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  • Development of a method for quantification of toluene diisocyanate and methylenediphenyl diisocyanate migration from polyurethane foam sample surface to artificial sweat by HPLC-UV-MS
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-12
    Aleksandra Donchenko; Simon Aubin; Sébastien Gagné; Mark Spence; Livain Breau; Jacques Lesage

    The US Environmental protection agency (EPA) has published guidance that includes test procedures for evaluating indoor exposure to chemicals from products. One of the test procedures represents the migration test for evaluating potential dermal exposure from home furniture. Such an evaluation involves the chemical measurement of the sweat which is currently unavailable in the literature. The objective of this project was to develop and validate an analytical method for quantification of migration of 4,4’-methylenediphenyl diisocyanate (MDI), 2,6-toluene diisocyanate (2,6-TDI) and 2,4-toluene diisocyanate (2,4-TDI) from a polyurethane (PU) flexible foam to artificial sweat that meets the recommendations of the EPA test protocol. Following the EPA protocol, six synthetic sweat solutions were prepared and used in evaluation of isocyanate recovery performance. The migration tests were conducted using five foam types that were chosen and supplied by PU foam manufacturers to represent the types most commonly found in commercial products, and with formulations anticipated to have the highest potential residual TDI or MDI. Migration tests were conducted using glass fiber filters (GFF) coated with 1-(2-methoxyphenyl)piperazine (1,2-MP) and analyzed using HPLC equipped with a UV detector for quantification and a MS detector to qualify peaks. The detection limits of the method were 0.002µg/mL for 2,6-TDI, 0.011µg/mL for 2,4-TDI, and 0.003µg/mL for MDI. Quantification limits were 0.006µg/mL, 0.037µg/mL, and 0.010µg/mL, respectively. The recovery tests on a Teflon surface for 5 of the 6 EPA-recommended synthetic sweat solutions indicate the recovery percentage was approximately 80% for diisocyanates. Recovery for the sixth sweat solution was low, approximately 30%. TDI and MDI migration was not observed when testing was conducted on foam samples.

    更新日期:2020-02-12
  • What’s in a whisker? High-throughput analysis of twenty-eight C19 and C21 steroids in mammalian whiskers by ultra-performance convergence chromatography-tandem mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-12
    Nico Lübcker; Liezl M. Bloem; Therina du Toit; Pieter Swart; P.J. Nico de Bruyn; Amanda C. Swart; Robert P. Millar

    Obtaining longitudinal endocrinological data from free-ranging animals remains challenging. Steroid hormones can be extracted sequentially from non-invasively sampled biologically inert keratinous tissues, such as feathers, nails, hair and whiskers. However, uncertainty regarding the type and levels of steroids incorporated into such tissues complicates their utility in wildlife studies. Here, we developed a novel, comprehensive method to analyze fourteen C19 and fourteen C21 steroids deposited chronologically along the length of seal whiskers in a single, 6-minute chromatographic step, using ultra-performance convergence chromatography-tandem mass spectrometry. The limits of detection and quantification ranged from 0.01 to 2 ng/mL and from 0.1 to 10 ng/mL, respectively. The accuracy and precision were within acceptable limits for steroids at concentrations ≥2 ng/mL. The recovery (mean = 107.5% at 200 ng/mL), matrix effect and process efficiency of steroids evaluated, using blanked whisker matrix samples, were acceptable. The method was applied to the analysis of steroid hormone levels in adult female whisker segments obtained from southern elephant seals (Mirounga leonina), n = 10, and two fur seal species, Antarctic fur seals (Arctocephalus gazella; n = 5) and subantarctic fur seals (Arctocephalus tropicalis; n = 5), sampled between 2012–2017. In the whisker subsamples analyzed (n = 71), the median concentration of steroid hormones detected above the LOQ ranged from 2.0–273.7 pg/mg. This was the first extraction of multiple C19 and C21 steroids, including their C11-oxy metabolites, from the whiskers of mammals. Measuring hormones sequentially along the whisker lengths can contribute to our understanding of the impact of stress associated with environmental/climate changes that affect the health, survival of organisms, as well as to delineate the reproductive cycles of free-living mammals with cryptic life stages.

    更新日期:2020-02-12
  • Post-column detection of cadmium chelators by high-performance liquid chromatography using 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-12
    Chia-Shang Chen; Shinya Ogawa; Yuki Imura; Michio Suzuki; Etsuro Yoshimura

    Cd(II) is toxic to many species, including humans, because it inactivates a number of enzymes and induces cytopathic effects in the liver, kidney, and skeletal tissues in humans. Metallothionein and glutathione (GSH) play a major role in the protection against Cd(II)-induced toxicity in mammalian cells. In this study, a relatively simple method for detecting trace amounts of Cd(II) chelators was developed by using 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid (TPPS). The TPPS–Cd(II) complex was added to the elutions of high-performance liquid chromatography. The Cd(II) chelators separated by column chromatography were mixed with Cd(II)-bound TPPS (TPPS-Cd(II)). Cd(II) from TPPS-Cd(II) was chelated by the eluted Cd(II) chelators, resulting in the formation of free TPPS. The absorbance of TPPS shifted from 434 nm (TPPS–Cd(II)) to 414 nm (TPPS), and this characteristic shift was used to estimate the quantity and affinity of the Cd(II) chelators. This new method was compared with the bathocuproine disulfonate (BCS) method developed in our previous study. Instead of BCS-Cu(I), TPPS-Cd(II) was used as the colorimetric reagent. The experimental setup of the TPPS-based method is more general, and the preparation of the colorimetric solution is also much simpler than the BCS method. To verify the efficacy of this new method, we determined the actual Cd(II)-chelating ability of GSH in horse blood; the obtained concentration was in good agreement with the previously reported value.

    更新日期:2020-02-12
  • Quantitation of Phenanthrene Dihydrodiols in the Urine of Smokers and Non-smokers by Gas Chromatography-Negative Ion Chemical Ionization-Tandem Mass Spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-07
    Kai Luo; J. Bradley Hochalter; Steven G. Carmella; Stephen S. Hecht

    Polycyclic aromatic hydrocarbons (PAH) are well-established environmental carcinogens likely to be causative agents for some human cancers. Bay-region diol epoxides are ultimate carcinogenic metabolites of multiple PAH. Dihydrodiols are the important intermediate products of this pathway and can be further oxidized to form diol epoxides. We quantified two dihydrodiol metabolites of phenanthrene (Phe), the simplest PAH with a bay-region, in the 6 h urine of smokers (N=25) and non-smokers (N=25) using a newly developed and validated analytical method. After hydrolysis by ß-glucuronidase and sulfatase, and solid phase extraction, the sample was silylated and analyzed by gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). Levels (nmol/6 h urine) of Phe-1,2-dihydrodiol (Phe-1,2-D) and Phe-3,4-dihydrodiol (Phe-3,4-D) were 2.04±1.52 and 0.51±0.35 , respectively, in smokers, significantly higher than those in non-smokers (1.35±1.11 of Phe-1,2-D, p<0.05; 0.27±0.25 of Phe-3,4-D, p<0.005). Cigarette smoking also influenced the regioselective metabolism of Phe, presenting as a significant difference in the urinary distribution pattern of Phe-1,2-D and Phe-3,4-D between smokers and non-smokers: the ratio Phe-3,4-D: Phe-1,2-D increased from 0.20 in non-smokers to 0.28 in smokers (p<0.01), which can be explained by the induction of the phenanthrene metabolizing enzymes CYP1A2 and CYP1B1 by cigarette smoke. The method described here is the first example of facile quantitation of an intact human dihydrodiol metabolite of any PAH with three or more aromatic rings and will be applicable in clinical and molecular epidemiology studies of PAH metabolism and cancer susceptibility.

    更新日期:2020-02-07
  • Lipid alterations in the skeletal muscle tissues of mice after weight regain by feeding a high-fat diet using nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-07
    Jung Yong Eum; Gwang Bin Lee; Sun Shin Yi; Il Yong Kim; Je Kyung Seong; Myeong Hee Moon

    This study investigated lipid alterations in muscle tissues [gastrocnemius (Gas) and soleus (Sol)] of mice under different diet programs (weight gain, weight maintenance, weight regain, and controls) by nanoflow ultrahigh pressure liquid chromatography-electrospray ionization-tandem mass spectrometry. Since overloaded lipids in the skeletal muscle tissues by excessive fat accumulation are related to insulin resistance leading to type II diabetes mellitus, analysis of lipid alteration in muscle tissues with respect to high-fat diet (HFD) is important to understand obesity related diseases. A total of 345 individual lipid species were identified with their molecular structures, and 184 lipids were quantified by selected reaction monitoring method. Most triacylglycerol (TG) and phosphatidylethanolamine (PE) species displayed a significant (>2-fold, p < 0.01) increase in both the Gas and Sol and to a larger degree in the Gas. However, lipid classes involved in insulin resistance and anti-inflammatory response, including lysophosphatidylcholine (18:0), diacylglycerol (16:0_18:1, 16:0_18:2, and 18:1_18:1), ceramide (d18:1/24:0 and d18:1/24:1), and phosphatidylinositol (18:0/20:4), showed a significant accumulation in the Sol exclusively after HFD treatment. In addition, the lipid profiles were not significantly altered in mice that were fed HFD only for the last 4 weeks (weight gain group), suggesting that consuming HFD in the younger age period can be more effective in the Gas. This study reveals that lipid classes related to insulin resistance accumulated more in the Sol than in the Gas following HFD treatment and the weight regain program perturbed lipid profiles of the Sol to a greater extent than that by the other diet programs, confirming that the Sol tissue is more influenced by HFD than Gas.

    更新日期:2020-02-07
  • 更新日期:2020-02-07
  • Upgrading analytical methodology through comparative study for screening of 267 pesticides/metabolites in five representative matrices using UPLC-MS/MS
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-06
    Tae-Woong Na; Md. Musfiqur Rahman; Sung-Woo Kim; Md. Ershadul Haque; Jong-Bang Eun; Jae-Han Shim
    更新日期:2020-02-06
  • Method development and validation of dissolution testing for nicotine release from smokeless tobacco products using flow-through cell apparatus and UPLC-PDA
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-04
    John H. Miller; Tim Danielson; Yezdi B. Pithawalla; Anthony P. Brown; Celeste Wilkinson; Karl Wagner; Fadi Aldeek
    更新日期:2020-02-04
  • Determination of Four Nitrofuran Metabolites in Gelatin Chinese Medicine using Dispersive Solid Phase Extraction - Solid Phase Extraction - Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-03
    Jinyan Gong; Jiong Li; Haina Yuan; Bingquan Chu; Weijie Lin; Qingwen Cao; Qiqi Zhao; Ruosi Fang; Ling Li; Gongnian Xiao

    This study established a validated analytical method for the first time on the determination of nitrofuran metabolites, including 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolinone (AMOZ), semicarbazide hydrochloride (SEM) and 1-aminohydantoin (AHD) in gelatin Chinese medicine. A C18 column with the mobile phase consisting of (A) acetonitrile and (B) 5 mmol/L ammonium acetate in water was used to separate these nitrofuran metabolites. The limit of detection of SEM, AHD, AOZ and AMOZ using LC-MS were found to be 0.2 µg/L, 0.3 µg/L, 0.2 µg/L and 0.2 µg/L, whereas their limit of quantification were 0.6 µg/L, 0.8 µg/L, 0.6 µg/L and 0.5 µg/L. These nitrofuran metabolites exhibited a good linear standard curve (regression coefficients above 0.99) with a concentration range of 2 µg/L to 100 µg/L. Regarding extraction procedure, gelatin Chinese medicine was pre-treated with pepsin and then extracted using 5% formic acid (v/v) in acetonitrile. The resultant extract was extracted through dispersive solid phase extraction using 1,000 mg anhydrous sodium sulfate, 300 mg octadecyl carbon silica gel sorbent absorbent and 500 mg ethylenediamine-N-propyl carbon silica gel absorbent), and then purified on Oasis PRiME HLB cartridges. The matrix effect was effectively eliminated after the extraction procedure as confirmed by comparing the ratio of standard curves prepared by standards dissolved in both matrix solvent and 5 mmol/L ammonium acetate in 95: 5 (v/v water: acetonitrile). The recoveries of these nitrofuran metabolites under the 2 µg/kg, 5 µg/kg and 20 µg/kg spiking levels were between 85.6% and 95.6%. These metabolites after the extraction were stable at 4°C for 24 hours. The validated method was used to analyze the residue level of these nitrofuran metabolites in 25 gelatin Chinese medicines. Results showed that only one Colla Corii Asini sample contained SEM (2.52 µg/kg) and AOZ (6.27 µg/kg), whereas one Testudinis Carapacis et Plastri sample had SEM (1.27 µg/kg) and AMOZ (9.53 µg/kg).

    更新日期:2020-02-03
  • Simultaneous detection of 24 oral antidiabetic drugs and their metabolites in urine by liquid chromatography–tandem mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-03
    Ying Hoo Lam; Mei Tik Leung; Chor Kwan Ching; Tony Wing Lai Mak

    Drugs are the most frequent cause of hypoglycemia. Though the drug history is usually obvious in diabetic patients, the diagnosis could be a challenge in patients without a history of such exposure. Screening for oral antidiabetic drugs has been recommended as part of the hypoglycemia workup in patients without diabetes. Many published analytical methods of oral antidiabetic agents were usually of limited coverage and restricted to parent drugs only. In the current study, a liquid chromatography–tandem mass spectrometry (LC-MS/MS) analytical system for the simultaneous detection of 24 oral antidiabetic drugs and their metabolites in urine was established and validated. The method covered both conventional as well as the newer antidiabetic drugs such as dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter-2 inhibitors. Following sample preparation by solid phase extraction, analytes were detected by LC-MS/MS with multiple reaction monitoring triggered enhanced product ion scan. The method was successfully applied to 233 cases of unexplained hypoglycemia, with 83 oral antidiabetic drugs detected in 51 of the urine samples.

    更新日期:2020-02-03
  • Quantitative assessment of secondary metabolites and cancer cell inhibiting activity by high performance liquid chromatography fingerprinting in Dendrobium nobile
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-03
    Zheng Shigang; Hu Yadong; Zhao Ruoxi; Zhao Tingmei; Li hongjie; Rao Dan; Chun Ze

    Dendrobium nobile is an important medicinal food beneficial for human health, well known for polysaccharides and dendrobine. For fast, accurate, and comprehensive comparison of its quality, high performance liquid chromatography (HPLC) fingerprinting method was constructed. Firstly, spring frost stressed D. nobile herb was observed for assessment. Decreased leaf thickness, chlorophyll, and drying rate, and increased free-proline indicated heavy damages on growth. But, the content of polysaccharides increased significantly in during-frost (DF), and dropped significantly in after-frost (AF). The content of dendrobine accumulated significantly in AF. Then, low similarity among HPLC fingerprints of before-frost (BF), DF, and AF, and 75.82% of significantly variant peaks indicated the changing of much more components. Especially, some less-polar components increased significantly in DF, but not in AF. Moreover, the highest suppression rates (SRs) to A549 lung cancer cells were up to 33.08% in DF, but only 15.63% and 12.12% in BF and AF. After association analysis, eleven less-polar components were found to be significantly and positively correlated to SRs under relatively high concentration. The result shows that frost stress not only causes damages to plant growth, but also promotes the accumulation of some health-beneficial bioactive metabolites. HPLC based fingerprinting method shows good applicability on quality evaluation and bioactivity correlation analysis of complexed agricultural products.

    更新日期:2020-02-03
  • 更新日期:2020-02-03
  • An improved cytochrome P450-phenotyping cocktail with a simplified and highly sensitive UHPLC-MS/MS assay in human plasma
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-01
    Shane K. Eagles; Xiaosuo Wang; Annette S. Gross; Andrew J. McLachlan

    Measuring in vivo changes in the drug metabolizing activity of cytochrome P450 (CYP) enzymes is critical to understanding and assessing drug-drug, drug-diet and drug-disease interactions. The sensitivity and specificity of ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) makes it an ideal tool for analyzing drugs and their metabolites in biological matrices, and has demonstrated utility in CYP phenotyping across varied applications. Published CYP phenotyping cocktail assays often require large plasma sample volumes (0.5-1 mL), have relatively low sensitivity and multi-step complex sample preparation and extraction procedures. Further, variability exists in the way that recovery and matrix effects are investigated and reported, and some studies fail to report these data altogether. Therefore, the aim of this study was to develop, validate and optimize a simplified assay for the probe drugs caffeine (metabolized by CYP1A2), omeprazole (CYP2C19), losartan (CYP2C9), dextromethorphan (CYP2D6), midazolam (CYP3A4) and their respective enzyme-specific metabolites in small volumes (100 μL) of human plasma, that addresses the issues noted. Analyte extraction involved protein precipitation with acetonitrile and solid-phase extraction (SPE). Samples were analyzed using an Agilent 1290 infinity LC system in tandem with 6460A triple quadrupole mass spectrometers. The assay met FDA guideline-recommended requirements for specificity, sensitivity (analyte LLOQs 0.78-23.4 ng/mL), accuracy (intra-day RE % nominal concentration 90.7-110.2%; inter-day RE % 87.0-110.5%) and precision (intra-day analyte RSD % 0.46-11.4%; inter-day RSD % 1.36-11.2%). Recovery and matrix effects were thoroughly investigated and excluded as potential interferers with assay performance. This assay has been used successfully to phenotype CYP activity in a human clinical trial participant. Importantly, the authors provide a contemporary commentary on commonly found issues in the CYP phenotyping cocktail assay literature, and make recommendations concerning best-practice approaches and the standardization of data reporting in this area.

    更新日期:2020-02-03
  • A Rapid, Simple and Highly Sensitive UPLC-MS/MS Method for Quantitation of Pimavanserin in Plasma and Tissues: Application to Pharmacokinetics and Brain Uptake Studies in Mice
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-01
    Essam Ezzeldin; Muzaffar Iqbal; Yousif A. Asiri; Azza A Ali; Toqa Elnahhas

    Pimavanserin is a new drug approved by the FDA for Parkinson's disease psychosis and other neurological disorders such as Alzheimer's disease. In this study, we developed a UPLC-MS/MS method to quantify pimavanserin disposition in the brain and its pharmacokinetics in mice. Vilazodone was used as the internal standard. Pimavanserin and IS were extracted by liquid-liquid extraction using tert-butyl methyl ether and separated using an Acquity UPLC BEH™ C18 column. The mobile phase consisted of solvent A (0.1% formic acid in acetonitrile) and B (0.1% formic acid in 20 mM ammonium acetate buffer) (A: B, 70:30 v/v) at a flow rate of 0.25 ml/min. The multiple reaction monitoring transitions were performed at m/z 428.23→98.15 for pimavanserin and m/z 441.70 > 155.03 for the IS. The developed method was found to be sensitive, fast, and reproducible. The linearity of the method was ˃0.99 over the range of 0.1- 300ng/mL in plasma and 0.25 -300 ng/g in the brain homogenate. Precision and accuracy were within the acceptance range. The method was applied to pharmacokinetics and brain uptake studies, which showed that pimavanserin penetrates the blood-brain barrier and reaches a Cmax of 21.9 ± 6.66 ng/g in 2.0 h. We also found that pimavanserin brain to plasma ratio (Kbrain/plasma) is 0.16 ± 0.05 and it is rapidly eliminated.

    更新日期:2020-02-03
  • Pharmacokinetics and tissue distribution study of liposomal albendazole in naturally Echinococcus granulosus infected sheep by a validated UPLC-Q-TOF-MS method
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-01
    Haibo Zhang; Jun Zhao; Bei Chen; Yunfang Ma; Zhiqiang Li; Xi Shou; Limei Wen; Yuan Yuan; Huijing Gao; Jie Ruan; Hongling Li; Shuai Lu; Yuehong Gong; Jianhua Wang; Hao Wen

    Albendazole (ABZ) is the first-line drug in treating echinococcosis, which is recommended by WHO. To address the poor bioavailability of albendazole, liposomal albendazole was formulated and is available in our hospital for many years. In this study, a sensitive, reliable and accurate UPLC-Q-TOF-MS method was developed and validated for the determination of albendazole and its metabolites, albendazole sulfoxide (ABZSO), albendazole sulfone (ABZSO2) and albendazole-2-aminosulfone (ABZSO2NH2) in naturally echinococcus granulosus (E. granulosus) infected sheep plasma and tissues with mebendazole (MBZ) as the internal standard (IS). Plasma and tissues samples were prepared by protein precipitation method. The separation was performed on an ACQUITY UPLC® BEH C18 column (2.1×50 mm, 1.7 μm) with a gradient mobile phase consisting of methanol and water containing 0.1% formic acid at 0.4 mL/min. The detection was performed on a quadrupole time-of-flight (Q-TOF) high-resolution mass spectrometer using positive electrospray ionization (ESI) source with a chromatographic run time of 6.0 min. The detection was operated using target ions of [M+H]+ at m/z 266.096 for ABZ, m/z 282.091 for ABZSO, m/z 298.086 for ABZSO2, m/z 240.081 for ABZSO2NH2 and m/z 296.104 for IS in selective ion mode, respectively. This method was validated in terms of selectivity, linearity, precision, accuracy, recovery, matrix effect, dilution effect, carryover effects, stability, calibration curve and LLOQ. All validation parameter results were within the acceptable range described in guideline for bioanalytical method validation. This method has been successfully applied to the pharmacokinetic study following single and multiple oral dose of 10 mg/kg liposomal albendazole, and tissue distribution study following multiple oral dose of 10 mg/kg, with emulsion albendazole as the reference preparation. The results in the article will provide valuable information for use in clinical applications of liposomal albendazole and also be beneficial for further development of liposomal albendazole in future studies.

    更新日期:2020-02-03
  • An immunochromatographic test system for the determination of lincomycin in foodstuffs of animal origin
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-02-01
    Olga D. Hendrickson; Elena A. Zvereva; Demid S. Popravko; Anatoly V. Zherdev; Chuanlai Xu; Boris B. Dzantiev
    更新日期:2020-02-03
  • An Easy and Simple Separation Method for Fc and Fab Fragments from Chicken Immunoglobulin Y (IgY)
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-31
    Xin Zhou; Yanru Wang; Dong Uk Ahn; Zhaoxia Cai

    Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.

    更新日期:2020-01-31
  • Microfluidic immobilized metal affinity chromatography based on Ti(IV)-decorated silica microspheres for purification of phosphoproteins
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-31
    Duygu Yıldırım; Çiğdem Kip; Khaliunsarnai Tsogtbaatar; İlkay Koçer; Eda Çelik; Ali Tuncel

    A silica-based immobilized metal affinity chromatography (IMAC) sorbent with the morphological properties suitable for purification of large phosphorylated biomolecules was synthesized. The sorbent was designed in the form of monodisperse-porous silica microspheres, 5.3 μm in size, having bimodal pore size distribution with a large median pore size (40 nm) and high surface area (163 m2/g) decorated with Ti(IV) cations (i.e. Ti(IV)@[email protected]2 microspheres). The decoration of silica microspheres with Ti(IV) cations was made by using 3-(trihydroxysilyl)propyl methylphosphonate (THSPMP) as a bifunctiontional linker, by preserving their bimodal pore size distribution. The mesopores provided a large surface area for parking of adsorbed phosphoproteins as large phosphorylated biomolecules while the intraparticular transport of phosphoproteins was facilitated by the macropores providing a large median pore size. High equilibrium adsorption capacity and high desorption yield in the purification of phosphoproteins were obtained using Ti(IV)@[email protected]2 microspheres as the sorbent in batch- and microfluidic-IMAC systems. The phosphoproteins, α-casein and β-casein were isolated from milk and human serum with almost quantitative yields and high purity in the batch IMAC system. The appropriate microcolumn permeability (3.66x10-14 m2) originating from its appropriate average diameter (5.3 μm), high porosity (0.948 cm3/g) and high surface area (163 m2/g) of Ti(IV)@[email protected]2 microspheres makes the synthesized sorbent a promising stationary phase for dynamic chromatography. Hence, a new phosphoprotein enrichment format, a microfluidic IMAC system was constructed and successfully operated for highly selective purification of phosphoproteins from non-fat milk as a complex sample. The microfluidic-IMAC system is a promising tool particularly for phosphoproteomic applications performed using samples in microliter or nanoliter scale, also involving an on-line connection of purification unit to LC-MS for the identification of large phosphorylated biomolecules enriched.

    更新日期:2020-01-31
  • Hybrid Mode of Hydrophobic Interaction Chromatography of Monoclonal Antibodies and Related Biomolecules: Influence of elution conditions on chromatographic performance using poly (alkyl aspartamide) silica columns
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-28
    Madhavi Srikoti; Mark S. Bolgar; Yuri Kazakevich

    A hybrid mode of hydrophobic interaction chromatography (HHIC) is an emerging analytical technique for the separation of biomolecules under non-denaturing conditions that combines elements of conventional hydrophobic interaction and reversed-phase chromatography. This article explores the impact of mobile phase composition such as salt concentration and organic modifier on the separation of therapeutic monoclonal antibodies and related large biomolecules using poly (alkyl aspartamide) silica HIC columns. The initial mobile phase salt concentration had a significant impact on the separation of a mixture of large biomolecules demonstrating that the relationship of elution and salt concentration was more complex than in conventional HIC. In general, the earlier eluting components exhibited greater retention at higher salt concentration as is typical of HIC separations. Conversely, the later eluting components showed greater retention at lower initial salt concentration. This differential is useful for improving the overall separation by widening the elution window for components of a mixture. In addition, no significant unfolding of the proteins was detected by intrinsic fluorescence or electrospray mass spectrometry. The impact of linear velocity and gradient steepness was also evaluated.

    更新日期:2020-01-29
  • Metabolite identification and profile of endosulfan sulfate in three human liver preparations using liquid chromatography-high resolution mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-28
    Hwa-Kyung Lee; Tae Yeon Kong; Won-Gu Choi; Ju-Hyun Kim; Yongho Shin; Hye Suk Lee; Yong Sang Lee; Jeong-Han Kim

    In this study, we performed the metabolism of endosulfan sulfate in human liver preparations (human liver microsomes, S9 fractions and hepatocytes) to identify new metabolites using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Endosulfan sulfate is a major oxidized metabolite of the organochlorine insecticide endosulfan, and it exhibits a similar toxicity to endosulfan. Six metabolites, including 5 novel metabolites of endosulfan sulfate, were identified in the three different human liver reaction mixtures and metabolic pathways of endosulfan sulfate were proposed. The phase I metabolites M1 and M2 were observed in human liver microsomes, S9 fractions and hepatocytes. M1 was suggested to be an endosulfan diol monosulfate and M2 was identified as (1,4,5,6,7,7-hexachloro-3-formylbicyclo[2,2,1]hept-5-en-2-yl)methyl hydrogen sulfate through the interpretation of the HRMS spectrum. The phase II metabolite M3 was produced as an endosulfan sulfate-GSH conjugate in those three liver preparations and transformed to M5 (dipeptide) in S9 fractions and hepatocytes. M3 was the most predominant metabolite identified in the three liver preparations. M4 was only detected in microsomes as an M2-GSH conjugate and was metabolized to M6 (monopeptide) in hepatocytes. These results are different from the metabolic pathway of endosulfan and suggest the possible detoxification metabolic reaction of endosulfan sulfate in living organisms.

    更新日期:2020-01-29
  • Proof of Site-Specificity of Antibody-Drug Conjugates Produced by Chemical Conjugation Technology: AJICAP First Generation
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-27
    Yutaka Matsuda; Maria-Christina Malinao; Veronica Robles; James Song; Kei Yamada; Brian A. Mendelsohn
    更新日期:2020-01-27
  • Dual-fiber solid-phase microextraction coupled with gas chromatography–mass spectrometry for the analysis of volatile compounds in traditional Chinese dry-cured ham
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-24
    Huan Liu; Junlong Huang; Qingkun Hu; Yan Ping Chen; Keqiang Lai; Jianqiao Xu; Gangfeng Ouyang; Yuan Liu
    更新日期:2020-01-24
  • 更新日期:2020-01-21
  • Extraction of benzoylurea pesticides from tea and fruit juices using deep eutectic solvents
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-20
    Xinya Liu; Mengyuan Chen; Zilin Meng; Heng Qian; Sanbing Zhang; Runhua Lu; Haixiang Gao; Wenfeng Zhou

    In this study, low-density deep eutectic solvent combined with dispersive liquid-liquid microextraction was applied to the extraction of five benzoylurea insecticides (BUs, including diflubenzuron, triflumuron, hexaflumuron, flufenoxuron, and chlorfluazuron) from beverages. Then the extracted and concentrated samples were analyzed and detected using the high-performance liquid chromatography combined with an ultraviolet detector. The DESs were synthesized by [P14,6,6,6]Cl as hydrogen bond acceptor and tetradecyl alcohol as hydrogen bond donor, and then characterized by Fourier transform infrared spectroscopy. In the experiment, the key factors affecting the extraction efficiency were screened by Plackett-Burman design and optimized with the central composite design. The extraction recovery rates were 85.91∼95.12%. The limits of detection and correlation coefficients of the method were 0.30∼0.60 μgL-1 and 0.9992∼0.9997. Finally, the method was applied to determine the BUs in four beverage samples, and satisfactory recoveries, within the range of 76.87∼101.19% were achieved. The present method has the potential to be applied to the detection of BUs in aqueous samples.

    更新日期:2020-01-21
  • LC-MS/MS analysis to study serum profile of short and medium chain fatty acids
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-18
    Michele Dei Cas; Rita Paroni; Anna Saccardo; Eleonora Casagni; Sebastiano Arnoldi; Veniero Gambaro; Marina Saresella; Clerici Mario; Francesca La Rosa; Ivana Marventano; Federica Piancone; Gabriella Roda

    Short and medium fatty acids derived from either dietary sources, gut microbiota, and liver production might play a role in the modulation of metabolism and inflammation. The outcome of different autoimmune or inflammatory diseases could be related to microbiota composition and consequently fatty acids production. Their analytical detection, historically completed by GC, was herein investigated using a sensitive approach of LC-MS/MS with straightforward chemical derivatization, using 3-NPH, to the respective acylhydrazines. An isopropanol protein precipitation coupled to LC-MS/MS analysis allowed to separate and quantify butyric, valeric, hexanoic acid and their branched forms. The serum physiological ranges of short and medium chain fatty acids were determined in a heterogeneous healthy population (n=54) from 18-85 years findings a concentration of 935.6 ± 246.5 (butyric), 698.8 ± 204.7 (isobutyric), 62.9 ± 15.3 (valeric), 1155.0 ± 490.4 (isovaleric) and 468.7 ± 377.5 (hexanoic) ng/mL respectively (mean ± SD). As expected, the biological levels in human serum are reasonably wide-ranging depending on several factors such as body-weight, gut microbiome dysbiosis, gut permeability, cardiometabolic dysregulation, and diets.

    更新日期:2020-01-21
  • Amalgamation of Stress Degradation and Metabolite Profiling in Rat Urine and Feces for Characterization of Oxidative Metabolites of Flibanserin using UHPLC-Q-TOF-MS/MS, H/D Exchange and NMR Technique
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-18
    Manish Kumar Sharma; Ravi P Shah; Pinaki Sengupta

    Flibanserin (FLB) is the first FDA approved drug showed to have significant activity against sexual desire disorder of premenopausal and postmenopausal women. Unfortunately, FLB is used as an adulterant in dietary supplement products as a performance enhancer in sports. Identification of FLB and its metabolites in the biological samples requires an authenticated analytical technique. The aim of this study was to identify N-oxide metabolite of FLB in microsomal and S9 human liver enzyme fractions, rat urine and feces. There are several N-oxide reported as genotoxic impurity or reactive metabolites based on position of N-oxide in piperazine ring. This study also describes the strategy to utilize degradation chemistry for isolation of N-oxide and its step-wise characterization. An LC-MS method has been developed and employed for identifying the N-oxide metabolite of FLB. The targeted N-oxide metabolite in the extracted ion chromatogram of the in vitro and in vivo samples has been confirmed by analyzing the changes in observed mass at m/z 407.1693. Major distinguished abundant ions at m/z 243.1104, 190.0974, 161.0705, 119.0601 confirmed the structure of the metabolite. This study will help to understand the oxidative potential of FLB in toxicokinetic study. The developed method can be useful to identify FLB or its N-oxide metabolite in dope testing in future. This is the first time to report a strategy to utilize degradation chemistry for N-oxide metabolite characterization. In this study, isolated N-oxidative degradation product was used to confirm N-oxide metabolite which was characterized by LC-MS through H/D exchange and structure was ensured by NMR spectroscopy (1H, COSY).

    更新日期:2020-01-21
  • Automated On-Line Packed Fiber Solid Phase Extraction for Determination of Urinary Catecholamines
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-18
    Chen LiQin; Tang Yi; Xu Bei; Xu Zhen; Shen Jun; Zhang WanQi
    更新日期:2020-01-21
  • Development and Validation of a GC-FID Method for the Analysis of Short Chain Fatty Acids in Rat and Human Faeces and in Fermentation Fluids
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-13
    Serena Scortichini; Maria Chiara Boarelli; Stefania Silvi; Dennis Fiorini

    Short-chain fatty acids (SCFAs) are gut microbiota metabolites recognized for their beneficial effects on the host organism. In this study, a simple and rapid sample preparation method combined to SCFAs analysis by direct injection and gas chromatography coupled with flame ionization detection (GC-FID), for the determination and quantification of eight SCFAs (acetic, propionic, i-butyric, butyric, i-valeric, valeric, i-caproic and caproic acids) in rat, mice and human faeces and in fermentation fluids samples, has been developed and validated. The method consists of extraction of the SCFAs by ethyl ether after acidification of the samples. The effect of the number of extractions has been assessed in order to optimize the procedure and to obtain a satisfactory yield for all the analyzed SCFAs. The increase of the extracted analytes quantity was significant passing from 1 to 2 and from 2 to 3 extractions (P < 0.05), while no significant differences were found performing 3, 4 or 5 extractions (P > 0.05). The SCFAs extracted are directly analyzed by GC-FID without derivatization and separated on a polyethylene glycol nitroterephthalic acid modified coated capillary column, with a chromatographic run time of 13 min. The proposed method showed good sensitivity, with limits of quantifications in the range 0.14-0.48 µM for SCFAs from propionic to caproic acids and 2.12 µM for acetic acid; recovery was between 80.8 and 108.8% and intraday and interday repeatability in the range 0.6-5.0% of precision (RSD, %) The optimized method is suitable for the quantitative analysis of SCFAs in real samples of rat, mouse and human faeces and in fermentation fluids, and it can be applied also to very small amount of faecal sample (20 mg).

    更新日期:2020-01-13
  • Hollow-fibre liquid-phase microextraction and gas chromatography-mass spectrometric determination of amphetamines in whole blood
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-13
    Henrique Silva Bombana; Marcelo Filonzi dos Santos; Daniel Romero Muñoz; Vilma Leyton

    Here, we present a fully validated method using a hollow-fibre liquid-phase microextraction technique for the determination by gas chromatography-mass spectrometry (GC-MS) of amphetamine (AMP), methamphetamine (MET), fenproporex (FEN), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxyethylamphetamine (MDEA) in whole blood. The validation parameters presented successful values within those recommended by the Scientific Working Group for Forensic Toxicology (SWGTox) in the Standard Practices for Method Validation in Forensic Toxicology. The limits of detection ranged from 1 to 3 ng/mL, and the limits of quantification ranged from 2 to 5 ng/mL. The determination coefficients (r2) ranged from 0.990 to 0.997, and the method presented good intraday and interday accuracy (from 90.4% to 97.2%) and satisfactory recovery (from 68% to 110%). No carryover was observed. The heteroscedasticity was tested, and only AMP presented homoscedasticity. Weighting factors were applied to correct the linearity of MET (1/x2), MDA (1/x), FEN (1/x1/2), MDMA (1/x2) and MDEA (1/y). Dilution integrity was tested at ratios of 1:2, 1:5 and 1:10, and all maintained intraday precision (from 94.9% to 99.3%) and interday precision (from 89.4% to 94.9%). The validated method was applied to six real whole blood samples from individuals suspected of consuming ecstasy, and MDMA, MDA and amphetamine were successfully identified and quantified.

    更新日期:2020-01-13
  • Spectrum-Effect Relationship Study Between HPLC Fingerprints And Antioxidant Activity of Sabia parviflora
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-11
    Yinrui Chen; Guoji Pan; Wenfen Xu; Qingwen Sun; Bo Wang; Ye Zhang; Tianjin Yang

    Nowadays, the increased prevalence of sub-health significantly affects human health worldwide. Suppressed sub-healthy by dietotherapy/herbal remedy which showed excellent safety profile, low cost and effectiveness, is an effective way. In this research, the fingerprint and antioxidant activity of Sabia parviflora were obtained by HPLC instrument and DPPH, ABTS, FRAP heatmap assays. And the antioxidant active substances were selected by spectrum-effect relationship. The results showed that significant differences in chemical compositions of samples from different sources, and EW, EE extracts had strong antioxidant activity. The antioxidant activity of Sabia parviflora was mainly determined by the complex interaction of various flavonoids (promoting, competing or antagonizing). These findings revealed that the abundant flavonoids in Sabia parviflora had significant antioxidant activity and could be potential antioxidants. With therapeutic potential for sub-health, this special tea might provide dietotherapy/herbal to remedy sub-healthy population.

    更新日期:2020-01-13
  • Identification of Metabolites and Potential Biomarkers of Kratom in Urine
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-11
    Stephanie Basiliere; Sarah Kerrigan

    Mitragyna speciosa (kratom) is a drug that is increasingly used recreationally and “therapeutically”, in the absence of medical supervision. The drug has been associated with a growing number of fatalities, and although its medicinal properties as an atypical opioid require further study, there are legitimate concerns regarding its unregulated use. Mitragynine is the most widely reported alkaloid within the plant, although more than forty other alkaloids have been identified. 7-Hydroxymitragynine is reported to have greater abuse liability due to its increased potency relative to mitragynine. In this report, biomarkers for mitragynine were investigated using liquid chromatography-quadrupole/time of flight mass spectrometry (LC-Q/TOF-MS). Speciociliatine and speciogynine were identified as alternative biomarkers, often exceeding the concentration of mitragynine in unhydrolyzed urine. 9-O-Demethylmitragynine and 7-hydroxymitragynine were identified in unhydrolyzed urine in 75% and 63% of the cases. Deconjugation of phase II metabolites using chemical hydrolysis was not suitable due to degradation of the Mitragyna alkaloids. Enzymatic hydrolysis was evaluated using three traditional glucuronidases, four sulfatases and four recombinant enzymes. Although enzymatic hydrolysis increased the concentration of 16-carboxymitragynine, it had nominal benefit for other metabolites. Deconjugation of urine was not necessary due to the abundance of parent drug (mitragynine), its diastereoisomers (speciociliatine and speciogynine) or metabolites (9-O-demethylmitragynine and 7-hydroxymitragynine).

    更新日期:2020-01-13
  • Label-free quantitative proteomics positions the effects and mechanisms of Herba Lysimachiae on synovial diseases based on biolabel-led research pattern
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-08
    Xu-Zhao Li; Hua-Jin Huang; Shuai-Nan Zhang; Qi Liu; Yu-Mei Wang

    Some previous studies have demonstrated that Herba Lysimachiae (HL) has a certain protective effect on synovial lesion. But the synovial diseases HL is suitable for treating have remained unclear, as well as the mechanisms involved. To investigate the therapeutic potentials of HL in synovial diseases based on the biolabel-led research pattern. Label-free quantitative proteomics analysis was used to screen the potential biolabels responsible for the intervention of HL on synovium. The effects of HL on the joint swelling and synovial platelet aggregation in osteoarthritis model was applied to confirm the biolabels analysis results. Totally, 140 common proteins were differentially expressed after treatment with HL, out of which 23 were involved in 4 key pathways and considered as the potential biolabels responsible for the interventions of HL on synovium. Biolabels analysis showed that HL increased the levels of the proteins promoting platelet aggregation in physiological situations. The potential biolabels and their related pathways were mainly associated with the pathogenesis of osteoarthritis. In osteoarthritis model, HL inhibited the joint swelling and the overexpression of Itga2b and Itgb3 in synovium to some extent. This study reveals that HL is suitable to treat osteoarthritis. Additionally, HL may produce the dual effects on platelet aggregation in synovium.

    更新日期:2020-01-09
  • A simple and rapid liquid chromatography-mass spectrometry method to assay cabozantinib in plasma: Application to therapeutic drug monitoring in patients with renal cell carcinoma
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-07
    Florent Ferrer; Caroline Solas; Madeline Giocanti; Bruno Lacarelle; Jean-Laurent Deville; Gwenaelle Gravis; Joseph Ciccolini

    Cabozantinib is a novel multi-target tyrosine kinase inhibitor recently approved in metastatic renal cell carcinoma (mRCC) leading to frequent severe toxicities requiring empirical dose reduction. Therapeutic drug monitoring (TDM) could help to predict the risk for severe toxicities by quickly detecting overexposed patients followed by prospective adaptive dosing strategy. To achieve this goal, a simple and rapid assay to monitor cabozantinib plasma concentration was developed and validated. After a single precipitation step with 87% recovery, cabozantinib was assayed by liquid chromatography tandem mass spectrometry (electrospray ionization interface) over a 25- 5000 ng/ml range covering usual plasma levels in clinical setting. For cabozantinib and cabozantinib 2H4 used as internal standard, quantification was performed using the m/z 502 → m/z 323 and m/z 506 → m/z 323 transitions, respectively.Analytical runtime was 5 minutes. Both inter-days and intra-day accuracy and precision were <15%. When tested in routine clinical practice in a subset of mRCC patients treated with standard 60 mg quaque die (QD) dosing, the method proved to be fully adapted and neither analytical interferences nor matrix effect was observed. Results showed that cabozantinib trough levels were highly variable among patients (i.e., 973 ±501 ng/ml, CV = 52%), calling for implementing TDM in patients with mRCC to monitor exposure levels and evaluate concentration-response relationship.

    更新日期:2020-01-07
  • Development and validation of a novel UPLC-MS/MS method for quantification of delafloxacin in plasma and aqueous humour for pharmacokinetic analyses
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-03
    Muzaffar Iqbal; Essam Ezzeldin; Rasheed Naji Herqash; Md. Khalid Anwer; Faizul Azam
    更新日期:2020-01-04
  • Generic and rapid determination of low molecular weight organic chemical contaminants in protein powder by using ultra-performance liquid chromatography-tandem mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-03
    Jia Zhan; Xi-zhi Shi; Xu-wen Xu; Guo-zhou Cao; Xian-feng Chen

    A rapid, simple, and generic analytical method that could simultaneously determine 291 undesirable low molecular weight chemical contaminants from different drug families in protein powder, such as veterinary drugs and pesticides, etc, had been developed. This method comprised the extraction with acetonitrile-dimethyl sulfoxide (DMSO), clean-up through dispersive solid phase extraction (D-SPE) and low temperature filtration, and analysis by ultra-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry at multiple-reaction monitoring mode. Acetonitrile-DMSO was more generic than acetonitrile or methanol for the extraction of large-scale organic chemical contaminants with different polarities in protein powder. Most interferences in the extract were eliminated by the combination of D-SPE and low temperature filtration, which simultaneously provided satisfactory recoveries of both hydrophobic and hydrophilic analytes. In particular, besides the purification function, the sorbent of D-SPE also played an important role in grinding samples to improve extraction efficiency during homogenization. This streamlined approach allowed the processes of extraction and the main purification were carried out in one-step, and dramatically reduced sample preparation turnaround times and solvent consumption. For quantification, matrix-fortified calibration curves showed competent linearity for most of the target compounds with linear regression coefficients (r) higher than 0.9900, except for two analytes. The limits of quantification ranged from 0.1 μg/kg to 50 μg/kg, which was usually sufficient to verify the compliance of products with legal tolerances. The average recoveries for spiked protein powder ranged from 65.6% to 142.2% with associated RSD values between 0.5% and 28.5%. For over 90% of the analytes, the recoveries were between 70% and 120% with RSD values in the range of 1%–15%. Applying this method in routine monitoring programs would drastically reduce both effort and time.

    更新日期:2020-01-04
  • Fast LC-MS quantification of ptesculentoside, caudatoside, ptaquiloside and corresponding pterosins in bracken ferns
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-03
    Vaidotas Kisielius; Dan Nybro Lindqvist; Mikkel Boas Thygesen; Michael Rodamer; Hans Christian Bruun Hansen; Lars Holm Rasmussen
    更新日期:2020-01-04
  • Minor compounds and potential interferents in gas chromatographic analyses of human serum fatty acids
    J. Chromatogr. B (IF 2.813) Pub Date : 2020-01-02
    Chen-Chen Lin; Amarjargal Sengee; Svein A. Mjøs

    Fatty acids from 100 randomly selected human serum samples were esterified to fatty acid methyl esters and analyzed by gas chromatography with flame ionization detector. A subset of the 20 samples that spans the variation in the original set of 100 samples were thereafter analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS data was acquired using capillary columns with two different stationary phases, BP20 (polyethylene glycol) and BPX70 (cyanopropyl polysilphenylene-siloxane). Equivalent chain lengths on the two columns are reported for 69 compounds that constituted more than 0.1% of the chromatographic area in at least one sample. Of these, 39 compounds were identified as regular fatty acid methyl esters. The remaining 30 compounds were decomposition products from cholesterol, dimethylacetals, three compounds that have been linked to poor kidney function, and 13 compounds that are currently unidentified. The retention index patterns showed that on both columns there were 16 compounds that were separated by less than 0.05 equivalent chain length units from the nearest neighbor, meaning that they were overlapping or poorly resolved. The relationship between the peak threshold level and the number of peaks found above the level predicts a dramatic increase in the number of peaks that have to be resolved if the threshold is lowered below 0.1%.

    更新日期:2020-01-02
  • A systematic and methodical approach for the efficient purification of recombinant protein from silkworm larval hemolymph
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-31
    Robert Minkner; Jian Xu; Holger Zagst; Hermann Wätzig; Tatsuya Kato; Imke Oltmann-Norden; Enoch Y. Park

    The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.

    更新日期:2019-12-31
  • Proteomic profiling of salmon skin mucus for the comparison of sampling methods
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-31
    C.K. Fæste; H. Tartor; A. Moen; A.B. Kristoffersen; A.K.S. Dhanasiri; J.H. Anonsen; T. Furmanek; S. Grove

    The epidermal mucus protects fish against harmful environmental factors and the loss of physiological metabolites and water. It is an efficient barrier between the fish and the biosphere. The integrity of the skin mucus is thus of vital importance for the welfare and survival of the fish. Since excreted proteins and small molecules in the mucus can mirror the health status of the fish, it is a valuable matrix for monitoring stress, pathogen exposure, and nutritional effects. Several methods for sampling epidermal mucus from different fish species have previously been described, but information about their efficiency or the comparability of mucus analyses is lacking. In the present study, skin mucus from farmed Atlantic salmon was therefore sampled by three methods, including absorption with tissue, wiping, and scraping with a blunt blade, and the mucus proteome was analyzed by ultra-high pressure liquid chromatograph high-resolution mass spectrometry. The measured protein content, numbers, compositions and the observed data quality were compared between sampling methods. Furthermore, functional annotation and classification of the identified proteins was performed. The results showed that the three skin mucus sample types differed qualitatively as well as quantitatively. The absorbed mucus was the least tainted by proteins resulting from damage inflicted to the fish epidermis by the sampling procedure. Wiped mucus showed a better protein yield than absorbed and delivered a larger proteome of identifiable proteins, with less contamination from epithelial proteins than observed for scraped mucus. We recommend that future research of mucus should use the absorption method in cases, where it is important that the mucus is devoid of proteins from the underlying epithelium, and the wiping method, when protein yield is crucial or when the proteome of the outer epithelium is of interest.

    更新日期:2019-12-31
  • Quantitative bio-analysis of pitavastatin and candesartan in rat plasma by HPLC-UV: Assessment of pharmacokinetic drug-drug interaction
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-30
    Misari Patel; Charmy Kothari

    A novel, precise, accurate and rapid HPLC-UV method was developed, optimised and fully validated for simultaneous estimation of pitavastatin (PIT) and candesartan (CAN) in rat plasma using telmisartan as an internal standard. Following liquid-liquid extraction of the analytes from plasma, chromatographic separation was accomplished on a Waters Reliant C18 column (4.6 × 250 mm, 5 µm) using ACN-5 mM Sodium acetate buffer (80:20, v/v; pH adjusted to 3.5 with acetic acid) as mobile phase at a flow rate of 0.8 mL/min and wavelength of 234 nm. The calibration curves were linear over the concentration ranges of 2-400 ng/mL and 3-400 ng/mL for pitavastatin and candesartan respectively. The method when validated as per US-FDA guidelines was found to be precise as well as accurate. Extraction recovery observed for both analytes was above 90% as well as reproducible and consistent. Stability studies showed the samples to be stable over a long period covering from sample collection to final analysis. The method was successfully applied to investigate pharmacokinetic interaction between PIT and CAN in wistar rats. The mean plasma concentration-time curves of PIT and CAN showed that single PIT as well as CAN show similar pharmacokinetic properties to those obtained when co-administrated with each other (P value > 0.05). Hence, there is no evidence for a potential drug-drug interaction between PIT and CAN. This information provides evidence for clinical rational use of CAN and PIT in cardiovascular patients.

    更新日期:2019-12-30
  • Efficient enrichment of total flavonoids from Pteris ensiformis Burm. extracts by macroporous adsorption resins and in vitro evaluation of antioxidant and antiproliferative activities
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-30
    Mengyang Hou; Wenzhong Hu; Zhilong Xiu; Yusheng Shi; Kexin Hao; Duo Cao; Yuge Guan; Hanlin Yin

    The aim of this work is to develop an efficient and economical method for the enrichment of total flavonoids from Pteris ensiformis Burm. extracts. Resin screening, adsorption kinetics, adsorption isotherms and thermodynamics were successively researched prior to the dynamic adsorption and desorption tests. NKA-II resin was chosen as the best adsorbent, and the adsorption data were best described by the pseudo-second-order kinetics model and Langmuir isotherm model. The optimum enrichment conditions were as follows: for adsorption the total flavonoids concentration, flow rate and volume of sample were 1.84 mg/mL, 2 BV/h and 5 BV, respectively, and for desorption the flavonoids-loaded NKA-II resin column was desorbed by 7 BV of 50% ethanol at a rate of 2 BV/h. The product had a 6.63-fold higher total flavonoids content than crude extracts, and the recovery yield of total flavonoids was 80.65%. Furthermore, flavonoids-enriched extracts exhibited higher in vitro scavenging activity against superoxide anion radical and hydroxyl radical than crude extracts. In addition, higher antiproliferative activity of flavonoids-enriched extracts against MCF-7 and HepG-2 cell lines was also found as compared to the crude extracts. The developed method is appropriate for large-scale enrichment of total flavonoids from Pteris ensiformis Burm. extracts in the food and pharmaceutical industries.

    更新日期:2019-12-30
  • Quantification of metronidazole in human bile fluid and plasma by liquid chromatography-tandem mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-28
    Jimin Yoon; Sung Wook Kang; Wang-Seob Shim; Jun Kyu Lee; Dong Kee Jang; Namyi Gu; Sae Kyul Kim; Kyung-Tae Lee; Eun Kyoung Chung

    This study was conducted to develop a highly selective, sensitive, and validated method for quantifying metronidazole in human plasma and bile fluid. Metronidazole and metronidazole-d4 (internal standard) were extracted from 100 μL of plasma and bile fluid by liquid-liquid extraction. Liquid chromatography with a Hydrosphere C18 column (50 × 2.0 mm) was performed using 10 mM ammonium formate (pH 4.0) and acetonitrile (20:80, v/v) as the mobile phase. Triple quadrupole mass spectrometry was operated with an electrospray ionization interface in multiple reaction monitoring and positive ion modes. The calibration curves were linear for bile and plasma samples over the range of 50 to 20000 ng/mL (r2 > 0.999). The intra- and inter-day coefficients of variation (CVs) for plasma ranged from 2.50% to 7.85% and 3.11% to 16.9%, respectively; for bile, the intra-and inter-run precision (CVs) ranged from 2.76% to 13.2% and 3.16% to 11.5%, respectively. The mean extraction recovery for metronidazole ranged from 76.5% to 82.1% in plasma and from 78.8% to 87.8% in bile, respectively. Our proposed analytical method was successfully applied to determine metronidazole concentrations in bile as well as in plasma at multiple time points in a patient with acute cholangitis.

    更新日期:2019-12-29
  • Potential Urinary Extracellular Vesicle Protein Biomarkers of Chronic Active Antibody-mediated Rejection in Kidney Transplant Recipients
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-26
    Hee-Yeon Jung; Chan-Hyeong Lee; Ji-Young Choi; Jang-Hee Cho; Sun-Hee Park; Yong-Lim Kim; Pyong-Gon Moon; Moon-Chang Baek; Jae Berm Park; Yeong Hoon Kim; Byung Ha Chung; Sang-Ho Lee; Chan-Duck Kim

    The aim of this study was to identify potential proteomic biomarkers for chronic active antibody-mediated rejection (CAMR) in kidney transplant recipients (KTRs). Among 385 KTRs enrolled in a cross-sectional multicenter study, 26 KTRs with biopsy-proven CAMR, 57 KTRs with long-term graft survival (LGS), and 10 rejection-free matched KTRs were included. A proteomic approach was employed to measure urinary extracellular vesicle (EV) changes in the KTRs. The urinary EVs were trypsin-digested using a gel-assisted protocol and quantified by label-free liquid chromatography with tandem mass spectrometry, using a data-dependent acquisition (DDA) mode. Western blot analysis was performed to confirm the protein levels for each candidate biomarker. Analysis of the isolated EV proteins revealed 93 and 97 proteins in the CAMR and LGS patients, respectively. Proteins that were identical in both groups were excluded and only high-significance proteins with a fold change of at least 1.5 were selected as candidate biomarkers. Six proteins (APOA1, TTR, PIGR, HPX, AZGP1, and CP) that were distinguishable between CAMR and LGS were selected. The proteins were confirmed by immunoblot analyses using independently acquired urinary EV samples. AZGP1 in particular was found to be a CAMR-specific proteomic biomarker that was distinguishable from the rejection-free control group with matching kidney function, duration of transplantation, and age. We identified and validated six proteomic biomarkers for CAMR and clarified one CAMR-specific proteomic biomarker in KTRs. Further clinical trials are needed before these rejection-specific biomarkers can be applied for the early prediction, diagnosis, and monitoring of the clinical response of KTRs to the treatment of CAMR.

    更新日期:2019-12-27
  • Quantification of aromatic amines derived from azo colorants in textile by ion-pairing liquid chromatography tandem mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-23
    Ádám Tölgyesi; Virender K. Sharma

    Azo dyes can metabolize back to precursor aromatic amines (arylamines), which are potentially carcinogenic. The ISO 14362-1:2017 standard requires the determination of the arylamines, produced from textile samples, preferably by using the gas chromatography mass spectrometric (GC-MS) method. This paper presents an ion-pairing high performance liquid chromatography tandem mass spectrometric (LC-MS/MS) method for determining aromatic amines, derived from azo colorants, in both natural and synthetic textiles. The separation enables adequate apparent retention factor (k’ > 2.1 for the most hydrophilic compounds) and has appropriate sensitivity without sample clean-up procedure. The background matrix constituents influence the quantification in even a 100-fold diluted sample, therefore, calibration requires background compensation. The method allows fast confirmation and quantification of twenty-five arylamines in complex matrices of textiles. The method was validated with success and used to both natural and synthetic textile proficiency test (PT) samples.

    更新日期:2019-12-23
  • Selective isolation of sesquiterpene coumarins from asafoetida using dummy molecularly imprinted solid phase extraction method
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-23
    Shima Eidi; Mehrdad Iranshahi; Arash Mohammadinejad; Mahdieh Sadat Mohsenzadeh; Faegheh Farhadi; Seyed Ahmad Mohajeri

    Due to the biological features of sesquiterpene coumarins and incomparable interest in therapeutics application of natural products, extraction of sesquiterpene coumarins from asafoetida have gained highly attention. One of the most important problems is removal of sulfur containing compounds which are co-existed with sesquiterpene coumarins. So employment of new methods for selective extraction and cleanup of sesquiterpene coumarins is very substantial. In this study using dummy molecularly imprinting technique, 7-hydroxycoumarin-imprited polymer was synthesized and after evaluation of binding properties of polymers, the optimum one was used as sorbent in solid phase extraction. Afterwards dummy molecularly imprinting solid phase extraction (DMISPE) method was calibrated for simultaneous extraction of galbanic acid, 7-isopentenyloxy coumarin and auraptene from aqueous media before high performance liquid chromatography with UV detector (HPLC-UV) analysis. The recovery was in the range of 68.32%-84.69%, which were in the acceptable range compared to previous works. Finally, the calibrated DMISPE method was used for extraction and cleanup of sesquiterpene coumarins from asafetida plant. The concentration of isosamarcandin, kellerin and farnesiferol in asafoetida extract was obtained 0.8, 2.7, and 5 µg/mL, respectively, using standard addition method.

    更新日期:2019-12-23
  • Simultaneous determination of deuterated vortioxetine and its major metabolite in human plasma by UPLC-MS/MS and application to a pharmacokinetic study in healthy volunteers
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-23
    Yingyue Yi; Guanghui Ren; Ming Zheng; Di Zhao; Ning Li; Xijng Chen; Yang Lu

    JJH201501, a deuterated modification for multi-target antidepressant vortioxetine, is currently in phase I clinical trial. This study aimed to establish a sensitive and rapid UPLC-MS/MS method that was capable of simultaneously detecting JJH201501 and its major metabolite JJH201501-01 quantitatively in human plasma. The pretreatment was achieved by protein precipitation using 4-fold(v:v) acetonitrile with 0.05 ng/mL fluoxetine as internal standard precipitant. For method validation, the method was investigated in terms of the selectivity, inter- and intra-run precision and accuracy, matrix effect, extraction recovery and stability. The total running time was 3 min, and the retention time of JJH201501 and JJH201501-01 was 1.17 min and 1.05 min, respectively. The linear concentration range for JJH201501 and JJH201501-01 was 0.2 to 50 ng/mL and 0.4 to 100 ng/mL, respectively. The results showed that this method was in line with the guidelines for bioanalytical method proposed by FDA. In addition, the method was successfully applied to a plasma pharmacokinetic study of JJH201501 tablets in healthy volunteers which was part of the phase I trial.

    更新日期:2019-12-23
  • Rapid sample preparation for ganglioside analysis by liquid chromatography mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-23
    Spiro Khoury; Elodie Masson; Estelle Sibille; Stéphanie Cabaret; Olivier Berdeaux

    Gangliosides (GG) are glycosphingolipids, composed of a ceramide moiety (fatty acid and long chain base) linked to an oligosaccharide chain containing one (or more) molecule of sialic acid. After lipid extraction from biological matrices, quantification of GG by liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI/MS) can be impacted by ion suppression effects due to co-elution with more abundant lipids in the matrix. In this study, a simple, rapid and efficient method to purify GG from biological samples by Phree columns is proposed. This approach proved to be useful in eliminating phospholipids (PL) from the matrix and thus increasing the signal of GG classes and molecular species in rat brain samples during LC-ESI/MS analysis.

    更新日期:2019-12-23
  • HPLC-MS/MS measurement of lidocaine in rat skin and plasma. Application to study the release from medicated plaster
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-18
    C. Matteo; G. Dovrtelova; A. Di Clemente; R. Frapolli; A. Passoni; T. Ceruti; G. Marsella; L. Cervo; M. Zucchetti

    A simple, sensitive HPLC-MS/MS method was developed and validated for the determination of lidocaine in skin and plasma of rats. The methods were established and validated assessing lower limit of quantitation (LLOQ), linearity, intra and inter-day precision and accuracy, selectivity, recovery and matrix effect. Chromatography was done on a Gemini column embedded with C18 stationary phase (50 mm x 2.0 mm, 5 µm particle size), using a gradient with mobile phases consisting of 0.1% HCOOH in bidistilled water and 0.1% HCOOH in acetonitrile. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring, using target ions m/z 235.10 for lidocaine and m/z 245.10 for lidocaine-d10, used as internal standard. Results the linearity of the method was in the ranges of lidocaine concentrations 10.0-200.0 ng/mL for skin homogenate (accuracy 94.1-105.5%; R2 ≥0.998) and 0.025–2 ng/mL for plasma (accuracy 96.2-104.8%; R2 ≥0.996). The intra- and inter-day precision and accuracy determined on three quality control samples (20, 75 and 170 ng/mL for skin and 0.075, 0.4 and 1.5 ng/mL for plasma) were ≤ 4.2% and 103.8-108.2% for skin and ≤12.4% and 95.5-101.4% for plasma. The LLOQ was 10 ng/mL in skin homogenate and 0.025 ng/mL in plasma. The applicability of the method was demonstrated by measuring lidocaine in skin and plasma after exposure to medicated patches containing 5% lidocaine.

    更新日期:2019-12-19
  • Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-16
    Brooke Koshel; Robert Birdsall; Weibin Chen

    Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in one-dimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dye-conjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP – IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the two-dimensional analysis can be detected at low nanogram levels, comparable to results achieved in a one-dimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques.

    更新日期:2019-12-17
  • Liquid Chromatography-Ion Mobility Spectrometry-Mass Spectrometry Analysis of Multiple Classes of Steroid Hormone Isomers in a Mixture
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-16
    Alana L. Rister; Eric D. Dodds

    Methods for the analysis of steroids have long been of interest due to the multiple uses for such methods in medical applications, sports monitoring, and environmental science. The analysis of steroids involves inherent analytical hurdles due to their low biological concentrations, poor ionization efficiencies, and frequent occurrence of isomerism. One analytical technique that has been recently applied to steroid analysis is ion mobility spectrometry (IMS). While previous work has focused on the use of metal adduction and multimer formation to enhance separation through IMS analysis coupled to mass spectrometry (MS), this work furthers this approach by coupling IMS-MS with liquid chromatography (LC). Three different LC methods with varying tradeoffs between chromatographic resolution and run time were developed, with one of these achieving a resolution above 1.5 for all steroid isomers. These results also indicate that the coupling of LC to IMS-MS can increase the overall resolution of steroid isomers relative to what can be achieved by either LC or IMS alone. Furthermore, the use of LC and IMS in concert can allow for a more rapid analysis of steroid isomers than can be achieved by LC-MS alone. Finally, the IMS dimension provided for measurements of ion-neutral collision cross sections (CCSs), which were found to be in good agreement with previously reported measurements. Thus, this approach provides three complementary quantitative parameters (retention time, CCS, and mass-to-charge ratio) that can contribute the identification of analytes. Overall, the work presented here demonstrates the potential of coupling LC, IMS, and MS for the analysis of isomeric steroid hormones.

    更新日期:2019-12-17
  • Simultaneous determination of eight arginine-related metabolites in cellular extracts using liquid chromatography-tandem mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-15
    Sarmila Amatya; Yumi Shin; Jeong Yeop Ha; Su-Jun Lee; Sang Wook Kang; Byungsuk Kwon; Dong Hyun Kim

    A simple, sensitive, and rapid liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of arginine and its pathway-related metabolites (ornithine, proline, citrulline, glutamate, agmatine, spermidine, and spermine) in cellular extracts. Cells were lysed and cellular proteins precipitated by the addition of acetonitrile followed by ultra-sonication. Supernatants were analyzed using a Chromolith High Resolution RP-18 endcapped column (100 × 4.6 mm, 1.15 μm, 150 Å), with mobile phases of 0.1% formic acid solution and 0.1% formic acid in acetonitrile. Detection was carried out in multiple reaction monitoring (MRM) mode. Calibration curves showed linearity (r2 > 0.99) for all metabolites over the calibration ranges used. The intra- and inter-day precision was less than 13.5%, and the accuracy was between 91.3 and 114.7%. The method developed in this study was successfully applied to measure arginine and its pathway-related metabolites, which are related to nitric oxide synthase/arginase pathways in mouse bone marrow-derived dendritic cells (BMDCs). The ability to simultaneously measure arginine and its pathway-related metabolites is valuable for better understanding local and systemic inflammatory processes.

    更新日期:2019-12-17
  • Liquid chromatography-tandem mass spectrometry bioanalytical method for the determination of kavain in mice plasma: application to a pharmacokinetic study
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-13
    Juliana Veloso Ferreira; Alysson Vinícius Braga; Renes de Resende Machado; Deborah Michel; Gerson Antônio Pianetti; Anas El-Aneed; Isabela Costa César

    A simple and fast bioanalytical method for the quantification of kavain in mice plasma was developed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A full method validation was performed, according to regulatory guidelines, employing isotopically labeled kavain as the internal standard (racemic-kavain-d3). For the quantification, [M+H]+ was formed using an electrospray ionization (ESI) source in the positive ion mode and multiple reaction monitoring (MRM) was employed using a quadrupole-linear ion trap (4000 QTRAP®) instrument. The monitored MRM transitions were 231.0→115.1 and 231.0→152.8 for kavain; and 234.2→199.2 for the internal standard. A linear response was obtained at the concentration range of 10 to 200 ng/mL with intra- and inter-day variations within the acceptable criteria for all quality control samples. After validation, the method was successfully applied for the quantification of kavain in mice plasma after oral administration of the kavain standard and Kava-kava extract. The plasma concentration over time results were applied for a pharmacokinetics study. The obtained pharmacokinetic parameters indicated a considerably higher bioavailability for kavain when Kava-kava extract was administered due to a pharmacokinetic synergism between the analyte and the other compounds present in the extract.

    更新日期:2019-12-13
  • Metabolic Study of Vardenafil analogues: Pseudovardenafil and Hydroxyvardenafil
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-13
    Dayoung Heo; Jae Seon Kang; Sanggil Choe; Sooyeun Lee; Kang Min Kim; Jaesung Pyo

    Vardenafil, a remedy for erectile dysfunction, is easily modified, facilitating the creation of analogues that have been illegally added to functional foods and counterfeit medications. However, the medical profile of these analogues, including their safety, efficacy, safe drug combinations, metabolism and excretion, has not been completely evaluated, which could cause serious health problems. In this study, two representative vardenafil analogues, pseudovardenafil and hydroxyvardenafil, were metabolized with in-vitro model (human liver microsome) and in-vivo model (rats). The metabolized samples were extracted and characterized, using liquid chromatography quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS). Some imprecise interpretations were evaluated with tandem mass spectrometry (LC-Q-TOF-MS/MS) for mass fragmentation analysis. A total of 11 metabolites of pseudovardenafil and 13 metabolites of hydroxyvardenafil that were identified have never been reported. These new metabolites could be usefully applied to forensic science and other metabolic fields. Furthermore, they could serve as principal references for the toxicity, danger, and side effects of unlawful vardenafil counterfeits.

    更新日期:2019-12-13
  • Quantification of anidulafungin and micafungin in human body fluids by high performance-liquid chromatography with UV-detection
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-12
    René Welte; Herbert Oberacher; Bernhard Schwärzler; Michael Joannidis; Romuald Bellmann

    The echinocandins anidulafungin (ANID) and micafungin (MICA) are recommended for treatment of invasive Candida infections. As target-site concentrations of antimicrobial agents are crucial for eradication of pathogens, we established and validated high-performance liquid chromatography-UV detection (HPLC-UV) assays for quantification of ANID and MICA in human plasma, ascites fluid, pleural effusion, and in cerebrospinal fluid (CSF). Sample pre-purification was performed by protein precipitation with acetonitrile followed by solid phase extraction. For both assays, intra- and interday precision, and accuracy fulfilled the requirements for bioanalytical methods issued by the European Medicine Agency (EMA). The lower limit of quantification was 0.01 mg/L for both drugs. At 25 °C, ANID and MICA concentrations declined by up to 70 % within 24 h. Concentrations remained stable over 24 hours at 4 °C and over four weeks at -80°C. In conclusion, the developed methods are fit for the assessment of target-site pharmacokinetics of ANID and MICA in clinical studies.

    更新日期:2019-12-13
  • Determination of bisphenol A in canned food by microwave assisted extraction, molecularly imprinted polymer-solid phase extraction and liquid chromatography-mass spectrometry
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-12
    Niki C. Maragou; Nikolaos S. Thomaidis; Georgios A. Theodoridis; Eugenia N. Lampi; Michael A. Koupparis

    Bisphenol A (BPA), a known potential endocrine disrupting compound (EDC) is expected to be present in low quantities in canned food due to its migration from the inner surface coating of cans made of epoxy resins. A selective and confirmatory analytical method, based on microwave assisted extraction (MAE), molecularly imprinted solid phase extraction (MISPE) using a polymer prepared by a non-covalent molecular imprinting technique and liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI/MS) was developed for the determination of BPA in canned pineapple, tuna and mushrooms. First, the effect of the loading medium of hydro- organic solutions on the binding of BPA and its deuterated analogue on the MISPE sorbent was investigated. Subsequently, the effects of the experimental conditions of the microwave assisted extraction (solvent, sample mass/solvent volume, time and temperature) on the obtained recovery of BPA from canned food were assessed and the parameters were optimized to provide maximum recovery and selectivity. It was demonstrated that the combination of MAE with MISPE permits the use of a selective extraction solvent (methanol/water, 4/6, v/v), simplifying the sample preparation steps and enhancing sample clean-up of complex food matrices. The method was validated in different food matrices, using BPA-d16 as internal standard and quantitative relative recoveries were determined. The precision (RSD %) of the method ranged from 7% to 10% and the limit of detection was at low ng/g level for all food matrices. The determined concentration of BPA in commercial canned samples ranged between 7.3 – 42.3 ng/g.

    更新日期:2019-12-13
  • Comparative analysis of the human serum N-glycome in lung cancer, COPD and their comorbidity using capillary electrophoresis
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-12
    Brigitta Mészáros; Gábor Járvás; Anna Farkas; Márton Szigeti; Zsuzsanna Kovács; Renáta Kun; Miklós Szabó; Eszter Csánky; András Guttman

    Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) are prevalent ailments with a great challenge to distinguish them based on symptoms only. Since they require different treatments, it is important to find non-invasive methods capable to readily diagnose them. Moreover, COPD increases the risk of lung cancer development, leading to their comorbidity. In this pilot study the N-glycosylation profile of pooled human serum samples (90 patients each) from lung cancer, COPD and comorbidity (LC with COPD) patients were investigated in comparison to healthy individuals (control) by capillary gel electrophoresis with high sensitivity laser-induced fluorescence detection. Sample preparation was optimized for human serum samples introducing a new temperature adjusted denaturation protocol to prevent precipitation and increased endoglycosidase digestion time to assure complete removal of the N-linked carbohydrates. The reproducibility of the optimized method was <3.5%. Sixty-one N-glycan structures were identified in the pooled control human serum sample and the profile was compared to pooled lung cancer, COPD and comorbidity of COPD with lung cancer patient samples. One important findings was that no other sugar structures were detected in any of the patient groups, only quantitative differences were observed. Based on this comparative exercise, a panel of 13 N-glycan structures were identified as potential glycobiomarkers to reveal significant changes (>33% in relative peak areas) between the pathological and control samples. In addition to N-glycan profile changes, alterations in the individual N-glycan subclasses, such as total fucosylation, degree of sialylation and branching may also hold important glycobiomarker values.

    更新日期:2019-12-13
  • Vancomycin and creatinine determination in dried blood spots: analytical validation and clinical assessment
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-10
    Letícia Scribel, Alexandre P. Zavascki, Douglas Matos, Francine Silveira, Talitha Peralta, Natalia Gonçalves Landgraf, Priscila Lamb Wink, Anne Caroline Cezimbra da Silva, Nadine Bordin Andriguetti, Letícia Loss Lisboa, Marina Venzon Antunes, Rafael Linden

    This study aims to develop a liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for vancomycin and creatinine measurement in dried blood spots (DBS) and to evaluate its clinical application. The analytes were extracted from DBS and analyzed by LC-MS/MS. Vancomycin and creatinine DBS and plasma concentrations were compared in 54 and 35 samples, respectively, from 29 patients. Accuracy was 94.4-102.6%, intra-assay precision was 2.1-5.6%, and inter-assay precision was 3.5-7.0%. Patients vancomycin plasma to DBS concentration ratios were highly variable (1.148 to 5.022), differently from creatinine (0.800 to 1.283). The assay has adequate analytical performance. Plasma concentrations can be satisfactorily predicted from DBS measurements for creatinine, but not for vancomycin, which limits its clinical application.

    更新日期:2019-12-11
  • 更新日期:2019-12-11
  • Determination and Validation of Mycophenolic Acid by a UPLC-MS/MS Method: Applications to Pharmacokinetics and Tongue Tissue Distribution Studies in Rats
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-09
    Xiuqing Gao, Robert Y.L. Tsai, Jing Ma, Parnit K. Bhupal, Xiaohua Liu, Dong Liang, Huan Xie

    Mycophenolic acid (MPA) has being used clinically for organ rejection prophylaxis. Recent studies have revealed that MPA can also act as a chemo-sensitizing agent when used in combination with various chemotherapeutic agents in a cancer type-specific manner, including with oxaliplatin on oral squamous cell carcinoma (OSCC) cells. To prepare for the analysis of a novel drug delivery route for MPA absorption via oral mucosa as a potential therapeutic product, it is essential to develop and validate a highly sensitive analytical method for the quantification of MPA in biological samples for pharmacokinetic and tissue distribution studies. Herein, we report a sensitive, specific and reproducible UPLC-MS/MS method to do so. Blank rat plasma or tongue tissue homogenates coupled with griseofulvin, as internal standard, was used for generating standard curves ranging from 0.5 – 1000 ng/mL (r > 0.9990) for both plasma and tongue tissue homogenates. The chromatographic separation was achieved by a reverse phase ACE Excel 2 Super C18 column with a flow rate of 0.4 mL/min under gradient elution. Mass detection was performed under positive ionization electrospray. Inter- and intra-day accuracy and precision of the assay were ≤15% in both plasma and tongue tissue homogenates. The matrix effect was non-significant and extraction recovery rates were within 87.99% and 109.69% in plasma and tongue homogenates, respectively. The validity of this assay has been confirmed by measuring MPA in rat plasma for pharmacokinetics following intravenous administration of 0.5 mg/kg of mycophenolate sodium, as well as monitoring MPA in rat tongues for tissue distribution and detecting MPA that diffused into systemic circulation following a 4-hr transmucosal delivery of 357 μg/cm2 of mycophenolate sodium.

    更新日期:2019-12-09
  • Determination of acrylamide in gingerbread and other food samples by HILIC-MS/MS: A dilute-and-shoot method
    J. Chromatogr. B (IF 2.813) Pub Date : 2019-12-09
    Ádám Tölgyesi, Virender K. Sharma

    This paper describes a hydrophilic interaction liquid chromatography tandem mass spectrometric (HILIC-MS/MS) method for determining acrylamide in food. The method primary optimised for gingerbread samples that have high sugar contents. A new sample preparation process was optimized using an experiment design based on a central composition design (CCD). A mixture of acidified aqueous acetonitrile was established as a suitable extraction medium. The extracts were further diluted and separation of acrylamide on TSKgel Amide-80 HILIC column was carried out within 8 min. The method was validated using naturally contaminated quality check (QC) as well as spiked samples. The developed method showed acceptable accuracy (101% - 105%) and precision (2.9% - 7.6%). The limit of quantification was 20 µg/kg. The method was also tested by analysing acrylamide in other food samples (bread, roasted coffee, instant coffee, cappuccino powder and fried potato). The acrylamide concentrations found in samples were between 20 µg/kg and 667 µg/kg, which were lower than the benchmark levels set by the European Union (EU). The main advantage of the newly developed method over the standard methods included the easier sample preparation and faster analysis with reduced ion suppression.

    更新日期:2019-12-09
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