Characterization of Human Serum Albumin isoforms by ion exchange chromatography coupled on-line to native mass spectrometry J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-18 Y. Leblanc, N. Bihoreau, G. Chevreux
Human Serum Albumin is the most abundant protein of the plasma and displays a wide range of non-oncotic properties such as antioxidant activity, distribution in tissues and organs of binding molecules and clearance of toxic compounds. Albumin is susceptible to numerous post-translational modifications and particularly related to its free thiol group at Cys34 which is the main circulating scavenger of reactive oxygen species. The characterization of these modifications is of high interest for the diagnosis and treatment of patients with liver diseases and for the structural integrity assessment of albumin as a therapeutic protein.In this study, an ion exchange chromatographic method coupled on-line to native mass spectrometry was developed in order to bridge an effective charge variants separation method with a powerful identification technique for a detailed characterization of albumin isoforms. The chromatographic performance of the method allows the separation of 9 different isoforms that were on-line characterized by MS as oxidized, glycated, deamidated and N/C-terminal truncated forms. The method is also able to detect Cu(II) ions binding to the N-terminal site of the protein which is an important antioxidant feature of albumin. Finally, the method showed preliminary good performance parameters in term of linearity, precision and sensitivity for characterization of purified albumin as well as albumin from raw plasma for clinical and pharmaceutical purposes.
Xanthohumol C, a minor bioactive hop compound: Production, purification strategies and antimicrobial test J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-18 Simon Roehrer, Juergen Behr, Verena Stork, Mara Ramires, Guillaume Médard, Oliver Frank, Karin Kleigrewe, Thomas Hofmann, Mirjana Minceva
Hop has been attracting scientific attention due to its favorable bioactivity properties. It is thus desirable to relate these properties to the specific hop compounds and extract these compounds in highly purified form in order to enhance the effect. The aim of the present study is the isolation of a sufficient amount of the highly purified prenylated minor hop compound xanthohumol C (XNC) for characterizing its bioactivity. Two strategies for the production of XNC were evaluated. The first strategy involved a capture of natural XNC from a xanthohumol (XN)-enriched hop extract (XF) by countercurrent chromatography. In the second approach, a one-step semi-synthesis of XNC was performed starting from XN, which had previously been separated from a natural XN-enriched hop extract. Both methods delivered XNC in sufficient amount and purity (>95%, HPLC), whereas the second strategy was preferable in terms of purity (>99%, HPLC) as well as productivity and solvent consumption. The methods were validated by identifying and quantifying XNC using LC-MS, LC-MS/MS and 1H NMR analysis. The XNC obtained in this way was supplied to several bacterial, yeast and fungal cultures in order to evaluate its antimicrobial effects. For comparison, microorganisms were also treated with the natural XN-enriched hop extract, as well as the prenylated hop compound XN. While still reducing cell proliferation, XNC was found to be less effective than both XF and XN for all studied bacteria and yeasts. Furthermore, for Bacillus subtilis, a strongly pH-dependent minimal inhibition concentration was observed for all three bioactive compounds, lowest at a pH of 5 and highest at a pH of 7.
Quantitative analysis of dextran in rat plasma using Q-Orbitrap mass spectrometry based on all ion fragmentation strategy J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-17 Hao Wang, Honglei Chen, Jie Geng, Yi Zheng, Zhongyu Zhang, Lin Sun, Guihua Tai, Yifa Zhou
Dextran is a biocompatible glucose-based polymer that is widely used clinically as a plasma volume expander, anti-thrombotic agent, macromolecular carrier, peripheral blood flow enhancer, and artificial tears promoter. Because dextran has polydisperse molecular weights and tends to produce innumerable multi-charged precursor ions in the ion source, it is difficult to analysis this polymer using conventional liquid chromatography mass spectrometry. In this assay, all ion fragmentation strategy is used to solve this problem by allowing all dextran precursor ions generated in the ion source to enter the collision cell. Then serial dextran-specific fragments can be effectively generated from all precursor ions by higher energy collision dissociation and scanned by Orbitrap detector. These high resolution fragment ions provide high specificity and sensitivity for the quantitation of dextran in rat plasma. Here, we report a new quantitative method using size exclusion chromatography combined with Q Exactive mass spectrometry based on an all ion fragmentation strategy. Assay validation showed that this method is linear over the concentration range from 3 to 300 μg/mL. Our approach has been successfully applied to a pharmacokinetic study of dextran in rat and will promote the development of studies with other polysaccharides.
1H NMR based pharmacometabolomics analysis of metabolic phenotype on predicting metabolism characteristics of losartan in healthy volunteers J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-17 Chenjie He, Yongbin Liu, Yicheng Wang, Jie Tang, Zhirong Tan, Xi Li, Yao Chen, Yuanfei Huang, Xiaoping Chen, Dongsheng Ouyang, Honghao Zhou, Jingbo Peng
Inter-individual variability in drug metabolism and disposition is common in both preclinical and clinical researches. Losartan and its active metabolite EXP3174 present a high degree of inter-individual differences in blood concentrations that affect drug efficacy and side effect. Pharmacometabolomics has been increasingly applied on predicting the drug responses by analyzing the differences in metabolic profile. A pre-dose metabolic phenotype was investigated to interpret inter-individual variations in the metabolism characteristics of losartan. 1H Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolic profiles were performed on 36 healthy Chinese male volunteers by measuring their pre-dose plasma samples. After oral administration of losartan, the concentrations of losartan and its bioactive metabolite EXP3174 were monitored by liquid chromatography-mass spectrometry (LC-MS). Orthogonal partial least-squares (O-PLS) model was conducted to select potential biomarkers that substantially contributed to the inter-individual variations in the metabolism features via analyzing the ratio of pharmacokinetics (PK) parameters of its metabolite to parent drug. Potential metabolites such as glycine, phosphorylcholine, choline, creatine, creatinine, lactate, citrate, α-glucose, and lipids showed strong correlations with metabolism features of losartan. In addition, the pathway analysis revealed that baseline lipid metabolism, the glycine, serine and threonine pathway, and glycolysis or gluconeogenesis metabolism pathway were significantly associated with the ratio of PK parameters of EXP3174 to losartan. Step-wise multiple linear regression (MLR) was constructed to investigate the potential roles of the selected biomarkers in predicting individualized metabolism characteristics of losartan. These results showed that the pre-dose individual metabolic traits may be a useful approach for characterizing individual differences in losartan metabolism characteristics and therefore for expediting personalized dose-setting in further clinical studies.
Chemometric approach to correlations between retention parameters of non-polar HPLC columns and physicochemical characteristics for ampholytic substances of biological and pharmaceutical relevance J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-17 Urszula Judycka, Karolina Jagiello, Maciej Gromelski, Leszek Bober, Jerzy Błażejowski, Tomasz Puzyn
The correlations between the retention parameters of forty ampholytic, biologically active and/or pharmaceutically relevant substances (obtained for three non-polar HPLC columns at various compositions of mobile phases and pH conditions: 2.5, 7.0, 11.5) and their thirty-two physicochemical (calculated/spectral) characteristics were investigated by applying chemometric methods of analysis. In three cases (among seven cases considered), Quantitative Property-Retention Relation (QPRR) models meeting the predictive capability criteria were developed (the values of R2, Q2CV, Q2Ext were higher than 0.76, 0.66 and 0.67, respectively, while values of RMSEC, RMSECV and RMSEExt were lower than 0.51, 0.65 and 0.65 in each developed model). These models create a useful platform for predicting retention parameters of untested chemicals and, to some extent, gaining pharmaceutically valuable information on the biologically active ampholytic substances based on their properties and the conditions of chromatographic separation.
Simultaneous detection of the novel anti-tumor drug MDH-7 and 5-fluorouracil in rat plasma by LC-MS/MS: Application to pharmacokinetic interactions J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-17 Juan Li, Yu Chen, Xiaorang Chen, Qingli Wang, Piao Zhou, Qiqi Fan, Yingqian Su, Yanyan Diao, Meili Guo, Mengchen An, Hongmin Liu
MDH-7 (2,3,9-tri-O-acetyl-5,6- dideoxy-1,10-di-[N4′-pentoxycarbonyl-5′-fluoro cytosine]-4-ulose 1,4: 7,10- difuranose-4,8-pyranose) is a novel anti-tumor drug candidate. To study the pharmacokinetic interaction between MDH-7 and 5-fluorouracil (5-FU), a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine the concentrations of MDH-7 and 5-fluorouracil (5-FU) in rat plasma. Plasma samples were prepared by simple liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters XBridge™ C18 column (5 μm, 2.1 mm × 150 mm) with the mobile phase of methanol and H2O (80:20, v/v). The ESI positive and negative ion switch was operated in the multiple reactions monitoring (MRM) mode. The calibration curves showed good linearity (r2 > 0.99) over the ranges of 50–8000 ng/ml for MDH-7 and 10–2000 ng/ml for 5-FU, respectively. The lower limit of quantitations (LLOQs) were 50 ng/mL (MDH-7) and 10 ng/mL (5-FU) with relative standard deviation (RSD) < 13.0%. The proposed method was successfully applied to simultaneous assessment of pharmacokinetic drug-drug interaction between MDH-7 and 5-FU in rats.
Comparing phospholipid profiles of mitochondria and whole tissue: Higher PUFA content in mitochondria is driven by increased phosphatidylcholine unsaturation J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-10 Cyrus E. Kuschner, Jaewoo Choi, Tai Yin, Koichiro Shinozaki, Lance B. Becker, Joshua W. Lampe, Junhwan Kim
Phospholipids content in cellular and mitochondrial membranes is essential for maintaining normal function. Previous studies have found a lower polyunsaturated fatty acid (PUFA) content in mitochondria than whole tissue, theorizing decreased PUFA protects against oxidative injury. However, phospholipids (PPLs) are uniquely difficult to quantify without class separation and, as prior approaches have predominately used reverse-phase HPLC or shotgun analysis, quantitation of PPL classes may have been complicated due to the existence of numerous isobaric and isomeric species. We apply normal-phase HPLC with class separation to compare whole tissue and mitochondrial PPL profiles in rat brain, heart, kidney, and liver. In addition, we establish a novel method to ascertain PPL origin, using cardiolipin as a comparator to establish relative cardiolipin /PPL ratios. We report a higher PUFA content in tissue mitochondria driven by increased phosphatidylcholine unsaturation, suggesting mitochondria purposefully incorporate higher PUFA PPLs.
Asymmetrical flow field-flow fractionation in purification of an enveloped bacteriophage phi6 J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-10 Mirka Lampi, Hanna M. Oksanen, Florian Meier, Evelin Moldenhauer, Minna M. Poranen, Dennis H. Bamford, Katri Eskelin
Basic and applied virus research requires specimens that are purified to high homogeneity. Thus, there is much interest in the efficient production and purification of viruses and their subassemblies. Advances in the production steps have shifted the bottle neck of the process to the purification. Nonetheless, the development of purification techniques for different viruses is challenging due to the complex biological nature of the infected cell cultures as well as the biophysical and -chemical differences in the virus particles. We used bacteriophage phi6 as a model virus in our attempts to provide a new purification method for enveloped viruses. We compared asymmetrical flow field-flow fractionation (AF4)-based virus purification method to the well-established ultracentrifugation-based purification of phi6. In addition, binding of phi6 virions to monolithic anion exchange columns was tested to evaluate their applicability in concentrating the AF4 purified specimens. Our results show that AF4 enables one-hour purification of infectious enveloped viruses with specific infectivity of ~1 × 1013 PFU/mg of protein and ~65–95% yields. Obtained purity was comparable with that obtained using ultracentrifugation, but the yields from AF4 purification were 2–3-fold higher. Importantly, high quality virus preparations could be obtained directly from crude cell lysates. Furthermore, when used in combination with in-line light scattering detectors, AF4 purification could be coupled to simultaneous quality control of obtained virus specimen.
Affinity purification of the avidin protein family, based on crystal structures of avidin-HABA complexes J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-11 S. Vetter, T. Miron, M. Wilchek
Since the importance of the high affinity between avidin and biotin, Kd = 3 × 10−16 M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4′-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, Kd = 6 × 10−6 M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent.
Determination of gentamicin C components in fish tissues through SPE-Hypercarb-HPLC-MS/MS J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-11 Xiaojie Sun, Yuanhao Yang, Qiangbing Tian, Derong Shang, Jun Xing, Yuxiu Zhai
In this work, an HPLC/MS/MS method for determination of gentamicin C components in fish tissues was developed based on strong cation exchange solid-phase extraction (SPE) purification coupled with Hypercarb chromatographic column in separation mode. Sample was extracted using trichloroacetic acid aqueous solution containing EDTA. Ion-pairing reagents were not needed because of the “graphite polarity retention effect” of the Hypercarb chromatographic column. HPLC-MS/MS was performed in multiple reaction monitoring (MRM) mode for simultaneous qualitative and quantitative analyses (using matrix external standard) of gentamicin C components in fish tissues. Good linearity was obtained for the target analytes within the concentration range from 0.0100 to 0.500 mg/L. The limits of quantification (LOQ) of this method were 10.0, 20.0, and 20.0 μg/kg for C1, C1a, and sum of C2 + C2a, respectively. The average recoveries of gentamicin C components were 80.0%–110% when spiked at three levels with the blank carp (Cyprinus carpio) matrix, and the relative standard deviations (RSD) were all less than 15% (n = 6). In addition, for the features of simple operation, high sensitivity and good reproducibility, the proposed method has been successfully applied for detection of gentamicin residues in fish tissues during actual breeding.
Quantification of ondansetron, granisetron and tropisetron in goat plasma using hydrophilic interaction liquid chromatography-solid phase extraction coupled with hydrophilic interaction liquid chromatography-triple quadrupole tandem mass spectrometry J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-11 Cunying Huang, Huihui Wan, Jing Zhang, Hongmin Zhong, Juan Li, YuMing Sun, Qing Wang, Hua Zhang
An assay method to quantify ondansetron (OND), granisetron (GRA) and tropisetron (TRO) in goat plasma has been successfully developed and validated. This method procedure for the analysis of OND, GRA and TRO was involved of extracting samples with hydrophilic interaction liquid chromatography (HILIC) solid phase extraction (SPE) and determination by liquid chromatography coupled to tandem mass spectroscopy. An SPE method for the simultaneous extraction of OND, GRA and TRO with high efficiency and selectivity was developed. Prior to HPLC-MS/MS analysis, most of the sources of interference present in the supernatant after protein precipitation of plasma proteins was efficiently removed from the samples by the HILIC SPE treatment. For the quantification of OND, GRA and TRO in the samples, tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring was used. The calibration curve was performed in the range of 0.2–20 ng/mL for the target OND, GRA and TRO in goat plasma samples. The precision of the intra- and inter-day assay for OND, GRA and TRO were 1.84–6.23% and 3.89–5.31%, 2.63–6.29% and 3.76–5.31%, 1.99–5.67% and 2.64–4.70%, respectively. The accuracy of the intra- and inter-day assay for OND, GRA and TRO were 89.15–97.39% and 89.46–95.17%, 91.08–100.82% and 91.24–99.47%, 92.30–100.74% and 94.21–97.90%, respectively. For the determination of OND, GRA and TRO in plasma samples, no significant matrix effects were observed. The mean absolute recoveries were 103–150%, 115–121%, and 98–141% for OND, GRA and TRO, respectively. Furthermore, the mean process efficiency values of silica SPE were 98–135%, 92–124%, and 72–109% for OND, GRA and TRO, respectively.
Applying novel approaches for GC × GC-TOF-MS data cleaning and trends clustering in VOCs time-series analysis J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-12 Luca Narduzzi, Elena Franciosi, Silvia Carlin, Kieran Tuohy, Alberto Beretta, Franco Pedrotti, Fulvio Mattivi
Phytothermotherapy (“grass baths”) is a traditional phytotherapy for rheumatism consisting of taking baths in hot fermenting grass. Scientific studies have demonstrated its efficiency in treating several rheumatic diseases. However the efficiency and repeatability of the therapy is dependent on the wild fermentations, determining sometimes the appearance of unpleasant conditions leading to the early abandonment of the therapy. The metabolism undergoing in the grass baths is unknown and there is not an established method to evaluate and predict grass baths quality. The aim of this study is to establish a simple VOCs profiling method able to evaluate the grass baths, predicting their evolution, through the identification of marker volatiles related to the best conditions and/or the spoilage. After replicating in real scale the traditional grass baths, the volatile profiles were measured using passive diffusion samplers injected in a thermal desorption-comprehensive GC × GC-TOF-MS. The high dimensionality of the data coupled with the limited number of time points, required a rigorous method development for the analysis of the data, achieved through the development of a novel R package for variable selection in GC × GC data matrices. The further application of a fuzzy clustering approach demonstrated to be a useful tool dealing with short time series, allowing to discard un-trending volatiles and giving a clear snapshot of the main trends in the data. A broad coverage of the volatolome was provided, thus suitable to describe the main metabolic changes ongoing in the grass baths. Coupling this data with the temperature and pH, and comparing it to the data from similar processes, like silage and compost, we demonstrated that the established method can be helpful to evaluate short time series, allowing us to obtain a list of volatiles as candidate markers for the quality of the grass baths. The established method gave a list of markers applicable to real scale grass baths to predict spoilage; furthermore it provides a list of volatiles where to search for candidate markers with reported health-related effects and can be used to generate hypothesis on the mechanisms of action of the treatment.
PRiME pass-through purification of lignans in Silybum marianum and UPLC−MS/MS analysis J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-05 Manman Zhang, Kang Chen, Zhiyan Hu, Qing Shen, Haixing Wang
A PRiME (process, robustness, improvements, matrix effects, ease of use) pass-through cleanup procedure was developed for the extraction and purification of silychristins A and B, silybins A and B, isosilybins A and B, and silydianin in Silybum marianum. After optimizing the extracting solvent types and the sample loading volume, the crude extract was diluted to 3 mL with 95% acetonitrile and then loaded on the PRiME cartridge. The eluate was analyzed by ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). All the target analytes were deprotonated as [M-H]− at m/z 481 by conducting collision-induced dissociation (CID), and the major fragment ions were m/z 463 ([M-H2O-H]−), 453 ([M-CO-H]−), 355 ([M-C6H6O3-H]−), 301 ([M355-CO2-H]−), and 179 ([C10H11O3]−). Afterwards, this method was validated in terms of linearity (R2 > 0.9990), intra-day precision (1.02%–3.79%), inter-day precision (1.59%–4.87%), sensitivity (LOD ≤ 0.45 μg·kg−1 and LOQ ≤ 1.50 μg·kg−1), and recovery (76.9–103.4%, RSD < 8.90%). Finally, the proposed protocol was successfully applied to eight batches of S. marianum samples. The total content of the seven active compounds varied amongst the batches from different places of origin.
Determination of methyl isopropyl hydantoin from rat erythrocytes by gas-chromatography mass-spectrometry to determine methyl isocyanate dose following inhalation exposure J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-05 Brian A. Logue, Zhiling Zhang, Erica Manandhar, Adam L. Pay, Claire R. Croutch, Eric Peters, William Sosna, Jacqueline S. Rioux, Livia A. Veress, Carl W. White
Methyl isocyanate (MIC) is an important precursor for industrial synthesis, but it is highly toxic. MIC causes irritation and damage to the eyes, respiratory tract, and skin. While current treatment is limited to supportive care and counteracting symptoms, promising countermeasures are being evaluated. Our work focuses on understanding the inhalation toxicity of MIC to develop effective therapeutic interventions. However, in-vivo inhalation exposure studies are limited by challenges in estimating the actual respiratory dose, due to animal-to-animal variability in breathing rate, depth, etc. Therefore, a method was developed to estimate the inhaled MIC dose based on analysis of an N-terminal valine hemoglobin adduct. The method features a simple sample preparation scheme, including rapid isolation of hemoglobin, hydrolysis of the hemoglobin adduct with immediate conversion to methyl isopropyl hydantoin (MIH), rapid liquid-liquid extraction, and gas-chromatography mass-spectrometry analysis. The method produced a limit of detection of 0.05 mg MIH/kg RBC precipitate with a dynamic range from 0.05–25 mg MIH/kg. The precision, as measured by percent relative standard deviation, was <8.5%, and the accuracy was within 8% of the nominal concentration. The method was used to evaluate a potential correlation between MIH and MIC internal dose and proved promising. If successful, this method may be used to quantify the true internal dose of MIC from inhalation studies to help determine the effectiveness of MIC therapeutics.
Electrochemical simulation of three novel cardiovascular drugs phase I metabolism and development of a new method for determination of them by liquid chromatography coupled with tandem mass spectrometry J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-06 Małgorzata Szultka-Młyńska, Sylwia Bajkacz, Magdalena Kaca, Irena Baranowska, Bogusław Buszewski
In this study electrochemistry (EC) coupled with electrospray ionization mass spectrometry (ESI-MS) was used to study the metabolic fate of three novel cardiovascular drugs: rivaroxaban (RIV), aliskiren (ALS), and prasugrel (PRS). Mimicry of the oxidative phase I metabolism was achieved in a simple amperometric thin-layer cell equipped with a boron-doped diamond (MD) working electrode. Structures of the electrochemically-generated metabolites were elucidated from MS/MS experiments. Additionally, a sensitive, specific, and rapid ultra-high performance liquid chromatography–tandem mass spectrometer (UHPLC–MS/MS) method has been developed and validated for the selected drugs in human urine samples. Three different sample preparation methods were compared and finally, sample preparation was accomplished through an ultrasound-assisted emulsification microextraction process (USAEME). The drugs were detected using a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source with positive ionization mode (ESI(+)). The results obtained by EC–MS were compared with conventional in vivo studies by analyzing urine samples from patients. Data from in vivo experiments showed good agreement with the data from electrochemical oxidation. Thus, EC–MS is very well-suited for the simulation of the oxidative metabolism of rivaroxaban, aliskiren, and prasugrel as well. Moreover, electrochemical conversion of target compounds appears to be a new in vitro technology for the prediction of potential metabolites.
Validation of analytical methods for chlordecone and its metabolites in the urine and feces of ewes J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-03 Maïlie Saint-Hilaire, Thomas Bertin, Chanthadary Inthavong, Gwenaëlle Lavison-Bompard, Thierry Guérin, Agnès Fournier, Cyril Feidt, Guido Rychen, Julien Parinet
Chlordecone (CLD) is a Persistent Organic Pollutant used between 1972 until 1993 in the French West Indies (FWI). Due to its persistence and extensive application, a quarter of the total local agricultural acreage is still moderate to heavily polluted. In consequence, livestock may be contaminated at various levels. This is a major public health concern, particularly for local consumers. In order to better understand the fate of CLD in livestock organisms, in vivo studies are required. There is no information available about its metabolism and elimination in ruminants, common livestock in the FWI. To be able to monitor the fate of chlordecone and its metabolites in livestock and to assess if the compounds could be released in the environment, urinary and fecal samples were logically targeted. In order to reach this goal, robust and validated analytical methods are required. For this purpose, Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction methods were validated to analyze CLD and its metabolites in the urine and feces. The analysis was carried using liquid phase chromatography with tandem mass spectrometry and validated according to French standard NF V03-110 and SANTE guidelines. Matrix effect, Accuracy, within-laboratory repeatability, specificity, Q/q relative ion intensities and uncertainty were reported. Recoveries between 70% and 120% were obtained from urine and feces. The limits of quantification (LOQ) in urine samples were 0.1 μg CLD L−1, 0.1 μg total CLD (CLD and its conjugates)·L−1, 1.3 μg CLDOH L−1 and 2.4 μg total CLD (chlordecol and its conjugates) L−1 of urine. LOQ in fresh feces were 3.2 μg CLD kg−1 and 5.8 μg CLDOH kg−1. Contaminated urinary and fecal samples from ewes were analyzed to confirm the relevance of the methods. In urine, CLD and conjugated CLDOH could be quantified whereas only free CLD and free CLDOH were found in feces. These methods are essential for future toxicokinetic studies and also to estimate the environmental contamination.
Recent development of chromatographic methods to determine parabens in breast milk samples: A review J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-03 Caroline Fernandes Grecco, Israel D. Souza, Maria Eugênia Costa Queiroz
Parabens have been widely used as antimicrobial preservatives in food, drugs, and cosmetics for over 60 years. These endocrine disruptors can alter both the wildlife and the human hormone function. Determining these compounds in human milk is important because breast milk plays an important role in infant growth and in neurocognitive development. This article summarizes the current state-of-the-art of chromatographic methods to determine parabens in breast milk samples. Apart from the conventional and modern microextraction sample preparation techniques described herein, the authors discuss the chromatographic systems, primarily LC-MS/MS, and the concentration ranges at which parabens have been detected in milk samples obtained from lactating women over the past few years.
Isolation of recombinant human antithrombin isoforms by Cellufine Sulfate affinity chromatography J. Chromatogr. B (IF 2.441) Pub Date : 2018-07-02 Tsutomu Sugihara, Shinji Fujiwara, Saori Ishioka, Tomonari Urakubo, Toshiyuki Suzawa
Human antithrombin (hAT) is a major serine protease inhibitor that regulates blood coagulation in human plasma and has been applied for the treatment of antithrombin (AT) deficiency and disseminated intravascular coagulation (DIC). In the past, hAT for therapeutic use has been obtained from human plasma; however, hAT can now be sourced from transgenic animals and Chinese hamster ovary (CHO) cells by recombinant technology. The dominant form of hAT in plasma is the α form, which is glycosylated with four oligosaccharides and sialylated at its terminals. However, it would be preferable to reduce the poorly sialylated α form, the minor β form with unoccupied glycosylation sites, and inactive forms from hAT. Cellufine Sulfate, a heparin-mimic affinity resin made of cellulose and modified with sulfate groups, has an affinity for heparin-binding proteins and can be used for cation exchange chromatography. Based on these properties, hAT could be separated and purified in the α and β forms depending on the rate of sialylation. Consequently, Cellufine Sulfate was used to enrich the highly sialylated α form with a high step yield, which was considerably higher than that obtained using conventional heparin-immobilized resins. Furthermore, subsequent hydrophobic interaction chromatography could eliminate inactive forms. These results suggest that Cellufine Sulfate is more effective than heparin-immobilized resins in purifying the highly sialylated α form of hAT for therapeutic applications.
Isolation and identification of a novel algicidal peptide from mackerel muscle hydrolysate J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-30 Ja Young Cho, Joong Kyun Kim
To help remedy damage from harmful algal blooms, an attempt was made to isolate an algicidal substance previously observed to be present in mackerel muscle hydrolysate. Crude extract was obtained by cold acetone precipitation, and it dissolved best in water. Through molecular weight cut-off determination and tricine-SDS PAGE, the algicidal substance was determined to be a peptide of <1 kDa. Based on this result, purification was first performed using size exclusion chromatography and preparative reverse phase high-performance liquid chromatography. Then, the active algicidal fraction was applied to an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry system, followed by MS/MS analysis. The algicidal peptide had linear structure consisting of amino acids with sequence NH-KMNF-COOH. Its calculated properties were: molecular weight 538.66 g/mol; isoelectric point 9.91; net charge +1 at pH 7.0; and 50% hydrophobicity. Algicidal ability of the identified peptide was confirmed using synthesized peptide. The LC50 values toward four harmful algal blooming species were 0.69, 0.83, 0.85 and 1.24 mg/ml for Alexandrium fundyense, A. catenella, Heterocapsa triquetra, and Prorocentrum minimum, respectively. There was no coincidence in the sequence of the identified peptide with those of known metabolites in the APD, Norine, CAMP, UniProt and METLIN databases. Consequently, this algicidal substance originating from mackerel protein was deduced to be a novel peptide that can usefully be applied to relieve harmful algal blooms.
Simultaneous detection of foodborne bacteria in milk by microchip electrophoresis combined with multiplex PCR amplification J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-30 Luqi Zhu, Yating Zhang, Pingang He, Yan Zhang, Qingjiang Wang
Foodborne bacteria are some of the most important human pathogens and can cause many diseases. In this study, we report the simultaneous detection of Staphylococcus aureus, Proteus mirabilis, and Enterobacter sakazakii in milk by multiplex PCR amplification combined with microchip electrophoresis (MCE). Three pairs of primers were specially designed for the amplification of target genes from three bacteria, and the target gene was the specific gene of these bacteria. The DNA fragments were extracted simultaneously, then three pairs of specific primers were added for multiplex PCR amplification. The PCR products of the three food-borne pathogens were separated within 135 s and the limits of detection were 1.2–2.2 ng μL−1, (S/N = 3). The limits of detection were calculated as 53 CFU mL−1 for Enterobacter sakazakii, 32 CFU mL−1 for Proteus mirabilis, 28 CFU mL−1 for Staphylococcus aureus, respectively. This simultaneous detection method based on multiplex PCR was then applied to detection of the three foodborne bacteria in milk samples and satisfactory results were obtained. Our method confers the advantages of less sample consumption, high selectivity, and high sensitivity.
Inter-laboratory study of the skin distribution of 4-n-butyl resorcinol in ex vivo pig and human skin J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-27 Yang Liu, Haiyan Ni, William Wargniez, Sébastien Grégoire, Ingrid Durand, Laurène Roussel-Berlier, Joan Eilstein, Qiang Jie, Tian Ma, Teng Shen, Yingyin Wang, Jie Qiu
4-n-butyl resorcinol (4-nBR) is a highly effective tyrosinase inhibitor, and can be used in cosmetic product for depigmentation purpose. Its efficacy correlates with 4-nBR that absorbed by skin. In this study, skin distribution of 4-nBR within either human or pig skin ex vivo was studied and compared by three independent laboratories. Good agreement was observed in each compartment considering usual inter-lab variability. This study supports the use of pig skin as an alternative source of skin when the availability of human skin is a limiting factor.
A sensitive method determination of the gender difference of neuroactive metabolites in tryptophan and dopamine pathways in the mouse serum and brain by UHPLC-MS/MS J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-25 Jiaxi Yao, Haihua Lu, Zhonghe Wang, Tingwei Wang, Fangfang Fang, Jun Wang, Jing Yu, Rong Gao
Tryptophan (TRP) and dopamine (DA) pathways are of great importance for their related pathology and physiology. In the present study, a new reliable and sensitive analytical method was developed and validated for 12 neuroactive metabolites in TRP and DA pathways in mouse serum and brain by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). The method exhibited good sensitivity as the lower limit of detections ranged from 0.10 to 0.50 ng/ml and the lower limit of quantifications ranged from 0.20 to 2.00 ng/ml by derivatization with dansyl chloride (DNS-Cl) following solid phase extraction (SPE) on C18 cartridges. Good linearity (R2 > 0.99), intra-day precision (<9.8% in serum and <8.8% in brain), inter-day precision (<8.9% in serum and <8.5% in brain) and accuracy (90.3%–110.3% in serum and 86.5%–114.0% in brain) were obtained. The method was successfully applied in measuring 12 neuroactive metabolites in TRP and DA pathways in serum and brain samples of male and female mice to explore the differences between genders. As a result, DA and the turnover of DA to 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxtryptamine (5-HT) to TRP and 5-hydroxyindole acetic acid (5-HIAA) to 5-HT in the serum and norepinephrine (NE) in the brain were significantly different between genders.
A new method for measuring thyroid hormones using nano-LC-MS/MS J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-25 Suvi Ruuskanen, Bin-Yan Hsu, Arttu Heinonen, Minna Vainio, Veerle M. Darras, Tom Sarraude, Anne Rokka
This paper describes a novel mass spectrometry based analytical method for analyzing thyroid hormones (THs). Thyroid hormones play a critical role in the regulation of many biological processes such as growth, metabolism and development. Several analytical methods using liquid chromatography-mass spectrometry (LC–MS) and tandem mass spectrometry (LC–MS/MS) have previously been developed to measure THs, especially in humans. For biomedical and toxicological research using small animal models, and in ecophysiological research using wild species where sample volume is limiting, sensitive methods are needed. In this study, we developed a nano-LC-MS/MS method enabling quantification of low concentrations of two key THs, thyroxine (T4) and 3,3′,5-triiodothyronine (T3). The method was tested with egg yolk samples. We used a low flow rate (300 nl/min) to obtain maximal sensitivity of the method. The limit of quantitation was 10.6 amol for T4 and 17.9 amol for T3. The method shows good linearity (r > 0.99), repeatability and reproducibility (CVs <10%). We also reanalyzed yolk samples with radioimmunoassay for a comparison of the newly developed and previously used methods. Finally, we applied the methodology to measure hormones in egg yolk extracts in multiple avian species, and report interesting variation in maternal TH deposition. The newly developed nano-LC-MS/MS method is thus suitable for measuring THs in low concentrations and across species.
Determination of 12 commonly found compounds in DUID cases in whole blood using fully automated supported liquid extraction and UHPLC-MS/MS J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-25 L. Kristoffersen, M. Langødegård, K.I. Gaare, I. Amundsen, M. Nilsen, D.H. Strand
A high-throughput UHPLC-MS/MS method for the most frequently found compounds; tetrahydrocannabinol (THC), amphetamine, methamphetamine, MDMA, clonazepam, diazepam, nordiazepam, oxazepam, alprazolam, nitrazepam, morphine, and codeine, in driving under the influence of drugs (DUID) cases in whole blood, is presented. Automated sample preparation by 96-well supported liquid extraction (SLE) plates with ethyl acetate + heptane (80 + 20, v/v) as organic solvent was carried out on a Freedom Evo 200 platform from Tecan. An aliquot of 100 μL whole blood was used. Sample preparation time for 96 samples was 1.5 h. Compounds were separated with gradient elution on a C18 column (50 × 2.1 mm, 1.7 μm) with a mobile phase consisting of 5 mM pH 10.2 ammonium formate and methanol. The run time was 4.5 min and 1 μl was injected on an Acquity UPLC I-Class system with a Xevo TQS tandem-quadrupole mass spectrometer in multiple-reaction monitoring mode (MRM) from Waters. Isotope labelled, 13C, internal standards (ISs) were used for all compounds except for alprazolam and morphine, which had deuterated analogs. Quantification was carried out with calibrators without whole blood matrix. Full validation was carried out according to international guidelines, and a new approach for evaluation of process efficiency (PE) has been presented. Linear or quadratic weighted (1/x) calibration curves were used with R2 ≥ 0.999. The method showed satisfactory deviations ±16% when compared to the existing methods, and satisfactory agreement with proficiency testing control samples (z-score −1.6 to 1.8, n = 16 samples). The precision, estimated as the relative standard deviation (RSD) of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤7.2% in antemortem and ≤9.3% in postmortem samples. Recovery was ≥85% for all the compounds, except morphine ≥62% and THC ≥ 50%. PE was satisfactory for all the compounds with low variation in IS response, RSD ≤ 16% (THC 27%) in antemortem samples and ≤34% (THC 66%) in postmortem samples. To the best of our knowledge, this is the first automated 96-well SLE UHPLC-MS/MS method developed for the simultaneous determination of these 12 compounds in whole blood covering the concentration ranges found in forensic samples. The method has been used in routine work during the last ten months, analysing about 9900 antemortem and 1000 postmortem whole blood samples, and has proven to be robust and reliable.
Simultaneous determination of nine anticoagulant rodenticides by ultra-performance liquid chromatography–tandem mass spectrometry with ultrasound-assisted low–density solvent dispersive liquid–liquid microextraction J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-25 Zhi Yan, Hongge Li, Hui Li, Guoyin Lai, Junhao Chu, Hao Guo, Qingbiao Zhao
A sensitive determination method is developed for nine anticoagulant rodenticides (ARs) in urine samples by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) with ultrasound-assisted low–density solvent dispersive liquid-liquid microextraction (UA–LDS–DLLME) pretreatment. The target analytes are brodifacoum, bromadiolone, warfarin, coumachlor, coumatetralyl, difenacoum, pindone, diphacinone and chlorophacinone. The parameters that influence the extraction recovery in the UA–LDS–DLLME were systematically investigated and optimized. With the optimized extraction parameters, recoveries ranging from 64.6%–124.2% were obtained for the target analytes. The linear range for all analytes was 0.1–100 ng/mL with correlation coefficients higher than 0.99. Very low LODs ranging in 0.003–0.03 ng/mL were obtained. LOQs were in the range of 0.01–0.1 ng/mL for the nine target analytes. The accuracy that was expressed as mean relative error was within ±5.8% while the precision expressed as relative standard error was less than 5.9%. The combination of UA–LDS–DLLME with UPLC–MS/MS is a feasible, sensitive and rapid analytical approach for the determination of ARs in urine matrix, which is particularly suitable for clinical and forensic purposes.
Application of a simple methodology to analyze Hydroxypropyl-β-Cyclodextrin in urine using HPLC–LS in early Niemann–Pick disease type C patient J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-25 Adrián Matencio, María Agustina Alcaráz-Gómez, Francisco García Carmona, Begoña Arias, José Manuel López-Nicolás
A new methodology, based on high resolution liquid chromatography with light scatterin detector, is applied to analyze Hydroxypropyl-beta-Cyclodextrin (HPβCD) in urine samples of a child affected by Niemann-Pick Type C disease. The treatment not only stopped disease progression, but has also increased the life expectancy and quality of our patient. The pharmacokinetic of HPβCD in the patient was studied with a 92.8% of HPβCD recovered. At 88 h, no HPβCD was found in the urine. During the treatment, HPβCD has not shown toxicity. Before application of the new treatment, injections were given every two weeks but, we have demonstrated that this can be increased to every four days.
Rapid analysis of neomycin in cochlear perilymph of guinea pigs using disposable SPE cartridges and high performance liquid chromatography-tandem mass spectrometry J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-26 Mian Zu, Jinan Jiang, Hui Zhao, Shili Zhang, Yan Yan, Shiwei Qiu, Shuolong Yuan, Jiawei Han, Yue Zhang, Weiwei Guo, Shiming Yang
Irreversible hearing loss induced by aminoglycoside in human through local or systemic administration route negatively impacts quality of life. The aim of this work was to develop and validate an analytical method suitable for the detection and quantification of neomycin in cochlear perilymph of guinea pig after local application. The SupelMIP SPE column was used for the pre-treatment of matrix. Chromatographic separation was conducted by a reversed phase ODS column (100 × 2.1 mm, 3 μm) at 40 °C in gradient mode with 0.2‰ (v/v) HFBA in water and 0.2‰ (v/v) HFBA in acetonitrile as mobile phase, at a flow rate of 0.30 mL/min, with retention time of 3.50 and 3.62 min for internal standard tobramycin and analyte neomycin, respectively. The MS was performed with positive ionization mode, with data acquisition in Multi Reaction Monitor (MRM) mode. This method was proved to be specific, accurate (97.1–115% of nominal values) and precise (CV% < 15%). Calibration curves for matrix matched standard of neomycin ranged from 1.25 to 200 μg/mL, with LOD and LLOQ of 0.625 and 1.25 μg/mL in blank matrix. The matrix effect was corrected to (−0.1) - 1.33 by adding internal standard. The relative SPE recovery values were ≥98.9% in low, medium and high QC samples. Neomycin in matrix proved to be stable under room temperature - and −20 °C, or under three freeze-thawing cycles, or under processing as well. Finally, the proposed method was successfully applied to a toxicokinetics study of neomycin in perilymph after round window membrane (RWM) administration, which was in accordance with threshold shift of auditory brainstem response (ABR) test related to hearing loss.
Determination of voriconazole and co-administered drugs in plasma of pediatric cancer patients using UPLC-MS/MS: A key step towards personalized therapeutics J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-27 Medhat A. Al-Ghobashy, Samah M. Kamal, Ghada M. El-Sayed, Ali K. Attia, Mohamed Nagy, Ahmed ElZeiny, Marwa T. Elrakaiby, Mohammed M. Nooh, Maggie Abbassi, Ramy K. Aziz
Untreated invasive aspergillosis results in high mortality rate in pediatric cancer patients. Voriconazole (VORI), the first line of treatment, requires strict dose monitoring because of its narrow therapeutic index and individual variation in plasma concentration levels. Commonly co-administered drugs; either Esomeprazole (ESO) or Ondansetron (OND) have reported drug-drug interaction with VORI that should adversely alter therapeutic outcomes of the latter. Although VORI, ESO and OND are co-administered to pediatric cancer patients, the combined effect of ESO and OND on the plasma concentration levels of VORI has not been fully explored. In this study, an accurate, reliable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of VORI, ESO, and OND in ultra-low sample volumes (25 μL) of plasma of pediatric cancer patients. Based on the physicochemical properties of the studied drugs and internal standard, liquid-liquid extraction was successfully adopted with methyl t-butyl ether. Consistent and reproducible recovery of the three drugs and the internal standard were calculated using plasma and matrix matched samples (RE% > 72.97%, RSD < 8.29%). Chromatographic separation was carried out using UPLC with C18 column and a mobile phase of acetonitrile:water:methanol (70:25:5 V/V/V) at 0.3 mL/min. Mass spectrometric determination at positive electrospray ionization in the MRM mode was employed. The analysis was achieved within 4 min over a linear concentration range of 1.00–200.00 ng/mL for the three drugs. The assay validity was assessed as per the Food and Drug Administration guidelines for bioanalytical method validation, and satisfactory results were obtained. The accuracy and precision were within the acceptable limits for the three drugs in both quality control and incurred plasma samples. Matrix effect and process efficiency were investigated in neat solvent, post-extraction matrix, and plasma. Correlation of the plasma concentration levels of the three drugs revealed differences from the reported drug-drug interactions. This confirmed the need for simultaneous determination of VORI and co-administered drugs in order to achieve optimal therapeutic outcomes. To achieve this, analysis results of this study, genetic polymorphisms in CYP2C19 and clinical data will be used to establish one model incorporating all possible factors that might lead to variation in therapeutic outcomes.
An effective and high-throughput analytical methodology for pesticide screening in human urine by disposable pipette extraction and gas chromatography – mass spectrometry J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-23 Anderson Luiz Oenning, Josias Merib, Eduardo Carasek
In this paper, the use of disposable pipette extraction (DPX) for the determination of pesticides in human urine in possible cases of poisoning is proposed for the first time. The pesticides studied were oxamyl, propoxur, carbofuran, 3‑hydroxycarbofuran, carbaryl, methiocarb, terbufos, parathion methyl, malathion, chlorpyrifos and endosulfan. The pipette tip used for the extraction of these compounds was commercially acquired. It has a capacity of 5 mL and contains 20 mg of sorbent material (styrene-divinylbenzene). The optimization of the main parameters that can influence the extraction efficiency of this sample preparation technique was performed with univariate and multivariate approaches. The analytes were separated and identified by gas chromatography coupled to mass spectrometry (GC–MS). The optimal extraction conditions were 5 extraction cycles of 30 s and 5 desorption cycles of 15 s with 250 μL of ethyl acetate. Elution of the extract was performed in a vial containing 100 mg of anhydrous sodium sulfate. The method developed was validated, providing correlation coefficients higher than 0.9955 for all analytes, limits of detection (LOD) of 0.76 to 1.52 μg L−1, limits of quantification (LOQ) of 2.5 to 5.0 μg L−1, relative recoveries of 63 to 118%, intra-day precision of 0.7 to 15.3% and inter-day precision of 4.9 to 13.1%. An effective and rapid method for the analysis of human urine for the identification of possible cases of poisoning by pesticides was successful developed.
Determination of three tetracyclines in bovine milk using magnetic solid phase extraction in tandem with dispersive liquid-liquid microextraction coupled with HPLC J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-23 Nour Al-Afy, Hassan Sereshti, Akram Hijazi, Hamid Rashidi Nodeh
A novel analytical method namely magnetic solid phase extraction in tandem with dispersive liquid-liquid microextraction was developed and used for the extraction/preconcentration of tetracycline (TCN), oxytetracycline (OTC) and doxycycline (DC) from bovine milk sample before HPLC-UV analysis. The β-cyclodextrin functionalized silica-coated magnetic graphene oxide (Fe3O4@SiO2@GO-β-CD) was used as an adsorbent. The adsorbent was fully characterized using FT-IR, SEM, EDX and Zeta potential techniques. The effective parameters on the performance of the method such as extraction solvent type and volume, adsorbent amount, desorption solvent type and volume, disperser solvent type, desorption time, ionic strength and pH value were investigated. The limit of detection (LOD) and limit of quantification (LOQ) were obtained in the ranges of 1.8–2.9 μg L−1 and 6.1–9.7 μg L−1, respectively. The linearity was in the range of 10.0–200.0 μg L−1 with satisfactory determination coefficients (R2) higher than 0.9929 and a good precision (RSD < 8.8%). The recovery percentages for the analytes in real samples (bovine milk and water) were achieved in a range from 70.6 to 121.5%.
Development of a selective and sensitive high-performance liquid chromatography-tandem mass spectrometry assay to support pharmacokinetic studies of LY-487,379 in rat and marmoset J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-19 Fleur Gaudette, Adjia Hamadjida, Dominique Bédard, Stephen G. Nuara, Jim C. Gourdon, Véronique Michaud, Francis Beaudry, Philippe Huot
Drugs modulating the metabotropic glutamate type 2 receptor (mGluR2) activity may have therapeutic benefits in treating a large spectrum of neuro-psychiatric disorders, from schizophrenia to Parkinson's disease, both as a symptomatic therapy and potential disease-modifying paradigm. LY-487,379 is a highly selective mGluR2 positive allosteric modulator that is widely used to study mGluR2 function using experimental animal models. The common marmoset is a small primate that has long been used in neuroscience. However, given its small size and small circulating blood volume, conducting studies to determine the PK profile of LY-487,379 is challenging. We developed and validated a sensitive and selective analytical method that enables quantification of LY-487,379 using a limited volume of plasma (10 μL). The analytical method consists of protein precipitation followed by high-performance liquid chromatography with heat assisted electrospray ionization mass spectrometry (HPLC-HESI-MS/MS). The chromatographic separation was achieved using gradient elution with a mobile phase consisting of acetonitrile and 0.01% formic acid in water on a Thermo Scientific Aquasil C18 analytical column (100 × 2.1 mm I.D., 5 μm) operating at 40 °C and at a flow rate of 300 μL/min. The method displays a linear relationship ranging from 0.2 to 100 ng/mL. Intra- and inter-day relative standard deviations are <1.4% and 7.9%, respectively and the relative error ranged from −6.9 to 9.7%. The method was used to quantify LY-487,379 in both rat and marmoset plasma, and PK parameters were determined after a single subcutaneous dose of 1.0 mg kg−1 in both species and significant differences in Cmax, AUC and T1/2 were observed.
Ultrasound-assisted emulsification microextraction for rapid determination of unmetabolized synthetic polycyclic and nitro-aromatic musks in human urine J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-20 Ting-An Chen, Wu-Hsun Chung, Erica M.C. Ding, Wang-Hsien Ding
An effective method to rapidly determine the presence of seven unmetabolized synthetic musks in human urine samples is developed. The target musks are five synthetic polycyclic musks (i.e., celestolide (ADBI), phantolide (AHMI), traseolide (ATII), galaxolide (HHCB), tonalide (AHTN)), and two nitro-aromatic musks (i.e., musk xylene (MX) and musk ketone (MK)). The method involved an ultrasound-assisted emulsification microextraction (USAEME) coupled with gas chromatography–mass spectrometry (GC–MS). The factors that affect USAEME efficiency were optimized in detail, and the optimized procedure involved the rapid injection of 50 μL of carbon tetrachloride into 1.0 mL of urine sample (contained 0.1-g of sodium chloride) in a conical bottom glass tube. After 1.0 min ultrasonication and 3 min centrifugation (at 7000 rpm), the sedimented extract 10 μL was directly injected into the GC–MS system. The limits of quantitation (LOQs) varied from 0.1 to 0.5 ng/mL. The precisions for both repeatability and reproducibility were <8%. The trueness varied from 79 to 96% with the RSD ranging from 2 to 8%. The total concentrations of the seven unmetabolized target musks in collected human urine samples were in the range from 0.93 to 3.74 ng/mL. HHCB and AHTN were detected in all the collected samples, and the daily excretion doses were evaluated.
Serum metabonomics study on antidiabetic effects of fenugreek flavonoids in streptozotocin-induced rats J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-20 Wenyue Jiang, Lihui Si, Pengdong Li, Bing Bai, Jiale Qu, Bao Hou, Hui Zou, Xing Fan, Zhiqiang Liu, Zhongying Liu, Lu Gao
Fenugreek is a well-known medicinal plant used for treatment of diabetes. In this study, the antidiabetic effect of fenugreek flavonoids was investigated by metabonomics based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Fenugreek flavonoids were purified using polyamide resin and D101 macroporous adsorption resin, characterized by UPLC-Q-TOF-MS, and administered to streptozotocin (STZ)-induced diabetic rats for 28 days. Pharmacological study results indicated that fenugreek flavonoids exerted a strong antidiabetic effect characterized by significant reduction of fasting blood glucose (P < 0.01), increase in serum insulin level (P < 0.01) and liver glycogen content (P < 0.01), attenuation of weight loss, and improvement of pancreatic islet and kidney conditions. The antidiabetic effect of fenugreek flavonoids was further analyzed by metabonomics. Serum samples of health and diabetic rats treated or not with fenugreek flavonoids were evaluated by UPLC-Q-TOF-MS, followed by principal component analysis (PCA) and orthogonal projection to latent structures squares-discriminant analysis (OPLS-DA). The PCA model revealed significant differences among the animal groups, and OPLS-DA identified fenugreek flavonoids-induced changes of 11 potential biomarkers involved in lipid metabolism (docosahexaenoic acid, arachidonic acid, sphinganine, sphingosine‑1‑phosphate, and lysophosphatidylcholines 20:4, 18:2, 16:0, and 20:2), amino acid metabolism (hippuric acid and tryptophan), and kidney function-related metabolism (2‑phenylethanol glucuronide). Our study demonstrates that flavonoids are bioactive components of fenugreek with potent antidiabetic activity, which exert their therapeutic effects by multiple mechanisms, including reducing insulin resistance, improving gluconeogenesis, and protecting islet cells and kidneys from damage.
Development and validation of analytical methodology by GC-FID using hexadecyl propanoate as an internal standard to determine the bovine tallow methyl esters content J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-20 Evandro Pereira, Amanda Napp, João V. Braun, Marcus Seferin, Jeane Ayres, Rosane Ligabue, Luiz A.M. Fontoura, Luciane M.P. Passaglia, Marilene H. Vainstein
Analysis of terephthalate metabolites in human urine by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-20 Shedrack R. Nayebare, Rajendiran Karthikraj, Kurunthachalam Kannan
Owing to their toxicity, phthalate plasticizers are currently being replaced with terephthalates in many consumer products. Nevertheless, data on human exposure to and toxicity of terephthalates are still scarce. In this study, we developed a robust analytical method for the measurement of six terephthalate metabolites (TPhMs) in human urine through their successful separation from phthalate metabolites (PhMs). Target analytes were identified, using commercially available standards, and quantified with isotopically labeled internal standards (IS). The limit of quantification (LOQ) of TPhMs were in the range of 0.12 to 0.4 ng/mL, with the exception of 2.8 ng/mL for terephthalic acid (TPA) and 3.75 ng/mL for mono-(2-ethylhexyl) terephthalate (mEHTP), which were found in procedural blanks at notable levels. The method developed in this study showed excellent accuracy (recoveries: 86–117%) and precision (RSD: 0.6–12.2%) for TPhMs. The method was successfully applied for the analysis of 30 human urine samples collected from individuals with no known history of occupational exposure. The detection frequencies (df %) of TPhMs in urine ranged between 26.6 and 100%. This is one of the first studies that report a method for the analysis of emerging class of environmental chemicals in human specimens.
Affinity adsorption of bovine hyaluronidase with ligands targeting to active site J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-20 Yu Xin, Mengyao Hao, Guangming Fan, Yao Zhang, Mengling Zheng, Liang Zhang
Four affinity ligands were designed from 6-chloromethyluracil and 2-aminobenzimidazole and simulated for the interaction with bovine hyaluronidase-1. Regarding sequence alignment, bovine hyaluronidase-1 precursor showed circa 83.6% similarity with human hyaluronidase-1. Regarding structural modeling and molecular docking, bovine hyaluronidase-1 interacted with ligands in the active site. Using epichlorohydrin, 1,3-propanediamine and cyanuric chloride as spacers, 6-chloromethyluracil and 2-aminobenzimidazole were composed to Sepharose beads. The modified Sepharose beads were then subjected to adsorption analysis with bovine hyaluronidase. After one step of affinity adsorption, the samples extracted from bovine testes were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and activity assay. As calculated, the densities of four ligands on sorbents (entitled as L-1, L-2, L-3 and L-4) were 37.7 ± 2.3, 36.4 ± 3.2, 42.4 ± 4.2 and 33.7 ± 2.3 μmol/g wet gel; the theoretical maximum adsorption (Qmax) of bovine hyaluronidase on the four sorbents were 63.6 ± 1.6, 72.0 ± 0.7, 111.0 ± 4.1 and 121.7 ± 2.3 mg/g wet gel, respectively; the dissociation constants (Kd) of the four sorbents were 18.5 ± 0.8, 48.1 ± 4.3, 35.0 ± 3.0, 40.6 ± 2.7 μg/g wet gel, respectively. After optimization, the proteins captured by sorbents attaching 2-aminobenzimidazole based ligands (L-3 and L-4) revealed the main single band at approximately 50 kDa, and the purities were about 85.2 and 96.4%; the bioactivity recoveries were 83.5 and 89.4%. In addition, the bands on SDS-PAGE gel were also extracted and confirmed with linear trap quadropole mass spectrometry (LTQ-MS) analysis.
Forced degradation of l-(+)-bornesitol, a bioactive marker of Hancornia speciosa: Development and validation of stability indicating UHPLC-MS method and effect of degraded products on ACE inhibition J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-22 José Hugo de Sousa Gomes, Grazielle Caroline da Silva, Steyner F. Côrtes, Rodrigo Maia de Pádua, Fernão Castro Braga
The antihypertensive activity of the medicinal plant Hancornia speciosa has been previously demonstrated by us, being the activity ascribed to polyphenols and cyclitols like l-(+)-bornesitol. We herein evaluated the stability of the bioactive marker bornesitol submitted to forced degradation conditions. Bornesitol employed in the study was isolated from H. speciosa leaves. An UHPLC-ESI-MS/MS method was developed to investigate bornesitol stability based on MRM (Multiple Reaction Monitoring) acquisition mode and negative ionization mode, employing both specific (m/z 193 → 161 Da) and confirmatory (m/z 193 → 175 Da) transitions. A gradient elution of 0.1% formic acid in water and acetonitrile was performed on a HILIC column. The method was validated and showed adequate linearity (r2 > 0.99), selectivity, specificity, accuracy, and precision (RSD < 2.9%). The method was robust for deliberate variations on dessolvation temperature, but not for changes in the flow rate and dessolvation gas. The results from the stability studies allowed us to classify bornesitol as labile for acidic and alkaline hydrolysis, but as very stable for oxidative and neutral hydrolysis exposure. Bornesitol was categorized as practically stable under photolysis degradation, whereas a considerable reduction on its contents was induced by metal ions and thermolysis exposure. Degraded samples from neutral hydrolysis and thermolysis were assayed in vitro for ACE inhibition and showed a substantial decrease in biological activity as compared to intact bornesitol. myo-Inositol was identified as the major degradation products in both matrices. This is the first report on bornesitol stability under different stress conditions and the obtained data are relevant for the development and quality control of standardized products from H. speciosa leaves.
Achievements in robotic automation of solvent extraction and related approaches for bioanalysis of pharmaceuticals J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-19 Michal Alexovič, Yannis Dotsikas, Peter Bober, Ján Sabo
Currently, the growing demand on quick, easy and ecological sample pretreatment methods is unquestionable. Such challenge involves also approaches focusing on the analysis of pharmaceuticals and other endogenous compounds in biological matrices, termed as Bioanalysis. Solvent extraction such as liquid-liquid extraction (LLE), derived liquid phase microextraction (LPME) and related approaches such as solid liquid extraction (SLE), proved to be applicable in bioanalysis, as numerous papers have been published in this field. However, their manual performances may suffer from a long-term and laborious preparation, due to the inherent complexity of the biological samples. A high sample-throughput (enabling measurement of tens or hundreds of samples on a daily basis) can be achieved when automation of sample pretreatment is performed, resulting in decreased imprecision and low waste production of hazardous solvents and risky biological materials. Here, robotic systems have a key role, especially when multiple processing (e.g., 96-well plate format) and coupling to modern analytical instrumentation (e.g. LC-MS) is done. A thorough overview on the up-to-date automations of LLE, LPME, SLE and solid LLE via robotics, is therefore presented. Pharmaceuticals and related compounds determined in classical liquid biological samples (i.e. plasma/serum, whole blood, urine, saliva etc.) and modern dried matrix spots (DMS) were considered as analytes of interest. The methodologies were critically compared to manual setups and among themselves.
Detection of tobacco smoke emanating from human skin surface of smokers employing passive flux sampler – GCMS system J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-19 Yoshika Sekine, Shodai Sato, Keita Kimura, Hiroshi Sato, Satoshi Nakai, Yukio Yanagisawa
Cigarette smoking is a significant risk factor for higher incidences of numerous adverse health consequences. Related health disorders are also found in non-smokers exposed to secondhand smoke (SHS). To investigate the influence of cigarette smoking and exposure to SHS on the composition of human skin gas, a trace biogas emanating from human skin, dermal emissions of volatile compounds were semi-quantitatively measured for volunteers who smoke a cigarette and those exposed to SHS. This was performed using a passive flux sampler (PFS) coupled with gas chromatography mass spectrometry (GCMS). Numerous chemicals were detected, including acetaldehyde, toluene, 3-methyl furan (3-MF), 2,5-dimethyl furan (2,5-DMF), 3-ethenyl pyridine (3-EP), and nicotine, in the samples collected from the smokers after a smoking event, and a remarkable increase in the amount of chemicals collected was observed just after smoking. These chemicals were also found in the samples collected from volunteers exposed to SHS. Assessment of current smoking status is important for managing the negative effects of active and passive smoking, and for the development of public health policy. The tobacco specific chemicals such as 3-MF, 2,5-DMF, 3-EP, and nicotine, emanating from human skin surfaces, represent a potential non-invasive biomarker for monitoring current smoking status of active and passive smokers after establishing a more quantitative procedure.
Dry Blood Spot sample collection for post-exposure monitoring of chemical warfare agents – In vivo determination of phosphonic acids using LC-MS/MS J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-19 Lilach Yishai Aviram, Miriam Magen, Shira Chapman, Adi Neufeld Cohen, Shlomi Lazar, Shai Dagan
Phosphonic acids are the direct and immediate metabolites of organophosphorus chemical warfare agents (OP-CWAs). Accordingly, their detection serves for evaluating exposure to OP-CWAs in a terror or war scenario. After exposure, phosphonic acids are present in the blood; however, blood drawing must be carried out by medical personnel, hence the number of samples that can be drawn in a mass-casualty event is limited. Herein, we describe a new approach developed for the determination of phosphonic acids in blood using Dry Blood Spots (DBSs) on a filter paper. The method is based on a simple sample preparation protocol, followed by LC-MS-MS targeted (MRM) analysis. The detection limits of Soman (GD), Cyclosarin (GF) and VX metabolites in whole blood were as low as 1 ng/ml, while the detection limits were 0.3 ng/ml for the GF metabolite and 0.5 ng/ml for the Sarin (GB) metabolite. Good recoveries were obtained in the range of 1–100 ng/ml for GB and GD metabolites, and 3–100 ng/ml for GF, VX and RVX metabolites, with a linear response (R2 = 0.99). The method has proven to be reliable even with DBS samples stored up to 35 days at room temperature before analysis. This method was implemented in a 24 h time-course determination of the Sarin metabolite in an in - vivo experiment, after rat exposure to 1 LD50 of Sarin. This technique is simple, rapid, sensitive, robust, long lasting and compatible with field collection and storage; hence, it can serve for large-scale sampling and reliable monitoring of potential OP-CWAs casualties. Since DBS sampling is amenable to nonprofessionals, including self-sampling, this technique is highly suitable for mass-casualty incidents.
A sensitive liquid chromatography-tandem mass spectrometry method for the quantification of valacyclovir and its metabolite acyclovir in mouse and human plasma J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-19 Jian Shi, Yongjun Hu, David E. Smith, Hao-Jie Zhu
It is very challenging to conduct a pharmacokinetic (PK) study on mice due to the limited amount of plasma one can obtain, which is also true for some clinical studies. Here, we developed and validated a simple, sensitive and robust LC-MS/MS method for measuring the prodrug valacyclovir (VACV) and its metabolite acyclovir (ACV) in mouse and human plasma. This assay utilized an acetonitrile protein precipitation method with isotope-labeled internal standards (IS) and enabled precise and accurate quantification of VACV and ACV from 10 μl plasma samples with a nine min gradient. The analytes were separated on a Waters Atlantis T3 C18 column. The precursor-product ion transitions for VACV (m/z 325.2 > 152.1), ACV (m/z 226.2 > 152.1), VACV-D4 (m/z 329.2 > 152.1, IS) and ACV-D4 (m/z 230.2 > 152.1, IS) were detected in a multiple reaction monitoring (MRM) positive ion mode using an API4000 LC-MS/MS system. The lower limit of quantification (LLOQ) was 2 nM for both VACV and ACV. The linear range was validated over the concentration ranges of 2–200 nM and 200–5000 nM for both compounds. The matrix effect and stability of VACV and ACV were also evaluated. This assay was successfully applied to a PK study in mice.
High-throughput lipidomics characterize key lipid molecules as potential therapeutic targets of Kaixinsan protects against Alzheimer's disease in APP/PS1 transgenic mice J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-18 Hong-Lei Gao, Ai-Hua Zhang, Jing-Bo Yu, Hui Sun, Ling Kong, Xiang-Qian Wang, Guang-li Yan, Liang Liu, Xi-Jun Wang
Alzheimer's disease (AD) is still a major problem nowadays. Under the circumstance of many chemical drugs have poor effects on AD, traditional Chinese medicine has become a hot spot for us due to its multi-target and multi-path advantages. To explore the potential therapeutic targets of Kaixinsan (KXS) protects against AD in APP/PS1 transgenic mice model. All mice were divided into three groups: control group, model group and KXS group. Orally given KXS from two month old, and the control and model groups were given the same dose of distilled water. We collected all mice's serum samples at the 12th month age to determine the lipid markers of AD by compare with the model and control groups in full-scan analysis based on high-throughput serum lipidomics technology. Then we found the lipid molecules called back by KXS from the KXS protects against AD. Compared with the control group, the metabolic profile of the model mice was obviously disordered, and we identified 16 lipid-related biomarkers associated with AD. After KXS treatment, the metabolic profiles of these disorders tended to recover compared with the model group. And we identified eight key lipid molecules, of which four had statistical significance. We found that the main perturbation pathways related to AD were linoleic acid metabolism, arachidonic acid metabolism and sphingolipid metabolism. All these metabolic pathways showed different degrees of rotation after KXS administration. Through the pathways analysis, we found 4 lipids molecules with significant differences, which could be used as new targets for the treatment of AD.
Unraveling the mysteries of modern size exclusion chromatography - the way to achieve confident characterization of therapeutic proteins J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-18 Alexandre Goyon, Szabolcs Fekete, Alain Beck, Jean-Luc Veuthey, Davy Guillarme
Modern size exclusion chromatography (SEC) can be defined by the use of relatively small columns (e.g., 150 × 4.6 mm) packed with sub-3 μm particles, allowing a 3- to 5-fold increase in method throughput compared to that of conventional SEC. The quick success of the first sub-2 μm SEC column introduced in 2010 led to the development of numerous ultra-high performance (UHP)-SEC columns for the analysis of therapeutic monoclonal antibody (mAb)-based products. Aggregates also known as high-molecular-weight species (HMWS) are indeed one of the most important critical quality attributes (CQAs) of mAbs, as HMWS may decrease the product efficacy or cause immunogenicity effects. Therefore, the confident characterization of mAbs requires strong knowledge of not only modern SEC performance (i.e., selectivity and efficiency) but also the inherent limitations caused by non-specific interactions more likely to occur with complex antibody drug conjugates (ADCs) and some commercial mAb products. This review discusses the importance of liquid chromatographic (LC) instrumentation in order to exploit the full potential of modern SEC columns and current trends to hyphenate SEC to mass spectrometry (MS). Recent applications for antibody-based products (i.e., mAbs, ADCs, Fc-Fusion proteins and bispecific antibodies) are presented. Finally, tips and tricks are provided to further optimize SEC separations and maintaining their performance over time with better understanding of unexpected SEC results.
Simultaneous quantification of straight-chain and branched-chain short chain fatty acids by gas chromatography mass spectrometry J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-18 Liqing He, Md Aminul Islam Prodhan, Fang Yuan, Xinmin Yin, Pawel K. Lorkiewicz, Xiaoli Wei, Wenke Feng, Craig McClain, Xiang Zhang
Biomedical research in areas such as metabolic disorders, neuromodulatory, and immunomodulatory conditions involves lipid metabolism and demands a reliable and inexpensive method for quantification of short chain fatty acids (SCFAs). We report a GC–MS method for analysis of all straight-chain and branched-chain SCFAs using pentafluorobenzyl bromide (PFBBr) as derivatization reagent. We optimized the derivatization and GC–MS conditions using a mixture containing all eight SCFA standards, i.e., five straight-chain and three branched-chain SCFAs. The optimal derivatization conditions were derivatization time 90 min, temperature 60 °C, pH 7, and (CH3)2CO:H2O ratio 2:1 (v:v). Comparing the performance of different GC column configurations, a 30 m DB-225ms hyphenated with a 30 m DB-5ms column in tandem showed the best separation of SCFAs. Using the optimized experiment conditions, we simultaneously detected all SCFAs with much improved detection limit, 0.244–0.977 μM. We further applied the developed method to measure the SCFAs in mouse feces and all SCFAs were successfully quantified. The recovery rates of the eight SCFAs ranged from 55.7% to 97.9%.
New, simple and sensitive HPTLC method for simultaneous determination of anti-hepatitis C Sofosbuvir and ledipasvir in rabbit plasma J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-18 Samia M. El-Gizawy, Salwa R. El-Shaboury, Noha N. Atia, Mohammad Nabil Abo-Zeid
Sofosbuvir (SOF) and ledipasvir (LDS) represent anti-hepatitis C binary mixture. Herein, a fast high-performance thin-layer chromatography (HPTLC) method was developed, validated and applied for simultaneous determination of SOF and LDS in biological matrix. An innovative strategy was designed which based on coupling dual wavelength detection with HPTLC. This strategy enabled sensitive, specific, high sample throughput and cost-effective determination of the SOF-LDS binary mixture. The developed HPTLC procedure based on a simple liquid–liquid extraction, enrichment of the analytes and subsequent separation with UV detection. Separations were performed on HPTLC silica gel 60 F2 5 4 aluminum plates with a mobile phase consisting of ethyl acetate–glacial acetic acid (100:5, v/v). The Rf values for SOF and LDS were 0.62 and 0.30, respectively. Dual wavelength scanning was carried out in the absorbance mode at 265 and 327 nm for SOF and LDS, respectively. The linear ranges were 40–640 and 9–144 ng/band for SOF and LDS, respectively with correlation coefficients of 0.9998. The detection limits were 10.61 and 2.54 ng/band and the quantitation limits were 32.14 and 7.70 for SOF and LDS, respectively indicating high sensitivity of the proposed method. Consequently, this permits in vitro and in vivo application of the proposed method in rabbit plasma with good percentage recovery (95.68–103.26%). Validation parameters were assessed according to ICH guidelines. The proposed method represents a simple, high sample throughput and economic alternative to the already existing more complicated reported LC-MS/MS techniques. The method would afford an efficient tool for therapeutic drug monitoring and bioavailability studies of SOF and LDS.
Identification of active ingredients mediating anti-platelet aggregation effects of BuyangHuanwu decoction using a platelet binding assay, solid phase extraction, and HPLC-MS/MS J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-18 Fengyun Liao, Aiming Yu, Jianye Yu, Ding Wang, Yinai Wu, Huazhu Zheng, Yingjiao Meng, Dongmei He, Xiao Shen, Lisheng Wang
BuyangHuanwu decoction (BHD) is widely used as a traditional herbal medicine because of its antithrombotic effect, which is attributed to the inhibition of platelet aggregation; however, its active compounds remain unknown. In this study, we developed a method involving platelet binding, solid-phase extraction, and HPLC-MS/MS for screening BHD compounds with potential anti-platelet aggregation properties. Five compounds showing platelet binding affinity were identified as 6-hydroxykaempferol-di-O-glucoside, paeoniflorin, calycosin-7-O-β-d-glucoside, galloylpaeoniflorin, and formononetin-7-O-β-d-glucoside. The results of anti-platelet aggregation experiments in vitro confirmed that these compounds inhibited adenosine diphosphate-induced platelet aggregation. Our results suggest that a platelet binding assay combined with solid-phase extraction and HPLC-MS/MS is an effective method for screening anti-platelet aggregation agents in traditional Chinese medicines.
Improved resolution of fluoroquinolones using cetyltrimethyl ammonium bromide–micellar electrokinetic chromatography and its application to residue analysis in surface water J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-18 Brompoj Prutthiwanasan, Leena Suntornsuk
Development and validation of an UHPLC-MS/MS method for simultaneous quantification of ibrutinib and its dihydrodiol-metabolite in human cerebrospinal fluid J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-15 D. Beauvais, J.-F. Goossens, E. Boyle, B. Allal, T. Lafont, E. Chatelut, C. Herbaux, F. Morschhauser, S. Genay, P. Odou, C. Danel
Ibrutinib is an orally administered first-in-class irreversible Bruton's tyrosine kinase (BTK) covalent inhibitor for the treatment of patients with B-cell malignancies. Several isolated clinical observations reported its efficacy in central nervous system dissemination. Herein, we described the development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) procedure for the quantification of ibrutinib and its active metabolite PCI-45227 in cerebrospinal fluid (CSF). This is the first complete validated method for quantification of ibrutinib and PCI-45227 in CSF. The compounds were eluted on a Waters BEH C18 column (50.0 × 2.1 mm; 1.7 μm) using a gradient elution with a mobile phase composed of ammonium formate buffer 5 mM pH 3.2 and acetonitrile +0.1% formic acid with a flow rate of 400 μL·min−1. Two deuterated internal standards were used to obtain the most accurate quantification. The CSF samples were prepared by a simple and rapid dilution. The method was validated by testing the selectivity, response function, intra-day and inter-day precisions, trueness, limits of detection (LOD) and lower limits of quantification (LLOQ). The validation results proved that the methods were suitable to quantify ibrutinib and PCI-45227 in real biological CSF samples from 0.50 (ibrutinib) or 1.00 (PCI-45227) to 30.00 ng·mL−1. Furthermore, the developed method was adapted to allow the quantification of both compounds in plasma and the results were compared to those reported in literature. The plasmatic samples were treated by protein precipitation and the method was validated to quantify ibrutinib and PCI-45227 in real biological plasmatic samples from 5.00 to 491 ng·mL−1. Lastly, for both matrices, accuracy profiles were plotted from the trueness and precision results using a 20% α-risk (β = 80%) and the tolerance intervals were comprised within the acceptance limits fixed at ±25% for the LLOQ and ±15% for the other concentrations. Finally, these methods were successfully applied to quantify ibrutinib and PCI-45227 in real human CSF and plasma samples.
Human odor and forensics: Towards Bayesian suspect identification using GC × GC–MS characterization of hand odor J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-15 Vincent Cuzuel, Roman Leconte, Guillaume Cognon, Didier Thiébaut, Jérôme Vial, Charles Sauleau, Isabelle Rivals
A new method for identifying people by their odor is proposed. In this approach, subjects are characterized by a GC × GC–MS chromatogram of a sample of their hand odor. The method is based on the definition of a distance between odor chromatograms and the application of Bayesian hypothesis testing. Using a calibration panel of subjects for whom several odor chromatograms are available, the densities of the distance between chromatograms of the same person, and between chromatograms of different persons are estimated. Given the distance between a reference and a query chromatogram, the Bayesian framework provides an estimate of the probability that the corresponding two odor samples come from the same person. We tested the method on a panel that is fully independent from the calibration panel, with promising results for forensic applications.
Simultaneous quantification of antibiotics in wastewater from pig farms by capillary electrophoresis J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-15 Carlos A. Díaz, J. Francisco Hernández-Chávez, Gabriela Ulloa-Mercado, Pablo Gortáres-Moroyoqui, Rosario Martínez Macías, Edna Meza-Escalante, Denisse Serrano-Palacios
Pig farming is an important activity in the economic development of Mexico with millions of tons of meat produced annually. Antibiotics are used in therapeutic dose to prevent diseases, and sometimes as growth promoters. These compounds are not completely metabolized; they are carried into the environment in its active form at concentrations that could induce antibiotic resistance in bacteria, which could be transferred to human pathogens by horizontal gene transfer. The objective of this work was to develop methods of analysis for simultaneous quantification of the antibiotics Oxytetracycline (OXT), Chlortetracycline (CLT), Enrofloxacin (ENRO) and Ciprofloxacin (CIPRO) by field-amplified sampling injection in capillary zone electrophoresis (FASI-CZE). The method was validated by parameters of (1) linearity, obtaining a lineal range of 0.05 at 1 μg mL−1 for ENRO and CIPRO, and from 0.1 to 1 μg mL−1 for OXT and CLT; (2) precision, obtaining values <5% of standard deviation for CIPRO and ENRO and <10% of standard deviation for OXT and CLT; (3) accuracy, with recovery values from 93 to 115%; (4) selectivity, with values of resolution >2 for the all antibiotics tested. To prove the method, a sample of wastewater from a local pig farm was analyzed, detecting a concentration of 0.140 ± 0.009 for OXT. This concentration was higher than the minimal selective concentration, indicating the point in which resistance to a determined antibiotic could develop. The methods were validated with precision and sensitivity comparable to chromatographic methods, which can be used to analyze wastewater from pig farms directly.
Separation of stereoisomers of 7-oxa-bicyclo[2.2.1]heptene sulfonate (OBHS), a Selective Estrogen Receptor Modulator (SERM), via chiral stationary phases using SFC/UV and SFC/MS J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-15 Penagaluri Balasubramanyam, Mehdi Ashraf-Khorassani, Jatinder S. Josan
The enantiomeric separation of a racemate of 7-oxa-bicyclo[2.2.1]heptene sulfonate (OBHS) derivatives, a Selective Estrogen Receptor Modulator (SERM), was obtained using supercritical fluid chromatography in tandem with UV and mass spectrometry (SFC/UV and SFC/MS, respectively). Supercritical CO2 modified with methanol or isopropyl alcohol was used with isopropylamine (IPAm), trimethylamine (TEA), or trifluoroacetic acid (TFA) as an additive to obtain the enantiomers separations. Both Chiralpak IC and IA were evaluated for the separation of enantiomers. Results showed enantiomers separation can be achieved in less than 5 min with a resolution greater than 1 and 0.9, respectively, for the different OBHS derivatives (compounds A and B) using supercritical CO2 modified with 40% isopropyl alcohol containing 0.25% IPAm and IC column applying isocratic conditions. Similar conditions were used with the semi-preparative Chiralpak IC column to isolate more than 50 mg of each enantiomer. SFC/MS and SFC/UV results showed pure enantiomers were isolated. Method development via SFC was much simpler than those reported in the literature using HPLC.
Development of a UPLC-MS/MS method for quantitation of metronidazole and 2-hydroxy metronidazole in human plasma and its application to a pharmacokinetic study J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-14 Stephani Stancil, Leon van Haandel, Susan Abdel-Rahman, Robin E. Pearce
An ultra-performance liquid-chromatography mass-spectrometry (UPLC-MS/MS) method for simultaneous quantitation of metronidazole and 2-hydroxymetronidazole in human plasma was developed and validated. Metronidazole and 2-hydroxymetronidazole were extracted from a small volume of human plasma (10 μL) by hydrophilic lipophilic balanced solid phase extraction on 96-well μ-elution plates. Chromatographic separation of analytes was achieved on an Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) using gradient elution with a blend of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.25 ml/min. Mass spectrometric detection was achieved using multiple reaction monitoring (MRM) in positive-ion electrospray-ionization (ESI) mode. Ion transitions were optimized at m/z 171.85–>127.9 for metronidazole and m/z 187.9–>125.9 for 2-hydroxymetronidazole. The assay was linear for both analytes over the concentration range of 0.1–300 μM; intra- and inter-assay precisions and accuracies were <13%. Recoveries for metronidazole and 2-hydroxymetronidazole ranged from 88 to 99% and 78 to 86%, respectively. Matrix effects for metronidazole and 2-hydroxymetronidazole in plasma ranged from 102 to 105% and 99 to 106%, respectively. The method was successfully applied to determine metronidazole and 2-hydroxymetronidazole plasma concentrations in a pharmacokinetic study conducted in adults administered an oral dose of 500 mg metronidazole. Pharmacokinetic parameters were comparable to previously reported values. By design, this method is amenable to high sample throughput and has the potential to be automated.
A new cause of false positive voriconazole levels: Watch your collection tubes! ☆ J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-13 Almudena Burillo, Alicia Gómez-López, Pilar Escribano, Alicia Galar, Beatriz Salinas, Nadire Díaz-Pérez, Patricia Muñoz, Emilio Bouza
We communicate the interference of a compound of the blood collection tube with the accuracy of a validated high-pressure liquid chromatography method with ultraviolet detection for quantifying voriconazole levels, which led to false positive results. This could have serious consequences for patient management. Our advice is to implement external assessment programs.
Supercritical fluid chromatography coupled to mass spectrometry – A metabolomics perspective J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-12 Vladimir Shulaev, Giorgis Isaac
Metabolomics as a global analysis of a large number of cellular metabolites relies heavily on the new developments in separation science and technology. None of the existing analytical techniques can simultaneously separate and measure all the cellular metabolites due to complexity of cellular metabolome and, therefore, a combination of analytical techniques must be used. Currently NMR, GC–MS and LC-MS are most often used in metabolomics. Novel separation methods such as supercritical fluid chromatography (SFC), which can increase metabolome coverage while decreasing cost and analysis time, can provide alternative to other analytical techniques. As a result of major improvements in instrumentation and development of a new diverse column chemistries SFC-MS is increasingly used in a variety of biomedical applications and is becoming an attractive compliment to other major analytical platforms in metabolomics. Despite its potential and advantages, SFC-MS application in metabolomics is limited. Here we provide a brief overview of the latest developments of SFC-MS for metabolomics applications.
Preconcentration and GC–MS determination of caffeine in tea and coffee using homogeneous liquid–liquid microextraction based on solvents volume ratio alteration J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-12 Tooraj Amini, Payman Hashemi
Simultaneous quantification of 33 active components in Notopterygii Rhizoma et Radix using ultra high performance liquid chromatography with tandem mass spectrometry J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-08 Xiu-Wen Wu, You-Bo Zhang, Lei Zhang, Xiu-Wei Yang
Searching for synergistic calcium antagonists and novel therapeutic regimens for coronary heart disease therapy from a Traditional Chinese Medicine, Suxiao Jiuxin Pill J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-08 Wei Lei, Jianan Ni, Xueting Xia, Min Jiang, Gang Bai
Coronary heart disease is a vital cause of morbidity and mortality worldwide, and calcium channel blockers (CCBs) are important drugs that can be used to treat cardiovascular diseases. Suxiao Jiuxin Pill (SX), a traditional Chinese medicine, is widely used as an emergency drug for coronary heart disease therapy. However, understanding its potential mechanism in intracellular calcium concentration ([Ca2+]i) modulation remains a challenge. To identify the active pharmacological ingredients (APIs) and reveal a novel combination therapy for ameliorating cardiovascular diseases, the ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) combined with a dual-luciferase reporter [Ca2+]i assay system was applied. Ligustrazine, ferulic acid, senkyunolide I, senkyunolide A and ligustilide were identified as potential calcium antagonists in SX, and the combination of ligustrazine and senkyunolide A showed synergetic calcium antagonistic activity. Additionally, the synergetic mechanism was further investigated by live-imaging analysis with the Ca2+ indicator fluo-4/AM by monitoring fluorescence changes. Our results indicated that ligustrazine can block voltage-operated Ca2+ channels (VDCCs) effectively and senkyunolide A can exert an inhibition effect mostly on ryanodine receptors (RYRs) and partly on VDCCs. Finally, an arterial ring assay showed that the combination of ligustrazine and senkyunolide A exerted a better vasodilatation function than using any components alone. In this study, we first revealed that a pair of natural APIs in combination acting on VDCCs and RYRs was more effective on vasodilatation by regulating [Ca2+]i.
Determination of aflatoxin and zearalenone analogs in edible and medicinal herbs using a group-specific immunoaffinity column coupled to ultra-high-performance liquid chromatography with tandem mass spectrometry J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-07 Shujuan Sun, Kai Yao, Sijun Zhao, Pimiao Zheng, Sihan Wang, Yuyang Zeng, Demei Liang, Yuebin Ke, Haiyang Jiang
Six aflatoxins (AFs; AF B1, B2, G1, G2, M1 and M2) and six zearalenone (ZEN) analogs (ZEN, zearalanone, α-zeralanol, β-zeralanol, α-zearalenol, and β-zearalenol) were simultaneously extracted from edible and medicinal herbs using a group-specific immunoaffinity column (IAC) and then identified by ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). The IAC was prepared by coupling N-hydroxysuccinimide-activated Sepharose 4B Fast Flow gel with two group-specific monoclonal antibodies. The column capacities to six AFs and six ZEN analogs ranged from 100.2 ng to 167.1 ng and from 59.5 ng to 244.4 ng, respectively. The IAC–UPLC–MS/MS method was developed and validated with three different matrices (Chinese yam [Dioscorea polystachya], Platycodon grandiflorum and coix seed [Semen Coicis]). Recoveries of twelve analytes from edible and medicinal herbs were in the range of 64.7%–112.1%, with relative standard deviations below 13.7%. The limits of quantification were in the range from 0.08 μg kg−1 to 0.2 μg kg−1. The method was proven to be sensitive and accurate, and suitable for the determination of real samples.
MS-based quantification of RhoA/B and RhoC ADP-ribosylation J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-07 Anke Schröder, Anastasia Benski, Anne Oltmanns, Ingo Just, Astrid Rohrbeck, Andreas Pich
Mono ADP-ribosylation is a common characteristic of bacterial toxins resulting to aberrant activation or inactivation of target proteins. The C3 exoenzyme of Clostridium botulinum (C3bot) ADP-ribosylates the small GTPases RhoA, RhoB and RhoC, leading to inactivation of these important regulators and impaired down-stream signaling. Quantification of ADP-ribosylation using gel migration assays, antibodies, and radioactivity-based methods are limited. Therefore a novel LC-MS-based method to specifically determine and quantify ADP-ribosylation of Rho GTPases was established. A heavy labeled protein standard that contained ADP-ribosylation specific peptides in similar amounts in ADP ribosylated and non ADP ribosylated form was used for relative quantification in vivo. In a proof of principle experiment HT22 cells were treated with C3bot and the kinetics of RhoA/B and RhoC ADP-ribosylation were determined in vivo.
Effect of seaweed supplementation on tocopherol concentrations in bovine milk using dispersive liquid-liquid microextraction J. Chromatogr. B (IF 2.441) Pub Date : 2018-06-07 Andrew Quigley, Siobhán W. Walsh, Eva Hayes, Damian Connolly, Wayne Cummins
A dispersive liquid-liquid microextraction (DLLME) method, combined with HPLC-UV detection, was developed for the extraction and preconcentration of δ-tocopherol from bovine milk. This method was used to study the effect of supplementing cow feed with the seaweed Ascophyllum nodosum on vitamin content in milk. The optimal experimental conditions were determined: 200 μL of chloroform (extraction solvent), 1.0 mL of ethanol (dispersive solvent), 5 mL of water (aqueous phase). Under these optimal conditions the DLLME method provided linearity in the range 0.01 μg/mL to 8 μg/mL with R2 values of 0.998. Limit of detection (LOD) was 0.01 μg/mL, while the enrichment factor was 89. Cow feed that was supplemented with Ascophyllum nodosum was shown to increase δ-tocopherol levels from 3.82 μg/mL to 5.96 μg/mL.
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