in the range from 1 to 1000 μM (r2 > 0.999) and their recoveries were 98.4% and 96.0%, respectively. Due to the simple sample preparation and easy detection, this method is suitable for the study of the conversion efficiency of CD/5-FC suicide gene system. It aims to intuitively evaluate CD/5-FC systems and helps to guide the improvement of more effective CD/5-FC suicide gene systems.

    更新日期:2018-01-07
  • Simultaneous accelerated solvent extraction and hydrolysis of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide in meconium samples for gas chromatography–mass spectrometry analysis
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-07
    Cinthia de Carvalho Mantovani, Jefferson Pereira e silva, Guilherme Forster, Rafael Menck de Almeida, Edna Maria de Albuquerque Diniz, Mauricio Yonamine

    Cannabis misuse during pregnancy is associated with severe impacts on the mother and baby health, such as newborn low birth weight, growth restriction, pre-term birth, neurobehavioral and developmental deficits. In most of the cases, drug abuse is omitted or denied by the mothers. Thus, toxicological analyzes using maternal-fetal matrices takes place as a suitable tool to assess drug use. Herein, meconium was the chosen matrix to evaluate cannabis exposure through identification and quantification of 11-nor- Δ9-tetrahydrocannabinol-9-carboxylic (THCCOOH). Accelerated solvent extraction (ASE) was applied for sample preparation technique to simultaneously extract and hydrolyze conjugated THCCOOH from meconium, followed by a solid-phase extraction (SPE) procedure. The method was developed and validated for gas chromatography–mass spectrometry (GC–MS), reaching hydrolysis efficiency of 98%. Limits of detection (LOD) and quantification (LOQ) were, respectively, 5 and 10 ng/g. The range of linearity was LOQ to 500 ng/g. Inter and intra-batch coefficients of variation were <8.4% for all concentration levels. Accuracy was in 101.7–108.9% range. Recovery was on average 60.3%. Carryover effect was not observed. The procedure was applied in six meconium samples from babies whose mothers were drug users and showed satisfactory performance to confirm fetal cannabis exposure.

    更新日期:2018-01-07
  • RP-HPLC method for simultaneous estimation of vigabatrin, gamma-aminobutyric acid and taurine in biological samples
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-07
    Anitha Police, Vijay Kumar Shankar, S. Narasimha Murthy

    Vigabatrin is used as first line drug in treatment of infantile spasms for its potential benefit overweighing risk of causing permanent peripheral visual field defects and retinal damage. Chronic administration of vigabatrin in rats has demonstrated these ocular events are result of GABA accumulation and depletion of taurine levels in retinal tissues. In vigabatrin clinical studies taurine plasma level is considered as biomarker for studying structure and function of retina. The analytical method is essential to monitor taurine levels along with vigabatrin and GABA. A RP-HPLC method has been developed and validated for simultaneous estimation of vigabatrin, GABA and taurine using surrogate matrix. Analytes were extracted from human plasma, rat plasma, retina and brain by simple protein precipitation method and derivatized by naphthalene 2, 3‑dicarboxaldehyde to produce stable fluorescent active isoindole derivatives. The chromatographic analysis was performed on Zorbax Eclipse AAA column using gradient elution profile and eluent was monitored using fluorescence detector. A linear plot of calibration curve was observed in concentration range of 64.6 to 6458, 51.5 to 5150 and 62.5 to 6258 ng/mL for vigabatrin, GABA and taurine, respectively with r2 ≥ 0.997 for all analytes. The method was successfully applied for estimating levels of vigabatrin and its modulator effect on GABA and taurine levels in rat plasma, brain and retinal tissue. This RP-HPLC method can be applied in clinical and preclinical studies to explore the effect of taurine deficiency and to investigate novel approaches for alleviating vigabatrin induced ocular toxicity.

    更新日期:2018-01-07
  • Quantitation of trans-fatty acids in human blood via isotope dilution-gas chromatography-negative chemical ionization-mass spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-06
    Heather C. Kuiper, Na Wei, Samantha L. McGunigale, Hubert W. Vesper

    Trans-fatty acids (TFA) are geometric isomers of naturally occurring cis-fatty acids. High dietary TFA intake has been associated with risk factors for cardiovascular disease. However, little is known about TFA levels in humans. To address this data need, we developed and validated a new isotope dilution-gas chromatography-negative chemical ionization-mass spectrometry (ID-GC-NCI-MS) method for quantitation of 27 fatty acids (FA) including 4 major TFA in human plasma, serum, and red blood cells (RBC) from 66 donors. Quantitation was performed with 18 isotope labeled internal standards and results are presented in μM and % of total FA. This method has high sensitivity and specificity due to use of pentafluorobenzyl-bromide derivatization combined with NCI-MS and a 200 m column to optimize positional and geometric FA isomer separation. The four major TFA, palmitelaidic acid, elaidic acid, trans-vaccenic acid, and linoelaidic acid, were detected in all samples, with median total TFA concentrations of 17.7 μM in plasma, 19.6 μM in serum, and 21.5 μM in RBC. The % of total FA for the TFA was 0.20% in plasma, 0.20% in serum, and 0.30% in RBC. Patterns for % FA are similar to those reported in other studies. We developed a highly specific, ID-GC-NCI-MS method to quantitate TFA and other FA in humans.

    更新日期:2018-01-06
  • Cloud point extraction coupled with microwave-assisted back-extraction (CPE-MABE) for determination of Eszopiclone (Z-drug) using UV–Visible, HPLC and mass spectroscopic (MS) techniques: Spiked and in vivo analysis
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-06
    Shivpoojan Kori, Ankush Parmar, Jony Goyal, Shweta Sharma

    A procedure for the determination of Eszopiclone (ESZ) from complex matrices i.e. in vitro (spiked matrices), as well as in vivo (mice model) was developed using cloud point extraction coupled with microwave-assisted back-extraction (CPE-MABE). Analytical measurements have been carried using UV–Visible, HPLC and MS techniques. The proposed method has been validated according to ICH guidelines and legitimate reproducible and reliability of protocol is assessed through intraday and inter-day precision < 3.61% and < 4.70%, respectively. Limit of detection has been obtained as 0.083 μg/mL and 0.472 μg/mL respectively, for HPLC and UV–Visible techniques, corresponding to assessed linearity range. The coaservate phase in CPE was back extracted under microwaves exposure, with isooctane at pre-concentration factor ~ 50 when 5 mL of sample solution was pre-concentrated to 0.1 mL. Under optimized conditions i.e. Aqueous-Triton X-114 4% (w/v), pH 4.0, NaCl 4% (w/v) and equilibrium temperature of 45 °C for 20 min, average extraction recovery has been obtained between 89.8 and 99.2% and 84.0–99.2% from UV–Visible and HPLC analysis, respectively. The method has been successfully applied to the pharmacokinetic estimation (post intraperitoneal administration) of ESZ in mice. MS analysis precisely depicted the presence of active N‑desmethyl zopiclone in impales as well as in mice plasma.

    更新日期:2018-01-06
  • Size exclusion chromatography (SEC-HPLC) as an alternative to study thrombin inhibition
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-06
    Emmanuel Pérez-Escalante, Luis Guillermo González-Olivares, Alma Elizabeth Cruz-Guerrero, Carlos Andrés Galán-Vidal, Ma. Elena Páez-Hernández, Giaan Arturo Álvarez-Romero
    更新日期:2018-01-06
  • The determination of 5-methoxytryptophan in human plasma
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-05
    George M. Anderson, Joshua T. Eighmie

    Methoxyindoles have been of continued interest due to their biological effects. Recent studies using enzyme-linked immunosorbent assays (ELISAs) have reported micromolar levels of 5-methoxytryptophan (MeOTRP) in human plasma. This prompted our development of method for the determination of MeOTRP in human plasma that involved solid phase extraction (SPE) followed by HPLC. The identity of a putative MeOTRP peak was confirmed through chromatographic, voltammometric and fluorometric characterization. Using a standard addition approach, actual mean human plasma MeOTRP levels were found to be approximately 2 ng/mL, 20 to 100 times lower than previously reported. Prior studies on the physiological and possible biomarker roles of MeOTRP need to be reconsidered and future studies of MeOTRP need to take the presented findings into consideration.

    更新日期:2018-01-06
  • Determination of semicarbazide in fish by molecularly imprinted stir bar sorptive extraction coupled with high performance liquid chromatography
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-04
    Tang Tang, Fangdi Wei, Xu Wang, Yujie Ma, Yueyue Song, Yunsu Ma, Quan Song, Guanhong Xu, Yao Cen, Qin Hu

    A novel molecularly imprinted stir bar (MI-SB) for sorptive extraction of semicarbazide (SEM) was prepared in present paper. The coating of the stir bar was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, dynamic adsorption and static adsorption tests. The saturated adsorption of MI-SB was about 4 times over that of non-imprinted stir bar (NI-SB). The selectivity of MI-SB for SEM was much better than NI-SB. A method to determine SEM was established by coupling MI-SB sorptive extraction with HPLC-UV. The liner range was 1–100 ng/mL for SEM with a correlation coefficient of 0.9985. The limit of detection was about 0.59 ng/mL, which was below the minimum required performance limit of SEM in meat products regulated by European Union. The method was applied to the determination of SEM in fish samples with satisfactory results.

    更新日期:2018-01-05
  • Preliminary investigation of human exhaled breath for tuberculosis diagnosis by multidimensional gas chromatography – Time of flight mass spectrometry and machine learning
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-04
    Marco Beccaria, Theodore R. Mellors, Jacky S. Petion, Christiaan A. Rees, Mavra Nasir, Hannah K. Systrom, Jean W. Sairistil, Marc-Antoine Jean-Juste, Vanessa Rivera, Kerline Lavoile, Patrice Severe, Jean W. Pape, Peter F. Wright, Jane E. Hill

    Tuberculosis (TB) remains a global public health malady that claims almost 1.8 million lives annually. Diagnosis of TB represents perhaps one of the most challenging aspects of tuberculosis control. Gold standards for diagnosis of active TB (culture and nucleic acid amplification) are sputum-dependent, however, in up to a third of TB cases, an adequate biological sputum sample is not readily available. The analysis of exhaled breath, as an alternative to sputum-dependent tests, has the potential to provide a simple, fast, and non-invasive, and ready-available diagnostic service that could positively change TB detection. Human breath has been evaluated in the setting of active tuberculosis using thermal desorption-comprehensive two-dimensional gas chromatography–time of flight mass spectrometry methodology. From the entire spectrum of volatile metabolites in breath, three random forest machine learning models were applied leading to the generation of a panel of 46 breath features. The twenty-two common features within each random forest model used were selected as a set that could distinguish subjects with confirmed pulmonary M. tuberculosis infection and people with other pathologies than TB.

    更新日期:2018-01-04
  • A sensitive and selective immunoaffinity column clean up coupled to UPLC-MS/MS for determination of trace methyl-3-quinoxaline-2-carboxylic acid in animal tissues
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-03
    Peipei Li, Xiaojun Zhang, Juanmei Zhang, Zhongyong Yan, Shuai Zhang, Si Chen, Yi Fang

    This paper described a reliable and simple method for the selective determination of MQCA in animal tissues using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A highly targeted immunoaffinity column was used for sample purification after enzymatic hydrolysis. The purified extracts were analyzed by reversed-phase HPLC–MS/MS in positive ESI and multiple reaction monitoring mode. The calibration curves showed good linearity with correlation coefficient (r2) larger than 0.995. The average recoveries at the spiked levels of 0.5, 2.0 and 20 μg kg− 1 were 90.2% to 103.5% with intra-day and inter-day relatives standard deviations (RSD, n = 6) ranging from 1.8% to 6.7% and 3.5% to 7.6% respectively. The limit of quantification (LOQ) was 0.5 μg kg− 1, which can fulfil the maximum residue level (MRL) of 4.0 μg kg− 1 stipulated by the Agricultural Minister of China and the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC. The method is sensitive, accurate, convenient and rapid, and has been successfully applied in real samples.

    更新日期:2018-01-04
  • Fluorous-assisted metal chelate affinity extraction for nucleotides followed by HILIC-MS/MS analysis
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-03
    Ena Kiyokawa, Tadashi Hayama, Hideyuki Yoshida, Masatoshi Yamaguchi, Hitoshi Nohta

    We herein developed a selective method for the determination of nucleotides by fluorous-assisted metal chelate affinity extraction followed by hydrophilic interaction liquid chromatography (HILIC) combined with tandem mass spectrometric (MS/MS) analysis. In this study, the nucleotides were selectively chelated by Fe(III)-immobilized perfluoroalkyliminodiacetic acid, and the resulting chelates were subsequently extracted into a fluorous solvent. The nucleotides present in the fluorous solvent were then back-extracted into a non-fluorous solution, such as a solution of ammonia in aqueous acetonitrile. The resulting non-fluorous solution containing the nucleotides was then directly injected into an amide-type HILIC column using a mixture of acetonitrile and aqueous ammonium bicarbonate as the mobile phase for gradient elution, and the nucleotides were detected using the negative electrospray ionization MS/MS mode. In this method, the extraction recoveries of the nucleotides ranged from 44.3 to 94.7% within a relative standard deviation of 17%. This method enabled the determination of intracellular concentrations of nucleotides.

    更新日期:2018-01-03
  • Urinary metabolomics study the mechanism of Taohong Siwu Decoction intervention in acute blood stasis model rats based on liquid chromatography coupled to quadrupole time-of-flight mass spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-02
    Xiaosong Zhang, Pengling Li, Yongli Hua, Peng Ji, Wanling Yao, Qi Ma, Ziwen Yuan, Yanqiao Wen, Chaoxue Yang, Yanming Wei

    Taohong Siwu Decoction (TSD) is a classic prescription in traditional Chinese medicine and is widely used to promote blood circulation to remove blood stasis. However, the effect mechanisms are not yet well understood. Here, a urinary metabolomic approach based on liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS) was conducted to explore the changes in the endogenous metabolites and to assess the integral efficacy of TSD on acute blood stasis model rats. Then, parameters for hemorheology and coagulation functions were detected. Principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA) was used to investigate the global metabolite alterations and to evaluate the preventive effects of TSD in rats. Potential metabolite markers were found using OPLS-DA and t-test. Furthermore, metabolic pathway analysis was performed to construct metabolic networks. The results showed that TSD could significantly decrease whole blood viscosity and plasma viscosity. It also significantly prolonged partial thromboplastin time (APPT) and prothrombin time (PT), increased thrombin time (TT) and lowered fibrinogen content (FIB). Moreover, 24 potential metabolite markers of acute blood stasis were screened, and the levels were all reversed to different degrees after TSD administration. In metabolic networks, amino acid metabolism (arginine and proline metabolism; histidine metabolism; alanine, aspartate, and glutamate metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis; phenylalanine metabolism) and lipid metabolism (glycerophospholipid metabolism; linoleic acid metabolism; alpha-linolenic acid metabolism) were closely related with the intervention mechanism of TSD on acute blood stasis. The urinary metabolomic approach can be applied to clarify the mechanism of TSD in promoting blood circulation to remove acute blood stasis and to provide the theoretical basis for further research on the therapeutic mechanism of TSD in clinical practice.

    更新日期:2018-01-03
  • Effect of aspirin on the pharmacokinetics and absorption of panax notoginseng saponins
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-02
    Zhihao Tian, Huanhuan Pang, Qiang Zhang, Shouying Du, Yang Lu, Lin Zhang, Jie Bai, Pengyue Li, Danqi Li, Mengdi Zhao, Xiaonan Chen

    Background Panax notoginseng saponins, a traditional Chinese medicine extraction, and aspirin are both widely used to treat cerebral infarction in China. Good results in clinical practice have been achieved, when Panax notoginseng saponins was taken together with aspirin. Methods To investigate the interaction of the two drugs in vivo, the concentration of notoginsenoside R1, ginsenoside Rg1, Rb1, Re and Rd. in blood were simultaneously measured by UPLC/MS/MS. Sample preparation was carried out by the protein precipitation technique with an internal standard saikosaponin A standard. The separation of six components was achieved by using an ACQUITY UPLC ®BEH C18 column (1.7 μm 2.1 × 100 mm) by gradient elution using water (containing 0.2% formic acid) and acetonitrile (containing 0.2% formic acid) as the mobile phase at a flow rate of 0.2 mL/min. The pharmacokinetic parameters were determined using non-compartmental analysis. The transport of notoginsenoside R1, ginsenoside Rg1, Rb1, Re and Rd. in MDCK -MDR1 cell monolayer was also used to verify the conclusion of pharmacokinetic drug-drug interaction and study the mechanism of drug interaction. Results The concentrations of the five components increased in a certain extent when the two drugs administered together in rats. The values of apparent permeability coefficients were significantly increased when the two drugs were used together. Aspirin and salicylic acid could destroy the tight junction protein and open the intercellular space to increase the absorption of Panax notoginseng saponins. Conclusion Pharmacokinetic drug-drug interaction in vivo existed between Panax notoginseng saponins and aspirin. The drug-drug interaction mainly occurred in the process of absorption.

    更新日期:2018-01-03
  • Development of a method to determine axitinib, lapatinib and afatinib in plasma by micellar liquid chromatography and validation by the European Medicines Agency guidelines
    J. Chromatogr. B (IF 2.603) Pub Date : 2018-01-02
    Jaume Albiol-Chiva, Josep Esteve-Romero, Juan Peris-Vicente

    A method based on micellar liquid chromatography to quantify the tyrosine kinase inhibitors axitinib, lapatinib and afatinib in plasma is reported. The sample pretreatment was a simple 1/5-dilution in a pure micellar solution, filtration and direct injection, without requiring extraction or purification steps. The three drugs were resolved from the matrix in 17 min, using an aqueous solution of 0.07 M sodium dodecyl sulfate – 6.0% 1-pentanol, buffered at pH 7 with 0.01 M phosphate salt as mobile phase, running under isocratic mode at 1 mL/min through a C18 column. The detection was performed by absorbance at 260 nm. An accurate mathematical relationship was established between the retention factor of each drug and the surfactant/organic solvent concentration in the mobile phase, achieved with a limited number of experiments, in order to optimize these factors. A binding behavior of the analytes face to the micelles was found out. The method was successfully validated by the guidelines of the European Medicines Agency in terms of: selectivity, linearity (r2 > 0.9995), calibration range (0.5 to 10 mg/L), limit of detection (0.2 mg/L), carry-over effect, accuracy (− 8.1 to + 6.9%), precision (< 13.8%), dilution integrity, matrix effect, stability and robustness. The procedure was found reliable, practical, economic, accessible, short-time, easy-to-handle, inexpensive, environmental-friendly, safe, useful for the analysis of many samples per day. Finally, the method was applied to the analysis of incurred, using quality control samples in the same analytical run, with adequate results. Therefore, it can be implementable for routine analysis in clinical laboratories.

    更新日期:2018-01-02
  • C18 core-shell column with in-series absorbance and fluorescence detection for simultaneous monitoring of changes in stilbenoid and proanthocyanidin concentrations during grape cane storage
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-29
    Vania Sáez, Camila Gayoso, Sebastián Riquelme, Julie Pérez, Carola Vergara, Claudia Mardones, Dietrich von Baer

    Grape canes, the residues from the annual pruning of vines, contain high levels of inducible (E)-resveratrol and also oligomeric stilbenoids and proanthocyanidins. These two families of phenolic compounds are bioactive, but to quantify them in a single chromatographic run using only ultraviolet detection is a difficult task. To overcome this limitation, a chromatographic method was developed using a core shell column for separation, an ultraviolet-visible diode array detector (DAD) and a fluorescence (FL) detector connected in series for quantification, with an electrospray ionization interface (ESI) and a triple quadrupole mass spectrometric detector (MS/MS) added for identification of the analytes. The proanthocyanidins (+)-catechin, (−)-epicatechin, procyanidins B1, B2, and C1, an unknown dimer and trimer, two prodelphinidin dimers, and monogallate procyanidin dimers were detected in the tested grape cane samples. The stilbenoids detected were (E)-resveratrol, (E)-piceatannol, (E)-piceid, (E)-ε-viniferin, vitisin B, a glycosylated monomer, three oxidized dimers, an unknown dimer and a tetramer, pallidol, hopeaphenol, (E)-δ-viniferin, and (E)-ω-viniferin. However, this method required 60 min for each analysis. A faster and more efficient method for quantitative analysis was developed based on HPLC-DAD-FL, reducing the time required to 24 min for the simultaneous quantification of proanthocyanidins and stilbenoids in Cabernet Sauvignon, Pinot Noir, and Tintorera grape canes stored at controlled temperatures and relativity humidities for 134 days after pruning. To the best of our knowledge, this is the first time a prodelphinidin dimer has been quantified in grape canes. The incorporation of fluorescence detection in series with DAD not only allowed the quantification of proanthocyanidins, it also improved the detectability of some minor stilbenoids present in the canes, such as (E)-piceid. The (E)-resveratrol and (E)-piceatannol levels increased significantly during cane storage, while those of (E)-ε-viniferin and ampelopsin A did not show significant increases. The relative humidity had a determining effect on the levels of (E)-resveratrol and (E)-piceatannol in the canes of all varieties studied; their concentrations were higher at a relative humidity of 60% than at 70%. This is the first time that the proanthocyanidin profiles of canes stored after pruning were monitored. The concentration of (−)-epicatechin decreased during storage under both relative humidities. Furthermore, the levels of proanthocyanidin B1 and the prodelphinidin dimer also decreased to a certain extent.

    更新日期:2017-12-31
  • Determination of IMM-H004 and its active glucuronide metabolite in rat plasma and Ringer's solution by ultra-performance liquid chromatography-tandem mass spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-29
    Jianwei Jiang, Ziqian Zhang, Xiaowen Zou, Rui Wang, Jie Bai, Shengyu Zhao, Xiaoqing Fan, Li Sheng, Yan Li

    IMM-H004 is a novel neuroprotective agent and its glucuronide metabolite IMM-H004G has similar protective effects against cerebral ischemic injury in vivo and in vitro. A specific and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was established and validated for determination of IMM-H004 and IMM-H004G simultaneously in rat plasma and Ringer's solution. Plasma samples containing IMM-H004, IMM-H004G and internal standard propranolol were prepared by direct protein precipitation in a sample-to-solvent ratio of 1:2:6 (plasma: water: acetonitrile), whereas no protein precipitation was required for Ringer's solution samples. Separation was performed with a gradient mobile phases of methanol/water with 0.5% formic acid (v/v) on Eclipse Plus C18 column (2.1 × 50 mm, 3.5 μm) at a flow rate of 0.3 mL/min. The detection was operated on a triple quadrupole mass spectrometer in positive ion multiple reaction monitoring (MRM) mode. The monitored transitions were 305.1 → 248.1 for IMM-H004, 481.3 → 305.1 for IMM-H004G and 260.1 → 183.1 for propranolol. The linear ranges of IMM-H004 and IMM-H004G were 5 to 3000 ng/mL and 10 to 3000 ng/mL for plasma method and 0.5 to 500 ng/mL for Ringer's solution method. All the intra-day and inter-day precision and accuracy for the two analytes in rat plasma were below 7.5% and the intra-day precision and accuracy for analytes in Ringer's solution were within ± 14.7%. There was no obvious matrix effect and the recoveries of the analytes were higher than 94.2%. IMM-H004 and IMM-H004G were stable during one analytic process. The established method was applied successfully to plasma pharmacokinetic and brain microdialysis studies of IMM-H004 and IMM-H004G in rats after a single intravenous administration of IMM-H004.

    更新日期:2017-12-31
  • Bioanalysis of a panel of neurotransmitters and their metabolites in plasma samples obtained from pediatric patients with neuroblastoma and Wilms' tumor
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-28
    Lucyna Konieczna, Anna Roszkowska, Teresa Stachowicz-Stencel, Anna Synakiewicz, Tomasz Bączek

    This paper details the quantitative analysis of neurotransmitters, including dopamine (DA), norepinephrine (NE), epinephrine (E), and serotonin (5-HT), along with their respective precursors and metabolites in children with solid tumors: Wilms' tumor (WT) and neuroblastoma (NB). A panel of neurotransmitters was determined with the use of dispersive liquid-liquid microextraction (DLLME) technique combined with liquid-chromatography mass spectrometry (LC-MS/MS) in plasma samples obtained from a group of pediatric subjects with solid tumors and a control group of healthy children. Next, statistical univariate analysis (t-test) and multivariate analysis (Principal Component Analysis) were performed using chromatographic data. The levels of tyrosine (Tyr) and tryptophan (Trp) (the precursors of analyzed neurotransmitters) as well as 3,4-dihydroxyphenylacetic acid (DOPAC) (a product of metabolism of DA) were significantly higher in the plasma samples obtained from pediatric patients with WT than in the samples taken from the control group. Moreover, statistically significant differences were observed between the levels of 5-HT and homovanillic acid (HVA) in the plasma samples from pediatric patients with solid tumors and the control group. However, elevated levels of these analytes did not facilitate a clear distinction between pediatric patients with WT and those with NB. Nonetheless, the application of advanced statistical tools allowed the healthy controls to be differentiated from the pediatric oncological patients. The identification and quantification of a panel of neurotransmitters as potential prognostic factors in selected childhood malignancies may provide clinically relevant information about ongoing metabolic alterations, and it could potentially serve as an adjunctive strategy in the effective diagnosis and treatment of solid tumors in children.

    更新日期:2017-12-31
  • Development and single-laboratory validation of a UHPLC-MS/MS method for quantitation of microcystins and nodularin in natural water, cyanobacteria, shellfish and algal supplement tablet powders
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-28
    Andrew D. Turner, Julia Waack, Adam Lewis, Christine Edwards, Linda Lawton

    A simple, rapid UHPLC-MS/MS method has been developed and optimised for the quantitation of microcystins and nodularin in wide variety of sample matrices. Microcystin analogues targeted were MC-LR, MC-RR, MC-LA, MC-LY, MC-LF, LC-LW, MC-YR, MC-WR, [Asp3] MC-LR, [Dha7] MC-LR, MC-HilR and MC-HtyR. Optimisation studies were conducted to develop a simple, quick and efficient extraction protocol without the need for complex pre-analysis concentration procedures, together with a rapid sub 5 min chromatographic separation of toxins in shellfish and algal supplement tablet powders, as well as water and cyanobacterial bloom samples. Validation studies were undertaken on each matrix-analyte combination to the full method performance characteristics following international guidelines. The method was found to be specific and linear over the full calibration range. Method sensitivity in terms of limits of detection, quantitation and reporting were found to be significantly improved in comparison to LC-UV methods and applicable to the analysis of each of the four matrices. Overall, acceptable recoveries were determined for each of the matrices studied, with associated precision and within-laboratory reproducibility well within expected guidance limits. Results from the formalised ruggedness analysis of all available cyanotoxins, showed that the method was robust for all parameters investigated. The results presented here show that the optimised LC-MS/MS method for cyanotoxins is fit for the purpose of detection and quantitation of a range of microcystins and nodularin in shellfish, algal supplement tablet powder, water and cyanobacteria. The method provides a valuable early warning tool for the rapid, routine extraction and analysis of natural waters, cyanobacterial blooms, algal powders, food supplements and shellfish tissues, enabling monitoring labs to supplement traditional microscopy techniques and report toxicity results within a short timeframe of sample receipt. The new method, now accredited to ISO17025 standard, is simple, quick, applicable to multiple matrices and is highly suitable for use as a routine, high-throughout, fast turnaround regulatory monitoring tool.

    更新日期:2017-12-31
  • Metal-organic framework UiO-66 for rapid dispersive solid phase extraction of neonicotinoid insecticides in water samples
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-21
    Xiaolin Cao, Zejun Jiang, Shanshan Wang, Sihui Hong, Hui Li, Chao Zhang, Yong Shao, Yongxin She, Fen Jin, Maojun Jin, Jing Wang

    UIO-66 crystals were explored for the first time to adsorb neonicotinoid insecticides in environmental water samples. HPLC coupled with tandem MS was used for quantification and determination of neonicotinoid insecticides. UiO-66 crystals was successfully synthesized by a simple constant-temperature bath method. Synthesized UiO-66 was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetry and nitrogen adsorption porosimetry (NAP), which demonstrated a uniform particle size, a large Brunauer–Emmett–Teller (BET) surface area and high thermostability. The adsorbing results showed that UIO-66 crystals could be used as a promising adsorbents for rapid extraction of neonicotinoid insecticides and be reused at least 10 times. Under the optimized conditions, the limits of detection (LODs, S/N = 3) and limits of quantification (LOQs, S/N = 10) for the five insecticides were found to be 0.02–0.4 ng/mL and 0.05–1.0 ng/mL, respectively. This developed approach not only provided more simple and sensitive method, as well as possessing satisfactory recovery for neonicotinoid insecticides, but also for other traces in environmental samples.

    更新日期:2017-12-22
  • Comparative metabolism of two major compounds in Fructus Corni extracts by gut microflora from normal and chronic nephropathy rats in vitro by UPLC-Q-TOF/MS
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-18
    Jin-hua Tao, Min Zhao, Shu Jiang, Xue-lian Pu, Xiao-yan Wei

    Herbal medicines are widely used as therapeutic products in many countries. Fructus Corni, a traditional herb medicine, has been clinically used to cure chronic nephropathy for thousands of years. It could be converted by gut microflora in vivo to shape its pharmacological profiles. Thus, metabolic profiles of major active constituents in Fructus Corni extracts by gut microflora from rats in healthy and nephropathy state were firstly investigated in vitro by ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) in this study. According to the features of protonated ions, five metabolites (M1, M2, M3, M5 and M6) were found and preliminarily authenticated. Intestinal bacteria were capable of converting N0 (loganin) to its aglycone M1 (loganetin). The latter was further hydrogenated to the corresponding M2 (hydrogenated loganetin) and subsequently to M3 (hydrogenated and demethylated loganetin) by demethylation; While M5 (demethylated morronisid aglycone) and M6 (dehydroxylated morronisid aglycone) were identified as the two metabolites of N4 (morronisid) through demethylation and dehydroxylation. Gut microflora from healthy and nephropathy rats could degrade loganin and morronisid to the above metabolites. However, healthy rat intestinal bacteria showed more powerful degradation and much more amounts of M1 and M6 were obtained in their samples. Additionally, this work demonstrated that UPLC-Q-TOF/MS approach connected with MetaboLynx™ analysis software was rapid and reliable for screening and authentication of natural product metabolites.

    更新日期:2017-12-18
  • Aptamer facilitated purification of functional proteins
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-16
    Stanislav S. Beloborodov, Jiayin Bao, Svetlana M. Krylova, Agnesa Shala-Lawrence, Philip E. John-son, Sergey N. Krylov

    DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method’s ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies.

    更新日期:2017-12-18
  • 更新日期:2017-12-16
  • Simultaneous determination of Vitamin A, 25-hydroxyl vitamin D3 α-tocopherol in small biological fluids by liquid chromatography-tandem mass spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-15
    Haifeng Zhang, Li Quan, Pei Pei, Ye Lin, Chao Feng, Hongyan Guan, Fang Wang, Ting Zhang, Jianxin Wu, Junsheng Huo

    A sensitive method for the simultaneous analysis of vitamin A (VA), 25-hydroxyl vitamin D3 (25-OH VD3) and α-tocopherol (VE) in children plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Sample preparation chose the solid phase extraction. 100 μL of plasma was mixed with 300 μL ethanol contained 4 μL isotope-labelled analytes. After a series operation, the supernatant was applied to the solid phase extraction (SPE) plate (HLB μElution plate). The eluate was evaporated, and reconstituted in 100 μL methanol. And then, 6 μL reconstituted sample was injected into LC-MS/MS. Quantitative analysis was carried out by multiple reaction monitoring (MRM) with a positive mode electrospray (ESi + ). Separations of VA, 25-OH VD3 and VE were performed on an Acquity UPLC reversed-phase Phenyl-Hexyl analytical column (CSH, 2.1 × 50 mm, 1.7 μm). Gradient elution was used at a flow rate of 0.4 mL/min with a mobile phase consisting of 0.1% formic acid solution and 0.1% formic acid, 5 mM ammonium formate in acetonitrile. The total time of analysis was 10 min. The method had a limit of quantification (LOQ) of 10.03, 1.20, and 0.04 ng/mL for VA, 25-OH VD3 and VE in methanol, respectively. The linear calibration curves were fitted over the range of 0.14–14.32 μg/mL, 1.80–180.29 ng/mL, and 6.03–602.99 ng/mL for VA, 25-OH VD3 and VE in methanol. The correlation coefficients were greater than 0.998 for all analytes. The recoveries for all analytes were between 80–120% with the inter- and intra-day precisions (presented as relative standard deviation, RSD%) less than 10.0%. Analysis of VA, 25-OH VD3 and VE in recurrent respiratory tract infection children plasma and anemic infants’ fingertip blood was then carried out using this method and statistical analysis of the data with statistic package for social science 20.0 (SPSS 20.0). Using this method, multiple fat-soluble vitamins could be detected at the same time. Solid phase extraction was used to simplify sample pretreatment. μElution plate used here could reduce the sample volume, only 100 μL sample was used in this method, and 6 μL reconstituted sample was injected into LC-MS/MS. This makes the method appropriate for larger sample pretreatment, and suitable for children, especially infants and newborns’ sample detection, in whom the circulation blood was low.

    更新日期:2017-12-15
  • Determination of Pyrethrin and Pyrethroid Residues in Animal Fat using Liquid Chromatography coupled to Tandem Mass Spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-13
    M. Moloney, S. Tuck, A. Ramkumar, A. Furey, M. Danaher

    A method was developed for the confirmatory and quantitative analysis of one pyrethrin and 18 pyrethroid residues in animal fat. Fat was extracted was collected from adipose tissue melted in an oven at 65 °C for 2 h. Fat samples (1 g) were dispersed with deactivated Florisil® sorbent and extracted with MeCN. Sample extracts were purified by cold temperature precipitation at −30 °C for 4 h and further purified using dispersive solid-phase extraction (d-SPE) clean-up in tubes 500 mg of Z-SEP+ and 125 mg of PSA bonded silica. Purified samples were analysed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) detection. Chromatographic separation was carried out on a Acquity C8 BEH column, using a binary gradient separation comprising of mobile phase A, 5 mM ammonium formate in water:MeOH (80:20, v/v,) and mobile phase B, 5 mM ammonium formate in MeOH. The mass spectrometer was operated in the positive electrospray ionisation mode (ESI(+)). Validation was performed following the 2002/657/EC guidelines. Trueness ranged between 84% and 143% and precision ranged between 3.9% and 29%. The developed method is particularly advantageous because the sample preparation procedure does not require complex sample extraction equipment and uses less solvent compared to other sample preparation

    更新日期:2017-12-15
  • UPLC-MS/MS method for the simultaneous quantification of sofosbuvir, sofosbuvir metabolite (GS-331007) and daclatasvir in plasma of HIV/HCV co-infected patients
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-12
    Stefania Notari, Massimo Tempestilli, Gabriele Fabbri, Raffaella Libertone, Andrea Antinori, Adriana Ammassari, Chiara Agrati

    Direct-acting antiviral agents (DAAs) represent the major advance in hepatitis C virus (HCV) infection treatment leading to extremely high eradication rates in HCV mono- and HIV/HCV co-infected patients. In this scenery, availability of therapeutic drug monitoring (TDM) is of interest to assess plasma concentrations to prevent either therapeutic failure due to suboptimal medication adherence and drug-drug interactions or avoid adverse events. Aim of this study was to develop and validate an ultra-performance liquid chromatography mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of sofosbuvir, sofosbuvir metabolite (GS-331007), and daclatasvir in human plasma. A simple protein precipitation was applied by adding 200 μL acetonitrile with internal standard 6,7-Dimethyl- 2,3-di(2-pyridyl) quinoxaline to 100 μL plasma sample. Drug separation was performed on analytical C-18 Luna Omega column (50 mm × 2.1 mm I.D.) with particle size of 1.6 μm. The mobile phase consisting of water containing 0.1% formic acid and acetonitrile at flow 0.4 mL/min and a gradient run time of 3.5 minutes. The injection volume was 10 μL. Anti-HCV drugs were detected in positive electrospray ionization mode. The full scan mass spectral analyses of sofosbuvir, GS-331007, daclatasvir and quinaxoline showed protonated molecule ions and transitions m/z: 530.098 → 243.02, 260.93 → 112.94, 739.4 → 339.27 and 313.03 → 77.99 respectively. The linearity of standard curves was excellent (r2 > 0.99), the absolute recovery of anti-HCV drugs ranged between 95 and 98%, and both imprecision and inaccuracy were <15% according to FDA guidelines. The UPLC-MS/MS method was applied to 16 plasma samples of as many HIV/HCV co-infected patients treated with sofosbuvir and daclatasvir. While sofosbuvir was not detectable in all samples, the median plasma concentrations of daclatasvir and GS-331007 were 223.6 ± 319.56 ng/mL and 537.11 ± 242.09 ng/mL, respectively. In conclusion, we describe an UPLC-MS/MS method allowing the simultaneous quantification of sofosbuvir, GS-331007 and daclatasvir in plasma samples. The method was sensitive, specific, robust, and time-saving.

    更新日期:2017-12-13
  • Comparison of salting-out and sugaring-out liquid-liquid extraction methods for the partition of 10-hydroxy-2-decenoic acid in royal jelly and their co-extracted protein content
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-12
    Xijuan Tu, Fanyi Sun, Siyuan Wu, Weiyi Liu, Zhaosheng Gao, Shaokang Huang, Wenbin Chen

    Homogeneous liquid-liquid extraction (h-LLE) has been receiving considerable attention as a sample preparation method due to its simple and fast partition of compounds with a wide range of polarities. To better understand the differences between the two h-LLE extraction approaches, salting-out assisted liquid-liquid extraction (SALLE) and sugaring-out assisted liquid-liquid extraction (SULLE), have been compared for the partition of 10-hydroxy-2-decenoic acid (10-HDA) from royal jelly, and for the co-extraction of proteins. Effects of the amount of phase partition agents and the concentration of acetonitrile (ACN) on the h-LLE were discussed. Results showed that partition efficiency of 10-HDA depends on the phase ratio in both SALLE and SULLE. Though the partition triggered by NaCl and glucose is less efficient than MgSO4 in the 50% (v/v) ACN-water mixture, their extraction yields can be improved to be similar with that in MgSO4 SALLE by increasing the initial concentration of ACN in the ACN-water mixture. The content of co-extracted protein was correlated with water concentration in the obtained upper phase. MgSO4 showed the largest protein co-extraction at the low concentration of salt. Glucose exhibited a large protein co-extraction in the high phase ratio condition. Furthermore, NaCl with high initial ACN concentration is recommended because it produced high extraction yield for 10-HDA and the lowest amount of co-extracted protein. These observations would be valuable for the sample preparation of royal jelly.

    更新日期:2017-12-12
  • Putative identification of components in Zengye Decoction and their effects on glucose consumption and lipogenesis in insulin-induced insulin-resistant HepG2 cells
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-12
    Zhenzhen Liu, Wenhua Kuang, Xi Xu, Dandan Li, Wufu Zhu, Zhou Lan, Xu Zhang

    Zengye Decoction (ZYD) is a well-known traditional medicine in China used for treating diseases associated with “Yin deficiency” such as diabetes. However, little information is available on its components, pharmacological effects and underlying mechanisms. This study was designed to identify its active components and evaluate the effects and mechanisms of ZYD on glucose consumption and lipogenesis in insulin-induced insulin-resistant (IR)-HepG2 cells. In this study, 45 compounds of ZYD were putatively identified, in which the iridoid glycosides such as catalpol, aucubin and harpagide were identified as the main components. The insulin-resistant (IR)-HepG2 cell model was established and the effect of ZYD at three doses (0.17, 0.5 and 1.5 μg/mL) on cell growth was evaluated with an IncuCyte™ live-cell imaging system. The effects of ZYD on glucose consumption and uptake were evaluated by glucose consumption and uptake assay. Meanwhile, the effect of ZYD on lipogenesis was investigated in IR-HepG2 cells by oil red O (ORO) staining. Western blot was applied to observe the changes in some of the key factors involved in glucose metabolism and lipogenesis. It was found that the ZYD at a dose of 1.5 μg/mL exhibited an inhibitory activity on IR-HepG2 cell growth. Besides, ZYD at doses of 0.5 and 1.5 μg/mL accelerated the glucose consumption, glucose uptake and reduced the lipogenesis in the IR-HepG2 cells. Western blot studies revealed that ZYD phosphorylated AMP-activated protein kinase α subunits (AMPKα), upregulated hexokinase (HK), phosphorylated acetyl-CoA carboxylase alpha (pACC1) and carnitine palmitoyltransferase 1A (CPT1A) in the IR-HepG2 cells. These results indicate ZYD promotes glucose consumption and uptake, and attenuates lipogenesis in IR-HepG2 cells, which may be involved in activating AMPK and regulating its downstream factors including HK, pACC1 and CPT1A.

    更新日期:2017-12-12
  • Efficient purification of Apolipoprotein A1 (ApoA1) from plasma by HEA HyperCel™: an alternative approach
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-11
    Gopalarajan Arun Govind, Agamudi Shivasankaran Kamalanathan, Mookambeswaran Arunachalam Vijayalakshmi, Krishnan Venkataraman

    HDL-ApoA1 plays a pivotal role in the prevention of atherosclerosis and cardiovascular diseases. ApoA1 purification from blood plasma has always remained tedious, involving multiple steps, large volumes of plasma and substantial loss in the final yield of pure ApoA1. In this study, a two-step method has been developed and optimized for the purification of ApoA1 from plasma. Plasma was first subjected to 60% ammonium sulphate (NH4)2SO4 precipitation and subsequently, ApoA1 was recovered using mixed mode chromatographic sorbent, HEA HyperCel™. ApoA1 was found to be enriched in 60% (NH4)2SO4 supernatant that was dialyzed and injected onto HEA sorbent with 50 mM phosphate buffer pH 7.4. The bound proteins were eluted by decreasing the pH in step-gradient from pH 7.4 to pH 4.0 and subsequently to pH 3.5 using 50 mM sodium acetate buffer. Gel electrophoresis showed elution of homogeneous apoA1 at pH 3.5, with purity and yield of 63%. An interesting feature of this approach is that the purified ApoA1 was monomeric with a mass of 28,079.30 Da as confirmed by MS analysis. This simple and efficient method of purification of apoA1 serves as an alternative method which can be combined with traditional approaches and has a great potential for biochemical and clinical studies.

    更新日期:2017-12-11
  • A novel liquid chromatography/mass spectrometry method for determination of neurotransmitters in brain tissue: Application to human tauopathies
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-09
    Andrea Forgacsova, Jaroslav Galba, Ralph M. Garruto, Petra Majerova, Stanislav Katina, Andrej Kovac

    Neurotransmitters, small molecules widely distributed in the central nervous system are essential in transmitting electrical signals across neurons via chemical communication. Dysregulation of these chemical signaling molecules is linked to numerous neurological diseases including tauopathies. In this study, a precise and reliable liquid chromatography method was established with tandem mass spectrometry detection for the simultaneous determination of aspartic acid, asparagine, glutamic acid, glutamine, γ −aminobutyric acid, N-acetyl-L-aspartic acid, pyroglutamic acid, acetylcholine and choline in human brain tissue. The method was successfully applied to the analysis of human brain tissues from three different tauopathies; corticobasal degeneration, progressive supranuclear palsy and parkinsonism-dementia complex of Guam. Neurotransmitters were analyzed on ultra-high performance chromatography (UHPLC) using an ethylene bridged hybrid amide column coupled with tandem mass spectrometry (MS/MS). Identification and quantification of neurotransmitters was carried out by ESI+ mass spectrometry detection. We optimized sample preparation to achieve simple and fast extraction of all nine analytes. Our method exhibited an excellent linearity for all analytes (all coefficients of determination >0.99), with inter-day and intra-day precision yielding relative standard deviations 3.2 % to 11.2 % and an accuracy was in range of 92.6 % to 104.3 %. The present study, using the above method, is the first to demonstrate significant alterations of brain neurotransmitters caused by pathological processes in the brain tissues of patient with three different tauopathies.

    更新日期:2017-12-10
  • Measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine in Cerebrospinal Fluid by Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-09
    Allan Weimann, Anja Hviid Simonsen, Henrik E. Poulsen

    Increased levels of nucleosides modified by oxidation in human cerebrospinal fluid (CSF) have several times been reported in Alzheimer patients and patients suffering from Parkinson’s disease. The focus has especially been on nucleosides containing the 8-hydroxylation of guanine. Only few reports on quantification of the ribonucleoside 8-oxo-7,8-dihydro-guanosine (8oxoGuo) in CSF have been published, whereas more have been published on the quantification of the deoxy-ribonucleoside 8-oxo-7,8-dihydro-2′-deoxyguanosine (8oxodGuo). The reports on the quantification of 8oxodGuo concentrations in CSF report absolute concentrations varying by a factor >105 in healthy humans. This could indicate that there is a serious specificity problem in some of the methods. In this paper an isotope-dilution UPLC-MS/MS method with high specificity and sensitivity for the quantification of 8oxoGuo and 8oxodGuo in CSF is presented. LLOQ for the two analytes is determined to 4pM and 2pM, respectively. The calibration curves has been tested to be linear in the range from 4–3,000pM for 8oxoGuo and between 2 and 3,000pM for 8oxodGuo. Using a weighting factor of 1/x the correlation coefficient “r” for both analytes is >0.999.

    更新日期:2017-12-10
  • Development of a fast HPLC-DAD method for simultaneous quantitation of three immunosuppressant drugs in whole blood samples using intelligent chemometrics resolving of coeluting peaks in the presence of blood interferences
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-09
    Maryam Vosough, Sadaf Mosleh Tehrani

    The present study describes a fast high performance liquid chromatography-diode array detection analytical methodology for quantification of tacrolimus, everolimus and cyclosporine A in whole blood samples, with minimum sample preparation steps. A short isocratic chromatographic elution was coupled with second-order calibration using multivariate curve resolution to stablish a smart and green methodology. Due to presence of matrix effect, a sample-added calibration strategy was used for quantification purposes. The serious issues related to background drift, chromatographic shifts and co-elution of non-calibrated blood components, were resolved by a proper background correction and multivariate curve resolution-alternating least squares (MCR/ALS) methods The main features of this study were based on the fact that the acquired data matrices were handled intelligently and all features of the concerned target analytes were taken into account. Satisfactory resolution and quantification results in the presence of matrix interferences were achieved and the second-order advantage was fully exploited. The average recoveries in therapeutic concentration ranges were 102 ± 10%, 99 ± 11% and 104 ± 12% for TAC, EVR and CsA, with average relative prediction errors of less than 7%. Considering the advantages of the present strategy, such as increased selectivity, sensitivity and sufficiency of lower limit of quantification through multivariate advantage, simplicity of sample treatment steps, a fast elution pattern and also a low-cost instrumentation compared with LC-MS/MS, the proposed method has the significant merits as an alternative for simultaneous therapeutic monitoring of immunosuppressants.

    更新日期:2017-12-10
  • The application of dispersive liquid-liquid microextraction in the analyses of the fatty acid profile in bovine milk in response to changes in body condition score.
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-09
    Andrew Quigley, Damian Connolly, Wayne Cummins

    Dispersive liquid-liquid microextraction (DLLME) was used prior to gas chromatography flame ionization detection (GC-FID) for the extraction of five fatty acids from milk taken from cows with different body condition scores. Optimum extraction conditions were: 300 μL of chloroform (extraction solvent), and 1 mL methanol (dispersive solvent). The procedure was optimised using Design of Experiments (DoE). The analytes were separated on a GC capillary column containing a polyethylene glycol stationary phase (15 m × 0.53 mm × 1.2 μm). Enrichment factors were in the range of 8–15 and limit of detection (LOD) was 0.04 μg/mL. Calibration graphs showed good linearity with coefficients of determination higher than 0.994% and relative standard deviations lower than 7%. This method provided a simple and rapid derivatisation and extraction method for the determination of fatty acids in bovine milk. It showed that there was a significant difference in the palmitic acid content of milk from cows that had an optimum body condition score (10.85 mg/mL) compared to cows that had a high body condition score (5.73 mg/mL).

    更新日期:2017-12-10
  • Development and validation of an UHPLC-ESI-QTOF-MS method for quantification of the highly hydrophilic amyloid-β oligomer eliminating all-d-enantiomeric peptide RD2 in mouse plasma
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-08
    Michelle Hupert, Anne Elfgen, Elena Schartmann, Sarah Schemmert, Brigitte Buscher, Janine Kutzsche, Dieter Willbold, Beatrix Santiago-Schübel

    During preclinical drug development, a method for quantification of unlabeled compounds in blood plasma samples from treatment or pharmacokinetic studies in mice is required. In the current work, a rapid, specific, sensitive and validated liquid chromatography mass-spectrometric UHPLC-ESI-QTOF-MS method was developed for the quantification of the therapeutic compound RD2 in mouse plasma. RD2 is an all-d-enantiomeric peptide developed for the treatment of Alzheimer’s disease, a progressive neurodegenerative disease finally leading to dementia. Due to RD2’s highly hydrophilic properties, the sample preparation and the chromatographic separation and quantification were very challenging. The chromatographic separation of RD2 and its internal standard were accomplished on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm particle size) within 6.5 minutes at 50 °C with a flow rate of 0.5 mL/min. Mobile phases consisted of water and acetonitrile with 1% formic acid and 0.025% heptafluorobutyric acid, respectively. Ions were generated by electrospray ionization (ESI) in the positive mode and the peptide was quantified by QTOF-MS. The developed extraction method for RD2 from mouse plasma revealed complete recovery. The linearity of the calibration curve was in the range of 5.3 ng/mL to 265 ng/mL (r2 > 0.999) with a lower limit of detection (LLOD) of 2.65 ng/mL and a lower limit of quantification (LLOQ) of 5.3 ng/mL. The intra-day and inter-day accuracy and precision of RD2 in plasma ranged from −0.54% to 2.21% and from 1.97% to 8.18%, respectively. Moreover, no matrix effects were observed and RD2 remained stable in extracted mouse plasma at different conditions. Using this validated bioanalytical method, plasma samples of unlabeled RD2 or placebo treated mice were analyzed. The herein developed UHPLC-ESI-QTOF-MS method is a suitable tool for the quantitative analysis of unlabeled RD2 in plasma samples of treated mice.

    更新日期:2017-12-10
  • Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-07
    Annelie Hansson, Heather Knych, Scott Stanley, Emma Berndtson, Liora Jackson, Ulf Bondesson, Mario Thevis, Mikael Hedeland

    LGD-4033 has been found in human doping control samples and has the potential for illicit use in racehorses as well. It belongs to the pharmacological class of selective androgen receptor modulators (SARMs) and can stimulate muscle growth, much like anabolic steroids. However, SARMs have shown superior side effect profiles compared to anabolic steroids, which arguably makes them attractive for use by individuals seeking an unfair advantage over their competitors. The purpose of this study was to investigate the metabolites formed from LGD-4033 in the horse in order to find suitable analytical targets for doping controls. LGD-4033 was administered to three horses after which plasma and urine samples were collected and analyzed for metabolites using ultra high performance liquid chromatography coupled to a high resolution mass spectrometer. In horse urine, eight metabolites, both phase I and phase II, were observed most of which had not been described in other metabolic systems. Six of these were also detected in plasma. The parent compound was detected in plasma, but not in non-hydrolyzed urine. The longest detection times were observed for unchanged LGD-4033 in plasma and in urine hydrolyzed with β-glucuronidase and is thus suggested as the analytical target for doping control in the horse. The metabolite profile determined in the horse samples was also compared to those of human urine and fungal incubate from Cunninghamella elegans. The main human metabolite, dihydroxylated LGD-4033, was detected in the horse samples and was also produced by the fungus. However, it was a minor metabolite for horse and fungus, which highlights the importance of performing metabolism studies in the species of interest.

    更新日期:2017-12-10
  • Analysis of monoamine oxidase (MAO) enzymatic activity by high-performance liquid chromatography-diode array detection combined with an assay of oxidation with a peroxidase and its application to MAO inhibitors from foods and plants
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-06
    Tomás Herraiz, Andrea Flores, Lidia Fernández

    Monoamine oxidase (MAO) enzymes catalyze the oxidative deamination of biogenic amines and neurotransmitters and produce ammonia, aldehydes, and hydrogen peroxide which is involved in oxidative processes. Inhibitors of MAO-A and -B isozymes are useful as antidepressants and neuroprotectants. The assays of MAO usually measure amine oxidation products or hydrogen peroxide by spectrophotometric techniques. Those assays are often compromised by interfering compounds resulting in poor results. This research describes a new method that combines in the same assay the oxidative deamination of kynuramine to 4-hydroxyquinoline analyzed by HPLC-DAD with the oxidation of tetramethylbenzidine (TMB) (or Amplex Rex) by horseradish peroxidase (HRP) in presence of hydrogen peroxide. The new method was applied to study the inhibition of human MAO-A and -B by bioactive compounds including β-carboline alkaloids and flavonoids occurring in foods and plants. As determined by HPLC-DAD, β-carbolines, methylene blue, kaempferol and clorgyline inhibited MAO-A and methylene blue, 5-nitroindazole and deprenyl inhibited MAO-B, and all of them inhibited the oxidation of TMB in the same extent. The flavonoids catechin and cyanidin were not inhibitors of MAO by HPLC-DAD but highly inhibited the oxidation of TMB (or Amplex Red) by peroxidase whereas quercetin and resveratrol were moderate inhibitors of MAO-A by HPLC-DAD, but inhibited the peroxidase assay in a higher level. For some phenolic compounds, using the peroxidase-coupled assay to measure MAO activity led to mistaken results. The new method permits to discern between true inhibitors of MAO from those that are antioxidants and which interfere with peroxidase assays but do not inhibit MAO. For true inhibitors of MAO, inhibition as determined by HPLC-DAD correlated well with inhibition of the oxidation of TMB and this approach can be used to assess the in vitro antioxidant activity (less hydrogen peroxide production) resulting from MAO inhibition.

    更新日期:2017-12-06
  • Extraction protocol and liquid chromatography/tandem mass spectrometry method for determining micelle-entrapped paclitaxel at the cellular and subcellular levels: application to a cellular uptake and distribution study
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-06
    Nan Zheng, Bin Lian, Wenwen Du, Guobing Xu, Jiafu Ji

    Paclitaxel-loaded polymeric micelles (PTX-PM) are commonly used as tumor-targeted nanocarriers and display outstanding antitumor features in clinic, but its accumulation and distribution in vitro are lack of investigation. It is probably due to the complex micellar system and its low concentration at the cellular or subcellular levels. In this study, we developed an improved extraction method, which was a combination of mechanical disruption and liquid-liquid extraction (LLE), to extract the total PTX from micelles in the cell lysate and subcellular compartments. An ultra-performance liquid chromatography tandem mass spectroscopy (UPLC–MS/MS) method was optimized to detect the low concentration of PTX at cellular and subcellular levels simultaneously, using docetaxel as internal standard (IS). The method was proved to release PTX totally from micelles (≥95.93%) with a consistent and reproducible extraction recovery (≥75.04%). Good linearity was obtained at concentrations ranging from 0.2 to 20 ng/mL. The relative error (RE%) for accuracy varied from 0.68 to 7.56%, and the intra- and inter-precision (relative standard deviation, RSD%) was less than 8.64% and 13.14%, respectively. This method was fully validated and successfully applied to the cellular uptake and distribution study of PTX-loaded PLGA-PEG micelles in human breast cancer cells (MCF-7).

    更新日期:2017-12-06
  • Simultaneous UPLC-MS/MS analysis of two stable isotope labelled versions of sucrose in mouse plasma and brain samples as markers of blood-brain barrier permeability and brain vascular space
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-06
    Ekram Ahmed Chowdhury, Faleh Alqahtani, Raktima Bhattacharya, Reza Mehvar, Ulrich Bickel

    Blood Brain Barrier (BBB) permeability is frequently compromised in the course of diseases affecting the central nervous system (CNS). Sucrose is a low molecular weight, hydrophilic marker with slow permeability at the naive BBB and therefore one of the widely used indicators of barrier integrity. Our laboratory recently developed a highly sensitive UPLC-MS/MS method for stable isotope labelled [13C12]sucrose in biological matrices. Correction of total brain concentration for contribution of intravascular space is required in such experiments in order to accurately measure BBB permeability, and it is often accomplished by vascular perfusion with buffer solutions prior to brain sampling. The purpose of the present study was to develop a UPLC-MS/MS method, which allows simultaneous analysis of two different stable isotope labeled sucrose variants, one of which can be utilized as a vascular marker. The first analyte, [13C12]sucrose, serves to quantify brain uptake clearance as a measure of BBB permeability, while the second analyte, [13C6]sucrose, is administered just before termination of the animal experiment and is considered as the vascular marker. [2H2]sucrose is used as the internal standard for both 13C labeled compounds. Because the majority of recent studies on CNS diseases employ mice, another objective was to validate the new technique in this species. The UPLC-MS/MS method was linear (r2 ≥ 0.99) in the tested concentration ranges, from 10 to 1,000 ng/mL for both analytes in plasma, from 2 to 400 ng/g [13C12]sucrose in brain and from 10 to 400 ng/g [13C6]sucrose in brain. It was also validated in terms of acceptable intra and inter run accuracy and precision values (n = 5). The dual analyte technique was applied in a study in mice. One group received intravenous bolus injections of 10 mg/kg [13C12]sucrose at time 0, and 10 mg/kg [13C6]sucrose at 14.5 min, and subsequent terminal blood and brain sampling was performed at 15 min. For comparison, another group received an intravenous bolus dose of 10 mg/kg [13C12]sucrose and was submitted to transcardiac perfusion with buffer after 15 min. We demonstrate that the two alternative techniques to correct for intravascular content deliver equivalent values for brain concentration and brain uptake clearance.

    更新日期:2017-12-06
  • Development of an LC–MS method to quantify coproporphyrin I and III as endogenous biomarkers for drug transporter-mediated drug-drug interactions
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-06
    Emmanuel Njumbe Ediage, Lieve Dillen, Ann Vroman, Luc Diels, Annett Kunze, Jan Snoeys, Tom Verhaeghe

    Coproporphyrins are proposed as endogenous biomarkers of hepatic Organic Anion Transporting Polypeptide (OATP)1 B functional activity. In this study, a new sample extraction method based on a mixed-mode anion exchange sorbent (SPE clean-up using Oasis 30 mg Max 96 well plates) was developed for absolute quantification of coproporphyrin I and III (CP-I and CP-III) in human plasma. Chromatographic separation was performed with an Ace Excel 2 C18 PFP, 3 μm, 2.1 × 150 mm, maintained at 60 °C. A 10 mM ammonium formate containing 0.1% HCOOH and acetonitrile (100%) was used as mobile phase A and B, respectively. Mass transition, m/z 655.3 → 596.3 was selected to monitor CP-I and CP-III, while m/z 659.3 → 600.3 transition was used for the stable isotope labelled internal standard. Optimization of the liquid chromatography tandem mass spectrometry method ensured a lower limit of quantification (LLOQ) of 20 pg/mL. Both CP-I and CP-III had extraction recoveries of 70%. The calibration range was 0.02–100 ng/mL for both CP-I and CP-III, yielding calibration curves with correlation coefficients greater than 0.988. Inter day precision (CV < 9%) and accuracy (84.3–103.9%) complied with the recommendation of the European Bioanalytical Forum. The optimized method was used to analyse plasma samples originating from three independent clinical studies. Obtained CP-I and CP-III plasma baseline levels in healthy volunteers were in good agreement with previously published data. Moreover, CP-I and CP-III plasma levels in human subjects dosed with a clinically confirmed OATP inhibitor were significantly increased compared to their baseline levels. These data demonstrate the potential of CP-I and CP-III as endogenous biomarkers to predict the drug-drug interaction (DDI) related to hepatic OATP1 B inhibition. Stability of CP-I and CP-III in plasma and solvents under different processing and storage conditions was also evaluated.

    更新日期:2017-12-06
  • Rapid preparative separation of monoclonal antibody charge variants using laterally-fed membrane chromatography
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-05
    Rahul Sadavarte, Pedram Madadkar, Carlos DM Filipe, Raja Ghosh

    Monoclonal antibodies undergo various forms of chemical transformation which have been shown to cause loss in efficacy and alteration in pharmacokinetic properties of these molecules. Such modified antibody molecules are known as variants. They also display physical properties such as charge that are different from intact antibody molecules. However, the difference in charge is very subtle and separation based on it is quite challenging. Charge variants are usually separated using ion-exchange column chromatography or isoelectric focusing. In this paper, we report a rapid and scalable method for fractionating monoclonal antibody charge variants, based on the use of cation exchange laterally-fed membrane chromatography (LFMC). Starting with a sample of monoclonal antibody hIgG1-CD4, three well-resolved fractions were obtained using either pH or salt gradient. These fractions were identified as acidic, neutral and basic variants. Each of these fractions contained intact heavy and light chains and so antibody fragmentation had no role in variant generation. The separation was comparable to that using column chromatography but was an order of magnitude faster.

    更新日期:2017-12-06
  • An improved liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of dexmedetomidine concentrations in samples of human plasma
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-05
    Seyed Mojtaba Moosavi, Kiran Shekar, John F Fraser, Maree T Smith, Sussan Ghassabian

    Dexmedetomidine (DMET) is a sedative, analgesic and anxiolytic with minimum adverse respiratory effects. An LC-MS/MS bioanalytical method has been developed and validated to accurately measure DMET concentrations in samples of human plasma. The method overcomes difficulties in the extraction and quantification of DMET due to the fact that it binds strongly to glass and plastic tubes, as well as solid phase extraction (SPE) cartridges. Human plasma (50 μL) was mixed with the internal standard (IS) (DMET-d4) solution (100 μl) and 0.1% formic acid (50 μL) and extracted using Oasis HLB 1 CC (30 mg) solid phase extraction (SPE) cartridges (Waters®). The glass tubes were coated with bovine serum albumin (BSA) 0.5% (20 μL) before eluting DMET and the IS. After evaporation under nitrogen at room temperature, the analytes were reconstituted in 20% acetonitrile in 0.1% formic acid in water and transferred to silanized glass vials. An electrospray ionisation (ESI) mass spectrometry method in positive mode was created and the precursor/product transitions (m/z) were 201.1 → 95.0 (DMET) and 204.9 → 99.0 (IS). The method was robust and fully validated based on the 2012 EMEA guideline for bioanalytical method validation in the concentration range of 0.5–20 ng/mL. Using this assay, we showed that DMET binds strongly to Extracorporeal Membrane Oxygenation (ECMO) circuits, consistent with expectations for small lipophilic compounds.

    更新日期:2017-12-06
  • Influence of direct and sequential extraction methodology on metabolic profiling
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-05
    K.M. Maria John, James Harnly, Devanand Luthria

    A systematic comparison was made of the detected metabolite profiles for two plant materials (black beans and soybeans) and a dietary supplement (black cohosh) extracted using sequential (hexane, ethyl acetate, and 50% aqueous methanol) and direct extraction with three solvent systems (80% aqueous methanol, methanol/chloroform/water (2.5:1:1, v/v/v) and water). Extracts were analyzed by LC–MS (without derivatization) and GC-FID (with BSTFA/TMCS derivatizations). For sequential extraction, HPLC-UV and BSTFA/TMCS-derivatized GC-FID detection were more responsive to the polar molecules with a rough distribution of 10%, 10%, and 80% of the total signals in hexane, ethyl acetate, and 50% aqueous methanol, respectively. With HPLC–MS detection, the distribution of signals was more balanced, roughly 40%, 30%, and 30% for the same extracts (hexane, ethyl acetate, and 50% aqueous methanol). For direct extraction, HPLC-UV and BSTFA/TMCS-derivatized 4GC-FID provided signals between 60% and 150% of the total sequential extracted signals. The overlap of signals for the 3 sequential extracts ranged from 1% to 3%. The overlap of the signals for direct extraction with the total for sequential extraction ranged from 15% to 98%. With HPLC–MS detection, signals varied from 30% to 40% of the total signals for sequential extraction. Multivariate analysis showed that the components for the sequential and direct extracts were statistically different. However, each extract, sequential or direct, allowed discrimination between the 3 plant materials.

    更新日期:2017-12-06
  • Investigation of antioxidant ability of grape seeds extract to prevent oxidatively induced DNA damage by gas chromatography-tandem mass spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Önder Aybastıer, Sam Dawbaa, Cevdet Demir

    Phenolic compounds have been studied elaborately for their efficacy to improve health and to protect against a wide variety of diseases. Herein this study, different analysis methods were implemented to evaluate the antioxidant properties of catechin and cyanidin using their standard substances and as they found in the grape seeds extracts. Total phenol contents were 107.39 ± 8.94 mg GAE/g dw of grape seeds for grape seed extract (GSE) and 218.32 ± 10.66 mg GAE/g dw of grape seeds for acid-hydrolyzed grape seed extract (AcGSE). The extracts were analyzed by HPLC-DAD system and the results showed the presence of catechin, gallic acid, chlorogenic acid and ellagic acid in the processed methanolic extract and cyanidin, gallic acid and ellagic acid in the processed acidified methanolic extract. The protective abilities of catechin and cyanidin were tested against the oxidation of DNA. The results showed that cyanidin has better protection of DNA against oxidation than catechin. GSE and AcGSE were revealed to inhibit the oxidatively induced DNA damage. GSE decreased about 57% of damage caused by the Fenton control sample. This study could show new aspects of the antioxidant profiles of cyanidin and catechin.

    更新日期:2017-12-02
  • Electrospun polydimethylsiloxane/polyacrylonitrile/titanium dioxide nanofibers as a new coating for determination of alpha-linolenic acid in milk by direct immersion-solid phase nanoextraction
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Sahar Sadeghi, Afsaneh Mollahosseini

    In this study, polydimethylsiloxane/polyacrylonitrile/titanium dioxide (PDMS/PAN/TiO2) nanofibers were synthesized via electrospinning method to extract and quantify alpha-linolenic acid (ALA, C18:3), as a model analyte, in milk by direct immersion-solid phase nanoextraction (DI-SPNE) with gas chromatography–flame ionization detector (GC–FID). The affecting factors on the electrospinning process such as, PDMS concentration, amount of TiO2 nanoparticles (NPs), voltage, and electrospinning distance were optimized using Taguchi’s orthogonal design. The SPNE experimental conditions such as, extraction time, agitation rate, pH and salt concentration, were also optimized using response surface methodology (RSM) based on a central composite design (CCD). Under optimized conditions, the resulting calibration curve was linear in the range of 1–4000 ng mL−1 with R2 = 0.998. The intraday, interday, and fiber-to-fiber repeatability were calculated and the corresponding relative standard deviation was less than 9% in all the cases. The limit of detection and limit of quantification were found to be 0.2 and 0.6 ng mL−1, respectively. Omega-3 enriched milk was used as a real sample and the value of relative recoveries were measured to be in the range of 92-106%.

    更新日期:2017-12-02
  • Moxidectin residues in lamb tissues: Development and Validation of Analytical Method by UHPLC-MS/MS
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Michelle Del Bianchi A. Cruz, Maria A.M. Fernandes, Patricia A. de C. Braga, Alda L.G. Monteiro, Daniela Daniel, Felix G.R. Reyes

    The development and validation of a throughput method for the quantitation of moxidectin residues in lamb target tissues (muscle, kidney, liver and fat) was conducted using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). To achieve higher recovery of the analyte from the matrices, a modified QuEChERS method was used for sample preparation. The chromatographic separation was achieved using a Zorbax Eclipse Plus C18 RRHD column with a mobile phase comprising 5 mM ammonium formate solution +0.1% formic acid (A) and acetonitrile +0.1% formic acid (B) in a linear gradient program. Method validation was performed based on the Commission Decision 2002/657/EC and VICH GL49. To quantify the analyte, matrix-matched analytical curves were constructed with spiked blank tissues, with a limit of quantitation of 5 ng g−1 and limit of detection of 1.5 ng g−1 for all matrices. The linearity, decision limit, detection capability accuracy, and inter- and intra-day repeatability of the method are reported. The method was successfully applied to incurred lamb tissue samples (muscle, liver, kidney and fat) in a concentration range from 5 to 200 μg kg−1, which demonstrated its suitability for monitoring moxidectin residues in lamb tissues in health surveillance programs, as well as for pharmacokinetics and residue depletion studies.

    更新日期:2017-12-02
  • Correlating Charge Heterogeneity Data Generated by Agarose Gel Isoelectric Focusing and Ion Exchange Chromatography Methods
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Babita Saxena Parekh, Arvind Srivastava, Shanmuuga Sundaram, Ming Ching-Heish, Joel Goldstein, Michael Barry, Qinwei Zhou

    An isoelectric focusing method (IEF) has been used to assess the charge heterogeneity profile of a monoclonal antibody during the early stages of product development. A more precise and sensitive ion exchange chromatography (IEC/CEX) method was developed and implemented as development progressed and was used concurrently with IEF for lot release and to monitor charge heterogeneity. Charge variants resolved by both methods (IEC and IEF) were purified and characterized. Tryptic peptide mapping and N- linked oligosaccharide profile analyses of the IEC and IEF fractions indicated a structural correlation between the charge variants separated by these two methods The major sources of molecular heterogeneity were due to the variation in the sialyated carbohydrate structure and heavy chain C-terminal lysine truncation. By monitoring the rates of change in the charge heterogeneity profiles of the monoclonal antibody stored at elevated temperatures by the IEC and IEF methods, a positive correlation between the two methods was established. This approach enabled replacement of the IEF method with the more precise IEC method.

    更新日期:2017-12-02
  • Metabolism and pharmacokinetics of alantolactone and isoalantolactone in rats: Thiol conjugation as a potential metabolic pathway
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Bailun Zhou, Ji Ye, Niao Yang, Liping Chen, Zhiguo Zhuo, Ling Mao, Qun Liu, Gongcai Lan, Jing Ning, Guangbo Ge, Ling Yang, Yunheng Shen, Shumei Wang, Weidong Zhang

    Alantolactone (AL) and isoalantolactone (IAL), two major active sesquiterpene lactones isolated from Radix Inulae extract, have a wide range of pharmacological activities. The predominant metabolic pathway of AL and IAL observed was glutathione (GSH) conjugation in vitro, which could occur in the absence of metabolic enzymes. Non-enzymatic conjugation with cysteine (Cys) couldalso be observed. Four metabolites (AL-GSH, AL-Cys, IAL-GSH, IAL-Cys) were subsequently isolated and confirmed by nuclear magnetic resonance (NMR). The results indicated that the thiol of GSH or Cys can be reacted with the exomethylene carbon atoms of α, β-unsaturated carbonyl of AL and IAL. After intravenous administration in rats, AL and IAL were extensively metabolized, and the exposure, as measured by area under the concentration-time curve (AUC), for AL-GSH, AL-Cys, IAL-GSH, and IAL-Cys was approximately 1.54-, 0.96-, 1.50-, and 0.91-fold that of the parent drug, respectively. The AUC ratio of metabolites to parent compounds of oral administration was 3.66-, 9.19-, 12.97-, and 9.92-fold that of the parent drug for the above metabolites, respectively. The bioavailability of AL-total (AL, AL-GSH, AL-Cys) and IAL-total (IAL, IAL-GSH, IAL-Cys) was, respectively, 8.39% and 13.07%, which was 3.62- and 6.95- fold that of AL (2.32%) and IAL (1.88%), respectively. The oral exposure will be underestimated if the parent drugs are tested alone. These findings provide useful information for preclinical safety evaluation, and for predicting AL and IAL metabolism in humans.

    更新日期:2017-12-02
  • Cohort-Friendly Protocol for a Sensitive and Fast Method for Trihalomethanes in Urine using Gas Chromatography − Triple Quadrupole Mass Spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Pantelis Charisiadis, Konstantinos C. Makris

    A cohort-friendly biomonitoring protocol has been developed for measuring biomarkers of exposure to disinfection by-products (trihalomethanes, THM) in urine using small initial volume (3 mL) and short analysis time (∼10 minutes) that facilitates the throughput of a large number of samples. The objective of this study was to optimise a cohort-friendly biomonitoring protocol for the determination of four THM analytes in human urine using gas chromatography coupled with triple quadrupole mass spectrometry (GC-QqQ-MS/MS). The proposed methodology will facilitate the inclusion of such urinary THM measurements into large population health studies.

    更新日期:2017-12-02
  • Quantification of glycocholic acid in human serum by stable isotope dilution ultra performance liquid chromatography electrospray ionization tandem mass spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Cheng Guo, Cong Xie, Peili Ding, Guangming Qin, Weimin Mo, Xiaoji Cao, Shu Zheng

    A rapid, accurate and sensitive stable isotope dilution ultra performance liquid chromatography electrospray ionization tandem mass spectrometry (ID-UPLC-ESI-MS/MS) method for the determination of glycocholic acid (GCA) in human serum was developed and validated. Serum samples were spiked with D5-glycocholic acid and then pretreated with protein precipitation. The analysis was performed on a Waters BEH C18 column (100 mm × 2.1 mm, 1.7 μm), followed by ESI-MS/MS detection in negative ion mode under multiple reaction monitoring mode. The calibration curves covered a concentration range from 0.2 to 400 ng/ml. The limit of detection and limit of quantification was 0.01 ng/mL and 0.05 ng/mL, respectively. The method showed satisfactory precision on intra-day (2.3–6.1%) and inter-day (2.4–4.6%) analyses and achieved good recovery at three spiked levels (103.7–114.3%). Moreover, this established method was successfully applied for quantification of GCA in serum samples from healthy volunteers, patients with hepatocellular carcinoma (HCC) and patients with other cancers. We demonstrated that the level of GCA in patients with HCC was significantly higher not only than that in healthy controls, but also than that in patients with other cancer, whereas no significant difference of GCA level was observed between healthy control group and other cancers group.

    更新日期:2017-12-02
  • Determination of blood concentrations of main active compounds in Zi-Cao-Cheng-Qi decoction and their total plasma protein binding rates based on hollow fiber liquid phase microextraction coupled with high performance liquid chromatography
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Miaomiao Li, Xuan Chen, Shuang Hu, Runqin Wang, Xiaoli Peng, Xiaohong Bai

    Oil-in-salt hollow fiber liquid phase microextraction coupled with high performance liquid chromatography ultraviolet detection (HPLC-UV) was developed for determination of the blood concentrations of the main active compounds, hesperidin, honokiol, shikonin, magnolol, emodin and β,β’-dimethylacrylshikonin, after oral administration of Zi-Cao-Cheng-Qi decoction (ZCCQD) and their total plasma protein binding rates. In the procedure, a hollow fiber segment was immersed in organic solvent to fill the solvent in the fiber lumen and wall pore, and then the fiber was immersed into sodium chloride solution to cover a thin salt membrane on the fiber wall pore filling organic solvent. Various factors affecting the procedure, such as extraction solvent, sample phase pH, stirring rate, extraction time, NaCl concentration and fiber immersion time in the NaCl solution, were optimized. Under the optimum conditions, good linearities (r2 ≥ 0.9905), low limits of detection (0.7–2.5 ng/mL) or quantitation (1.2–12 ng/mL), satisfactory precision (2.6%-12.8%) and accuracy (81.0%–114.2%) of this method, were observed. The results showed that, after oral administration of a 25 g/kg dose, (1) the blood concentrations (at 0.5 h) of hesperidin, honokiol, shikonin, magnolol, emodin and β,β’-dimethylacrylshikonin were 0.45, 0.40, 0.48, 0.74, 0.11 and 1.11 μg/mL, respectively; (2) the total plasma protein binding rates of the six active compounds were 42.0% (hesperidin), 71.8% (honokiol), 64.6% (shikonin), 77.7% (magnolol), 75.3% (emodin) and 75.7% (β,β’-dimethylacrylshikonin), respectively. The proposed procedure coupled with HPLC shows obvious advantages, such as low solvent consumption, simple operation, high sensitivity and strong purifying and can be used for the determination of both the blood concentrations and total plasma protein binding rates of active compounds in traditional Chinese medicine.

    更新日期:2017-12-02
  • GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Volha Zabela, Timm Hettich, Götz Schlotterbeck, Laurin Wimmer, Marko D. Mihovilovic, Fabrice Guillet, Belkacem Bouaita, Bénédicte Shevchenko, Matthias Hamburger, Mouhssin Oufir

    In a screening of natural products for allosteric modulators of GABAA receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABAA and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization. Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds.

    更新日期:2017-12-02
  • Identification and Analysis of a Mercapturic Acid Conjugate of Indole-3-methyl Isothiocyanate in the Urine of Humans who Consumed Cruciferous Vegetables
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-02
    Pramod Upadhyaya, Adam T. Zarth, Naomi Fujioka, Vincent A. Fritz, Stephen S. Hecht

    Glucobrassicin, a quantitatively significant constituent of Brassica vegetables, gives rise to indole-3-carbinol (I3C) and its dimer di-indolylmethane (DIM) when the vegetables are chewed. I3C and DIM have been extensively studied with respect to their anti-carcinogenic properties. However, the presumed intermediate isothiocyanate in their formation, indole-3-methyl isothiocyanate (IMITC), has to our knowledge never been observed, despite the fact that isothiocyanates derived from cruciferous vegetables are known to have anti-carcinogenic properties. Therefore, we investigated the formation and presence in human urine of IMITC by analyzing for its N-acetylcysteine conjugate, IMITC-NAC, in order to gain a more complete understanding of the biochemical pathways leading to formation of I3C and DIM upon consumption of vegetables rich in glucobrassicin. Standard IMITC-NAC was synthesized and its structure confirmed by NMR and MS. IMITC-NAC was identified in extracts of Brussels sprouts chopped in the presence of N-acetylcysteine. An LC-ESI-MS/MS-SRM method for analysis of IMITC-NAC, with [13C,15N]IMITC-NAC as internal standard, was developed and validated. Then, ten subjects (7 females) consumed a salad of Brussels sprouts and cabbage (containing 100 to 500 μmol glucobrassicin) once daily for 3 days. Urine was collected at intervals up to 24 h after vegetable consumption. Levels of IMITC-NAC in the urine of these 10 subjects ranged from 0.2 to 30.2 pmol/mL urine. These results provide the first evidence for the presumed intermediacy of IMITC in the formation of I3C and DIM in humans who consumed Brussels sprouts and cabbage as a source of glucobrassicin.

    更新日期:2017-12-02
  • A simple, rapid and stability indicating validated method for quantification of lamotrigine in human plasma and dry plasma spot using LC-ESI-MS/MS: Application in clinical study
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-12-01
    Kuldeep Kumar Namdev, Jaya Dwivedi, Deepak Chandra Chilkoti, Swapnil Sharma

    Lamotrigine (LTZ) is a phenyltriazine derivative which belongs to anti-epileptic drugs (AEDs) class and prescribed as mono- or adjunctive-therapy in treatment of epilepsy. Therapeutic drug monitoring (TDM) of AEDs provides a valid clinical tool in optimization of overall therapy. However, TDM is challenging due to the high biological samples (plasma/blood) storage/shipment costs and the limited availability of laboratories providing TDM services. Sampling in the form of dry plasma spot (DPS) or dry blood spot (DBS) are suitable alternative to overcome these issues. We developed and validated a new method for quantification of LTZ in human plasma and DPS. The extraction of LTZ from plasma and DPS was performed using liquid-liquid extraction with diethyl ether and an extraction solution composed of diethyl ether- methyl tert–butyl ether– acetone (50:30:20, v/v/v), respectively. Lamotrigine- 13C3, d3 was used as internal standard (ISTD) and the chromatographic separation was achieved on Hypurity Advance C18 column (150 × 4.6 mm, 5 μm). Quantitative estimation of LTZ and ISTD was performed on a liquid chromatography tandem mass spectrometer coupled with electrospray ionization interface operated under positive mode of ionization. Calibration curves were linear (r2 > 0.99) over the concentration range of 10–3020 ng/mL for both plasma and DPS. Statistical analysis provides insignificant difference between LTZ concentration extracted from plasma and DPS samples. The method is found suitable for application in clinical study and in therapeutic monitoring of LTZ. To the best of our knowledge this is the first report which describing a validated stability indicating assay for quantification of LTZ in dry plasma spot.

    更新日期:2017-12-02
  • A novel adenine-releasing assay for ribosome-inactivating proteins
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-28
    Alexander Weng

    Ribosome-inactivating proteins (RIPs) are toxic enzymes that are mostly biosynthesized by plants. RIPs are N-glycosidases that cleave an essential adenine molecule from the 28S rRNA. This is followed by the irreversible inhibition of protein synthesis leading to cell death. By fusing RIPs to cancer cell specific targeting ligands RIPs have been utilized for targeted anti-tumor therapy. The anti-tumoral efficiency of such conjugates depends significantly on the N-glycosidase activity of the RIP domain.

    更新日期:2017-11-29
  • Multi-residue analysis of sedative drugs in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-28
    Liqun Zhang, Pinggu Wu, Quan Jin, Zhengyan Hu, Junlin Wang

    Background Sedative drugs are often used for the treatment of depression, anxiety and insomnia and they are involved in many forensic cases. This work established a method for the simultaneous determination of multi-groups of sedative drugs (benzodiazepines, barbitals, phenothiazines, tricyclic antidepressant and butyrophenone) in human plasma for forensic analysis. A large number of sedative drugs with forensic interest can be analyzed in a short time with lower limits of quantification by combination of SPE extraction and ultra performance liquid chromatography-tandem mass spectrometry. Methods Sample preparation consisted of extraction of the plasma by hydrochloric acid solution and further purified by SLW solid phase extraction column. Results The method was fully validated to cover large concentration ranges of 50–1000 μg/L for 5 Barbital drugs, while 1.0–50 μg/L for the other 14 sedative drugs. The average recoveries of the drugs spiked at three levels ranged from 65.3% to114.3% with the relative standard deviation between 1.2% and 13.2%. The lowest limits of quantification (LLOQ) were 100 μg/L for Barbital drugs and1.0 μg/L for all the other 14 drugs. Conclusions This validated method has been successfully used in emergency analysis of multi-residues of sedative drugs in plasma.

    更新日期:2017-11-29
  • Enhanced capillary electrophoretic screening of Alzheimer based on direct apolipoprotein E genotyping and one-step multiplex PCR
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-27
    Nain Woo, Su-Kang Kim, Yucheng Sun, Seong Ho Kang

    Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer’s disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer’s. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2 min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120–180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4–62.0 pM, which were approximately 100–100,000 times more sensitive than previous Alzheimer’s diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer’s patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer’s with high sensitivity and short analysis time even with direct use of whole blood.

    更新日期:2017-11-28
  • Microwave assisted synthesis for A2E and development of LC-ESI–MS method for quantification of ocular bisretinoids in human retina
    J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-26
    A. Kotnala, S. Senthilkumari, N. Halder, A. Kumar, T. Velpandian

    Purpose To develop a microwave assisted method for the rapid synthesis of A2E and also to develop a method to quantify N-retinylidene-N-retinylethanolamine(A2E), all-trans retinal dimer (ATRD), A2-glycerophospho ethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE) and monofuran A2E (MFA2E) in age matched retina. Methods The development of microwave assisted synthesis of A2E, its purification and Characterization for its utility in quantification in human retina. The semi-quantitative method development using LC-ESI–MS, LC-ESI–MS/MS and LC-APCI-MS/MS from pooled macula and peripheral retina for the bisretinoid analysis has been done. Results Maximum A2E conversion using microwave assisted process took place at 80 °C for 45 min with a yield of 55.01%. Highly sensitive and specific mass spectrometric method was developed using reverse phase C-18 separation with positive electrospray ionization and positive atmospheric phase chemical ionization of tandom mass spectrometry. A gradient mobile phase separation was achieved using water and methanol with 0.1% TFA. Multiple reaction monitoring acquisition for ESI and APCI was performed at ATRD m/z 551.2/522.2, A2GPE m/z 746.4/729.5, A2DHPEm/z 594.4/576.5, MFA2E m/z 608.2/591.2, A2E m/z 592.4/418.2. Method was validated using LC-ESI-SIM mode to determine selectivity, linearity, sensitivity, precision and accuracy. Conclusion An attempt towards optimization of the synthetic procedure of A2E was made so as to reduce the lengthy reaction time without compromising the yield. Developed method was capable enough for the detection of low level of bisretinids in retina.

    更新日期:2017-11-27
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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