Quantification of amlodipine in dried blood spot samples by high performance liquid chromatography tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-16 Gaoyun Chen, Feras Jirjees Sr, Abdel Al Bawab, James C. McElnay
A sensitive and specific method, utilising high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) was developed for the quantitative determination of amlodipine in dried blood spot (DBS) samples. Chromatographic separation was achieved using a Waters XBridge C18 column with gradient elution of a mixture of water and acetonitrile containing 0.1% formic acid (v/v). Amlodipine was quantified using a Waters Quattro Premier mass spectrometer coupled with an electro-spray ionization (ESI) source in positive ion mode. The MRM transitions of 408.9 m/z → 238.1 m/z and 408.9 → 294.0 m/z were used to quantify and qualify amlodipine, respectively. The method was validated across the concentration range of 0.5–30 ng/mL by assessing specificity, sensitivity, linearity, precision, accuracy, recovery and matrix effect according to the Food and Drug Administration (FDA) guidelines. This method was also validated clinically within a large pharmacoepidemiological study in which amlodipine blood concentration were determined in patients who had been prescribed this medication.
Determination of non-liposomal and liposomal doxorubicin in plasma by LC–MS/MS coupled with an effective solid phase extraction: In comparison with ultrafiltration technique and application to a pharmacokinetic study J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-16 Yaping Xie, Nan Shao, Yi Jin, Liang Zhang, Huan Jiang, Ningjie Xiong, Fangming Su, Haiyan Xu
Liposomal formulation of doxorubicin has been widely applied in clinic for treatment of various cancers. The separation and measurement of free drug (drug which is not entrapped in liposomes) and liposomal drug in the plasma after injection of liposomal doxorubicin is of prime importance due to toxicity and activity concerns. In this study, a rapid and convenient method was developed to isolate and determine the non-liposomal and liposomal drugs in plasma. Plasma samples were prepared by solid phase extraction (SPE) using Oasis HLB cartridges. Liposomal doxorubicin (L-DOX) was collected in the aqueous eluate with its internal standard (IS), metformin; and non-liposomal doxorubicin (NL-DOX) and its isotope labelling IS were eluted from the cartridge by methanol containing 0.5% formic acid. After SPE separation, L-DOX and NL-DOX were subsequently quantified by a validated sensitive LC–MS/MS method individually. The calibration curves were found to be linear for L-DOX in the range of 0.156–40.0 μg/mL and for NL-DOX in the range of 3.13–200 ng/mL. The extraction recovery was about 97% for L-DOX and about 65% for NL-DOX. This method was further applied to investigate the pharmacokinetics of doxorubicin in Beagle dogs after an intravenous dose of 1.0 mg/kg Doxil®. After injection of Doxil®, L-DOX was the predominant component circulating in plasma, whose amount was about 1000-fold higher than that of NL-DOX. The analytical method might be helpful in pharmacokinetics and toxicity assessment of liposomal formulation.
In vivo study of erysolin metabolic profile by ultra high performance liquid chromatography coupleded to Fourier transform ion cyclotron resonance mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-16 Binglong Li, Hui Zhou, Guochun Yang, Fei Han, Yanting Li, Yongfeng Gao, Jinwei Gao, Feng Zhang, Lixin Sun
An ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) method was developed for the first time to study the in vivo metabolism of erysolin, a compound derived from cruciferous plants which has a definite effect of anti-tumor and anti-nerve injury. In this research, the chromatographic separation was performed on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters, USA) and eluted by a gradient program, the identification work was achieved on a Bruker ultra-high resolution spectrometer in positive ion mode. Plasma, urine, feces and bile samples were collected from rats to screen metabolites after an intragastric administration of erysolin at the dose of 100 mg/kg. As a result, the parent drug and a total of six phase II metabolites were detected and preliminarily identified by analyzing their MS and MS/MS spectrometry profiles. Our results indicated that erysolin mainly metabolized via the mercapturic acid metabolic pathway, erysolin first react with glutathione to form glutathione conjugate, followed by taking off the glutamic acid and glycine to form cysteine conjugate, then the N-acetylation reaction occurs, the product would be excreted out of the body at last. In conclusion, results obtained in our study may contribute to a better understanding of the metabolism process and characteristics of erysolin in vivo, and provide an important reference for future research.
Determination of adrenaline, noradrenaline and corticosterone in rodent blood by ion pair reversed phase UHPLC-MS/MS J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-13 Marianne Skov-Skov Bergh, Inger Lise Bogen, Jannike Mørch Andersen, Åse Marit Leere Øiestad, Thomas Berg
A novel ion pair reversed phase ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of the stress hormones adrenaline, noradrenaline and corticosterone in rodent blood was developed and fully validated. Separations were performed on an Acquity HSS T3 column (2.1 mm i.d. × 100 mm, 1.8 μm) with gradient elution and a runtime of 5.5 min. The retention of adrenaline and noradrenaline was substantially increased by employing the ion pair reagent heptafluorobutyric acid (HFBA). Ion pair reagents are usually added to the mobile phase only, but we demonstrate for the first time that including HFBA to the sample reconstitution solvent as well, has a major impact on the chromatography of these compounds. The stability of adrenaline and corticosterone in rodent blood was investigated using the surrogate analytes adrenaline-d3 and corticosterone-d8. The applicability of the described method was demonstrated by measuring the concentration of stress hormones in rodent blood samples.
Determination of isoquercitrin in rat plasma by high performance liquid chromatography coupled with a novel synergistic cloud point extraction J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-12 Jun Zhou, Jiang Bing Sun, Qiao Feng Wang
A novel improved preconcentration method known as synergistic cloud point extraction was established for isoquercitrin preconcentration and determination in rat plasma prior to its determination by high performance liquid chromatography. Synergistic cloud point extraction greatly simplified isoquercitrin extraction and detection. This method was accomplished at room temperature (about 22 °C) in 1 min with the nonionic surfactant Tergitol TMN-6 as the extractant, n-octanol as cloud point revulsant and synergic reagent. Parameters that affect the synergistic cloud point extraction processes, such as the concentrations of Tergitol TMN-6, volume of n-octanol, sample pH, salt content and extraction time were investigated and optimized. Under the optimum conditions, the calibration curve for the analyte was linear in the range from 5 to 500 ng mL−1 with the correlation coefficients greater than 0.9996. Meanwhile, limit of detection (S/N = 3) was less than 1.6 ng mL−1 and limit of quantification (S/N = 10) was less than 5 ng mL−1. It demonstrated that the method can be successfully applied to the pharmacokinetic investigation of isoquercitrin.
First investigations for the characterization of glucosamine-6-phosphate synthase by capillary electrophoresis J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-12 Miriam Beneito-Cambra, Pierre Gareil, Bernard Badet, Marie-Ange Badet-Denisot, Nathalie Delaunay
The enzyme glucosamine-6-phosphate synthase (GlmS) is an important point of metabolic control in biosynthesis of amino sugar-containing macromolecules and is therefore a potential target in order to design antibacterial and antifungal drugs. It has two oligomerization states, which are the active dimer and the inactive hexamer. For the first time, the potential of CE to separate and quantify the two forms was studied. After incubating GlmS with the D-glucosamine 6-phosphate (GlcN6P) inhibitor, an electrolyte based on sodium phosphate at pH 7.2 and an ionic strength of 100 mM plus GlcN6P (either 2 or 20 mM) allowed the hexamer-dimer separation. However, the displacement of the dimer/hexamer equilibrium during the analysis time prevented any improvement of the resolution when varying the effective separation length or the temperature of the analysis. Therefore, the use of a short-end CE method allowed the decrease in the analysis time to about 1 min. Some parameters such as the temperature and the time of incubation and the ratio of the inhibitor and enzyme concentrations were studied. Then, it was also possible to test, very rapidly and with a very small amount, some molecules having an inhibition potential for the GlmS enzyme (arabinose-5-phosphate oxime, 2-amino-2-deoxy-D-glucitol 6-phosphate, and glucose-6-phosphate).
Direct analysis of ethylene glycol in human serum on the basis of analyte adduct formation and liquid chromatography-tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-12 Marek Dziadosz
The aim of this work was to develop a fast, cost-effective and time-saving liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method for the analysis of ethylene glycol (EG) in human serum. For these purposes, the formation/fragmentation of an EG adduct ion with sodium and sodium acetate was applied in the positive electrospray mode for signal detection. Adduct identification was performed with appropriate infusion experiments based on analyte solutions prepared in different concentrations. Corresponding analyte adduct ions and adduct ion fragments could be identified both for EG and the deuterated internal standard (EG-D4). Protein precipitation was used as sample preparation. The analysis of the supernatant was performed with a Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column and a mobile phase consisting of 95% A (H2O/methanol = 95/5, v/v) and 5% B (H2O/methanol = 3/97, v/v), both with 10 mmol L−1 ammonium acetate and 0.1% acetic acid. Method linearity was examined in the range of 100 ̶ 4000 μg/mL and the calculated limit of detection/quantification was 35/98 μg/mL. However, on the basis of the signal to noise ratio, quantification was recommended at a limit of 300 μg/mL. Additionally, the examined precision, accuracy, stability, selectivity and matrix effect demonstrated that the method is a practicable alternative for EG quantification in human serum. In comparison to other methods based on liquid chromatography, the strategy presented made for the first time the EG analysis without analyte derivatisation possible.
Graphene oxide reinforced ionic liquid-functionalized adsorbent for solid-phase extraction of phenolic acids J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-12 Xiudan Hou, Xiaofeng Lu, Sheng Tang, Licheng Wang, Yong Guo
An environmental friendly sorbent of polymeric ionic liquids modified graphene oxide-grafted silica (PILs@GO@Sil) was synthesized for solid-phase extraction (SPE) of phenolic acids. The sorbent was prepared via a chemical layer-to-layer fabrication including amidation reaction, surface radical chain-transfer polymerization and in situ anion exchange. After modification with PILs, the silica surface had higher positive potential so that it would exhibit stronger electrostatic interaction for acidic compounds compared with GO@Sil. The adsorption performance of phenolic acids was investigated through the theoretical calculation and static, kinetic state adsorption experiments. Under the optimized conditions, wide linear ranges were obtained with correlation coefficients ranging from 0.9912 to 0.9998, and limits of detection were in the range of 0.20–0.50 μg L−1. Compared with other reported methods, the proposed PILs@GO@Sil-SPE-HPLC showed higher extraction efficiency. Finally, the black wolfberry yogurt and urine were analyzed as real samples and good recoveries spiked with standard solution were obtained.
Coupling of On-column Trypsin Digestion – Peptide Mapping and Principal Component Analysis for Stability and Biosimilarity Assessment of Recombinant Human Growth Hormone J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-11 Sara M. Shatat, Basma M. Eltanany, Abeer A. Mohamed, Medhat A. Al-Ghobashy, Faten A. Fathalla, Samah S. Abbas
Peptide mapping (PM) is a vital technique in biopharmaceutical industry. The fingerprint obtained helps to qualitatively confirm host stability as well as verify primary structure, purity and integrity of the target protein. Yet, in-solution digestion followed by tandem mass spectrometry is not suitable as a routine quality control test. It is time consuming and requires sophisticated, expensive instruments and highly skilled operators. In an attempt to enhance the fuctionality of PM and extract multi-dimentional data about various critical quality attributes and comparability of biosimilars, coupling of PM generated using immobilized trypsin followed by HPLC-UV to principal component analysis (PCA) is proposed. Recombinant human growth hormone (rhGH); was selected as a model biopharmaceutical since it is available in the market from different manufacturers and its PM is a well-established pharmacopoeial test. Samples of different rhGH biosimilars as well as degraded samples: deamidated and oxidized were subjected to trypsin digestion followed by RP-HPLC-UV analysis. PCA of the entire chromatograms of test and reference samples was then conducted. Comparison of the scores of samples and investigation of the loadings plots clearly indicated the applicability of PM-PCA for: i) identity testing, ii) biosimilarity assessment and iii) stability evaluation. Hotelling’s T2 and Q statistics were employed at 95% confidence level to measure the variation and to test the conformance of each sample to the PCA model, respectively. Coupling of PM to PCA provided a novel tool to identify peptide fragments responsible for variation between the test and reference samples as well as evaluation of the extent and relative significance of this variability. Transformation of conventional PM that is largely based on subjective visual comparison into an objective statiscally-guided analysis framework should provide a simple and economic tool to help both manufacturers and regulatory authorities in quality and biosimilarity assessment of biopharmaceuticals.
Improving the selectivity and sensitivity for quantifying 8-α-hydroxy-mutilin in rabbit tissues by using basic mobile phases and negative ionization J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-11 Aimin Tan, Guifen Gu, Xuan Gui, Yanxin Wu, Gordon Bolger, Albert Licollari, John C. Fanaras
Previously reported LC–MS methods for quantifying 8-α-hydroxy-mutilin (a marker residue of tiamulin) in tissues all used a pseudo MRM transition (from protonated molecular ion to protonated molecular ion, m/z 337 → 337) due to difficulties in finding a product ion, leading to suboptimal selectivity and sensitivity for detection. By using electrospray negative ionization in a basic medium, we, for the first time, found a highly selective and sensitive true MRM transition for 8-α-hydroxy-mutilin, m/z 335 → 179. With this newly found MRM transition and the use of pleuromutitlin as the internal standard, a very sensitive, selective, and robust LC–MS/MS method has been developed and validated for quantifying 8-α-hydroxy-mutilin in rabbit tissues (muscle, liver, kidney, and fat). In comparison with the previously published methods, the selectivity and sensitivity were significantly improved. For the concentration range validated (0.2 to 10 ppm or 0.2 to 10 μg/g), the within-run and between-run accuracies (% bias) ranged from −5.0 to 3.1 and −4.9 to 3.0, respectively. The % CV ranged from 2.2 to 6.6 and 4.7 to 8.3 for within-run and between-run precisions, respectively. The validated method was successfully used to support two GLP tissue residue depletion studies in rabbits.
LIPOPHILICITY ASSESSEMENT IN DRUG DISCOVERY: EXPERIMENTAL AND THEORETICAL METHODS APPLIED TO XANTHONE DERIVATIVES J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-10 Álvaro Santos, José Xavier Soares, Sara Cravo, Maria E. Tiritan, Salette Reis, Carlos Afonso, Carla Fernandes, Madalena M.M. Pinto
For the last several years, searching of new xanthone derivatives (XDs) with potential pharmacological activities has remained one of the main areas of interest of our group. The optimization of biological activity and drug-like properties of hits and leads is crucial at early stage of the drug discovery pipeline. Lipophilicity is one of the most important drug-like properties having a great impact in both pharmacokinetics and pharmacodynamics processes. In this work, we describe the lipophilicity of a small library of bioactive XDs, previously synthesized by our group, using different methods: computational, vortex-assisted liquid-liquid microextraction coupled with high-performance liquid chromatography (VALLME-HPLC), reversed-phase high-performance thin layer chromatography (RP-HPTLC), reversed-phase high-performance liquid chromatography (RP-HPLC), and biomembranes model by the partition between micelles and aqueous phase. The different results obtained by the used methods: were compared and discussed. The methodologies and data gathered in this study will expand the investigation of lipophilicity of xanthones derivatives, an important class of compounds in medicinal chemistry.
Quantification of fluorescent dyes in organ tissue samples via HPLC analysis J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-10 S. Morsbach, A. García-Bardon, J. Kamuf, B. Müller, N. Beghersa, K. Mohr, K. Landfester
The determination of regional blood flow via the accumulation of fluorescent microspheres is a concept regularly used in medical research. Typically, the microbeads get extracted from the tissue of interest and are then quantified by measuring the absorption or fluorescence of the incorporated dyes without further separation from the medium. However, in that case the absorption spectra of different dyes can overlap when used simultaneously, leading to an overestimation of the concentration. Additionally, background absorption from the medium can be problematic. Therefore, a high performance liquid chromatography method for the simultaneous detection of four dyes (orange, crimson, yellow-green and red) incorporated in different microbeads in samples from biological media such as organ tissue (brain, heart and kidneys) was developed. Since for biological samples often a large sample size is required for sufficient statistics, the method was optimized to yield very short run times. With this method it was possible to detect very low concentrations of only one microsphere per gram of organ tissue. By applying this sensitive quantification technique, it was demonstrated that the application of microbeads for perfusion measurements might not be reliable due to different organ distributions in each animal.
Micro-QuEChERS extraction coupled to GG-MS for a fast determination of Bisphenol A in human urine J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-08 Luísa Correia-Sá, Sónia Norberto, Cristina Delerue-Matos, Conceição Calhau, Valentina F. Domingues
Bisphenol A (BPA) is considered an endocrine disruptor and public concern over BPA exposure has been raised. Several studies have assessed human exposure to this plasticizer, confirming its ubiquitous presence and highlighting children as a public of special concern. A simple, efficient, cheap and green analytical procedure is reported within this paper. This paper reports, for the first time, the development of a modified Micro-QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method coupled to gas chromatography-mass spectrometry (GC-MS) as a new strategy for the efficient extraction and determination of Bisphenol A in human urine samples. Several parameters that are known to influence extraction were optimized. Good linearity was achieved at the studied concentration range (1–50 μg/L), with a correlation coefficient (R2) of 0.998. The optimized method proved to be accurate (≥74% recovery), reproducible ( < 11% relative standard deviation) and sensitive for BPA determination (detection limit of 0.13 μg/L and quantification limit of 0.43 μg/L). The analytical procedure was applied to the analyses of 12 urine samples collected from children living in the North/Center region of Portugal. BPA was detected in all the analyzed samples in concentrations ranging from 1.5 μg/L to 48.9 μg/L. The proposed methodology is suitable for the determination of BPA in urine samples in the framework of biomonitoring studies and bioanalytical analyses, applying GC-MS detection.
Development and validation of modified QuEChERS method coupled with LC-MS/MS for simultaneous determination of cymiazole, fipronil, coumaphos, fluvalinate, amitraz, and its metabolite in various types of honey and royal jelly J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-08 Weijia Zheng, Jin-A Park, A.M. Abd El-Aty, Seong-Kwan Kim, Sang-Hyun Cho, Jeong-min Choi, Hee Yi, Soo-Min Cho, Amer Ramadan, Ji Hoon Jeong, Jae-Han Shim, Ho-Chul Shin
Over the past few decades, honey products have been polluted by different contaminants, such as pesticides, which are widely applied in agriculture. In this work, a modified EN − quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method was developed for the simultaneous quantification of pesticide residues, including cymiazole, fipronil, coumaphos, fluvalinate, amitraz, and its metabolite 2,4-dimethylaniline (2,4-DMA), in four types of honey (acacia, wild, chestnut, and manuka) and royal jelly. Samples were buffered with 0.2 M dibasic sodium phosphate (pH 9), and subsequently, acetonitrile was employed as the extraction solvent. A combination of primary secondary amine (PSA) and C18 sorbent was used for purification prior to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) analysis. The estimated linearity measured at six concentration levels presented good correlation coefficients (R2) ≥0.99. The recovery, calculated from three different spiking levels, was 62.06–108.79% in honey and 67.58–106.34% in royal jelly, with an RSD <12% for all the tested compounds. The matrix effect was also evaluated, and most of the analytes presented signal enhancement. The limits of quantification (LOQ) ranged between 0.001 and 0.005 mg/kg in various samples. These are considerably lower than the maximum residue limits (MRL) set by various regulatory authorities. A total of 43 market (domestic and imported) samples were assayed for method application. Among the tested samples, three tested negative and three tested positive for cymiazole residues. The residues in the rest of the samples were detected but not quantified. We concluded that the protocol developed in this work is simple and versatile for the routine detection of cymiazole, 2,4-DMA, fipronil, coumaphos, amitraz, and fluvalinate in various types of honey and royal jelly.
Non-targeted metabolomics-guided sildenafil metabolism study in human liver microsomes J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-07 Ju-Hyun Kim, Jun Hyun Jo, Kyung-ah Seo, Hayoung Hwang, Hye Suk Lee, Sangkyu Lee
Metabolomics combined with high-resolution mass spectrometry (HR-MS) and multivariate data analysis has broad applications in the study of xenobiotic metabolism. Although information about xenobiotic metabolism is essential to understand toxic mechanisms, pharmacokinetic parameters and excretion pathways, it is limited to predict all generated metabolites in biological fluids. Here, we revisited sildenafil metabolism in human liver microsomes using a metabolomics approach to achieve a global picture of sildenafil phase 1 metabolism. Finally, 12 phase 1 metabolites were identified in human liver microsomes; M1-M5 were previously known metabolites. The chemical structures of the novel metabolites were elucidated by MS2 fragmentation using an HR-MS system as follows: M6, reduced sildenafil; M7, N,N-deethylation and mono-oxidation; M8, demethanaime, N,N-deethylation and mono-hydroxylation; M9, demethanaime and N,N-deethylation; M10 and M11, mono-oxidation in the piperazine ring after N-demethylation; and M12, mono-oxidation. All metabolites, except M1, were formed by CYP3A4 and CYP3A5. In conclusion, we successfully updated the metabolic pathway of sildenafil in human liver, including 7 novel metabolites using metabolomics combined with HR-MS and multivariate data analysis.
Quantification of glycerophospholipids and sphingomyelin in human milk and infant formula by high performance liquid chromatography coupled with mass spectrometer detector J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-07 Isabelle Tavazzi, Patric Fontannaz, Le Ye Lee, Francesca Giuffrida
Phospholipids and sphingomyelin have a central role in infant nutrition, phospholipid acting as a nutrient carrier of long chain polyunsaturated fatty acids and sphingomyelin having an important role in cognitive function. However, analytical methods to precisely characterize and quantify these compounds in maternal milk are needed. Phospholipids and sphingomyelin were extracted using chloroform and methanol and separated on Polaris 3 Si column 250 × 2.0 mm from Varian and analyzed by high performance liquid chromatography (HPLC) coupled with mass spectrometer detector (MS). The analytical method was validated and repeatability, intermediate reproducibility, and recovery values were calculated. The relative standard deviation of repeatability (CV(r)) and intermediate reproducibility (CV(iR)) values ranged between 2.3 and 7.2% and 9.5 and 17.8%, respectively and the recovery values between 96 and 109%. Finally, the validated method was tested on breast milk samples and on infant formula which were analysed also by HPLC coupled with evaporative light scattering detector (ELSD). Sphingomyelin (9.28 mg 100 mL−1) was the most abundant compound, followed by phosphatidylcholine (5.39 mg 100 mL−1), phosphatidylethanolamine (2.85 mg 100 mL−1) and phosphatidylinositol (1.82 mg 100 mL−1).
Separation and purification of four phenolic compounds from persimmon by high-speed counter-current chromatography J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-07 Jinming Peng, Kaikai Li, Wei Zhu, Xiangyi Deng, Chunmei Li
An efficient method was established by high-speed counter-current chromatography (HSCCC) for preparation of four phenolic compounds from the depolymerization products of persimmon tannin. Using the two solvent systems of n-hexane/ethyl acetate/water (3:17:20, v/v/v) and ethyl acetate/methanol/water (50:1:50, v/v/v), the preparative isolation was successfully performed by a two-step separation. The yields of one run (150 mg crude sample) for gallic acid, methyl gallate, and epigallocatechin-3-gallate-(4β → 8, 2β → O → 7)-epigallocatechin-3-gallate dimer (A-type EGCG dimer) were 4.7, 44.2 and 5.9 mg, respectively. In addition, 4.6 mg epicatechin-3-gallate-(4β → 8, 2β → O → 7)-epicatechin-3-gallate dimer (A-type ECG dimer) was obtained by further preparative high-performance liquid chromatography (prep-HPLC). The purities of these compounds were all above 95.0% and their structures were identified by HPLC/ESI-MS. We found that HSCCC had definite advantages for the preparation of dimeric procyanidins compared with previous methods. Furthermore, it was shown that the four phenolic compounds possessed greater antioxidant activities than Trolox.
LC-MS/MS quantification of felinine metabolites in tissues, fluids, and excretions from the domestic cat (Felis catus) J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-06 Ayami Futsuta, Wataru Hojo, Tamako Miyazaki, Tetsuro Yamashita, Masao Miyazaki
Domestic cat urine contains large concentrations of the unusual amino acid, felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid). A felinine derivative, 3-mercapt-3-methylbutanol is a potential scent signal for letting other animals know that the scent owners are cats. 3-Methylbutanol-glutathione (MBG) is an upstream precursor of the biosynthetic pathway of felinine that may be produced in hepatocytes by conjugation of glutathione with isopentenyl pyrophosphate, an intermediate for cholesterol biosynthesis. However, little evidence exists to support the biosynthesis of MBG biosynthesis in the liver. This study examined the distribution of metabolites of the felinine biosynthetic pathway in multiple tissues, body fluids, and excretions of cats. MBG, the felinine precursor, 3-methylbutanol-cysteinylglycine (MBCG), felinine, and felinine N-acetyl derivative were quantified by liquid chromatography-electron spray ionization-tandem mass spectrometry. All compounds were detected in cat serum. In males, MBG and MBCG contents were significantly higher than felinine and N-acetylfelinine contents. MBG was detected in multiple tissues, including the salivary gland, heart, liver, spleen, gut, kidney, bladder, adipose tissue, and muscle. Sex differences in MBG levels were observed in the liver and other tissues. Felinine and N-acetylfelinine were also detected in those tissues. Furthermore, we detected all compounds in cat bile and fecal samples, indicating that felinine is excreted into the feces via bile from the liver. We conclude that MBG is synthesized in several tissues in a sex-dependent manner. These findings improve our understanding of felinine metabolism and function in cats.
Determination of perfluoroalkyl acid isomers in biosolids, biosolids-amended soils and plants using ultra-high performance liquid chromatography tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-06 Hongna Zhang, Bei Wen, Wen Wen, Yibing Ma, Xiaoyu Hu, Yali Wu, Lei Luo, Shuzhen Zhang
Isomer-specific analysis of perfluoroalkyl acids (PFAAs) is important to accurately assess their environmental source, fate, and human risks. In this study, a method was developed for the determination of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorohexane sulfonate (PFHxS) isomers in biosolids, biosolids-amended soils and plants using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The separation efficiencies of two chromatographic columns and extraction capacities of different methods were tested. Compared with the C18 column (ACQUITY UPLC BEH Shield RP18 column), the column with an alkyl perfluorinated C8 stationary phase (Epic FO LB column), in combination with the distinct MS/MS transitions of analytes, allowed better separation of most isomers. The ion-pair extraction method showed more effective matrix separation than that of the alkaline digestion method, with recoveries ranging from 79.6-105% for biosolids, 80.4-116% for soils, and 68.0-114% for plant tissues. The method detection limits ranged from 10-55, 3-13, and 8–58 pg/g dry weight for biosolids, soil, and plants, respectively. This method was applied successfully to quantify individual isomers in biosolids, biosolids-amended soils and plants. Six PFOA, eight PFOS, and two PFHxS isomers were found in the samples, with linear isomers being the dominant species. Further analysis revealed that the translocation potentials of branched isomers within plants were higher than those of linear isomers.
A rapid quantitative analysis of bile acids, lysophosphatidylcholines and polyunsaturated fatty acids in biofluids based on ultraperformance liquid chromatography coupled with triple quadrupole tandem massspectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-06 Zhangxiao Peng, Qian Zhang, Ziming Mao, Jie Wang, Chunying Liu, Xuejing Lin, Xin Li, Weidan Ji, Jianhui Fan, Maorong Wang, Changqing Su
Much evidence suggested that quantitative analysis of bile acids (BAs), lysophosphatidylcholines (LPCs), and polyunsaturated fatty acids (PUFAs) in biofluids may be very useful for diagnosis and prevention of hepatobiliary disease with a non-invasive manner. However, simultaneously fast analysis of these metabolites has been challenging for their huge differences of physicochemical properties and concentration levels in biofluids. In this study, we present a liquid chromatography–mass spectrometry method with a high throughput analytical cycle (10 min) to fast and accurately quantify fifteen potential biomarkers (eight BAs, four LPCs and three PUFAs) of hepatobiliary disease. The accuracy for the fifteen analytes in plasma and urine matrices was 80.45%–118.99% and 84.55%–112.66%, respectively. The intra- and inter- precisions for the fifteen analytes in plasma and urine matrices were all less than 20% and the lower limit of quantification (LLOQ) of analytes is up to 0.0283–8.2172 nmol/L. Therefore, this method is fast, sensitive and accurate for the quantitative analysis of BAs, LPCs and PUFAs in biofluids. Moreover, the stability and concentration differences of the analytes in plasma and serum were evaluated, and the results demonstrated that LPCs is stable, but PUFAs is very unstable in freeze and thaw cycles, and the concentrations of the analytes in serum were slightly higher than those in plasma. We suggested plasma may be a kind of better bio-sample than serum using for quantitative analysis of metabolites in blood, due to the characteristics of plasma are more close to blood than those of serum.
Simultaneous determination of nine kinds of dominating bile acids in various snake bile by ultrahigh-performance liquid chromatography with triple quadrupole linear iontrap mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-02 Jie Zhang, Yeqin Fan, Yajun Gong, Xiaoyong Chen, Luosheng Wan, Chenggao Zhou, Jiewen Zhou, Shuangcheng Ma, Feng Wei, Jiachun Chen, Jing Nie
Snake bile is one of the most expensive traditional Chinese medicines (TCMs). However, due to the complicated constitutes of snake bile and the poor ultraviolet absorbance of some trace bile acids (BAs), effective analysis methods for snake bile acids were still unavailable, making it difficult to solve adulteration problems. In present study, ultrahigh-performance liquid chromatography with triple quadrupole linear ion trap mass spectrometry (UHPLC-QqQ-MS/MS) was applied to conduct a quantitative analysis on snake BAs. The mass spectrometer was monitored in the negative ion mode, and multiple-reaction monitoring (MRM) program was used to determine the contents of BAs in snake bile. In all, 61 snake bile from 17 commonly used species of three families (Elapidae, Colubridae and Viperidae), along with five batches of commercial snake bile from four companies, were collected and detected. Nine components, Tauro-3α,12α-dihydroxy-7-oxo-5β-cholenoic acid (T1), Tauro-3α,7α,12α,23R-tetrahydroxy-5β-cholenoic acid (T2), taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), cholic acid (CA), Tauro-3α,7α-dihydroxy-12-oxo-5β-cholenoic acid (T3), and Tauro-3α,7α,9α,16α-tetrahydroxy-5β-cholenoic acid (T4) were simultaneously and rapidly determined for the first time. In these BAs, T1 and T2, self-prepared with purity above 90%, were first reported with their quantitative determination, and the latter two (T3 and T4) were tentatively determined by quantitative analysis multi-components by single marker (QAMS) method for roughly estimating the components without reference. The developed method was validated with acceptable linearity (r2 ≥ 0.995), precision (RSD ＜ 6.5%) and recovery (RSD ＜ 7.5%). It turned out that the contents of BAs among different species were also significantly different; T1 was one of the principle bile acids in some common snake bile, and also was the characteristic one in Viperidae and Elapidae; T2 was the dominant components in Enhydris chinensis. This quantitative study of BAs in snake bile is a remarkable improvement for clarifying the bile acid compositions and evaluating the quality of snake bile.
A fast, sensitive, and high throughput method for the determination of esomeprazole in dog plasma by UHPLC-MS/MS: application to formulation development of the compound preparation of esomeprazole J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-04 Ran Liu, Pei Li, Jie Xiao, Yidi Yin, Zheng Sun, Kaishun Bi, Qing Li
To investigate deeply into the preclinical pharmacokinetics and prescription design of esomeprazole, a sensitive, high throughput and robust UHPLC-MS/MS method had been developed and fully validated for the analysis of esomeprazole in dog plasma. Esomeprazole and diazepam (IS) were fast extracted from plasma by alkalified organic solvent, and separated on MP-C18 column with methanol and 0.1% formic acid. The quantification of esomeprazole and IS had been achieved using fragmentation transitions of m/z 346.1 → 198.1 and m/z 285.0 → 193.2 in MRM detection under positive ESI mode. The concentration of esomeprazole in dog plasma was linear with the range of 3.75–500 ng/mL. The precisions of intra- and inter-day were no more than 11.6%, while the accuracies were all within ±9.7% of the nominal values. The recovery was no more than 77.06%, and the matrix effect, stability, dilution integrity tests were all satisfied the currently criterion. Then the method was successfully performed to evaluating pharmacokinetics of esomeprazole and optimizing the prescription of modified esomeprazole with varied addition of sodium bicarbonate. Consequently, a pharmacokinetic study of three doses esomeprazole with the optimized addition of sodium bicarbonate in dogs has been successfully researched for the first time. It could be a promising approach to improve the stabilization of acid-labile esomeprazole and would provide a useful reference for the formulation design of esomeprazole.
A UPLC-MS/MS method for simultaneous determination of 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin in rat plasma and its application in pharmacokinetic and absolute bioavailability studies J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-04 Tingting Liang, Shun Liu, Fang Wang, Jiamei Gu, Yang Lu, Weike Chen, Cunyu Li, Yunfeng Zheng, Guoping Peng
A specific, sensitive, rapid, precise, and reliable UPLC–MS/MS-based method was designed for the first time for the simultaneous determination of 1-deoxynojirimycin (DNJ) and N-methyl-1-deoxynojirimycin (N-CH3-DNJ) in rat plasma. Miglitol was served as the internal standard (IS). An MN-NUCLEODUR HILIC column was assessed to separate the two compounds by isocratic elution using acetonitrile: water with 0.05% formic acid and 6.5 mM ammonium acetate (72:28, v/v) at a flow rate of 0.4 mL/min. A triple quadrupole mass spectrometer was operated in the positive ionization mode using multiple reaction monitoring (MRM), and it was employed to determine transitions of m/z 164.1 → 110.1, 178.1 → 100.1, and 208.1 → 146.1 for DNJ, N-CH3-DNJ, and IS, respectively. The method of the two constituents was validated and the results were acceptable. The absolute bioavailability of DNJ and N-CH3-DNJ in rats was 50 ± 9% and 62 ± 24%, respectively. The method was then successfully used for the first time to study the pharmacokinetic behavior and absolute bioavailability of DNJ and N-CH3-DNJ in rats after intravenous (10 mg/kg) and oral administration (80 mg/kg). The results of this study might provide more information on preclinical pharmacokinetics and a solid basis for assessing the clinical efficacy of DNJ and N-CH3-DNJ.
Comparative Assessment of the Effect of Glyco-engineering on the Pattern and Kinetics of Aggregate Formation of Darbepoetin Alfa using a Stability-Indicating Orthogonal Testing Protocol J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-04 Eman M. Moenes, Medhat A. Al-Ghobashy, Abeer A. Mohamed, Maissa Y. Salem
Darbepoetin alfa (DA); hyper-glycosylated Erythropoietin alfa (EPO) is an essential treatment of anemia in patients with chronic kidney failure and cancer. In this study, DA and EPO were subjected to physicochemical stress factors that might be encountered during production, transport and storage (pH, temperature, agitation, repeated freeze-thaw and oxidation). An orthogonal stability-indicating assay protocol comprised of SE-HPLC, RP-HPLC, ELISA and SDS-PAGE was developed and validated to investigate the effect of further glycosylation of DA on the pattern and kinetics of degradation. Results showed a relatively higher stability and lower tendency to form high molecular weight aggregates in the case of DA when compared to EPO, under equivalent stress conditions. Dimers and aggregates were formed for both drugs across the whole pH range and following incubation at temperatures higher than 2–8 °C or repeated freeze/thaw. The same observation was noted upon agitation of standard samples prepared in the formulation buffers at high speed and upon oxidation with hydrogen peroxide. The agreement between SE-HPLC, supported with spectral purity data and ELISA confirmed the specificity of both techniques for the intact drugs. Results of RP-HPLC and SDS-PAGE indicated that dimerization occurred through disulfide and bi-tyrosine covalent bonds in the case of pH and oxidation, respectively. It was evident that aggregation was significantly suppressed upon increasing the glycan size and under any of the studied stress factors loss of the glycan has not been observed. These observations supported with the slow kinetics of degradation confirmed the superiority of glyco-engineering over chemical pegylation to enhance the stability of EPO. Formation of such potentially immunogenic product-related impurities at all tested stress factors confirmed the need for orthogonal testing protocols to investigate the complex pattern of degradation of such sensitive products.
Separation of aflatoxin B1 from synthetic physiological fluids using talc and diatomite: kinetic and isotherm aspects J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-04 Myroslav Sprynskyy, Iwona Krzemień-Konieczka, Renata Gadzała-Kopciuch, Bogusław Buszewski
The objective of the study was to examine adsorption of the aflatoxin B1 from synthetic gastric fluid and synthetic intestinal fluid by talc, raw and calcined diatomite. The kinetic and equilibrium adsorption processes were studied in the batch adsorption experiments applying high performance liquid chromatography for the aflatoxin B1 determination. The kinetic study showed a very fast adsorption of the aflatoxin B1 onto the selected adsorbents from the both physiological fluids with reaching equilibrium within 1–15 min. The aflatoxin B1 was almost completely adsorbed in initial linear step of the kinetic process that can be described well by the zero-order kinetics model. The experimental data of the equilibrium adsorption were characterized using the Langmuir and Freundlich isotherm models. The high adsorption effectiveness was found in a range of 90%–100% and 60%–100% for the diatomite samples and the talc respectively at the initial concentrations of the aflatoxin B1 as 31–300 ng/mL. The possible mechanisms of the aflatoxin adsorption onto the used mineral adsorbents are also discussed in the work.
Simultaneous determination of saikosaponin a, paeonol, and imperatorin, components of DA-9805, in rat plasma by LC-MS/MS and application to a pharmacokinetic study J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-04 Mi Hye Kwon, Jin Seok Jeong, Jayoung Ryu, Young Woong Cho, Hee Eun Kang
DA-9805 is a new botanical antiparkinson drug candidate formulated using an ethanolic extract of the root of Bupleurum falcatum, the root cortex of Paeonia suffruticosa, and the root of Angelica dahurica. In this study, a sensitive and rapid LC-MS/MS method was developed to simultaneously determine, saikosaponin a, paeonol, and imperatorin, three active/representative ingredients of DA-9805, in rat plasma. Plasma was extracted by mixture of ethyl acetate and methyl tertiary butyl ether. Chromatographic separation was carried out using a C18 column and a gradient elution of mobile phases consisting of 5 mM formic acid in water and acetonitrile. Total chromatographic run time was 10.5 min. Multiple reaction monitoring mode was used for mass spectrometry; the transitions were m/z 779.5 → 617.2 for saikosaponin a in negative-ion mode, m/z 167 → 149 for paeonol and m/z 271.1 → 203 for imperatorin in positive-ion mode. Calibration curves were constructed in the range of 0.5–1000 ng/mL for saikosaponin a, 20–10000 ng/mL for paeonol, and 0.2–1000 ng/mL for imperatorin. All the validation data, including the selectivity, linearity, precision, accuracy, recovery, matrix effect, and stability satisfied the acceptance requirements. The method was successfully applied in a pharmacokinetic study of saikosaponin a, paeonol, and imperatorin following oral administration of DA-9805.
Fractionation separation of human plasma proteins using HPLC with a homemade iron porphyrin based monolithic column J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-04 Doudou Zhang, Yu Zhao, Dandan Lan, Xiaomin Pang, Ligai Bai, Haiyan Liu, Hongyuan Yan
Carbamazepine, lamotrigine, levetiracetam and valproic acid in dried blood spots with liquid chromatography tandem mass spectrometry; method development and validation. J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-04 Camilla Linder, Anna Hansson, Sara Sadek, Lars L. Gustafsson, Anton Pohanka
Monitoring of antiepileptic drugs in children with epilepsy require multiple visits at a clinic for blood collection. Dried blood spot sampling is an alternative way of collection, performed at home by self-collection and can save time and costs for patients and family members. The aim was to develop and validate an LC-MS/MS dried blood spot method for carbamazepine, lamotrigine, levetiracetam and valproic acid with the requirements of using standard equipment and material in a routine laboratory setting.Whatman-903 filter paper was utilized, and discs were punched into a 96 well plate with an automated puncher and barcode reading. Extraction with methanol/water solution including internal standards on an orbital shaker was followed by a vacuum centrifuge step and reconstitution in mobile phase. Bioanalytical validation was performed according to guidelines from European Medicines Agency and additional dried blood spot specific validation.Calibration curves of the four included drugs had R2 values ≥0.994. Therapeutic relevant concentrations were well within measuring ranges. Within and −between run precision had %CV:s of 2.9 to 10.5%. Accuracy (%bias) was between −16.5% (lower limit of quantification) to +7.4%. Blood spots in a volume range of 15–50 μL with hematocrit in expected ranges for this patient group were within precision and accuracy limits. To test the method, concentrations from dried blood spot venous and capillary patient samples (n = 50) were compared with plasma concentrations. Good correlations for all four drugs with R2 of >0.92 was shown.In summary, a fast method for dried blood spots based on a 96 well format was developed for four commonly prescribed antiepileptic drugs. This validated method with traceability in sample preparation by bar code reading makes it suitable for the clinical laboratory.
Determination of 3,5 – Dimethylpyrazolium Glyceroborate Nitrification Inhibitor in Nitrogen Fertilizer Samples: HPLC-DAD Method Development and Validation for 3,5 – Dimethylpyrazole J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-02 Serkan Şahan, Uğur Şahin, Mustafa Başaran, Oğuzhan Uzun, Adem Güneş
3,5–Dimethylpyrazolium Glyceroborate is a nitrification inhibitor (a member of pyrazole derivatives) used for the fixation of nitrogen into the soil. In this study, an HPLC-DAD method was developed and validated for determination of 3,5–Dimethylpyrazole in order to determine 3,5–Dimethylpyrazolium Glyceroborate in fertilizer samples. For method development, analytical parameters like type of eluent solution and column filling material and device parameters like eluent flow rate, column oven temperature and measurement wavelength were all optimized. For method validation, implementations were performed for linearity, limit of detection (LOD), limit of quantification (LOQ), specificity, stability, intra- and inter-day precision and accuracy. The developed and validated method was used for inhibitor detection in nitrogenous fertilizers. Sample analyses were performed with 95.6-103.3% recovery rates and 0–4.61% relative errors.
UPLC–PDA–ESI–qTOF-MS profiling and potent anti-HSV-II activity of Eucalyptus sideroxylon leaves J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-01 Mona M. Okba, Rania A. El Gedaily, Rehab M. Ashour
Eucalyptus is one of the most important and highly exploited genus in family Myrtaceae. An UPLC/PDA/ESI-qTOF-MS method was adopted to identify Eucalyptus sideroxylon Cunn. ex Woolls leaves phytoconstituents. Cytotoxicity of E. sideroxylon leaves phloroglucinol-rich extract (PGRE) on VERO cells was determined. The antiviral effect of PGRE against hepatitis A (HAV), herpes simplex type 1 (HSV-I), herpes simplex type 2 (HSV-II), coxsackie (CoxB4), and adenoviruses was in vitro evaluated using MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). UPLC-MS analysis allowed the identification of 70 metabolites including: 26 triterpenes, 13 phloroglucinols, 8 fatty acids, 5 flavonoids, 5 oleuropeic acid glucosides, 3 gallic acid derivatives, and 10 miscellaneous. Twenty four metabolites identified in the leaves of E. sideroxylon and four in the genus Eucalyptus are reported herein for the first time. PGRE was found to be non-cytotoxic; the concentration that reduced the cell viability by 50% (CC50) was 0.808 mg/mL. Maximum non-toxic concentration (MNTC) of PGRE on Vero cells was 0.312 mg/mL. The best antiviral activity was observed against HSV-II. Its mechanism was through decreasing the viral replication (IC50 189.36 μg/mL, 87.65% inhibition) and attachment on Vero cells (IC50 199.34 μg/mL, 83.13% inhibition) rather than virucidal effect (IC50 293.1 μg/mL, 50.68% inhibition). This study provides a complete map for E. sideroxylon leaves composition. It also suggests the plant as a source of new antiviral agents.
Quantitative analysis of clonidine and ephedrine by a microfluidic system: On-chip electromembrane extraction followed by high performance liquid chromatography J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-01 Mahroo Baharfar, Yadollah Yamini, Shahram Seidi, Monireh Karami
In this work, a microfluidic device was developed for on-chip electromembrane extraction of trace amounts of ephedrine (EPH) and clonidine (CLO) in human urine and plasma samples followed by HPLC-UV analysis. Two polymethylmethacrylate (PMMA) plates were used as substrates and a microchannel was carved in each plate. The microchannel channel on the underneath plate provided the flow pass of the sample solution and the one on the upper plate dedicated to a compartment for the stagnant acceptor phase. A piece of polypropylene sheet was impregnated by an organic solvent and mounted between the two parts of the chip device. An electrical field, across the porous sheet, was created by two embedded platinum electrodes placed in the bottom of the channels which were connected to a power supply. The analytes were converted to their ionized form, passed through the supported liquid membrane (SLM), and then extracted into the acceptor phase by the applied voltage. All the effective parameters including the type of the SLM, the SLM composition, pH of donor and acceptor phases, and the quantity of the applied voltage were evaluated and optimized. Several organic solvents were evaluated as the SLM to assess the effect of SLM composition. Other parameters were optimized by a central composite design (CCD). Under the optimal conditions of voltage of 74 V, flow rate of 28 μL min−1, 100 and 20 mM HCl as acceptor and donor phase composition, respectively, the calibration curves were plotted for both analytes. The limits of detection (LODs) were less than 7.0 and 11 μg L−1 in urine and plasma, respectively. The linear dynamic ranges (LDR) were within the range of 10–450 and 25–500 μg L−1 (r2 ˃ 0.9969) for CLO, and within the range of 20–450 and 30–500 μg L−1 (r2 ˃ 0.9907) for EPH in urine and plasma, respectively. To examine the capability of the method, real biological samples were analyzed. The results represented a high accuracy in the quantitative analysis of the analytes with relative recoveries within the range of 94.6-105.2% and acceptable repeatability with relative standard deviations lower than 5.1%.
Determination of tropane alkaloids by heart cutting reversed phase – Strong cation exchange two dimensional liquid chromatography J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-01 Zhen Long, Yanhai Zhang, Paul Gamache, Zhimou Guo, Frank Steiner, Nana Du, Xiaoda Liu, Yan Jin, Xingguo Liu, Lvye Liu
Current Chinese Pharmacopoeia (ChP) standards apply liquid liquid extraction combined with one dimensional liquid chromatography (1DLC) method for determining alkaloids in herbal medicines. The complex pretreatments lead to a low analytical efficiency and possible component loss. In this study, a heart cutting reversed phase – strong cation exchange two dimensional liquid chromatography (RP – SCX 2DLC) approach was optimized for simultaneously quantifying tropane alkaloids (anisodine, scopolamine and hyoscyamine) in herbal medicines and herbal medicine tablets without further treatment of the filtered extract. The chromatographic conditions were systematically optimized in terms of column type, mobile phase composition and flow rate. To improve peak capacity and obtain symmetric peak shape of alkaloids, a polar group embedded C18 column combined with chaotropic salts was used in the first dimension. To remove the disturbance of non-alkaloids, achieve unique selectivity and acquire symmetric peak shape of alkaloids, an SCX column combined with phosphate buffer was used in the second dimension. Method validation was performed in terms of linearity, precision (0.54–0.82%), recovery (94.1–105.2%), limit of detection (LOD) and limit of quantification (LOQ) of the three analytes varied between 0.067–0.115 mg L−1 and 0.195–0.268 mg L−1, respectively. The method demonstrated superiority over 1DLC method in respect of resolution (less alkaloid co-eluted), sample preparation (no pretreatment procedure) and transfer rate (minimum component loss). The optimized RP – SCX 2DLC approach was subsequently applied to quantify target alkaloids in five herbal medicines and herbal medicine tablets from three different manufactures. The results demonstrated that the developed heart cutting RP – SCX 2DLC approach represented a new, strategically significant methodology for the quality evaluation of tropane alkaloid in related herbal medicines that involve complex chemical matrix.
Metabolites identificaion of two bioactive constituents in Trollius ledebourii in rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-01 Man Liao, Xiaoye Cheng, Xinpeng Diao, Yupeng Sun, Lantong Zhang
Orientin and vitexin, 4’- hydroxyl-2-phenylchromen-4-one, are both major flavones derivatives found in Trollius ledebourii possessing definite pharmacological activities. In this study, in vitro metabolisms investigated on rat liver microsomes (RLMs) and in vivo metabolisms explored on Male Sprague Dawley rats of orientin and vitexin were tested, respectively. A systematic method based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was developed to characterize metabolites by means of electrospray ionization (ESI) mass spectrometry in positive ion mode. An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to observe probable relevant metabolites. By comparison of chromatographic behaviors with reference substances, exact protonated ions, MS/MS fragment ions and relevant literature, a total of 12 metabolites of orientin and 23 metabolites of vitexin were detected, respectively, which suggested that orientin is more metabolically stable than vitexin. Oxidation, methylation, acetylation, reduction, loss of C6H10O5 and glucuronide conjugation were the major biotransformation routes of both of them in rats. More significant, glutamine conjugation, loss of CO and loss of CH2O were the unique metabolic pathways of vitexin compared with that of orientin for the first time. Besides, most metabolites were observed in rat urine and feces, implying that urine and feces were the active metabolic places for flavones. This is the first study on metabolisms of orientin and vitexin in vitro and in vivo simultaneously and the proposed metabolic pathways of them might provide further understanding of their pharmacological mechanisms and later study on their excretion.
Comparison and characterization of volatile compounds as markers of oils stability during frying by HS–SPME-GC/MS and Chemometric analysis J. Chromatogr. B (IF 2.603) Pub Date : 2017-11-01 Ibtissem ben Hammouda, Flavia Freitas, Ammar Sonda, M.D.R. Gomes Da Silva, Mohamed Bouaziz
The formation and emission of volatile compounds, including the aldehydes and some toxic compounds of oil samples, ROPO pure (100%) and the blended ROPO/RCO (80-20%), were carried out during deep frying at 180 °C. The volatile profile of both oil samples was evaluated by an optimized HS–SPME-GC/MS method, before and after 20, 40 and 60 successive sessions of deep-frying. Actually, from 100 detected compounds, aldehydes were found to be the main group formed. In addition, the oil degradation under thermal treatment regarding the volatile compounds were evaluated and compared. Consequently, the blended ROPO/RCO revealed fewer formations of unsaturated aldehydes, including toxic ones, such as acrolein, and showed a greater stability against oxidative thermal degradation compared to ROPO pure.
Trace anti-inflammatory β-carboline alkaloid identified in Arenaria kansuensis by two-dimensional chromatography coupled with UniElut C18AEX based solid-phase extraction re-enrichment technology J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-31 Yulei Cui, Na Shen, Shuo Wang, Zenggen Liu, Lijuan Mei, Jun Dang, Yanduo Tao
Traditional Chinese medicine is important for discovery of drug precursors. However, information about trace chemical composition of them is very limited due to the lack of appropriate enrichment and chromatographic purification methods In our work, A. kansuensis was taken as an example, a novel two-dimensional reversed-phase/hydrophilic interaction liquid chromatography coupled with UniElut C18AEX solid-phase extraction re-enrichment method based on anti-inflammatory bioactivity-guided assay was developed for gathering and purifying trace β-carboline alkaloids with high purity from the ethyl acetate extract of A. kansuensis. Extraction with ethyl acetate as the first enrichment method, then, a UniElut C18AEX column was employed to re-enrich trace fraction which was hardly detected by diode array detector during high performance liquid chromatography analysis, eighteen grams of UniElut C18AEX was used as sorbent material to pack a 60 mL pipette tip for the extraction of β-carboline alkaloids from 100 mL of ethyl acetate sample. The whole extraction process was finished in 10 min, and the volume of eluent used was only 120 mL. The enriching fraction (100 mg) was used for the following two-dimensional purification. First-dimensional preparation was carried on a RP-Megress-C18 prep column, and four anti-inflammatory fractions were obtained from the 100 mg re-enriching sample with a recovery of 66.9%. A HILIC-XAmide prep column was selected for the second dimensional preparation. Finally, two pair of analogue β-carboline alkaloids and one other β-carboline alkaloid were purified from A. kansuensis. The purity of the isolated compounds was >98%, which indicated that the method was efficient to re-enrich and manufacture single trace β-carboline alkaloids with high purity from A. kansuensis. Additionally, this method showed great potential to serve as a good example for the purification and enrichment of analogue structure anti-inflammation carboline alkaloids from other plant materials.
Comparative study on the interaction between 3 CYP2C9 allelic isoforms and benzbromarone by using LC–MS/MS method J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-31 Lingli He, Chuan Li, Xuyuan Liu, Qingqing Yang, Haizhi Zhang, Weiren Xu, Luyong Zhang, Changxiao Liu
Benzbromarone is a uricosuric drug metabolized predominantly by cytochrome P450 2C9 from in vitro findings. Human CYP2C9 exhibits extensive genetic polymorphism and numbers of clinic studies have demonstrated that CYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of benzbromarone. But in vitro study on the interaction between CYP2C9 allelic isoforms and benzbromarone was rare. Here, an LC–MS/MS method was established and validated to determine the concentration of benzbromarone in different CYP2C9 enzyme incubation systems for the drug-enzyme interaction study. By selecting appropriate internal standard and optimizing separation system, including mobile phase, sample solvent and gradient elution condition, this LC–MS/MS method was developed with fine linearity (r2 ≥ 0.996), good reproducibility (RSD ≤ 6.6%), high stability (92.37–114.67%), efficient recovery (91.23–109.82%) and acceptable matrix effect (110.54–115.31%). Based on this method, the interaction between 3 CYP2C9 allelic isoforms and benzbromarone was researched by kinetics parameters (Km, Vmax, Clint). As a result, CYP2C9*1 displayed the highest metabolic activity towards benzbromarone, CYP2C9*2 showed a little lower catalytic activity than CYP2C9*1 (relative clearance/*1 = 85.86%), CYP2C9*3 showed the lowest catalytic activity (relative clearance/*1 = 21.57%). The result illustrated that various CYP2C9 allelic isoforms showed different enzymatic activities towards benzbromarone, which could offer effective consultation for personalized administration in clinic.
Multivariate optimization of solvent bar microextraction combined with HPLC-UV for determination of Trace amounts of vincristine in biological fluids J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-29 Mahsa Kiani, Mahnaz Qomi, Farshad Hashemian, Mehdi Rajabi
In the current work, an efficient method named solvent bar microextraction-high performance liquid chromatography-UV detection (HPLC-UV) was developed for preconcentration and determining the trace amount of vincristine (VCR) in biological samples such as plasma and urine. Briefly, VCR was extracted from an aqueous sample with pH 10.7 (donor phase) into 1-octanol as the supported liquid membrane (SLM) which is inserted into the pores of the hollow fiber and followed by back extraction into an aqueous receiving phase (pH = 3.1). Studying the factors affecting the extraction performance in order to achieve a high extraction efficiency, requires the design of experiments (DOE) approach. In this regards, diverse factors’ effects including the pH value of donor and acceptor phases, extraction time, extraction temperature, stirring rate and salt content of the donor phase were considered. The optimum experimental condition was as following: pH of the source phase, 10.7; pH of the receiving phase, 3.1; stirring rate, 1000 rpm; extraction temperature, 51 °C; extraction time, 60 min and 11.3% w/v NaCl in the sample solution. Under the optimal; extraction condition, a favorable preconcentration factor equal to 98.5 was achieved. The linearity range was obtained in the domain of 0.05–5 mg L−1. The limits of detection and quantification were 0.015 and 0.05 mg L−1. Within-day and between-day RSDs of the proposed SBME method were 4.1% and 12.5%, respectively. Finally, the applicability of the implemented SBME method was evaluated by the extraction and quantification of VCR from biological samples such as urine and plasma and satisfactory results were obtained.
A rapid and sensitive supercritical fluid chromatography/tandem mass spectrometry method for detection of ezetimibein dog plasma and its application in pharmacokinetic studies J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-28 Mengxiong Di, Zhenbao Li, Qikun Jiang, Tianyang Wang, Wenjuan Zhang, Zhisu Sun, Jin Sun, Xiaohong Liu
The aim of this study is to develop and validate a rapid, high-selective and sensitive supercritical fluid chromatography/tandem mass spectrometry (SFC-MS/MS) with a multiple reactions monitoring (MRM) mode method for the detection of ezetimibe in dog plasma. Several conditions were optimized systematically as follows: lipid-lipid extraction (LLE) performances were used to extract analytes from dog plasma; an ACQUITY HSS C18 SB (1.8 μm, 3.0 × 100 mm) column was employed to separate the target compounds; the triple-quadrupole mass spectrometry equipped with electrospray ionization (ESI) source was applied to detect ezetimibe. The method, which required a relatively small volume of plasma (100 μL), was obtained at concentration ranging from 1.0 to 100 ng/mL(r2 > 0.99). The lower limit of quantification (LLOQ)for ezetimibe was found to be as low as 1.0 ng/mL. In addition, the validations of the methodology including sensitivity, recovery, matrix effect, intra- and inter-day precision, accuracy and stability were all within acceptable limits. The Cmax, AUC0–inf and Tmax values obtained in our study were 52.2 ± 6.3, 820.6 ± 4.3 and 1.25 ± 0.35 for reference formulation; 61.8 ± 12.6, 924.2 ± 4.7 and 2.00 ± 0 for test formulation. In conclusion, the method developed in this study can be successfully applied to pharmacokinetic studies after oral administration of ezetimibe in dogs.
Purification and biochemical characterization of a 22-kDa stable cysteine- like protease from the excretory-secretory product of the liver fluke Fasciola hepatica by using conventional techniques. J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-28 Ahmed Hemici, Roumaila Sabrina Benerbaiha, Dalila Bendjeddou
This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40-60%) followed by a gel filtration through Sephadex G–100 and DEAE− cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9 U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS–PAGE to be 22 kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS–PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35–40 °C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The Km values obtained for this protease were 5.4, 13, 160 and approximately 1000 μM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5 mM) or iodoacetamide (10 mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease.
Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-27 Abdelbasset Chafik, Abdelkhalid Essamadi, Safinur Yildirim Çelik, Ahmet Mavi
Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37 U/mg were obtained. The native enzyme had a molecular weight of 268 kDa and was composed of four subunits of equal size (65 kDa). The enzyme showed optimal activity at a temperature of 45 °C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al3+, Cd2+ and Mg2+, whereas Ca2+, Co2+ and Ni2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The Km and Vmax of the enzyme for hydrogen peroxide were 37.31 mM and 6185157 U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with Ki value of 14.43 μM, the IC50 was found to be 16.71 μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert.
A validated UPLC-MS/MS method for biomonitoring the exposure to the fragrance 7-hydroxycitronellal J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-26 Markus Stoeckelhuber, Dusan Krnac, Nikola Pluym, Max Scherer, Edgar Leibold, Gerhard Scherer
7-Hydroxycitronellal is a synthetic fragrance (CAS No. 107-75-5) which is used commonly in cosmetics, washing- and cleaning agents and as flavoring in foods. Due to its broad application in various fields, 7-hydroxycitronellal was selected for the development of a biomonitoring method for the quantitative exposure assessment within the frame of the cooperation project of the German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the German Chemical Industry Association (VCI). For this purpose, an ultra performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) based method was developed for the determination of potential biomarkers of 7-hydroxycitronellal (7-HC) in human urine samples. 7-Hydroxycitronellylic acid (7-HCA) turned out to be the quantitatively most important metabolite of 7-HC in human urine, occurring in 100 times higher amounts than 7-hydroxycitronellol (7-HCO) or other potential metabolites. Therefore, an analytical method for 7-HCA was developed using stable isotope-labeled 7-HCA as internal standard (IS). The method includes a cleavage step of possible metabolite conjugates with an enzyme mix of ß-glucuronidase and arylsulfatase. Subsequent sample cleanup was performed by liquid-liquid extraction (LLE) with dichloromethane. The method was calibrated by calculating the linear regression between the analyte/IS ratio and the nominal 7-HCA concentrations in water. The method was validated according to approved standard guidelines and proved to be robust, reliable and sensitive for the human biomonitoring of 7-HC. The method was applied to urine samples of 40 adult volunteers from the general population. 7-HCA was quantifiable in urine of all subjects. Thus the developed method proved to be suitable for assessing the background exposure to 7-HC in the general population.
An UPLC-MS/MS Method for Quantifying Tetrandrine and Its Metabolite Berbamine in Human Blood: Application to a Human Pharmacokinetic Study J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-26 Guangyi Yang, Chenning Zhang, Pei Hu, Meiling Zhu, Ming Hu, Song Gao
Tetrandrine (TET) is approved by the China Food and Drug Administration (CFDA) for the treatment of silicosis. However, patients can’t use this effective drug chronically due to side effects. The purpose of this study is to develop an UPLC-MS/MS method to quantify TET and its major metabolite and determine the parent and metabolite concentrations in the blood in a single dose human pharmacokinetic study. A Restek UItra BiPh column (100 × 2.1 mm, 5 μm) was used with acetonitrile and 0.1% formic acid in water as the mobile phases. The mass analysis was performed in a Waters Xevo TQ mass spectrometer via multiple reaction monitoring (MRM) with positive scan mode. A one-step protein precipitation by acetonitrile was used to extract the analytes from blood sample. The method showed linearity in the concentration ranges of 2.05 to 1050.00 ng/mL for TET and 1.27 to 650.00 ng/mL for berbamine. The intra/inter-day precisions were less 15% for these two analytes. The extraction recoveries of these two analytes were from 75.6% to 107.8% and the matrix effects ranged from 92.4% to 110.4%. The stabilities of these compounds in plasma were evaluated by analyzing three different concentrations following storage at 25 °C for 6 h, and −80 °C for 30 days. All the samples displayed less than 15.0% variations. The validated method was applied to PK study in human and the PK parameters of TET and berbamine were determined. In conclusion, a robust and sensitive LC-MS/MS method was developed and validated. In addition, the results of human PK experiment showed that TET and berbamine could be accumulated and more study is needed to establish a reasonable dose segment.
Effective phospholipid removal from plasma samples by solid phase extraction with the use of copper (II) modified silica gel cartridges J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-26 Jolanta Flieger, Małgorzata Tatarczak-Michalewska, Anna Kowalska, Anna Madejska, Tomasz Śniegocki, Anna Sroka-Bartnicka, Monika Szymańska-Chargot
An UHPLC-MS/MS method for simultaneous quantification of human amyloid beta peptides Aβ1-38, Aβ1-40 and Aβ1-42 in cerebrospinal fluid using micro-elution solid phase extraction J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-23 Ping-ping Lin, Wei-li Chen, Fei Yuan, Lei Sheng, Yu-jia Wu, Wei-wei Zhang, Guo-qing Li, Hong-rong Xu, Xue-ning Li
Amyloid beta (Aβ) peptides in cerebrospinal fluid are extensively estimated for identification of Alzheimer’s disease (AD) as diagnostic biomarkers. Unfortunately, their pervasive application is hampered by interference from Aβ propensity of self-aggregation, nonspecifically bind to surfaces and matrix proteins, and by lack of quantitive standardization. Here we report on an alternative Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous measurement of human amyloid beta peptides Aβ1-38, Aβ1-40 and Aβ1-42 in cerebrospinal fluid (CSF) using micro-elution solid phase extraction (SPE). Samples were pre-processing by the mixed-mode micro-elution solid phase extraction and quantification was performed in the positive ion multiple reaction monitoring (MRM) mode using electrospray ionization. The stable-isotope labeled Aβ peptides 15N51- Aβ1-38, 15N53- Aβ1-40 and 15N55- Aβ1-42 peptides were used as internal standards. And the artificial cerebrospinal fluid (ACSF) containing 5% rat plasma was used as a surrogate matrix for calibration curves. The quality control (QC) samples at 0.25, 2 and 15 ng/mL were prepared. A “linear” regression (1/x2 weighting): y = ax + b was used to fit the calibration curves over the concentration range of 0.1–20 ng/mL for all three peptides. Coefficient of variation (CV) of intra-batch and inter-batch assays were all less than 6.44% for Aβ1-38, 6.75% for Aβ1-40 and 10.74% for Aβ1-42. The precision values for all QC samples of three analytes met the acceptance criteria. Extract recoveries of Aβ1-38, Aβ1-40 and Aβ1-42 were all greater than 70.78%, both in low and high QC samples. The stability assessments showed that QC samples at both low and high levels could be stable for at least 24 h at 4 °C, 4 h at room temperature and through three freeze-thaw cycles without sacrificing accuracy or precision. And no significant carryover effect was observed. This validated UHPLC/MS/MS method was successfully applied to the quantitation of Aβ peptides in real human CSF samples. Our work may provide a reference method for simultaneous quantitation of human Aβ1-38, Aβ1-40 and Aβ1-42 from CSF.
Homogenization-assisted cavitation hybrid rotation extraction and macroporous resin enrichment of dihydroquercetin from Larix gmelinii J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-23 Yu Xia, Yinhang Wang, Wei Li, Chunhui Ma, Shouxin Liu
A new reliable, transposable and cost-effective assay for absolute of total plasmatic bevacizumab by LC-MS/MS in human plasma comparing two internal standard calibration approaches J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-23 Rachel Legeron, Fabien Xuereb, Stephane Chaignepain, Alain-Pierre Gadeau, Stephane Claverol, Jean-William Dupuy, Sarah Djabarouti, Thierry Couffinhal, Jean-Marie Schmitter, Domonique Breilh
The quantification of monoclonal antibodies (mAbs) such as bevacizumab, a recombinant humanized immunoglobulin G1 (hIgG1), in biological fluids, is an essential prerequisite to any pharmacokinetic preclinical and clinical study. To date, reference techniques used to quantify mAbs rely on enzyme-linked immunosorbent assay (ELISA) lacking specificity. Furthermore, the commercially available ELISA kit to quantify bevacizumab in human plasma only assesses the free fraction of the drug. However, the conditions of storage and analysis of plasma samples could alter the physiological equilibrium between the free, bound and partially bound forms of bevacizumab and this could result in over- or underestimation of drug concentration. We developed a new assay for absolute quantification of total fraction of bevacizumab by liquid chromatography tandem mass spectrometry (LC–MS/MS) basing identification and quantification of bevacizumab on two specific peptides. In this report we compare our assay with two internal standard (IS) calibration approaches: one using a different human mAb (Trastuzumab) and the other using a stable isotope labeled specific peptide. After enrichment by affinity chromatography on protein A and concentration by ultrafiltration, human plasma samples were proteolyzed by trypsin. Linearity was established from 12.5 to 500 μg/mL with an interday accuracy ranging from 101.7 to 110.6% and precision from 7.0% to 9.9%.This study demonstrates the importance of the choice of the IS in quantifying bevacizumab in human plasma and highlights the difficulty of reaching a reliable proteolysis with a sufficient recovery. We developed a reliable and cost-effective LC-MS/MS method to quantify total plasmatic fraction of bevacizumab in human plasma. Through our development we proposed a generic methodology easily transposable to quantify all IgG1 subclass very useful for clinical pharmacokinetics studies.
Determination of benzotriazoles and benzothiazoles in human urine by UHPLC-TQMS J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-23 Jiufeng Li, Hongzhi Zhao, Yanqiu Zhou, Shunqing Xu, Zongwei Cai
Benzotriazole (BTR) and benzothiazole (BTH) derivatives are extensively applied in industrial processes and consumer products, and are thus frequently detected in the environmental matrices. Due to their potential estrogenic effects reported in animal studies, the assessment of human exposure to BTRs and BTHs is important. In this study, a method was developed and validated to determine six BTRs and five BTHs in human urine by using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-TQMS) in positive electrospray ionization mode. After de-conjugation by β-glucuronidase, urine samples were liquid-liquid extracted for the measurement of total concentrations of BTRs and BTHs. The linearity, detection limit, precision, recovery, accuracy and matrix effect were evaluated. The limits of detection were less than 0.5 ng/mL. The validated method demonstrated good precision (RSD% < 15%), linearity (R2 > 0.99), recovery (>80%) and accuracy (80–100%). The method was successfully applied for the analysis of 22 human urine samples. Four out of eleven targeted compounds were detected in more than half of participants at ng/mL levels.
Quantification of isoflavonoids and triterpene saponins in Astragali Radix, the root of Astragalus membranaceus, via reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-23 Seung-Min Lee, Ji-Seon Jeong, Ha-Jeong Kwon, Seon-Pyo Hong
Astragali Radix, the root of Astragalus membranaceus Bunge, is one of the most frequently used crude drugs in Asian medicine. We developed a quantification method for 6 components (calycosin, formononetin, astragaloside I–IV) of Astragali Radix and Hwanggi-gyeji-omul-tang (HGOT) using reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection (RP-HPLC-IPAD). The plants were extracted in 80% ethanol for 2 h. All target components were detected with good sensitivity using sodium hydroxide (as a post-column eluent). The limit of detection (S/N = 3) and limit of quantification (S/N = 10) of the target components ranged from 0.10–1.00 ng and from 0.30–3.00 ng, respectively. The coefficients of linear regression ranged from 0.9993–1.0000, all interday and intraday precision values were < 3.64%, and the average recovery ranged from 99.00–102.97% for Astragali Radix and 97.73–102.57% for HGOT. This method exhibited good selectivity, sensitivity, and reproducibility and can be used directly without any pretreatment steps. Our method will therefore be useful as a quality control measure for Astragali Radix.
Application of an UHPLC-MS/MS method to tissue distribution and excretion study of 2-(2-hydroxypropanamido) benzoic acid in rats J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-23 Qili Zhang, Jiao Guan, Shasha Li, Yunli Zhao, Zhiguo Yu
2-(2-Hydroxypropanamido) benzoic acid (HPABA) is a potential anti- inflammatory agent isolated from the marine fungus Penicillium chrysogenum. In vivo tissue distribution and excretion of HPABA were unknown. Therefore, the purpose of this paper was to establish an UHPLC-MS/MS method for the determination of HPABA and proceed to apply for the study of tissue distribution and excretion in rats. The results obtained indicated that HPABA in tissues was rapidly distributed and cleared. The highest level was found in intestine and stomach, followed by kidney, liver, lung and heart. These observations showed that HPABA was well distributed into abundant blood-supply tissues, suggesting that the blood flow or perfusion rate of organs plays a major role. A small amount of HPABA was detected in brain indicated that the ability of it in crossing the blood brain barrier (BBB) was very weak in rats. Excretion studies demonstrated that HPABA was mainly excreted by kidney. The total cumulative excretion of HPABA were 54.3% and 44.1% in male and female rats, respectively. In addition, gender-related differences in the excretion and distribution profile of HPABA were observed in rats.
Preparation of p-aminophenol modified superparamagnetic iron oxide nanoparticles for purification of α-amylase from the bovine milk J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-20 Hamed Farzi-Khajeh, Kazem D. Safa, Siavoush Dastmalchi
Currently, modified Fe3O4 magnetic nanoparticles frequently were used as nanocarriers for proteins and enzymes purification. In the present study, Fe3O4 magnetic nanoparticles were prepared, and their surfaces were modified with p-aminophenol affinity ligand immobilized by different linkers. The modified nanocarriers were used for the purification of α-amylase from the bovine milk (after precipitation the casein) by affinity purification. To evaluate the effectiveness of the p-aminophenol modified magnetic nanocarriers, the three different types of nanocarriers with different linkers having varying lengths were prepared. All nanocarriers were characterized and validated using FT-IR, SEM, EDX, VSM and XRD analysis methods According to our results, p-aminophenol ligand attached to the nanocarrier by long linkers better separates the α-amylase from the casein free skim milk with 49.66% recovery and 48.18-fold purification efficiency. The results of this study showed that our novel magnetic nanocarriers have the capacity to be used for fast, reproducible and cost-effective purification of α-amylase.
Determination of the R(−) and S(+)-enantiomers of vigabatrin in human plasma by ultra-high-performance liquid chromatography and tandem mass-spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-20 Paul Duhamel, Marwa Ounissi, Thomas Le Saux, Hugues Bienayme, Catherine Chiron, Vincent Jullien
An analytical method was developed for the quantification in plasma of the R and S enantiomers of vigabatrin (VGB), a drug used for the treatment of some refractory pediatric epileptic syndromes. After adding 50 μl of the internal standard, which consisted of a 15 mg/L solution of deuterated racemic VGB, and 100 μL of water to 100 μL of plasma samples, a protein precipitation was performed by adding 600 μL of methanol. The supernatant was evaporated to dryness under a stream of nitrogen and the dry residue was reconstituted with 500 μL of water. Then, 100 μL of 0.01 M o-phthaldialdehyde and 0.01 M N-acetyl-L-cysteine in borate buffer (0.1 M, pH = 9.5) were added for pre-column derivatization of the enantiomers as diastereomeric isoindoles. One μL of the resulting mixture was injected in the chromatographic system. The chromatographic separation was performed in gradient elution mode at a flow rate of 400 μL/min using a phenomenex EVO C-18 column with a mobile phase composed of 5 mM ammonium acetate and a methanol:acetonitrile (63:37 v/v) mixture. Detection was performed by mass spectrometry in selected reaction monitoring mode using heated electrospray ionization in positive mode as the ion source. Intra- and inter-day precision and accuracy were lower than 15% over the calibration range (0.2–50 mg/L for each enantiomer) and the method was successfully used to assess plasma concentrations of VGB in epileptic children.
Study on the discrimination between Corydalis Rhizoma and its adulterants based on HPLC-DAD-Q-TOF-MS associated with chemometric analysis J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-20 Xiaojiao Zheng, Wenlin Zheng, Jingjing Zhou, Xue Gao, Zhihui Liu, Na Han, Jun Yin
Corydalis Rhizoma (Yanhuso), is a crucial antianalgesic Traditional Chinese Medicine in clinic. Its adulterants (Northeast Yanhusuo), which are also from Corydalis and distributed in Northeast area of China, are used instead of Corydalis Rhizoma in the local places with a long history. In this paper, we compared the chemical differences of Corydalis Rhizoma and its seven adulterants by HPLC-DAD-Q-TOF-MS associated with chemometric analysis. 48 alkaloids are identified from them, and 14 alkaloids are reported for the first time based on the Mass data. The classification of different species is verified through PLS-DA and Hierarchical Clustering Heatmap analysis based on 26 samples. We find that large discrimination existing in the categories and content of the alkaloids between different species. C.y. is similar with C.t., which mainly contains protoberberine, tetrahydroprotoberberine and protopine types of alkaloids. Six other Northeast Yanhusuo, including C.r., C.w., C.a. as well as its three formas, can be classified into one category, because they mainly contained benzophenanthridines and aprophines.
Enrichment of chelidonine from Chelidonium majus L. using macroporous resin and its antifungal activity J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-20 Jialiang Pan, Yang Yang, Rui Zhang, Hanwen Yao, Kangkang Ge, Manyin Zhang, Ling Ma
Chelidonium majus L. (greater celandine) has been used as an herbal medicine for several centuries. This study investigated an efficient method to purify chelidonine from the extract of C. majus L. using macroporous adsorption resins and evaluated the antifungal activity of chelidonine against Botryosphaeria dothidea as a model strain. Static adsorption and desorption tests revealed that D101 was the optimal resin for chelidonine purification. Pseudo-second-order kinetics model and Freundlich equation model were the most suitable for evaluating the endothermic and spontaneous adsorption processes of chelidonine on D101. Dynamic adsorption and desorption tests on D101 columns showed that the concentration of chelidonine increased 14.16-fold, from 2.67% to 37.81%, with the recovery yield of 80.77%. The antifungal activity of enriched chelidonine products was studied with B. dothidea. The results showed that the EC50 of crude extracts, enriched chelidonine products, and chelidonine standard against B. dothidea were 3.24 mg/mL, 0.43 mg/mL, and 0.77 mg/mL, respectively. The result of antifungal activity test showed that chelidonine had the potential to be a useful antifungal agent. Moreover, the enrichment method of chelidonine was highly efficient, low cost, and harmless to the environment for industrial applications.
“ONE-POT” ETHYL CHLOROFORMATE DERIVATIZATION AND LIQUID LIQUID EXTRACTION OF REDUCED GLUTATHIONE IN ERYTHROCYTE AND ITS QUANTITATIVE GCMS ANALYSIS J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-20 Alessandra Manca, Eugenio Alladio, Paola Massarenti, M. Paola Puccinelli, Antonella De Francesco, Erika Del Grosso, Giulio Mengozzi, Marco Pazzi, Marco Vincenti
A simple “one-pot” derivatization and liquid liquid extraction (LLE) procedure was developed for GC-MS analysis of reduced glutathione (GSH) analysis in erythrocytes. The metabolite was extracted by 5% (w/v) TCA, the supernatant treated with ECF and ethanol − pyridine media and the derivative separated and detected by gas chromatography − mass spectrometry using a short non − polar capillary GC column at a high column − head pressure. Total analysis time was 11 minutes. The process was optimized by a Design of Experiment. The method was validated showing a good linearity over the 25.4-813.4 μM concentration range, providing satisfactory results in terms of intra-day and inter-day precision as well as an optimal accuracy. The new method was evaluated in a pilot study involving patients with severe protein malnutrition. Comparing this group with a group of healthy subjects revealed significantly lower GSH concentrations in erythrocytes in the former, proving thus that the described GC-MS method could be employed for fast and simple GSH analysis in clinical studies.
Comprehensive characterization of the in vitro and in vivo metabolites of limonin in human samples using LC-Q-TOF/MS J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-19 Shijia Liu, Peidong Chen, Nongshan Zhang, Luning Sun, Guoliang Dai, Lei Zhu, Changyin Li, Yang Zhao, Li Zhang, Haiyan Fu, Wenzheng Ju
Limonin is a bitter triterpenoid dilactone in the genus Citrus with potential medicinal value. In this study, the metabolism of limonin in human was characterized by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). A total of 7 metabolites were identified from human samples. Among them, 3 metabolites of M1, M2 and M4 were detected in urine and feces, and the others were found in intestinal bacteria sample. Notably, M1 and M3 were chemically synthesized, of which the structures were further confirmed by NMR spectra data. The metabolism of limonin involved three major pathways, namely, reduction, hydrolysis and methylation. The reduction and hydrolysis were commonly observed in ring D of limonin. The metabolites showed decomposition in ring A. This study provides data for the metabolism of limonin in humans, and will contribute to explain its biological activity.
Detailed phenolic composition of Vidal grape pomace by ultrahigh-performance liquid chromatography-tandem mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-16 Lanxin Luo, Yan Cui, Shuting Zhang, Lingxi Li, Hao Suoc, Baoshan Sun
Vidal Blanc grape (Vitis vinifera cv.) is the predominant white grape variety used for the production of icewine in China’s Liaoning province. In this paper, the development and validation of the method by ultrahigh-performance liquid chromatography-tandem mass spectrometry has been performed for determination of the detailed phenolic composition in the skin, seed and stem of Vidal grapes. The validation of the method was realized by calculating the linearity, repeatability, precision, stability and the limits of detection (LOD) and quantification (LOQ) of standard solutions. All the curves exhibited good linearity (r2 > 0.9997) and the LOD and LOQ were in the range of 0.002–0.025 and 0.006–0.086 μg/ml, respectively. Good repeatability (RSD < 4.3%) and stability (RSD < 3.7%) were also found. Results confirmed that the developed method was more effective and sensitive for simultaneous determination of the major phenolic compounds in Vidal grape pomace. The optimized and validated method of ultrahigh-performance liquid chromatography tandem two complementary techniques, fourier transform ion cyclotron resonance mass spectrometry and triple-quadrupole mass spectrometry, allowed to identify and quantify up to 35 phenolic compounds in Vidal grape pomace, which has, as far as we know, been reported for this grapevine variety for the first time. Seeds, skins and stems exhibited different qualitative and quantitative phenolic profiles. These results provided useful information for recovery of phenolic antioxidants from different parts of icewine pomace.
Determination of the authenticity of plastron-derived functional foods based on amino acid profiles analyzed by MEKC J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-16 Lin-Qiu Li, Joewel T. Baibado, Qing Shen, Hon-Yeung Cheung
Plastron is a nutritive and superior functional food. Due to its limited supply yet enormous demands, some functional foods supposed to contain plastron may be forged with other substitutes. This paper reports a novel and simple method for determination of the authenticity of plastron-derived functional foods based on comparison of the amino acid (AA) profiles of plastron and its possible substitutes. By applying micellar electrokinetic chromatography (MEKC), 18 common AAs along with another 2 special AAs — hydroxyproline (Hyp) and hydroxylysine (Hyl) were detected in all plastron samples. Since chicken, egg, fish, milk, pork, nail and hair lacked of Hyp and Hyl, plastron could be easily distinguished. For those containing collagen, a statistical analysis technique — principal component analysis (PCA) was adopted and plastron was successfully distinguished. When applied the proposed method to authenticate turtle shell glue in the market, fake products were commonly found.
An LC-MS/MS method for quantification of the active abiraterone metabolite Δ(4)-abiraterone (D4A) in human plasma J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-16 Merel van Nuland, Hilde Rosing, Jelle de Vries, Huib Ovaa, Jan H.M. Schellens, Jos H. Beijnen
Δ(4)-Abiraterone (D4A) is a recently discovered active metabolite of the oral anti-androgen drug abiraterone acetate. For quantification of this metabolite in human plasma, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated. Human plasma samples of patients treated with abiraterone acetate were prepared by protein precipitation with acetonitrile. The method was validated over a linear range of 0.2 to 20 ng/mL. Intra-assay and inter-assay variabilities were within ±15% of the nominal concentrations for quality control (QC) samples at medium and high concentrations and within ±20% at the lower limit of quantification (LLOQ), respectively. The described method for quantification of D4A was validated successfully and implemented to support therapeutic drug monitoring in patients treated with abiraterone acetate.
Simultaneous determination and pharmacokinetics of fourteen bioactive compounds in rat plasma by LC-ESI-MS/MS following intravenous injection of Gegen-Sanqi compatibility solution J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-16 Bin Ji, Xing Zhao, Peipei Yu, Lin Meng, Yunli Zhao, Zhiguo Yu
A high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and fully validated for simultaneous determination of puerarin, 3′-hydroxy puerarin, 6″-O-xylosyl puerarin, 3′-methoxy puerarin, mirificin, puerarin-7-O-glucoside, daidzin, daidzein, daidzein-7-O-glucoside, ginsenoside-Rd, notoginsenoside-R1, ginsenoside-Re, ginsenoside-Rg1, ginsenoside-Rb1 in rat plasma after intravenous injection of 5 mL/kg Gegen-Sanqi compatibility solution (containing Pueraria flavonid 10.8 mg/mL and Panax notoginsenosidum 5.4 mg/mL). After addition of internal standard (IS) baicalin, the analytes and IS were recovered from rat plasma by protein precipitation using acetonitrile containing 0.1% formic acid. Chromatographic separation was performed on a Capcell pak MG C18 column (3.0 mm × 75 mm, 3.0 μm) at 35 °C with a flow rate of 0.75 mL/min. Mass spectrometry was conducted using multiple reaction monitoring in positive mode. The method was linear for all of the analytes over 1000 times concentration range with correlation coefficients greater than 0.99. The precision and accuracy of the LC-MS/MS assay based on the three analytical quality control (QC) levels were well within the acceptance criteria from FDA guidance for bioanalytical method validation. Method recoveries were higher than 75% and the matrix effects were minimal. All analytes were stable under tested conditions. It was the first time to study the pharmacokinetics of daidzein-7-O-glucoside in rat plasma. To the best of our knowledge, this is the first study for simultaneous determination of so many analytes in rat plasma after intravenous injection of Gegen-Sanqi compatibility solution. It was expected that the present work would provide some helpful references for the apprehension of the mechanism of action and further clinical efficacy studies of Gegen-Sanqi herb-pair.
Oxalic acid quantification in mouse urine and primary mouse hepatocyte cell culture samples by ion exclusion chromatography-mass spectrometry J. Chromatogr. B (IF 2.603) Pub Date : 2017-10-16 Alexander Schriewer, Moritz Brink, Kathrin Gianmoena, Cristina Cadenas, Heiko Hayen
Due to medical relevance and a direct correlation with some diseases, accurate quantification of oxalic acid in different complex matrices is required. Effective chromatographic separation of this strong carboxylic acid was achieved by ion exclusion chromatography (IELC). Sensitive and selective detection was carried out by means of electrospray ionization-tandem mass spectrometry. Furthermore, it was shown that the isobaric interference of lactic acid is chromatographically resolved. Structurally similar compounds like glyoxylic acid and glycolic acid were baseline separated as well. The application of stable isotope dilution analysis with 13C2 oxalic acid facilitated precise quantification. The developed method was validated with a reference oxalate sample of human urine diluted to a range of 10 to 500 μM. Finally, the applicability of this method was demonstrated on complex matrices, like mouse urine and supernatants of primary mouse hepatocyte cell cultures.
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