A porous graphitized carbon LC-ESI/MS method for the quantitation of metronidazole and fluconazole in breast milk and human plasma J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-22 Ariadni Geballa-Koukoula, Irene Panderi, Konstantinos Zervas, Khalil Geballa-Koukoulas, Eirini Kavvalou, Eirini Panteri-Petratou, Panagiota Vourna, Dimitra Gennimata
Information on drug transfer into the breast milk is essential to protect the infant from undesirable adverse effects of maternal consumption of drugs and to allow effective pharmacological treatment of breastfeeding mothers. Metronidazole and fluconazole are two drugs frequently used in nursing women to treat various infections, thus questioning infant's safety due to drug exposure through breast milk. In this article a porous graphitized carbon LC/ESI-MS assay was developed for the quantitation of metronidazole and fluconazole in breast milk and human plasma. The assay was based on the use of 150 μL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the LC/ESI-MS system. All analytes and the internal standard, ropinirole, were separated by using a porous graphitized carbon analytical column (150 × 2.1 mm i.d., particle size 5 μm) with isocratic elution. The mobile phase consists of 55% acetonitrile in water acidified with 0.1% concentrated formic acid and pumped at a flow rate of 0.25 mL min−1. The assay was linear over a concentration range of 0.1 to 15 μg mL−1 for all analytes in both biological samples. Intermediate precision was found to be <8.4% over the tested concentration ranges. A run time of <5 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application for the analysis of metronidazole and fluconazole in both breast milk and human plasma and it can be used to support a wide range of clinical studies.
Quantitation of peptides from non-invasive skin tapings using isotope dilution and tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-21 Nichole Reisdorph, Michael Armstrong, Roger Powell, Kevin Quinn, Kevin Legg, Donald Leung, Rick Reisdorph
Previous work from our laboratories utilized a novel skin taping method and mass spectrometry-based proteomics to discover clinical biomarkers of skin conditions; these included atopic dermatitis, Staphylococcus aureus colonization, and eczema herpeticum. While suitable for discovery purposes, semi-quantitative proteomics is generally time-consuming and expensive. Furthermore, depending on the method used, discovery-based proteomics can result in high variation and inadequate sensitivity to detect low abundant peptides. Therefore, we strove to develop a rapid, sensitive, and reproducible method to quantitate disease-related proteins from skin tapings. We utilized isotopically-labeled peptides and tandem mass spectrometry to obtain absolute quantitation values on 14 peptides from 7 proteins; these proteins had shown previous importance in skin disease. The method demonstrated good reproducibility, dynamic range, and linearity (R2 > 0.993) when n = 3 standards were analyzed across 0.05–2.5 pmol. The method was used to determine if differences exist between skin proteins in a small group of atopic versus non-atopic individuals (n = 12). While only minimal differences were found, peptides were detected in all samples and exhibited good correlation between peptides for 5 of the 7 proteins (R2 = 0.71–0.98). This method can be applied to larger cohorts to further establish the relationships of these proteins to skin disease.
The utility of ultra-high performance supercritical fluid chromatography–tandem mass spectrometry (UHPSFC-MS/MS) for clinically relevant steroid analysis J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-20 Karl-Heinz Storbeck, Lorna Gilligan, Carl Jenkinson, Elizabeth S. Baranowski, Jonathan L. Quanson, Wiebke Arlt, Angela E. Taylor
Liquid chromatography tandem mass spectrometry (LC-MS/MS) assays are considered the reference standard for serum steroid hormone analyses, while full urinary steroid profiles are only achievable by gas chromatography (GC–MS). Both LC-MS/MS and GC–MS have well documented strengths and limitations. Recently, commercial ultra-high performance supercritical fluid chromatography–tandem mass spectrometry (UHPSFC-MS/MS) systems have been developed. These systems combine the resolution of GC with the high-throughput capabilities of UHPLC. Uptake of this new technology into research and clinical labs has been slow, possibly due to the perceived increase in complexity. Here we therefore present fundamental principles of UHPSFC-MS/MS and the likely applications for this technology in the clinical research setting, while commenting on potential hurdles based on our experience to date.
Major signal suppression from metal ion clusters in SFC/ESI-MS — Cause and effects J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-19 Alfred Haglind, Mikael Hedeland, Torbjörn Arvidsson, Curt E. Pettersson
The widening application area of SFC-MS with polar analytes and water-containing samples facilitates the use of quick and simple sample preparation techniques such as “dilute and shoot” and protein precipitation. This has also introduced new polar interfering components such as alkali metal ions naturally abundant in e.g. blood plasma and urine, which have shown to be retained using screening conditions in SFC/ESI-TOF-MS and causing areas of major ion suppression. Analytes co-eluting with these clusters will have a decreased signal intensity, which might have a major effect on both quantification and identification. When investigating the composition of the alkali metal clusters using accurate mass and isotopic pattern, it could be concluded that they were previously not described in the literature. Using NaCl and KCl standards and different chromatographic conditions, varying e.g. column and modifier, the clusters proved to be formed from the alkali metal ions in combination with the alcohol modifier and make-up solvent. Their compositions were [(XOCH3)n + X]+, [(XOH)n + X]+, [(X2CO3)n + X]+ and [(XOOCOCH3)n + X]+ for X = Na+ or K+ in ESI+. In ESI−, the clusters depended more on modifier, with [(XCl)n + Cl]− and [(XOCH3)n + OCH3]− mainly formed in pure methanol and [(XOOCH)n + OOCH]− when 20 mM NH4Fa was added. To prevent the formation of the clusters by avoiding methanol as modifier might be difficult, as this is a widely used modifier providing good solubility when analyzing polar compounds in SFC. A sample preparation with e.g. LLE would remove the alkali ions, however also introducing a time consuming and discriminating step into the method. Since the alkali metal ions were retained and affected by chromatographic adjustments as e.g. mobile phase modifications, a way to avoid them could therefore be chromatographic tuning, when analyzing samples containing them.
Fabric phase sorptive extraction-high performance liquid chromatography-photo diode array detection method for simultaneous monitoring of three inflammatory bowel disease treatment drugs in whole blood, plasma and urine J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-16 Abuzar Kabir, Kenneth G. Furton, Nicola Tinari, Laurino Grossi, Denise Innosa, Daniela Macerola, Angela Tartaglia, Valentina Di Donato, Cristian D'Ovidio, Marcello Locatelli
This paper reports a novel fabric phase sorptive extraction-high performance liquid chromatography-photodiode array detection (FPSE-HPLC-PDA) method for the simultaneous extraction and analysis of three drug residues (ciprofloxacin, sulfasalazine, and cortisone) in human whole blood, plasma, and urine samples, generally administered in human patients to treat inflammatory bowel disease (IBD). The drugs of interest were well resolved using a Luna C18 column (250 mm × 4.6 mm; 5 μm particle size) in gradient elution mode within 20 min. The analytical method was optimized and validated in the range 0.05–10 μg/mL for whole blood, 0.25–10 μg/mL for human plasma, and 0.10–10 μg/mL for human urine. Blank human whole blood, plasma, and urine were used as the sample matrix for the method development and validation; while methyl-p-hydroxybenzoate was used as the internal standard (IS). Weighted-matrix matched standard calibration curves showed a good linearity up to a concentration of 10 μg/mL. The intra- and inter-day accuracy values (precision and trueness) were found in the range from −10.9% to 12.3%, and the performances of the validated FPSE-HPLC-PDA were further tested on real IBD patient samples. This is the first FPSE procedure applied simultaneously to whole blood, plasma, and urine samples for the determination of residual IBD drugs, which possess a wide range of polarity (logP values ranging from 2.30 for Ciprofloxacin, to 1.66 for Cortisone, and 2.92 for Sulfasalazine). The new approach exhibits high potential for immediate adoptation as a rapid, robust and green analytical tool for future clinical and pharmaceutical applications.
Metabolism profiling of nevadensin in vitro and in vivo by UHPLC-Q-TOF-MS/MS J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-16 Caijuan Liang, Xia Zhang, Xinpeng Diao, Man Liao, Yupeng Sun, Lantong Zhang
Nevadensin is major constituents of Lysionotus pauciflorus Maxim. (Chinese name: Shidiaolan), which has a variety of pharmacological effects such as anti-mycobacterium tuberculosis activities, antitussive, anti-inflammatory and anti-hypertensive. In this paper, we investigated the metabolism of nevadensin in vitro and in vivo. A strategy was firstly developed to identify the metabolites of nevadensin by using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). An on-line data acquisition method a multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Furthermore, some assistant tools, such as key fragment ions (KFI), were employed for compound hunting and identification. Based on the proposed method, 23 metabolites were structurally characterized in vivo including 16 phase I and 7 phase II metabolites, and 12 metabolites were detected in vitro containing 10 phase I and 2 phase II metabolites. The results indicated that oxidation, hydrolysis, demethylation, methylation, sulfate conjugation and glucuronide conjugation were main metabolic pathways of nevadensin. In a word, this study maybe can provide reference and valuable evidence for further investigation of the metabolic mechanism of nevadensin.
Corrigendum to “Quantitative determination of cyclic phosphatidic acid and its carba analog in mouse organs and plasma using LC–MS/MS” [J. Chromatogr. B 1076 (2018) 15–21] J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-16 Yoshibumi Shimizu, Masaki Ishikawa, Mari Gotoh, Keiko Fukasawa, Shinji Yamamoto, Kensuke Iwasa, Keisuke Yoshikawa, Kimiko Murakami-Murofushi
A new magnetic nanodiamond/graphene oxide hybrid (Fe3O4@ND@GO) material for pre-concentration and sensitive determination of sildenafil in alleged herbal aphrodisiacs by HPLC-DAD system J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-15 Erkan Yilmaz, Halil İbrahim Ulusoy, Özge Demir, Mustafa Soylak
A sensitive analytical methodology was investigated to concentrate and determine of sildenafil citrate (SLC) present at trace level in herbal supplementary products. The proposed method is based on simple and sensitive pre-concentration of SLC by using magnetic solid phase extraction with new developed magnetic nanodiamond/graphene oxide hybrid (Fe3O4@ND@GO) material as a sorbent. Experimental variables affecting the extraction efficiency of SLC like; pH, sample volume, eluent type and volume, extraction time and amount of adsorbent were studied and optimized in detail. Determination of sildenafil citrate after magnetic solid phase extraction (MSPE) was carried out by HPLC-DAD system. The morphology, composition, and properties of the synthesized hybrid material was characterized by Fourier transform infrared spectrometry (FT-IR), Raman spectrometry (Raman), X-ray diffraction spectrometry (XRD), scanning electron microscopy (SEM), mapping photographs and BET surface area analysis. Under optimized conditions, linear range was ranged from 5.00 to 250.00 ng mL−1 with R2 of 0.9952. The limit of detection (LOD) was 1.49 ng mL−1 and the recoveries at two spiked levels were ranged from 94.0 to 104.1% with the relative standard deviation (RSD) < 7.1% (n = 5). The enhancement factor (EF) was 86.9. The results show that the combination MSPE with HPLC-DAD is a suitable and sensitive method for the determination of SLC in real samples.
Optimization of a two-dimensional liquid chromatography-supercritical fluid chromatography-mass spectrometry (2D-LC-SFS-MS) system to assess “in-vivo” inter-conversion of chiral drug molecules J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-15 Meenakshi Goel, Eli Larson, C.J. Venkatramani, Mohammad Al-Sayah
Enantioselective analysis is an essential requirement during the pharmaceutical development of chiral drug molecules. In pre-clinical and clinical studies, the Food and Drug Administration (FDA) mandates the assessment of “in vivo” inter-conversion of chiral drugs to determine their physiological effects. In-vivo analysis of the active pharmaceutical ingredient (API) and its potential metabolites could be quite challenging due to their low abundance (ng/mL levels) and matrix interferences. Therefore, highly selective and sensitive analytical techniques are required to separate the API and its metabolites from the matrix components and one another. Additionally, for chiral APIs, further analytical separation is required to resolve the API and its potential metabolites from their corresponding enantiomers. In this work, we demonstrate the optimization of our previously designed two-dimensional liquid chromatography-supercritical fluid chromatography-mass spectrometry (2D-LC-SFC -MS) system to achieve 10 ng/mL detection limit . The first LC dimension, used as a desalting step, could efficiently separate the API from its potential metabolites and matrix components. The API and its metabolites were then trapped/focused on small trapping columns and transferred onto the second SFC dimension for chiral separation. Detection can be achieved by ultra-violet (UV) or MS detection. Different system parameters such as column dimensions, transfer volumes, trapping column stationary phase, system tubing internal diameter (i.d.), and detection techniques, were optimized to enhance the sensitivity of the 2D-LC-SFC-MS system. The limit of detection was determined to be 10 ng/ml. An application is described where a mouse hepatocyte treated sample was analyzed using the optimized 2D-LC-SFC-MS system with successful assessment of the ratio of API to its metabolite (1D-LC), as well as the corresponding enantiomeric excess values (% e.e.) of each (2D-SFC).
Multiple analyte adduct formation in liquid chromatography-tandem mass spectrometry - Advantages and limitations in the analysis of biologically-related samples J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-15 Marek Dziadosz
Multiple analyte adduct formation was examined and discussed in the context of reproducible signal detection in liquid chromatography-tandem mass spectrometry applied in the analysis of biologically-related samples. Appropriate infusion solutions were prepared in H2O/methanol (3/97, v/v) with 1 mM sodium acetate and 10 mM acetic acid. An API 4000 QTrap tandem mass spectrometer was used for experiments performed in the negative scan mode (−Q1 MS) and the negative enhanced product ion mode (-EPI). γ‑Hydroxybutyrate and its deuterated form were used as model compounds to highlight both the complexity of adduct formation in popular mobile phases used and the effective signal compensation by the application of isotope-labelled analytes as internal standards.
Simultaneous determination of gemcitabine prodrug, gemcitabine and its major metabolite 2′, 2′-difluorodeoxyuridine in rat plasma by UFLC-MS/MS J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-14 Yilin Sun, Le Zhen, Ying Peng, Jiankun Wang, Fei Fei, A. Lixiang, Wenjiao Jiang, Xue Pei, Li Lu, Jie Liu, Guangji Wang, Kun Hao
To improve bioavailability and provide resistance to deamination, an array of gemcitabine (dFdC) prodrugs carrying the acyl modifications has been successful in the optimization of pharmacokinetic properties of dFdC, but the reports about 4-N-carbobenzoxy-dFdC (Cbz-dFdC), a dFdC prodrug bearing alkyloxycarbonyl modification, are relatively rare. Notably, in vivo enzymatic hydrolysis was an absolutely essential factor for the activation of these prodrugs, which is correlated with the anti-tumor activity. Therefore, detailed metabolism studies of Cbz-dFdC should be carried out for a more authentic pharmacodynamic evaluation. In order to detect the pharmacokinetic characteristics of Cbz-dFdC, a selective, sensitive and accurate method for the simultaneous determination of Cbz-dFdC, along with dFdC and its major metabolite dFdU in rat plasma was developed and validated using UFLC–MS/MS techniques. Column was at 40 °C for separation using an eluent with acetonitrile and 0.1% formic acid, 1 mM ammonium formate at a flow rate of 0.2 mL/min. Detection was performed using ESI source in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 398.1 → 202.2 (Cbz-dFdC), m/z 264.1 → 112.0 (dFdC), m/z 265.3 → 113.2 (dFdU) and m/z 246.1 → 112.0 (IS). Analytes were extracted by simple precipitation with acetonitrile containing internal standards followed by liquid-liquid extraction with ethyl acetate. The calibration curves of Cbz-dFdC, dFdC and dFdU were linear in the concentration range of 2 to 500 ng/mL, 2 to 500 ng/mL and 40 to 10,000 ng/mL, respectively. The assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. In conclusion, the sensitive analytical assay was selective and accurate for the determination of rat plasma concentrations of Cbz-dFdC, dFdC and dFdU from a single LC–MS/MS analysis and well-suited to support pharmacokinetic studies.
How much separation for LC–MS/MS quantitative bioanalysis of drugs and metabolites? ☆ J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-14 Aimin Tan, John C. Fanaras
LC–MS/MS has been the dominant analytical technology for quantitative bioanalysis of drugs and metabolites for more than two decades. Despite this, a very fundamental question like how much separation is required for LC–MS/MS quantitative bioanalysis of drugs and metabolites has not been adequately addressed. Some think that no or only very limited separation is necessary thanks to the unparalleled selectivity offered by tandem mass spectrometry. Others think that the more separation, the better, because of the potential detrimental impact of matrix effect (ion suppression or enhancement). Still others just use a rule-of-thumb approach by keeping the adjusted retention/capacity factor always between 2 and 5. The purpose of this article is to address this fundamental question through rational thinking together with various real case examples drawn from regulated bioanalytical laboratories.
Analysis of 10 β-agonists in pork meat using automated dispersive pipette extraction and LC-MS/MS J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-14 Kaylee R. Mastrianni, Kalmorat Metavarayuth, William E. Brewer, Qian Wang
Sensitive analysis and pharmacokinetic study of a novel gemcitabine carbamate prodrug and its active metabolite gemcitabine in rats using LC-ESI-MS/MS J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-14 Yi Zhou, Qingqing Chang, Wenjie Wang, Xiaofang Zhang, Fang Zhou, Jianguo Sun, Guangji Wang, Ying Peng
FY363 is a new chemical entity of gemcitabine analog, which has been shown to have a significant inhibitory effect on cell proliferation in a variety of tumor cell lines in vitro. As a carbamate derivative, FY363 would be converted to the active metabolite gemcitabine through enzyme action in vivo. In order to clarify the exposure of FY363 prototype and its metabolite gemcitabine in vivo after administration of FY363, a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and validated to simultaneously determine FY363 and gemcitabine in rat plasma after liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a highly stable polar column of Synergi 4u Polar-RP 80A (4 μm, 4.6 × 250 mm) which has a unique ether - phenyl bonded phase. Gradient elution was accomplished with mobile phase system consisting of 5 mM ammonium formate buffer containing 0.1% formic acid and mixed organic solvents containing methanol-acetonitrile (3:2, v/v). Multiple reaction monitoring transitions were performed on triple quadrupole mass spectrometric detection in positive-ion mode with an electrospray ionization source. The calibration curves showed good linearity (r > 0.99) over the established concentration range of 1.0–1000 ng/mL both for FY363 and gemcitabine. The assay was validated to be selective, robust and reproducible. This well validated method was successfully applied to demonstrate the pharmacokinetic behavior and the metabolic transformation of FY363 in rats. Results revealed that about 20% of FY363 were converted into its active metabolite gemcitabine in rats by comparing the exposure of gemcitabine after the FY363 administration with that after direct gemcitabine administration at equimolar dose.
Development, validation and utilization of a highly sensitive LC-MS/MS method for quantification of levonorgestrel released from a subdermal implant in human plasma J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-12 Lauren R. Cirrincione, Sujan Dilly Penchala, Kimberly K. Scarsi, Anthony T. Podany, Lee C. Winchester, David J. Back, Saye H. Khoo, Courtney V. Fletcher, Marco Siccardi, Laura J. Else
Levonorgestrel (LNG) is a synthetic progestin that is available in oral contraceptive tablets, a subdermal implant, and an intrauterine system for contraception. LNG pharmacokinetics are a pivotal determinant of contraceptive efficacy and essential in assessing drug-drug interactions influencing LNG exposure following different routes of LNG administration. A highly sensitive LC-MS/MS method was developed and validated to quantify levonorgestrel in human plasma. Liquid-liquid extraction was utilized with a sample volume of 500 μL to extract levonorgestrel from plasma. Chromatographic separation of LNG was achieved with a Fortis™ C18 (3 μm: 100 mm × 2.1 mm) reverse phase analytical column. The mobile phases consisted of de-ionized water plus 0.1% NH4OH (100:0.1%, v/v) (A), and methanol plus 0.1% NH4OH (100:0.1%, v/v) (B) delivered as a gradient at a flow rate of 400 μL/min. Detection of LNG and internal standard (D-(−)-norgestrel-d7) was achieved using positive polarity mode monitoring at 313.2–245.2 amu and 320.1–251.2 amu, respectively. The assay was linear over the calibration range of 49.6 to 1500 pg/mL. This method was used to quantify plasma LNG released by subdermal implant in support of a drug interaction study among women with HIV receiving efavirenz- or nevirapine-based antiretroviral therapy.
Complementation of UPLC-Q-TOF-MS and CESI-Q-TOF-MS on identification and determination of peptides from bovine lactoferrin J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-12 Hui Chen, Pujie Shi, Fengjiao Fan, Maolin Tu, Zhe Xu, Xianbing Xu, Ming Du
A novel, simplified and stability-indicating high-throughput ultra-fast liquid chromatography method for the determination of rosmarinic acid in nanoemulsions, porcine skin and nasal mucosa J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-12 Flávia N.S. Fachel, Marina C. Nemitz, Bruna Medeiros-Neves, Kleyton S. Veras, Valquíria L. Bassani, Letícia S. Koester, Amelia T. Henriques, Helder F. Teixeira
Currently, there is an increasing interest on the development of topical formulations containing rosmarinic acid (RA) due to its well-documented antioxidant activity. This study aimed to develop and validate a stability-indicating ultra-fast liquid chromatography (UFLC) method for the determination of RA in nanoemulsions, porcine skin and nasal mucosa intended to be applied in permeation/retention studies and for development of topical nanoemulsions. Chromatographic separation was carried out using a C18 column packed with 2.6 μm particle size in isocratic conditions using as mobile phase water:acetonitrile (83:17, v/v), acidified with 0.1% trifluoracetic acid (v/v), with a total time of analysis of 3.5 min and detection at 330 nm. RA analysis was specific in the presence of both non-biological (blank nanoemulsion and receptor fluid) and biological matrices (porcine ear skin and porcine nasal mucosa). No interference of degradation products of RA was verified after different stress conditions such as acidic, alkaline, oxidative, light exposure (UV-A and UV-C) and thermal demonstrating the method stability-indicating property. The analytical (0.1–10.0 μg·mL−1) and bioanalytical (0.5–10.0 μg·mL−1) linearity was proved by analysis of the calibration curves of RA and no matrix effect was observed. The method was sensitive, precise and accurate, and showed recovery higher than 85%. The method was considered robust as evaluated by a Plackett-Burman experimental design. In the validated conditions, the RA was determined in the nanoemulsions obtained by spontaneous emulsification procedure (1.007 ± 0.040 mg·mL−1), porcine ear skin (1.13 ± 0.19 μg·cm2 −1) and nasal mucosa (22.46 ± 3.99 μg·cm2 −1) after retention/permeation studies. Thus, a highly sensitive, simple, fast and stability-indicating method was developed for RA analysis during the development of topical nanoemulsions and bioanalytical assays in complex matrices.
Simultaneous determination of antiretroviral drugs in human hair with liquid chromatography-electrospray ionization-tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-11 Yan Wu, Jin Yang, Cailing Duan, Liuxi Chu, Shenghuo Chen, Shan Qiao, Xiaoming Li, Huihua Deng
The determination of the concentrations of antiretroviral drugs in hair is believed to be an important means for the assessment of the long-term adherence to highly active antiretroviral therapy. At present, the combination of tenofovir, lamivudine and nevirapine is widely used in China. However, there was no research reporting simultaneous determination of the three drugs in hair. The present study aimed to develop a sensitive method for simultaneous determination of the three drugs in 2-mg and 10-mg natural hair (Method 1 and Method 2). Hair samples were incubated in methanol at 37 °C for 16 h after being rinsed with methanol twice. The analysis was performed on high performance liquid chromatography tandem mass spectrometry with electronic spray ionization in positive mode and multiple reactions monitoring. Method 1 and Method 2 showed the limits of detection at 160 and 30 pg/mg for tenofovir, at 5 and 6 pg/mg for lamivudine and at 15 and 3 pg/mg for nevirapine. The two methods showed good linearity with the square of correlation coefficient >0.99 at the ranges of 416–5000 and 77–5000 pg/mg for tenofovir, 12–5000 and 15–5000 pg/mg for lamivudine and 39–50,000 and 6–50,000 pg/mg for nevirapine. They gave intra-day and inter-day coefficient of variation <15% and the recoveries ranging from 80.6 to 122.3% and from 83.1 to 114.4%. Method 2 showed LOD and LOQ better than Method 1 for tenofovir and nevirapine and matched Method 1 for lamivudine, but there was high consistency between them in the determination of the three drugs in hair. The population analysis with Method 2 revealed that the concentrations in hair were decreased with the distance of hair segment away from the scalp for the three antiretroviral drugs.
Application of liquid chromatography–tandem mass spectrometry to study the effect of docetaxel on pharmacokinetics and tissue distribution of apatinib in mice J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-10 Siqi Feng, Jingwei Zhang, Ying Wang, Runbin Sun, Dong Feng, Ying Peng, Na Yang, Yue Zhang, Haoxue Gao, Huilin Gu, Guangji Wang, Jiye Aa, Fang Zhou
Apatinib, a highly selective small-molecule inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2), has attracted many attentions due to its anticancer activity in various malignancies containing non-small-cell lung cancer (NSCLC). Our previous preclinical study confirmed the enhanced anti-tumor efficacy of combined treatment between apatinib and docetaxel for NSCLC. However, the effects of docetaxel on pharmacokinetics and tissue distribution of apatinib are not clear. In present study, a reliable HPLC-MS/MS method was established for determination of apatinib. This method had a good linearity in the range of 1–5000 ng/mL, and the recovery and matrix effect were 100.1–103.5%, 77.6–83.5%, respectively. Plasma exposure level of apatinib and the values of Cmax, AUC0–12h, T1/2, and MRT were not affected by multi-dose of docetaxel. The tissue distributions (kidney, heart, lung, spleen) of apatinib in combined treatment group were lower at 0.25 h but higher at 2 h, and that in intestine and liver were not significantly changed compared with control group. However, pre-treatment with docetaxel had no significant effect on AUC0–4h of apatinib in tissues in mice. In conclusion, plasma and tissues exposure levels of apatinib were not affected by long-termed treatment with docetaxel, indicating that docetaxel is less likely to increase the side effect of apatinib such as hypertension, hand-foot syndrome and so on.
Metabolism of the synthetic cannabinoids AMB-CHMICA and 5C-AKB48 in pooled human hepatocytes and rat hepatocytes analyzed by UHPLC-(IMS)-HR-MSE J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-10 Marie Mardal, Petur Weihe Dalsgaard, Bing Qi, Christian Brinch Mollerup, Pieter Annaert, Kristian Linnet
Evaluation of in-situ fatty acid extraction protocols for the analysis of staphylococcal cell membrane associated fatty acids by gas chromatography J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-10 Marcus J. Crompton, R. Hugh Dunstan
The composition and integrity of the bacterial cytoplasmic membrane is critical to the survival of staphylococci in dynamic environments and it is important to investigate how the cell membrane responds to changes in the environmental conditions. The staphylococcal membrane differs from eukaryotic and many other bacterial cell membranes by having a high abundance of branch fatty acids and relatively few unsaturated fatty acids. The range of available methods for extraction and efficient analyses of staphylococcal fatty acids was initially appraised to identify the best potential procedures for appraisal. Staphylococcus aureus subsp. aureus Rosenbach (ATCC® 29213) was grown under optimal conditions to generate a cell biomass to compare the efficiencies of three approaches to extract and prepare methyl esters of the membrane fatty acids: (1) acidic direct transesterification of lipids, (2) modified basic direct transesterification of membrane lipids with adjusted reaction times and temperatures, and (3) base catalysed hydrolysis followed by acid catalysed esterification in two separate chemical reactions (MIDI process). All methods were able to extract fatty acids from the cell mass effectively where these lipids represented approximately 5% of the cellular dry mass. The acidic transesterification method had the least number of steps, the lowest coefficient of variation at 6.7% and good resistance to tolerating water. Basic transesterification was the least accurate method showing the highest coefficient of variation (26%). The MIDI method showed good recoveries, but had twice the number of steps and a coefficient of variation of 16%. It was also found that there was no need to use an anti-oxidant such as BHT for the protection of polyunsaturated fatty acids when the GC–MS injection liner was clean. It was concluded that the acidic transesterification procedures formed the most efficient and reproducible method for the analyses of staphylococcal membrane fatty acids.
Simultaneous determination of eight bioactive compounds by LC-MS/MS and its application to the pharmacokinetics, liver first-pass effect, liver and brain distribution of orally administrated Gouteng-Baitouweng (GB) in rats J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-09 Xiaoting Tian, Zhou Xu, Mingcang Chen, Pei Hu, Fang Liu, Zhaolin Sun, Huan Liu, Xiaozheng Guo, Zhixiong Li, Chenggang Huang
Bioanalytical assay for the quantification of the ALK inhibitor lorlatinib in mouse plasma using liquid chromatography-tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-09 Claudia Spatari, Wenlong Li, Alfred H. Schinkel, Gaetano Ragno, Jan H.M. Schellens, Jos H. Beijnen, Rolf W. Sparidans
Quantification of methionine and selenomethionine in biological samples using multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS) J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-09 Dai Long Vu, Karolina Ranglová, Jan Hájek, Pavel Hrouzek
Quantification of selenated amino-acids currently relies on methods employing inductively coupled plasma mass spectrometry (ICP-MS). Although very accurate, these methods do not allow the simultaneous determination of standard amino-acids, hampering the comparison of the content of selenated versus non-selenated species such as methionine (Met) and selenomethionine (SeMet). This paper reports two approaches for the simultaneous quantification of Met and SeMet. In the first approach, standard enzymatic hydrolysis employing Protease XIV was applied for the preparation of samples. The second approach utilized methanesulfonic acid (MA) for the hydrolysis of samples, either in a reflux system or in a microwave oven, followed by derivatization with diethyl ethoxymethylenemalonate. The prepared samples were then analyzed by multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS). Both approaches provided platforms for the accurate determination of selenium/sulfur substitution rate in Met. Moreover the second approach also provided accurate simultaneous quantification of Met and SeMet with a low limit of detection, low limit of quantification and wide linearity range, comparable to the commonly used gas chromatography mass spectrometry (GC–MS) method or ICP-MS. The novel method was validated using certified reference material in conjunction with the GC-MS reference method.
Fragmentation patterns involving ammonium adduct fragment ions: A comparison of the determination of metaldehyde in human blood by HPLC-QqQ-MS/MS and UHPLC-Q-TOF-MS J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-08 Paweł Szpot, Grzegorz Buszewicz, Tomasz Jurek, Grzegorz Teresiński
This paper presents a rapid, sensitive and precise method for the determination of metaldehyde in human blood, using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry. Separation was performed with a Poroshell 120 EC-C18 column; 2.7 μm atrazine‑d5 (IS) and 200 mg NaCl were added to the blood sample. Proteins in human blood were precipitated using acetonitrile; the supernatant was then analyzed with the UHPLC-Q-TOF-MS or HPLC-QqQ-MS/MS system. The results of selectivity, linearity, accuracy, precision, limits of quantification, recovery, and matrix effects were sufficient to enable the measurement of metaldehyde in human blood samples. In addition, we proposed a fragmentation pathway involving ammonium adduct fragment ions for metaldehyde.
Fast quantification of short chain fatty acids and ketone bodies by liquid chromatography-tandem mass spectrometry after facile derivatization coupled with liquid-liquid extraction J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-08 Mingfei Zeng, Huachuan Cao
Short chain fatty acids (SCFA) and ketone bodies recently emerged as important physiological relevant metabolites because of their association with microbiota, immunology, obesity and other metabolic states. They were commonly analyzed by GC–MS with long run time and laborious sample preparation. In this study we developed a novel LC-MS/MS method using fast derivatization coupled with liquid-liquid extraction to detect SCFA and ketone bodies in plasma and feces. Several different derivatization reagents were evaluated to compare the efficiency, the sensitivity and chromatographic separation of structural isomers. O‑benzylhydroxylamine was selected for its superior overall performance in reaction time and isomeric separation that allowed the measurement of each SCFAs and ketone bodies free from interferences. The derivatization procedure is facile and reproducible in aqueous-organic medium, which abolished the evaporation procedure hampering the analysis of volatile short chain acids. Enhancement in sensitivity remarkably improved the detection limit of SCFA and ketone bodies to sub-fmol level. This novel method was applied to quantify these metabolites in fecal and plasma samples from lean and DIO mouse.
Use of RSM for the multivariate, simultaneous multiobjective optimization of the operating conditions of aliphatic carboxylic acids ion-exclusion chromatography column: Quantitative study of hydrodynamic, isotherm, and thermodynamic behavior J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-08 Tahereh Shojaeimehr, Farshad Rahimpour, Michael Schwarze, Jens-Uwe Repke, Hamid Reza Godini, Günter Wozny
The present study evaluates the capability of ion exclusion chromatography (IEC) of short chain aliphatic carboxylic acids using a cation exchange column (8% sulfonated cross-linked styrene-divinylbenzene copolymer) in different experimental conditions. Since one of the prerequisites to the development of an efficient carboxylic acid separation process is to obtain the optimum operational conditions, response surface methodology (RSM) was used to develop an approach to evaluate carboxylic acids separation process in IEC columns. The effect of the operating conditions such as column temperature, sulfuric acid concentration as the mobile phase, and the flow rate was studied using Central Composite Face (CCF) design. The optimum operating conditions for the separate injection of lactic acid and acetic acid is temperature of 75 °C, sulfuric acid concentration of 0.003 N for both acids and flow rate of 0.916 (0.886) mL/min for acetic acid (lactic acid). Likewise, the optimum conditions for the simultaneous injection of acetic and lactic acid mixture are the column temperature of 68 °C, sulfuric acid concentration of 0.0003 N, and flow rate of 0.777 mL/min. In the next step, the adsorption equilibria of acetic acid and lactic acid on the stationary phase were investigated through a series of Frontal Analysis (FA), Frontal Analysis by Characteristic Points (FACP), and using Langmuir isotherm model. The results showed an excellent agreement between the model and experimental data. Finally, the results of thermodynamic studies proved that the IEC process for separation of acetic and lactic acid is a spontaneous, feasible, exothermic, and random process with a physical adsorption mechanism. The results of the current paper can be a valuable information in the stages of designing IEC columns for separation of aliphatic carboxylic acids.
Development and application of a UPLC–MS/MS method for P-glycoprotein quantification in human tumor cells J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-08 Zhaohui Qiu, Jie Peng, Lingli Mou, Xiao Li, Fanqi Meng, Peng Yu
What are the current solutions for interfacing supercritical fluid chromatography and mass spectrometry? J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-07 Davy Guillarme, Vincent Desfontaine, Sabine Heinisch, Jean-Luc Veuthey
Mass spectrometry (MS) is considered today as one of the most popular detection methods, due to its high selectivity and sensitivity. In particular, this detector has become the gold standard for the analysis of complex mixtures such as biological samples. The first successful SFC-MS hyphenation was reported in the 80’s, and since then, several ionization sources, mass analyzers and interfacing technologies have been combined. Due to the specific physicochemical properties and compressibility of the SFC mobile phase, directing the column effluent into the ionization source is more challenging than in LC. Therefore, some specific interfaces have to be employed in SFC-MS, to i) avoid (or at least limit) analytes precipitation due to CO2 decompression, when the SFC mobile phase is not anymore under backpressure control, ii) achieve adequate ionization yield, even with a low proportion of MeOH in the mobile phase and iii) preserve the chromatographic integrity (i.e. maintaining retention, selectivity, and efficiency). The goal of this review is to describe the various SFC-MS interfaces and highlight the most favorable ones in terms of reliability, flexibility, sensitivity and user-friendliness.
Simultaneous determination of 21 trace perfluoroalkyl substances in fish by isotope dilution ultrahigh performance liquid chromatography tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-07 Yan Gao, Xiaomin Li, Xiuqin Li, Qinghe Zhang, Hongmei Li
Perfluoroalkyl substances (PFASs) have been identified as emerging environmental contaminants. In this study, an efficient and accurate ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous determination of 21 PFASs in fish. Acetonitrile was used for sample extraction. Solid phase extraction (SPE) by WAX cartridges and then freezing at −30 °C were adopted as cleanup strategies. Strict measurements were performed to control background contamination during the whole procedure. Matrix effects were evaluated by external standard and isotope dilution mass spectrometry (IDMS) methods. IDMS can compensate the matrix effects to a large extent. The method detection limits (MDLs) ranged from 2 pg/g to 10 pg/g except for PFBA (120 pg/g). The method quantitation limits (MQLs) ranged from 5 pg/g to 30 pg/g except for PFBA (300 pg/g). The matrix spiked recoveries of three spiked levels were in the range of 79.6%–109%. The intra-day relative standard deviation (RSD) and inter-day RSD were from 0.94% to 13.9% and 0.36% to 11.2% respectively. Two fish tissue reference materials were analyzed by the developed method. The results of reference materials were within the uncertainty of the given value. The quantitative results of IDMS and standard addition (SA) - IDMS were comparable. The developed UHPLC-MS/MS method was applied for PFASs detection in 20 marine fish samples. 9 PFASs were detected in the samples with the ∑9PFASs concentration range of 0.04 to 2.14 ng/g wet weight (ww).
Screening and isolation of potential neuraminidase inhibitors from leaves of Ligustrum lucidum Ait. based on ultrafiltration, LC/MS, and online extraction-separation methods J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-06 Yuchi Zhang, Yan He, Chengyu Liu, Chunming Liu, Sainan Li
Ultrafiltration liquid chromatography-mass spectrometry (ultrafiltration LC/MS) is introduced as an efficient method that can be applied to rapidly screen and identify ligands from the leaves of Ligustrum lucidum Ait. Using this method, we identified 13 compounds, including organic acids, flavonoids, and glycosides, as potent neuraminidase inhibitors. A continuous online method, employing pressurized liquid extraction followed by parallel centrifugal partition chromatography and preparative liquid chromatography PLE-(parallel-CPC/PLC), was developed for the efficient, scaled-up production of 12 compounds with high purities. The bioactivities of the separated compounds were assessed by an in vitro enzyme inhibition assay. The use of ultrafiltration LC/MS combined with PLE-(parallel-CPC/PLC), and an in vitro enzyme inhibition assay facilitated the efficient screening and isolation of neuraminidase inhibitors from complex samples, and could serve as an important platform for the large-scale production of functional ingredients.
Fluoroalcohol – Induced coacervates for selective enrichment and extraction of hydrophobic proteins J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-03 Amir Koolivand, Stephan Clayton, Halie Rion, Armin Oloumi, Ariel O'Brien, Morteza G. Khaledi
As previously reported, fluoroalcohols can induce coacervation in aqueous solutions of amphiphilic compounds with subsequent formation of two-phase systems, where one phase is enriched in amphiphile and fluoroalcohol and the other is primarily an aqueous – rich phase. This study focuses on the use of simple coacervates made of a single component amphiphile induced by a fluoroalcohol for extraction and enrichment of proteins. 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) and 2,2,2-trifluoroethanol (TFE) were used to induce coacervation in the aqueous solutions of a cationic amphiphile, cetyltrimethylammonium bromide (CTAB) or tetra-n-butylammonium bromide (TBAB). Cationic amphiphiles (CTAB, TBAB) formed two-phase coacervate systems in a basic pH and/or sufficient ionic strength depending on the strength of coacervator (HFIP or TFE). The phase diagrams for TBAB paired with HFIP or TFE coacervates were created. By increasing the concentration of coacervator (HFIP or TFE) at a constant surfactant concentration, transition from a single liquid phase to a two or multiple phase mixture, and then eventually to a single liquid phase was observed. TBAB/HFIP mixture without additives showed a unique three-phase system before transitioning to a two-phase system upon increasing HFIP concentration. However, salt addition eliminated this three-phase region and expanded the region of two-phase formation. Select two-phase systems composed of TBAB and a perfluoroalcohol (HFIP or TFE) were utilized to extract model proteins of ranging hydrophobicity. All coacervate phases extracted bacteriorhodopsin, a membrane protein, and gramicidin, a very hydrophobic polypeptide ion channel. The most hydrophilic protein in the mixture, ribonuclease A, remained in aqueous phases. The coacervates formed from TBAB/TFE/200 mM NaCl mixture and TBAB/HFIP mixture exhibited the most selectivity in extracting proteins of high hydrophobicity. The partition coefficient (P) for each protein was calculated using the ratio of the protein concentration in the coacervate to that in the aqueous-rich phases. TBAB (50 mM)/HFIP (8%, v/v) coacervate showed remarkable selectivity and a high partition coefficient (>100) for both bacteriorhodopsin and gramicidin. Thus, this system may potentially be beneficial for facile fractionation of hydrophobic and membrane proteins in proteomics applications.
Profiling and analysis of multiple constituents in Baizhu Shaoyao San before and after processing by stir-frying using UHPLC/Q-TOF-MS/MS coupled with multivariate statistical analysis J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-03 Yangyang Xu, Hao Cai, Gang Cao, Yu Duan, Ke Pei, Sicong Tu, Jia Zhou, Li Xie, Dongdong Sun, Jiayu Zhao, Jing Liu, Xiaoqi Wang, Lin Shen
Baizhu Shaoyao San (BSS) is a famous traditional Chinese medicinal formula widely used for the treatment of painful diarrhea, intestinal inflammation, and diarrhea-predominant irritable bowel syndrome. According to clinical medication, three medicinal herbs (Atractylodis Macrocephalae Rhizoma, Paeoniae Radix Alba, and Citri Reticulatae Pericarpium) included in BSS must be processed using some specific methods of stir-frying. On the basis of the classical theories of traditional Chinese medicine, the therapeutic effects of BSS would be significantly enhanced after processing. Generally, the changes of curative effects mainly result from the variations of inside chemical basis caused by the processing procedure. To find out the corresponding changes of chemical compositions in BSS after processing and to elucidate the material basis of the changed curative effects, an optimized ultra-high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry in positive and negative ion modes coupled with multivariate statistical analyses were developed. As a result, a total of 186 compounds were ultimately identified in crude and processed BSS, in which 62 marker compounds with significant differences between crude and processed BSS were found by principal component analysis and t-test. Compared with crude BSS, the contents of 23 compounds were remarkably decreased and the contents of 39 compounds showed notable increase in processed BSS. The transformation mechanisms of some changed compounds were appropriately inferred from the results. Furthermore, compounds with extremely significant differences might strengthen the effects of the whole herbal formula.
Factors affecting the separation performance of proteins in capillary electrophoresis J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-03 Yueping Zhu, Zhenqing Li, Ping Wang, Lisong Shen, Dawei Zhang, Yoshinori Yamaguchi
Capillary electrophoresis (CE) is an effective tool for protein separation and analysis. Compared with capillary gel electrophoresis (CGE), non-gel sieving capillary electrophoresis (NGSCE) processes the superiority on operation, repeatability and automaticity. Herein, we investigated the effect of polymer molecular weight and concentration, electric field strength, and the effective length of the capillary on the separation performance of proteins, and find that (1) polymer with high molecular weight and concentration favors the separation of proteins, although concentrated polymer hinders its injection into the channel of the capillary due to its high viscosity. (2) The resolution between the adjacent proteins decreases with the increase of electric field strength. (3) When the effective length of the capillary is long, the separation performance improves at the cost of separation time. (4) 1.4% (w/v) hydroxyethyl cellulose (HEC), 100 V/cm voltage and 12 cm effective length offers the best separation for the proteins with molecular weight from 14,400 Da to 97,400 Da. Finally, we employed the optimal electrophoretic conditions to resolve Lysozyme, Ovalbumin, BSA and their mixtures, and found that they were baseline resolved within 15 min.
Intestinal absorption differences of major bioactive compounds of Gegenqinlian Decoction between normal and bacterial diarrheal mini-pigs in vitro and in situ J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-03 Xiao Ling, Yuqiang Xiang, Feilong Chen, Qingfa Tang, Wei Zhang, Xiaomei Tan
Intestinal condition plays an important role in drug absorption and metabolism, thus the effects of varied gastrointestinal diseases such as infectious diarrhea on the intestinal function are crucial for drug absorption. However, due to the lack of suitable models, the differences of absorption and metabolism of drugs between the diarrheal and normal intestines are rarely reported. Thus, in this study, Escherichia coli diarrhea model was induced in mini-pigs and single-pass intestinal perfusion and intestinal mucosal enzyme metabolism experiments were conducted. A simple and rapid ultrahigh performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to determine the concentrations of 9 major components in Gegen Qinlian decoction (GQD). Samples were pretreated by protein precipitation with methanol and naringin and prednisolone were used as internal standards. The validated method demonstrated adequate sensitivity, selectivity, and process efficiency for the bioanalysis of 9 compounds. Results of intestinal perfusion showed that puerarin, daidzein, daidzin and baicalin and berberine were absorbed faster in diarrheal jejunum than in normal intestines (p < 0.05). However, puerarin, daidzin and liquiritin were metabolized more slowly in diarrheal intestine after incubation compared with the normal group (p < 0.05). The concentrations of daidzein in both perfusion and metabolism and wogonin in metabolism were significantly increased (p < 0.05). In conclusion, absorption and metabolism of GQD were significantly different between the diarrheal and normal intestines, which suggest that bacterial diarrheal mini-pigs model can be used in the intestinal absorption study and is worthy to be applied in the other intestinal absorption study of anti- diarrheal drugs.
A sensitive and robust HPLC method to quantify recombinant antibody fragments in E. coli crude cell lysate J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-02 Thomas Gundinger, Alexander Pansy, Oliver Spadiut
Antibody fragments (Fabs) represent a highly interesting class of biopharmaceuticals with a broad range of medical applications. They bind antigens comparable to full length monoclonal antibodies, but are smaller in size allowing increased tissue penetration, and lack the glycosylated Fc domain, which is why they can be produced in the prokaryotic expression host E. coli. Due to the presence of disulfide bonds Fabs are usually produced in the oxidative environment of the E. coli periplasm. Even though recombinant production in E. coli is cheaper and easier to realize than in mammalian cells, this intracellular production of the Fab in E. coli entails difficulties in subsequent product analytics. Whereas Fabs are produced extracellularly by mammalian cells and thus can be analyzed in the environment of a defined medium, recombinantly produced Fabs in E. coli have to be analyzed in crude cell lysate containing a lot of host cell proteins, host cell DNA and lipids. Thus, robust and sensitive HPLC analytics for Fab quantification is still scarce today and recombinant Fabs from E. coli are conventionally quantified by expensive and cumbersome immunoassays. In this study we developed a sensitive and robust affinity-based HPLC method for the quantification of recombinant Fabs in E. coli crude cell lysates with a limit of quantification down to 46 μg/mL and high reproducibility. This method will definitely be of key importance for strain generation as well as for early steps in upstream process development for recombinant E. coli producing Fabs.
UPLC-ESI-MS/MS method for the quantitative measurement of aliphatic diamines, trimethylamine N-oxide, and β-methylamino-l-alanine in human urine ☆ J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-02 Deepak Bhandari, Brett A. Bowman, Anish B. Patel, David M. Chambers, Víctor R. De Jesús, Benjamin C. Blount
Determination of acetyl coenzyme A in human whole blood by ultra-performance liquid chromatography-mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-02 Roberto Speziale, Camilla Montesano, Maria Lucia De Leonibus, Fabio Bonelli, Paola Fezzardi, Maria G. Beconi, Edith Monteagudo, Daniel Elbaum, Laura Orsatti
Development and validation of an ultra high performance liquid chromatography-electrospray tandem mass spectrometry method using selective derivatisation, for the quantification of two reactive aldehydes produced by lipid peroxidation, HNE (4-hydroxy-2(E)-nonenal) and HHE (4-hydroxy-2(E)-hexenal) in faecal water J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-02 S. Chevolleau, M.-H. Noguer-Meireles, I. Jouanin, N. Naud, F. Pierre, F. Gueraud, L. Debrauwer
Red or processed meat rich diets have been shown to be associated with an elevated risk of colorectal cancer (CRC). One major hypothesis involves dietary heme iron which induces lipid peroxidation. The quantification of the resulting reactive aldehydes (e.g. HNE and HHE) in the colon lumen is therefore of great concern since these compounds are known for their cytotoxic and genotoxic properties. UHPLC-ESI-MS/MS method has been developed and validated for HNE and HHE quantification in rat faeces. Samples were derivatised using a brominated reagent (BBHA) in presence of pre-synthesized deuterated internal standards (HNE-d11/HHE-d5), extracted by solid phase extraction, and then analysed by LC-positive ESI-MS/MS (MRM) on a TSQ Vantage mass spectrometer. The use of BBHA allowed the efficient stabilisation of the unstable and reactive hydroxy-alkenals HNE and HHE. The MRM method allowed selective detection of HNE and HHE on the basis of characteristic transitions monitored from both the 79 and 81 bromine isotopic peaks. This method was validated according to the European Medicines Agency (EMEA) guidelines, by determining selectivity, sensitivity, linearity, carry-over effect, recovery, matrix effect, repeatability, trueness and intermediate precision. The performance of the method enabled the quantification of HNE and HHE in concentrations 0.10–0.15 μM in faecal water. Results are presented on the application to the quantification of HNE and HHE in different faecal waters obtained from faeces of rats fed diets with various fatty acid compositions thus corresponding to different pro-oxidative features.
A three-point identity criteria tool for establishing product identity using icIEF method J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-01 Deepti Ahluwalia, Madesh Belakavadi, Tapan Das, Amit Katiyar
Product identity is one of the release testing requirements that needs to be established to ensure that there is no misidentification of drugs. Here, we demonstrated the challenges that can come across while establishing a product identity method for monoclonal antibody (mAb) and mAb-related products using icIEF method. A unique three-point identity criteria tool (visual comparison, pI of individual peaks and ΔpIs) was applied to distinguish mAb1 from the other in-house mAbs. A reduction approach followed by icIEF showed higher potential for establishing identity for mAb1 product as compared to native and enzymatic digestion approach. In general, icIEF method lacks specificity required to unequivocally establish the identity for mAbs, therefore, risk analysis is recommended before implementing icIEF as a stand-alone identity method for monoclonal antibodies.
Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling J. Chromatogr. B (IF 2.603) Pub Date : 2018-03-01 Matic Grom, Mirijam Kozorog, Simon Caserman, Andrej Pohar, Blaž Likozar
Stable isotope labeling - dispersive solid phase extraction - liquid chromatography - tandem mass spectrometry for quantitative analysis of transsulfuration pathway thiols in human serum J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-28 Ya-Lan Wang, Quan-Fei Zhu, Li-Ming Cheng, Shao-Ting Wang, Shan-Shan Qin, Shu-Jian Zheng, Hua-Ming Xiao, Juan-Juan Li, Song-Mei Liu, Bi-Feng Yuan, Yu-Qi Feng
Low-molecular-weight thiols play important roles in a variety of pathological processes and are closely associated with a wide range of diseases. In this study, a selective and sensitive method was developed for the simultaneous determination of all the 7 thiols occurring in the transsulfuration pathway (Cysteine (Cys), homocysteine (Hcys), glutathione (GSH), N-acetylcysteine (Nac), cysteinylglycine (CysGly), glutamylcysteine (GluCys) and cysteamine (CA)) in human serum by in-vitro stable isotope labeling - dispersive solid phase extraction - liquid chromatography - tandem mass spectrometry analysis (IL-DSPE-LC-MS/MS). In the proposed method, a pair of stable isotope-labeling reagents, BQB (ω-bromoacetonylquinolinium bromide) and BQB-D7, were utilized to label thiols in human serum samples and thiol standards, respectively. The BQB labeled thiols which carry a positive charge were extracted and purified with C8-SO3H-based DSPE followed by LC-MS/MS analysis. Good linearities for 7 thiols occurring in the transsulfuration pathway were obtained with the coefficient of determination (R2) >0.9901. The limits of detection (LODs) were in the range of 0.7–6.0 nmol/L. The method was further applied to investigate the contents change of 7 thiols in human serum samples of type 2 diabetes mellitus (T2DM) patients and breast cancer (BC) patients. The results showed that the contents of these thiols occurring in the transsulfuration pathway significantly changed and were highly diseases-related. In addition, partial least squares discriminant analysis (PLS-DA) suggested excellent classification performance between patients and healthy controls. The findings indicated that these significantly changed thiols occurring in the transsulfuration pathway in T2DM patients and BC patients might serve as the indicator for the diagnosis of T2DM and BC. Taken together, the developed IL-DSPE-LC-MS/MS method provides a promising tool for the sensitive analysis of thiols from complex biological samples, which may promote the in-depth investigation of the functions of thiols.
Validation of a routine gas chromatography mass spectrometry method for 2-hydroxyglutarate quantification in human serum as a screening tool for detection of idh mutations J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-28 Esther Fernández-Galán, Núria Massana, Marina Parra-Robert, Susana Hidalgo, Gregori Casals, Jordi Esteve, Wladimiro Jiménez
High circulating levels of 2-hydroxyglutarate (2HG) have been reported in patients with determinate isocitrate dehydrogenase (IDH) mutated tumors. Recent studies indicate that in malignancies such as acute myeloid leukemia (AML), measurements of 2HG in serum provide useful diagnostic and prognostic information and improve patient selection and monitoring of IDH-targeted treatments. In the current study, we validated a sensitive and specific gas chromatography mass spectrometry (GC–MS) method specifically intended to quantify serum levels of 2HG in routine clinical laboratories. Extraction was liquid-liquid with ethyl acetate, and derivatization was reduced to 3 min of microwave irradiation. The analytical method was linear over a wide dynamic range, presenting acceptable intraday and day-to-day precision and accuracy. The limit of quantification was 10 ng/mL, process efficiency ranged between 38% and 49%, and recovery of added 2HG was 99–105%. 2HG was found to be stable in serum for up to 48 h at both 4 °C and at ambient temperature, and after three freeze-thaw cycles. Microwave derivatizated extracts in the autosampler were found to be stable for up to 120 h. In summary, the present method is useful for quantification of 2HG serum levels in patients with IDH mutated malignancies in clinical laboratories.
Quantitative determination of metformin, saxagliptin and 5-hydroxy saxagliptin simultaneously by hydrophilic interaction liquid chromatography - electrospray ionization mass spectrometry and its application to a bioequivalence study with a single-pill combination in human J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-27 Ying Peng, Qingqing Chang, Na Yang, Shiyin Gu, Yi Zhou, Lifang Yin, A. Jiye, Guangji Wang, Jianguo Sun
A simple, sensitive and specific hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometric (HILIC–MS) method was developed and validated to determine the plasma concentrations of metformin, saxagliptin and 5-hydroxy saxagliptin simultaneously in clinical studies. Plasma samples were first acidified and then protein precipitated with acetonitrile. Chromatographic separation was achieved on a HILIC Chrom Matrix HP amide column (5 μm, 3.0 × 100 mm I.D.). The mobile phase consisted of acetonitrile and 5 mM ammonium formate buffer containing 0.1% formic acid. Multiple reaction monitoring transitions were performed on triple quadrupole mass spectrometric detection in positive-ion mode with an electrospray ionization source. The calibration curves showed good linearity (r ≥ 0.999) over the established concentration range of 1.0–1000 ng/mL for metformin and 0.1–100 ng/mL for saxagliptin and its active metabolite 5-hydroxy saxagliptin. The extraction recovery for all of the analytes was >92% and the matrix effect ranged from 91.0 to 110.0%. After validation, the method was successfully applied to a bioequivalence study with a single-pill combination (SPC) consisting of 5 mg saxagliptin and 500 mg metformin in 10 healthy Chinese subjects.
UPLC-G2Si-HDMS untargeted metabolomics for identification of metabolic targets of Yin-Chen-Hao-Tang used as a therapeutic agent of dampness-heat jaundice syndrome J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-27 Hui Sun, Le Yang, Meng-xi Li, Heng Fang, Ai-hua Zhang, Qi Song, Xing-yuan Liu, Jing Su, Meng-die Yu, Toshiaki Makino, Xi-jun Wang
Yin-Chen-Hao-Tang (YCHT), the classic formulae of traditional Chinese medicine (TCM), is widely used to treat dampness-heat jaundice syndrome (DHJS) and various liver diseases. However, the therapeutic mechanism of YCHT is yet to have an integrated biological interpretation. In this work, we used metabolomics technology to reveal the adjustment of small molecule metabolites in body during the treatment of YCHT. Aim to discover the serum biomarkers which are associated with the treatment of DHJS against YCHT. Pathological results and biochemical indicators showed that the hepatic injury and liver index abnormalities caused by DHJS was effectively improve after treatment with YCHT. On the basis of effective treatment, ultra-high performance liquid chromatography (UPLC-G2Si-HDMS) combined with the multivariate statistical analysis method was utilized to analyze the serum samples. Finally, 22 biomarkers were identified by using mass spectrometry and illuminated the correlative metabolic pathways which play a significant role and as therapeutic targets in the treatment of DHJS. This work demonstrated that mass spectrometry metabolomics provides a new insight to elucidate the action mechanism of formulae.
Multi-block analysis coupled with GC-FID and ATR-MIR for the evaluation of thermal degradation in vegetable oils J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-26 Valéria Rampazzo, Leomara Floriano Ribeiro, Poliana Macedo Santos, Maresa Custódio Molinari Ferreira, Evandro Bona, Giselle Maria Maciel, Charles Windson Isidoro Haminiuk
A comparison of the determination and speciation of inorganic arsenic using general HPLC methodology with UV, MS and MS/MS detection J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-26 Gregory Gilmartin, Diane Gingrich
The determination and speciation of arsenic in natural resources such as drinking water and agricultural soils has been a growing concern in recent years due to its many toxicological effects [, ; ]. To speciate and quantitate concentrations of <1 ppm of arsenic, typically an ion chromatograph (IC) interfaced to an inductively coupled plasma mass spectrometer (ICP-MS) is employed [, , , , ; ]. This methodology may be very robust and sensitive, but it is expensive and not as ubiquitous as high performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection or electrospray ionization mass spectrometry (ESI-MS). Anion exchange chromatography is a well-documented means of speciating arsenite (As(III), As2O3) and arsenate (As(V), AsO4) using UV , conductivity , or ESI-MS detection [12,13]. This paper demonstrates the utilization of common liquid chromatographic instrumentation to speciate and determines inorganic Arsenic compounds using UV or MS via selected ion recording (SIR) or multiple reaction monitoring (MRM) detection. This paper describes the analysis of arsenite and arsenate samples prepared using both deionized and ground water. The limit of quantitation for the techniques described in this paper for samples spiked in ground water were 454 ppb (As(III)) and 562 ppb (As(V)) for UV detection, 45.4 ppb (As(III)) and 56.2 ppb (As(V)) for SIR detection, and 4.54 ppb (As(III)) and 5.62 ppb (As(V)) for MRM detection.
Metformin impacts cecal bile acid profiles in mice J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-25 Nina Sillner, Alesia Walker, Wendelin Koch, Michael Witting, Philippe Schmitt-Kopplin
Bile acids (BAs) are major components of bile synthesized from cholesterol and take part in the digestion of dietary lipids, as well as having signaling functions. They undergo extensive microbial metabolism inside the gastrointestinal tract. Here, we present a method of ultra-high pressure liquid chromatography coupled to ion trap mass spectrometry for quantification of 45 BAs in mouse cecum. The system was validated in regard to sensitivity with limits of detection and quantification (0.6–24.9 nM), interday accuracy (102.4%), interday precision (15.2%), recovery rate (74.7%), matrix effect (98.2%) and carry-over effect (<1.1%). Afterwards, we applied our method to investigate the effect of metformin on BA profiles. Diabetic mice were treated with metformin for 1 day or 14 days. One day of treatment resulted in a significant increase of total BA concentration (2.7-fold increase; db/db metformin 5.32 μmol/g, db/db control mice 1.95 μmol/g), most notable in levels of 7-oxodeoxycholic, 3-dehydrocholic and cholic acid. We observed only minor impact on BA metabolism after 14 days of metformin treatment, compared to the single treatment. Furthermore, healthy wild type mice had elevated concentrations of allocholic and ω-muricholic acid compared to diabetic mice. Our method proved the applicability of profiling BAs in cecum to investigate intestinal BA metabolism in diabetes and pharmacological applications.
A rapid, LC-MS/MS assay for quantification of piperacillin and tazobactam in human plasma and pleural fluid; application to a clinical pharmacokinetic study J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-24 Natalia D. Popowicz, Sean J. O'Halloran, Deirdre Fitzgerald, Y.C. Gary Lee, David A. Joyce
Piperacillin, in combination with tazobactam is a common first-line antibiotic used for the treatment of pleural infection, however its pleural pharmacokinetics and penetration has not previously been reported. The objective of this work was to develop and validate a rapid and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay for quantification of piperacillin (PIP) and tazobactam (TAZ). PIP and TAZ were extracted from both human plasma and pleural fluid samples by protein precipitation in methanol containing the internal standards (IS) piperacillin-d5 (PIP-d5) and sulbactam (SUL). Briefly, 5 μL of sample was mixed with 125 μL of methanol containing IS, vortexed and centrifuged. Supernatant (50 μL) was diluted into 500 μL of mobile phase containing 10 mM of ammonium bicarbonate in LCMS grade water and transferred to the autosampler tray. Electrospray ionization in positive mode and multiple reaction monitoring (MRM) were used for PIP and piperacillin-d5 at the transitions m/z 518.2 → 143.2 and m/z 523.2 → 148.2 respectively, and electrospray ionization in negative mode and MRM were used for TAZ and SUL at the transitions m/z 299.1 → 138.1 and m/z 232.4 → 140.1. The chromatographic separation was achieved using an Acquity BEH C-18 column with gradient elution of mobile phase containing 10 mmol/L ammonium bicarbonate in water and methanol. A linear range was observed over the concentration range of 0.25–352 mg/L and 0.25–50.5 mg/L for PIP and TAZ respectively. Complete method validation was performed according to US FDA guidelines for selectivity, specificity, precision and accuracy, LLOQ, matrix effects, recovery and stability, with all results within acceptable limits. This method was successfully applied to two patients with pleural infection and is suitable for further pharmacokinetic studies and therapeutic drug monitoring.
Determination of the sulfate and glucuronide conjugates of levornidazole in human plasma and urine, and levornidazole and its five metabolites in human feces by high performance liquid chromatography–tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-23 Gaoli He, Beining Guo, Jing Zhang, Yi Li, Xiaojie Wu, Yaxin Fan, Yuancheng Chen, Guoying Cao, Jicheng Yu
Levornidazole is a novel third-generation nitroimidazoles antibiotic which metabolism and disposition in human are not well known. We have previously developed two methods to quantify levornidazole and its phase I metabolites, Ml (hydroxylation metabolite), M2 (N-dealkylation metabolite) and M4 (Oxidative dechlorination metabolite), in human plasma and urine. In this study, we developed three novel liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods and analyzed its phase II metabolites, sulfate conjugate (M6) and glucuronide conjugate (M16), in human plasma and urine, and the parent drug and above-mentioned five metabolites in human feces samples. Analytes and internal standard (IS) in human plasma were extracted by a solid-phase extraction procedure and separated on an ACQUITY UPLC CSH C18 column in gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase. The pretreatment procedures for urine and feces homogenate samples involved a protein precipitation followed by liquid-liquid extraction, and chromatographic separations were performed on the Atlantis T3 columns of different lengths and particle sizes (2.1 × 50 mm, 3 μm and 2.1 × 150 mm, 5 μm), respectively. The mobile phases consisted of formic acid and acetonitrile-methanol solution (v/v, 50:50) in gradient elution. The MS/MS analysis was conducted on TSQ Quantum triple quadrupole mass spectrometer using electrospray ionization with selected reaction monitoring (SRM) in the positive ion mode. The calibration curves for all analytes were linear and the validation ranges were as follows: 0.005–0.500 μg/mL for M6 and 0.005–2.500 μg/mL for M16 in plasma; 0.010–10.000 μg/mL for M6 and M16 in urine; 0.005–1.000 μg/mL for levornidazole, M2, M4 and M16, and 0.010–2.000 μg/mL for M1 and M6 in human feces homogenate. Across these matrices, mean intra- and inter- batch accuracy values were in the ranges of 80.0%–120.0%, and intra- and inter-batch precision values did not exceed 20%. It was fully validated including selectivity, linearity, matrix effect, extraction recovery, stability, dilution integrity, carryover and incurred sample analysis (ISR). These newly developed methods were successfully applied in pharmacokinetics, metabolism and disposition study of levornidazole in 12 healthy Chinese subjects.
Evaluation of a method for measuring the radioprotective metabolite WR-1065 in plasma using chemical derivatization combined with UHPLC-MS/MS J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-20 Eric S. Simon, Dawn Reyna, Richard J. Lister, Cheryl Harteg, Elke Lipka
Hypotension is the dose-limiting side effect of the radio-protective drug Amifostine and results from relaxation of the vascular smooth muscle, which is directly mediated by the active metabolite, WR-1065, of Amifostine. The route of administration (currently FDA-approved only for intravenous administration) and the rapid metabolic conversion of Amifostine combine to yield high systemic levels of WR-1065 and facilitate the onset of hypotension. Research efforts aiming to optimize the delivery of WR-1065 to maintain efficacy while reducing its peak, systemic concentration below levels that induce hypotension are underway. To fully characterize the effect of reduced dose levels and alternative routes of administration of Amifostine on systemic WR-1065 concentrations, improved analytical techniques are needed. We have developed and evaluated a highly sensitive method for measuring WR-1065 in rat plasma that employs chemical derivatization, protein precipitation and UPLC-MS/MS analysis. The method exhibits a limit of quantification (LOQ) of 7.4 nM in plasma, which is a significant improvement over conventional approaches that utilize LC-electrochemical detection (ECD) (LOQ 150 nM or higher). The method was assessed in a pharmacokinetics study in rats administered Amifostine intravenously and via direct jejunal injection (10 mg/kg each route). The bioavailability of WR-1065 was 61.5% after direct jejunal injection indicating rapid conversion and absorption of the metabolite in the intestinal tract. This demonstrates that an oral formulation of Amifostine designed for site-specific release of the drug in the upper GI tract can deliver systemic absorption/conversion to WR-1065, provided that the formulation protects the therapeutic from gastric decomposition in the stomach.
Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-20 Valentina D’Atri, Szabolcs Fekete, Dwight Stoll, Matthew Lauber, Alain Beck, Davy Guillarme
Brentuximab vedotin (Adcetris) is a cysteine-linked antibody-drug conjugate (ADC) used in the treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). In this study, the drug payload and glycan modifications of this ADC were simultaneously characterized using a unique LC-MS middle-up analysis, involving hydrophilic interaction chromatography (HILIC). This work demonstrates that HILIC is an effective and complementary analytical technique to reversed phase liquid chromatography (RPLC) for subunit-level characterization of immuno-conjugates.
A high-throughput UPC2-MS/MS method for the separation and quantification of C19 and C21 steroids and their C11-oxy steroid metabolites in the classical, alternative, backdoor and 11OHA4 steroid pathways J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-20 Therina du Toit, Maria A. Stander, Amanda C. Swart
In the present study an ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analytical method was developed and validated for the determination of 17 C19 and 14 C21 steroids, including C11-oxy C19 and C11-oxy C21 steroids. The limit of detection and limit of quantification ranged from 0.01 to 10 ng/mL and from 0.01 to 20 ng/mL, respectively, and the method shows the recovery, matrix effect and process efficiency of steroids isolated from a serum matrix to be within acceptable limits. Good accuracy, repeatability and reproducibility were also shown and the method provided excellent sensitivity and selectivity as stereoisomers and regioisomers were also resolved and quantified accurately. Clinical conditions such as congenital adrenal hyperplasia, polycystic ovary syndrome in females and disorders of sex development in neonates and in children, amongst others, are characterized by abnormal steroid levels. Steroid profiling is essential to accurately diagnose steroid levels in the above settings as well as in androgen excess or deficiency in adrenal-linked endocrine diseases. Our method, separating C19 and C21 steroids in a single chromatographic step, offers a reduced sample turnover rate in the clinical setting, while providing comprehensive steroid profiles of in vivo steroids in the nmol/L range. This is, to our knowledge, the first method reported to simultaneously separate C19 and C21 steroids, together with their C11-hydroxy and C11-keto metabolites –one which may hold promise in the identification of new steroid markers in steroid-linked endocrine diseases, in addition to profiling steroid metabolism and abnormal enzyme activity in patients.
Targeted and untargeted-metabolite profiling to track the compositional integrity of ginger during processing using digitally-enhanced HPTLC pattern recognition analysis J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-19 Reham S. Ibrahim, Hoda Fathy
Tracking the impact of commonly applied post-harvesting and industrial processing practices on the compositional integrity of ginger rhizome was implemented in this work. Untargeted metabolite profiling was performed using digitally-enhanced HPTLC method where the chromatographic fingerprints were extracted using ImageJ software then analysed with multivariate Principal Component Analysis (PCA) for pattern recognition. A targeted approach was applied using a new, validated, simple and fast HPTLC image analysis method for simultaneous quantification of the officially recognized markers 6-, 8-, 10-gingerol and 6-shogaol in conjunction with chemometric Hierarchical Clustering Analysis (HCA). The results of both targeted and untargeted metabolite profiling revealed that peeling, drying in addition to storage employed during processing have a great influence on ginger chemo-profile, the different forms of processed ginger shouldn't be used interchangeably. Moreover, it deemed necessary to consider the holistic metabolic profile for comprehensive evaluation of ginger during processing.
A novel technique for determination of the fructose, glucose and sucrose distribution in nectar from orchids by HPLC-ELSD J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-18 Dan Nybro Lindqvist, Henrik Ærenlund Pedersen, Lars Holm Rasmussen
The dominant components in floral nectar is fructose, glucose and sucrose. The concentration and the ratio between the sugars are indicative for plant species and play an important part in the interplay between plants and pollinators. In this paper we present a novel HPLC-ELSD based analytical method for sugar characterization of nectar from orchids. Nectar was collected on Whatman No. 1 paper and preserved in the field by 70 v/v% ethanol. The analytical method had a linear range up to at least 3000 mg L−1 for all 3 sugars with a precision of 1.5–1.7%. Correlation coefficients were 0.9999 to 1.0000. The LOD of all sugars were 5–7 mg L−1 and the LOQ were 17–19 mg L−1. Field samples were stable for min. 7 weeks at −18 °C. The technique was applied to two species of Platanthera (Orchidaceae) in order to test whether species-related differences in sugar composition could be observed. No differences were found between the two species, which were sucrose-dominant (53.5–100%) though with high variation within species and between individual flowers.
Synthesis and characterization of Ag+-decorated poly(glycidyl methacrylate) microparticle design for the adsorption of nucleic acids J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-18 Kadir Erol, Aytekin Uzunoglu, Kazım Köse, Büşra Sarıca, Emre Avcı, Dursun A. Köse
In this study, we report on the adsorption of RNA and DNA molecules by exploiting the high binding affinity of these nucleic acids to Ag+ ions anchored on magnetic poly(glycidyl methacrylate) (PGMA) microparticles. PGMA microparticles were synthesized and modified with nicotinamide which enabled to anchor Ag+ ions on the surface. The successful preparation of PGMA was confirmed by the presence of characteristic FTIR peaks. The ESR results showed that the incorporation of Fe Ni salt to the polymeric structure provided a magnetic property to the microparticles. The amount of nicotinamide and Ag+ ions used to modify the surface of the particles were found to be 1.79 wt% and 52.6 mg Ag/g microparticle, respectively. The high affinity of nucleic acids to Ag+ ions were exploited for the adsorption studies. At the optimum working conditions, the adsorption capacity of microparticles was found to be 40.1 and 11.48 mg nucleic acid/g microparticle for RNA and DNA, respectively. Our study indicated that the use of novel Ag+-decorated magnetic PGMA particles can be successfully employed as adsorbents for fast, easy, and cost-friendly adsorption of nucleic acids with high purity as well as high in quantity.
A UPLC-ESI-Q-TOF method for rapid and reliable identification and quantification of major indole alkaloids in Catharanthus roseus J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-17 Won Tae Jeong, Heung Bin Lim
We developed a novel ultra performance liquid chromatography-quadrupole time-of-flight (UPLC-Q-TOF) mass spectrometry method that allows sensitive, rapid, and reliable detection and identification of six representative indole alkaloids (vincristine, vinblastine, ajmalicine, catharanthine, serpentine, and vindoline) that exhibit physiological activity in Catharanthus roseus (C. roseus). The alkaloids were eluted on a C18 column with acetonitrile and water containing 0.1% formic acid and 10 mM ammonium acetate, and separated with good resolution within 13 min. Electrospray ionization-Q-TOF (ESI-Q-TOF) analysis was performed to characterize the molecules and their fragment ions, and the characteristic ions and fragmentation patterns were used as to identify the alkaloids. The proposed analytical method was verified in reference to the ICH guidelines and the results showed excellent linearity (R2 > 0.9988), limit of detection (1 ng/mL to 10 ng/mL), limit of quantification (3 ng/mL to 30 ng/mL), intra-day and inter-day precisions, and extraction recovery rates (92.8% to 104.1%) for all components. The validated UPLC-Q-TOF method was applied to the analysis of extracts from the root, stem, and leaves of C. roseus, allowing the identification of six alkaloids by comparison of retention times, molecular ions, and fragmentation patterns with those of reference compounds. Sixteen additional indole alkaloids were tentatively identified by comparison of chromatograms to chemical databases and literature reports. The contents of bis-indole alkaloids (vincristine and vinblastine) were high in the aerial parts, while the contents of mono-indole alkaloids (ajmalicine, catharanthine, serpentine, and vindoline) were high in the roots. The present results demonstrate that the proposed UPLC-Q-TOF method can be useful for the investigation of phytochemical constituents of medicinal plants.
Comparisons of the pharmacokinetic and tissue distribution profiles of withanolide B after intragastric administration of the effective part of Datura metel L. in normal and psoriasis guinea pigs J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-17 Lianrong Yang, Xin Meng, HaixueKuang
A simple, highly sensitive ultra-performance liquid chromatography- electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed to quantify of withanolide B and obakunone (IS) in guinea pig plasma and tissues, and to compare the pharmacokinetics and tissue distribution of withanolide B in normal and psoriasis guinea pigs. After mixing with IS, plasma and tissues were pretreated by protein precipitation with methanol. Chromatographic separation was performed on a C18 column using aqueous (0.1% formic acid) and acetonitrile (0.1% formic acid) solutions at 0.4 mL/min as the mobile phase. The gradient program was selected (0–4.0 min, 2–98% B; 4.0–4.5 min, 98–2% B; and 4.5–5 min, 2% B). Detection was performed on a 4000 QTRAP UPLC–ESI-MS/MS system from AB Sciex in the multiple reaction monitoring (MRM) mode. Withanolide B and obakunone (IS) were monitored under positive ionization conditions. The optimized mass transition ion-pairs (m/z) for quantitation were 455.1/109.4 for withanolide B and 455.1/161.1 for obakunone.
Supercritical fluid chromatographic-tandem mass spectrometry method for monitoring dissipation of thiacloprid in greenhouse vegetables and soil under different application modes J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-16 Runan Li, Zenglong Chen, Fengshou Dong, Jun Xu, Xingang Liu, Xiaohu Wu, Xinglu Pan, Yan Tao, Yongquan Zheng
A rapid, sensitive and effective supercritical fluid chromatographic-tandem mass spectrometry (SFC-MS/MS) method was developed to analyze thiacloprid for the first time. The SFC-MS/MS conditions were optimized with the ultra-performance convergence chromatography (UPC2) BEH column (100 mm × 3.0 mm, 1.7 μm particle size) and thiacloprid was eluted at 1.22 min in gradient mode with CO2/methanol as mobile phase. The 0.1% formic acid in methanol (v/v) was used as postcolumn compensation solution to improve sensitivity. The ABPR pressure, flow rate of mobile phase and flow rate of compensation pump were set at 1800 psi, 1.8 mL/min, and 0.1 mL/min, respectively. The average recoveries of thiacloprid in soil at four spiking levels (5, 10, 100, 1000 μg/kg) ranged between 78.8% and 107.1% with relative standard deviations (RSDs) lower than 12.2% and the limit of quantitation (LOQ) was 5 μg/kg. The proposed method can distinctly improve the analysis efficiency by 2–12 times and reduce the solvent consumption by 5%–95% compared with reported methods. It was applied to investigate the dissipation rates of thiacloprid in greenhouse vegetables and soil under different application modes. The half-lives of thiacloprid in cucumber and soil were 9.55–20.44 days and 3.74–9.14 days separately under different application modes, 10.60 days in tomato under foliar spraying. The residues in vegetables under root irrigation were all less than that under foliar spraying. The results could offer useful data for risk assessment of thiacloprid in agricultural production.
Micellar electrokinetic chromatography with laser induced fluorescence detection shows increase of putrescine in erythrocytes of Parkinson's disease patients J. Chromatogr. B (IF 2.603) Pub Date : 2018-02-13 L. Betancourt, P. Rada, L. Hernandez, H. Araujo, G.A. Ceballos, L.E. Hernandez, P. Tucci, Z. Mari, M. De Pasquale, D.A. Paredes
A highly sensitive method was developed to measure putrescine by micellar electrokinetic chromatography with laser induced fluorescence detection with excellent linearity in the 1 nM to 3 μM range. The technique was tested on a drop of blood from Parkinson's disease patients obtained by finger prick. The results showed a statistically significant increase of putrescine in the erythrocytes compared to controls and a non-significant increase in plasma. This high level of putrescine does not constitute by itself proof that putrescine and polyamines are directly related to Parkinson's disease. However, the present results and several others addressed in the discussion suggest that these compounds might be causally involved in the pathophysiology of Parkinson's disease. In addition, the analytical method reported here may help to find new biomarkers for many diseases including Parkinson's disease.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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