Journal of Chromatography B (v.1054, #C)

A simple and selective bioanalytical method was developed for simultaneous determination of levodopa and carbidopa in rat plasma by LC–MS/MS. Levodopa and carbidopa are small polar molecules, posing challenges in the development of selective and efficient chromatography conditions. Perfluoropentanoic acid (PFPA), a volatile ion-pairing agent, was utilized to enhance chromatographic characteristics of both compounds in the reversed-phase mechanism. The ion-pairing chromatography played an essential role in mitigating matrix effects and achieving adequate separation between interfering background peaks and those of the analytes of interest, especially for levodopa. A 96-well based, automated liquid-liquid extraction, via the use Hamilton NIMBUS liquid handlers, was developed. Butyl alcohol, when mixed with ethyl acetate, greatly increased the recovery of both levodopa and carbidopa. The addition of PFPA further enhanced recovery for both analytes. Sodium metabisulfite, an antioxidant, was used to stabilize levodopa and carbidopa in rat plasma. The method was validated in the ranges of 50–10,000 ng/mL and 25–5000 ng/mL for levodopa and carbidopa, respectively, using levodopa-d3 and carbidopa-d3 as internal standards. The validated method was successfully applied to analyze rat plasma samples from in-life studies.
Keywords: Levodopa and carbidopa; Perfluoropentanoic acid (PFPA); Ion-pairing agent reversed-phase chromatography; Automated liquid-liquid extraction (LLE); Matrix stability;

Viral particles are used in medical applications as vaccines or gene therapy vectors. In order to obtain product of high purity, potency and safety for medical use purification of virus particles is a prerequisite, and chromatography is gaining increased attention to meet this aim. Here, we report on the use of ion-exchange and hydrophobic interaction chromatography on monolithic columns for purification of mumps virus (MuV) and measles virus (MeV). Efficiency of the process was monitored by quantification of infective virus particles (by 50% cell culture infective dose assay) and total virus particles, and monitoring of their size (by Nanoparticle Tracking Analysis). Ion-exchange chromatography was shown to be inefficient for MuV and best results for MeV were obtained on QA column with recovery around 17%. Purification of MuV and MeV by hydrophobic interaction chromatography resulted in recoveries around 60%. Results showed that columns with small channels (d  = 1.4 μm) are not suitable for MuV and MeV, although their size is below 400 nm, whereas columns with large channels (6 μm) showed to be efficient and recoveries independent on the flow rate up to 10 mL/min. Heterogeneity of the virus suspension and its interday variability mostly regarding total-to-infective particle ratio was observed. Interestingly, a trend in recovery depending on the day of the harvest was also observed for both viruses, and it correlated with the total-to-infective particle ratio, indicating influence of the virus sample composition on the chromatography results.
Keywords: Mumps virus; Measles virus; Hydrophobic interaction chromatography; Monoliths; Nanoparticle tracking analysis; Virus heterogeneity;

Sanjie Zhentong capsule, a well-known traditional Chinese medicine prescription, are used for the treatment of endometriosis-related diseases. In this study, a simple, rapid and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method was developed for the simultaneous determination of ten bioactive constituents, including peimine, peiminine, peimisine, loureirin A, loureirin B, 7,4′-dihydroxyflavone, pterostilbene, ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 in rat plasma after oral administration of Sanjie Zhentong capsule. The sample preparations for protein removal was accomplished using a simple methanol precipitation method. The analytes were completely separated from the endogenous compounds on an Agilent Poroshell 120 SB-C18 column (4.6 mm × 150 mm, 2.7 μm) using an isocratic elution with methanol − 0.1% formic acid aqueous (4/1, v/v) as a mobile phase. The single-run analysis time was as short as 14.0 min. The inter-day and intra-day precision of the quality control samples exhibited relative standard deviations (RSD) <9.5% and the accuracy values ranged from −8.6% to 15.0%. The lower limits of quantification (LLOQ) were 10, 10, 10, 10, 10, 10, 5, 10, 10 and 20 ng/mL for peimine, peiminine, peimisine, loureirin A, loureirin B, 7,4′-dihydroxyflavone, pterostilbene, ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, respectively. The analytical method was successfully applied to a pharmacokinetic study of the multi-components after oral administration of Sanjie Zhentong Capsule in rats.
Keywords: LC–MS; MS; Non-compartment model; Pharmacokinetics; Sanjie zhentong capsule;

Metabolic profiling of newly reported two topoisomerase inhibitors namely fluorescein hydrazones (FLHs) were studied in rat liver microsomes (RLMs) and the data were acquired in a liquid chromatography (LC) ion trap mass spectrometry. (E)-3',6'-dihydroxy-2-((2-nitrobenzylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-one (1) was bio-transformed into two mono-hydroxylated, one double hydroxylated and a tetra hydroxylated metabolites, on the other hand, (Z)-3',6'-dihydroxy-2-((2-oxoindolin-3-ylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-one (2) was bio-transformed into eight possible metabolites by cleavage reduction, hydroxylation, cleavage reduction together with hydroxylated and azo-reduction in RLMs in presence of NADPH-generating system. No metabolites were generated in the absence of either NADPH or microsomes for the compounds (1 and 2), indicating a likely involvement of CYP450 enzymes and the cofactor NADPH in the metabolisms. The structures of those metabolite were elucidated by MS/MS fragmentation study.
Keywords: FLHs; RLMs; topoisomerase inhibitors; metabolic profiling;

Simultaneous determination of tryptophan and 8 metabolites in human plasma by liquid chromatography/tandem mass spectrometry by Lysiane Boulet; Patrice Faure; Patrice Flore; Julien Montérémal; Véronique Ducros (36-43).
Tryptophan (Trp) is an essential amino-acid and the precursor of many biologically active substances such as kynurenine (KYN) and serotonin (5HT). Its metabolism is involved in different physiopathological states, such as cardiovascular diseases, cancer, immunomodulation or depression. Hence, the quantification of Trp catabolites, from both KYN and 5HT pathways, might be usefulfor the discovery of novel diagnostic and follow-up biomarkers.We have developed a simple method for quantification of Trp and 8 of its metabolites,involved in both KYN and 5HT pathways, using liquid chromatography coupled to tandem mass spectrometry. We also validated the methodin human plasma samples, according to NF EN ISO 15189 criteria.Our method shows acceptable intra- and inter-day coefficients of variation (CV) (<12% and <16% respectively). The linearity entirelycovers the human plasma range. Stabilities of whole blood and of residues weredetermined, as well as the use of 2 different types of collectiontube, enabling us to adapt our process. Matrix effects and reference values showed good agreement compared to the literature.We propose here a method allowing the simultaneous quantification of a panel of Trp catabolites, never used before to our knowledge. This method, witha quickchromatographic runtime (15 min) and simple sample preparation, has beenvalidated according to NF EN ISO 15189 criteria. The method enables the detailed analysis of these metabolic pathways, which are thought to be involved in a number of pathological conditions.
Keywords: Tryptophan; Kynurenine; Anthranilic acid; 3-hydroxykynurenine; 5-hydroxytryptophan; Serotonin;

Centrifugal partition chromatography in the isolation of minor ecdysteroids from Cyanotis arachnoidea by Halima Meriem Issaadi; Yu-Chi Tsai; Fang-Rong Chang; Attila Hunyadi (44-49).
Phytoecdysteroids are known for their various beneficial bioactivities in mammals including a non-hormonal anabolic and adaptogenic effect. Cyanotis arachnoidea extracts are extensively utilized worldwide as ecdysteroid-rich materials for various purposes, e.g. food supplementation, use in agriculture and aquaculture, etc. Preparative chromatography of ecdysteroids requires extensive use of methods of different selectivity, and only a very limited number of papers are available on related application of modern liquid-liquid chromatographic techniques.In this work, a centrifugal partition chromatography (CPC) method was developed for the isolation of two minor ecdysteroids, dacryhainansterone and calonysterone, from a pre-purified commercial extract of Cyanotis arachnoidea. The biphasic solvent system was optimized by HPLC, and was composed of n-hexane – ethyl acetate – methanol – water (1:5:1:5, v/v/v/v). The isolated dacryhainansterone and calonysterone represented 99.1% and 99.7% purity, respectively.Calonysterone exerts a stronger effect on the protein kinase B (Akt) phosphorylation in mammalian skeletal muscle cells than the abundant 20-hydroxyecdysone, while no related data are available on dacryhainansterone. Despite their presence in food supplements, neither compound has appropriately been assessed for safety and efficacy. The reported method allows the gram scale isolation of these compounds, opening ways to their in-depth pharmacological investigation.
Keywords: Preparative liquid-liquid chromatography; Cyanotis arachnoidea extract; Anabolic food supplement; Ecdysterone; Dacryhainansterone; Calonysterone;

Characterization and application of restricted access carbon nanotubes in online extraction of anticonvulsant drugs from plasma samples followed by liquid chromatography analysis by Rodrigo Campos dos Santos; Adriana Kaori Kakazu; Mariane Gonçalves Santos; Fábio Antônio Belinelli Silva; Eduardo Costa Figueiredo (50-56).
Anticonvulsant drugs are often used in the treatment of epilepsy. However, their therapeutic monitoring is often necessary in order to obtain an appropriate dose adjustment, due to the proximity between their therapeutic and toxic ranges. The aim of this study was to carry out the synthesis, characterization and use of restricted access carbon nanotubes (RACNTs) in an online method for the analyses of phenobarbital and carbamazepine and primidone from untreated human blood plasma by column switching liquid chromatography. Therefore, the synthesis of RACNTs was carried out through coating commercial Carbon nanotubes with bovine serum albumin (BSA) to subsequently use them as adsorbents in a column switching system operating in the backflush mode. This material was evaluated through the construction of the kinetic and isotherm curves. The experimental data for the interaction of primidone with RACNTs were adequately adjusted to the chemisorption and Sips models for the kinetic and adsorption studies, respectively. The analytical curves ranged from 2.0 to 40.0 mg L−1, with correlation coefficients higher than 0.99, for all the analytes. The LODs of 0.1, 0.1 and 0.01 μg mL−1 were defined for PHB, PRM and CBZ, respectively. The relative standard deviation values ranged from 1.0% to 8.4% for the intra assay precision and from 2.7% to 7.6% for inter assay precision. The relative error values ranged from −13.4% to 7.7% for the intra assay accuracy and from −8.6% to 2.5% for the inter assay accuracy. The method was adequately used in the therapeutic monitoring of anticonvulsant drugs in human plasma samples.
Keywords: Restricted access carbon nanotubes; Column switching; Bi-dimensional chromatography; Protein exclusion; Anticonvulsant drugs;

Enzyme cross-linkers, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde (GA), have been used to improve the stability of immobilized enzymes. We have developed a relatively stable and high-activity immobilized trypsin through EDC and GA cross-linking. Carboxymethyl chitosan (CM-CTS)-functionalized magnetic nanoparticles (Fe3O4@CM-CTS) were prepared, and characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis, X-ray diffraction, and transmission electron microscopy. The CM-CTS loading of Fe3O4 @CM-CTS was 8.701%, and the CM-CTS coating did not cause any significant changes in the crystal structure of Fe3O4. The Fe3O4@CM-CTS nanoparticles exhibited superparamagnetic properties. Trypsin was successfully immobilized on Fe3O4@CM-CTS via EDC and GA cross-linking (Fe3O4@CM-CTS-EDC-TRY and Fe3O4@CM-CTS-GA-TRY, respectively). Trypsin immobilization was verified by FTIR and enzyme assays. Changes in the secondary structures of the immobilized trypsin were present in both Fe3O4@CM-CTS-EDC-TRY and Fe3O4@CM-CTS-GA-TRY. However, kinetic studies demonstrated that the immobilized trypsin retained efficient biocatalytic activity. Fe3O4@CM-CTS-EDC-TRY and Fe3O4@CM-CTS-GA-TRY both showed maximum catalytic activity at pH 8.4 and 45 °C, and retained 71% and 88.5%, respectively, of their initial activities after 6 usage cycles, and 80% and 88% of their initial activities after being stored for 14 d at 4 °C. The Fe3O4@CM-CTS-GA-TRY showed higher activity and conformational stability than Fe3O4@CM-CTS-EDC-TRY, which indicates that GA is effective for the immobilization of trypsin on Fe3O4@CM-CTS.
Keywords: Magnetic nanoparticles; Carboxymethyl chitosan; Trypsin; Enzyme immobilization; Cross-linkers;

Display OmittedIn this work, for the first time, a new hyphenated technique of stable isotope-labeling derivatization-ultrasound-assisted dispersive liquid–liquid microextraction has been developed for the simultaneous determination of monoamine neurotransmitters (MANTs) and their biosynthesis precursors and metabolites. The developed method was based on ultra high performance liquid chromatography tandem mass spectrometry detection using multiple-reaction monitoring mode. A pair of mass spectrometry sensitizing reagents, d0-10-methyl-acridone-2-sulfonyl chloride and d3-10-methyl-acridone-2-sulfonyl chloride, as stable isotope probes was utilized to facilely label neurotransmitters, respectively. The heavy labeled MANTs standards were prepared and used as internal standards for quantification to minimize the matrix effects in mass spectrometry analysis. Low toxic bromobenzene (extractant) and acetonitrile (dispersant) were utilized in microextraction procedure. Under the optimized conditions, good linearity was observed with the limits of detection (S/N > 3) and limits of quantification (S/N > 10) in the range of 0.002–0.010 and 0.015–0.040 nmol/L, respectively. Meanwhile, it also brought acceptable precision (4.2–8.8%, peak area RSDs %) and accuracy (recovery, 96.9–104.1%) results. This method was successfully applied to the simultaneous determination of monoamine neurotransmitters and their biosynthesis precursors and metabolites in rat brain microdialysates of Parkinson's disease and normal rats. This provided a new method for the neurotransmitters related studies in the future.
Keywords: In vivo microdialysis; Neurotransmitters; Triple quadrupole mass spectrometry; Sensitivity enhancement; Stable isotope-labeling derivatization; Targeted metabolomics;

In the present study, a novel sample preparation method based on magnetic core-mesoporous shell microspheres with C8-modified interior pore walls (C8-Fe3O4@mSiO2) was established for the identification of 20(S)-protopanaxadiol (PPD) metabolites in rat plasma by UPLC-Q-TOF-MS/MS analysis. C8-Fe3O4@mSiO2 allowed selective extraction of PPD metabolites from rat plasma by excluding macromolecules in the plasma owing to size exclusion effect. Five extraction conditions including the amount of C8-Fe3O4@mSiO2 microspheres used, extraction time, elution solvents, elution volume, and elution time were investigated and optimized. The present method was compared with two conventional sample preparation methods: protein precipitation and C8 solid phase extraction (C8-SPE). Our method provided higher UPLC intensity of result than protein precipitation method. While the resulting intensity of our method and that of C8-SPE were not significantly different, it consumed less processing time (15 min 55 s for C8-Fe3O4@mSiO2, and 27 min 30 s for C8-SPE). Finally, the proposed method was successfully applied in the identification of PPD metabolites in vivo, in which a total of 17 metabolites and the parent drug were identified in rat plasma.
Keywords: 20(S)-Protopanaxadiol; Metabolite; C8-modified interior pore-walls; UPLC-Q-TOF; Restricted access material;

The combined use of analytical tools for exploring tetanus toxin and tetanus toxoid structures by Caroline Bayart; Sébastien Peronin; Elisa Jean; Joseph Paladino; Philippe Talaga; Marc Le Borgne (80-92).
Display OmittedAldehyde detoxification is a process used to convert toxin into toxoid for vaccine applications. In the case of tetanus toxin (TT), formaldehyde is used to obtain the tetanus toxoid (TTd), which is used either for the tetanus vaccine or as carrier protein in conjugate vaccines. Several studies have already been conducted to better understand the exact mechanism of this detoxification. Those studies led to the identification of a number of formaldehyde-induced modifications on lab scale TTd samples. To obtain greater insights of the changes induced by formaldehyde, we used three industrial TTd batches to identify repeatable modifications in the detoxification process. Our strategy was to combine seven analytical tools to map these changes. Mass spectrometry (MS), colorimetric test and amino acid analysis (AAA) were used to study modifications on amino acids. SDS-PAGE, asymmetric flow field flow fractionation (AF4), fluorescence spectroscopy and circular dichroism (CD) were used to study formaldehyde modifications on the whole protein structure. We identified 41 formaldehyde-induced modifications across the 1315 amino acid primary sequence of TT. Of these, five modifications on lysine residues were repeatable across TTd batches. Changes in protein conformation were also observed using SDS-PAGE, AF4 and CD techniques. Each analytical tool brought a piece of information regarding formaldehyde induced-modifications, and all together, these methods provided a comprehensive overview of the structural changes that occurred with detoxification. These results could be the first step leading to site-directed TT mutagenesis studies that may enable the production of a non-toxic equivalent protein without using formaldehyde.
Keywords: Tetanus toxin; Tetanus toxoid; Formaldehyde; Analytical tools; Amino acid; Vaccine;

Metabolic profiling of newly reported five topoisomerase inhibitors namely ethyl fluorescein hydrazones (EtFLHs) were studied in rat liver microsomes (RLMs) and the data were acquired in a liquid chromatography (LC) ion trap mass spectrometry. Hydroxyl group containing EtFLHs derivatives (1–3) were bio-transformed into hydrolyzed, mono-hydroxylated and hydrolyzed together with mono-hydroxylated metabolites. On the other hand, nitro and methoxy groups containing EtFLHs derivatives (4–5) were bio-transformed into hydrolyzed, hydrolyzed together with mono-hydroxylated and azo-reductive metabolites in the presence of NADPH. No metabolites were observed in the absence of either NADPH or microsomes for the compounds (1–5), indicating a likely involvement of CYP450 enzymes and cofactor NADPH in the metabolisms.
Keywords: EtFLHs; RLMs; Topoisomerase inhibitors; Drug metabolism;

Separation and purification of polyphenols from red wine extracts using high speed counter current chromatography by Yuanyuan Li; Lingxi Li; Yan Cui; Shuting Zhang; Baoshan Sun (105-113).
Polyphenols are important compounds of red wine owing to their contribution to sensory properties and antioxidant activities. In this study, high-speed counter-current chromatography (HSCCC) coupled with semi-preparative HPLC was used for large-scale separation and purification of polyphenols from red wine extracts. With the solvent system of hexane-ethyl acetate-water (1-50-50), various oligomeric procyanidins including monomer catechin, epicatechin, dimers B1, B2; phenolic acids including coutaric acid, caftaric acid and other type of polyphenols were largely separated within 370 min and most of these compounds presented high yields (0.97 mg to 13.79 mg) with high purity (90.34% to 98.91%) after the semi-preparative HPLC isolation. Using the solvent system of Methyl tert-Butyl Ether (MTBE) − n-butyl alcohol- acetonitrile-water (1-40-1-50, acidified with 0.01% trifluoroacetic acid (TFA)) by one-step HSCCC of 100 mg of the red wine extracts, the major anthocyanins, i.e., malvidin-3-O-glucoside, delphinidin-3-O-glucoside and peonidin-3-O-glucoside, as well as two polymeric proanthocyanidin fractions were successfully separated one another within 320 min. The yields of malvidin-3-O-glucoside, delphinidin-3-O-glucoside and peonidin-3-O-glucoside were 12.12 mg, 1.78 mg and 11.57 mg with the purity of 92.74%, 91.03% and 91.21%, respectively. Thiolysis-UPLC analysis indicated that the two polymeric proanthocyanidin fractions presented high purity, with mean degree of polymerization of 7.66 ± 0.12 and 6.20 ± 0.09, respectively. The further experiments on the antioxidant activities by DPPH radical test, FRAP assay and ABTS method showed that all of the isolated procyandins and anthocyanins and the two polymeric proanthocyanidin fractions, with exception of phenolic acids possessed much greater antioxidant activities compared to standard Trolox and  l-ascorbic acid (2–14 times).
Keywords: Polyphenols; Procyanidins; Anthocyanins; HSCCC; Red wine; Antioxidant activity;