Application of immunocapture and nanoflow LC-MS/MS to overcome the barriers to the quantitation of oxytocin in plasma of women in third stage labour.

AIMS: Postpartum haemorrhage (PPH) is the leading cause of maternal mortality worldwide. To prevent PPH, the WHO recommends administration of oxytocin (OT) immediately after birth, i.e. during the third stage of labour (TSL). Previous studies demonstrate that methods to quantify OT in biological matrices, e.g. enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) lack the specificity and/or sensitivity to accurately quantify OT in plasma from women administered OT during TSL. This is due to increased metabolic clearance of OT in late-stage pregnancy and at the time of childbirth, resulting in extremely low OT plasma concentrations. This study describes the development of an ultra-sensitive bioanalytical method that overcomes the issues previously reported and enables accurate pharmacokinetic analyses of exogenously administered OT in TSL. METHODS: A selective and sensitive assay to quantify OT in TSL plasma was developed. Immunoprecipitation (IP) was applied to selectively extract OT from the TSL plasma, thereby generating clean extracts compatible with nanoflow LC (nLC). nLC-MS/MS was chosen for its high sensitivity and ability to differentiate between OT and potentially co-captured OT-like immunoreactive products. RESULTS: The presented methodology is accurate and precise, with a good linear fit between 100-10 000 fg mL-1 OT. TSL plasma samples from a clinical phase 1 study (NCT02999100) were analysed successfully, enabling OT quantification down to 100 fg mL-1. CONCLUSIONS: The presented IP-nLC-MS/MS method succeeded in overcoming the sensitivity challenge related to the assay of OT in TSL plasma and thereby revealing the PK profiles of OT in TSL plasma clinical study samples.

IP-LC-MSMS Enables Identification of Three Tau O-GlcNAcylation Sites as O-GlcNAcase Inhibition Pharmacodynamic Readout in Transgenic Mice Overexpressing Human Tau.

O-ß-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates tau phosphorylation and aggregation: the pharmacological increase of tau O-GlcNAcylation upon treatment with inhibitors of O-GlcNAc hydrolase (OGA) constitutes a potential strategy to tackle neurodegenerative diseases. Analysis of tau O-GlcNAcylation could potentially be used as a pharmacodynamic biomarker both in preclinical and clinical studies. The goal of the current study was to confirm tau O-GlcNAcylation at S400 as a pharmacodynamic readout of OGA inhibition in P301S transgenic mice overexpressing human tau and treated with the OGA inhibitor Thiamet G and to explore if additional O-GlcNAcylation sites on tau could be identified. As a first step, an immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) methodology was developed to monitor changes in O-GlcNAcylation around S400 of tau in mouse brain homogenate (BH) extracts. Second, additional O-GlcNAc sites were identified in in-house produced recombinant O-GlcNAcylated human tau at relatively high concentrations, thereby facilitating collection of informative LC-MS data for identification of low-concentration O-GlcNAc-tryptic tau peptides in human transgenic mouse BH extracts. This strategy enabled, for the first time, identification of three low abundant N-terminal and mid-domain O-GlcNAc sites of tau (at S208, S191, and S184 or S185) in human transgenic mouse BH. Data are openly available at data.mendeley.com (doi: 10.17632/jp57yk9469.1; doi: 10.17632/8n5j45dnd8.1; doi: 10.17632/h5vdrx4n3d.1).

In Vitro and In Vivo Study of the Gastrointestinal Absorption and Metabolisation of Hymenocardine, a Cyclopeptide Alkaloid.

Hymenocardine is a cyclopeptide alkaloid present in the root bark of Hymenocardia acida. In traditional African medicine, the leaves and roots of this plant are used to treat malaria, and moderate in vitro antiplasmodial activity has been reported for hymenocardine. However, in view of its peptide-like nature, potential metabolisation after oral ingestion has to be taken into account when considering in vivo experiments. In this study, the stability and small intestinal absorption of hymenocardine was assessed using an in vitro gastrointestinal dialysis model. In addition, potential liver metabolisation was investigated in vitro by incubation with a human S9 fraction. Moreover, hymenocardine was administered to rats per os, and blood and urine samples were collected until 48 and 24 h after oral administration, respectively. All samples resulting from these three experiments were analyzed by LC-MS. Analysis of the dialysate and retentate, obtained from the gastrointestinal dialysis model, indicated that hymenocardine is absorbed unchanged from the gastrointestinal tract, at least in part. After S9 metabolisation, several metabolites of hymenocardine could be identified, the major ones being formed by the reduction and/or the loss of an N-methyl group. The in vivo study confirmed that hymenocardine is absorbed from the gastrointestinal tract unchanged, since it could be identified in both rat plasma and urine, together with hymenocardinol, its reduction product.

Selection of chemical markers for the quality control of medicinal plants of the genus Cecropia.

CONTEXT: Several Cecropia (Cecropiaceae) species are traditionally used in Latin America for the treatment of a variety of diseases including diabetes, arterial hypertension, asthma, bronchitis, anxiety, and inflammation. At present, a number of commercial products based on these plants have been introduced into the market with very little information on methods for guaranteeing their quality and safety. OBJECTIVE: This work proposes potential chemical markers for the quality control of the raw materials of Cecropia obtusifolia Bertol., Cecropia peltata L., Cecropia glaziovii Snethl., Cecropia pachystachya Trécul, and Cecropia hololeuca Miq. METHODS: The Herbal Chemical Marker Ranking System (Herb MaRS) developed by the National Institute of Complementary Medicine (NICM) at the University of Western Sydney was used for selecting chemical markers for the quality control of selected medicinal species of Cecropia. This review covers the period from 1982 to 2016. RESULTS: Chlorogenic acid, flavonoidal glycosides (orientin, isoorientin, vitexin, isovitexin, and rutin), catechin, epicatechin, procyanidins (B2, B5, and C1), steroids (ß-sitosterol), and triterpenoids (α-amyrin, pomolic, tormentic and ursolic acids) were selected as chemical markers for the quality control of the leaves. CONCLUSION: It is necessary to establish comprehensive standards for guaranteeing quality, safety and efficacy of herbal drugs. The selection of adequate chemical markers for quality control purposes requires a good knowledge about the chemical composition of medicinal plants and their associated biological properties. To the best of our knowledge this review article is the first to address the identification and quantitative determination of the chemical markers for the genus Cecropia.

A First Step in the Quest for the Active Constituents in Filipendula ulmaria (Meadowsweet): Comprehensive Phytochemical Identification by Liquid Chromatography Coupled to Quadrupole-Orbitrap Mass Spectrometry.

Filipendula ulmaria (meadowsweet) is traditionally used for the treatment of inflammatory diseases and as a diuretic and antirheumatic. Extracts of Filipendulae herba are on the market in the European Union as food supplements. Nevertheless, its active constituents remain to be revealed. During this study, the phytochemical composition of Filipendulae Ulmariae Herba was comprehensively characterised for the first time with two complementary generic ultrahigh-performance liquid chromatography-photodiode array-accurate mass mass spectrometry methods. Selective ion fragmentation experiments with a hybrid quadrupole-orbital trap mass spectrometer significantly contributed to compound identification: a total of 119 compounds were tentatively identified, 69 new to F. ulmaria. A rich diversity of phenolic constituents was detected and only a few non-phenolic phytochemicals were observed. Metabolisation and pharmacological studies should be conducted to investigate which of these constituents or metabolites there of contribute to the activity of F. ulmaria after oral intake.

Saponins and Flavonoids from an Infusion of Herniaria hirsuta.

Stone diseases present a major health problem in the Western society, since both urinary and biliary stones occur with a relatively high prevalence of 10-12 % and 10-20 %, respectively, and demonstrate a high recurrence rate. At the moment treatment is mainly based on interventional procedures, or prophylactic and dissolution therapy. However, many of the current drugs cause severe side effects, and therefore, there is an increasing interest in natural medicines. At the moment no registered herbal medicinal products are available for treatment of gallstones. Since an infusion of Herniaria hirsuta L. has a proven efficacy against urolithiasis and cholelithiasis, its phytochemical composition has been investigated. Two previously undescribed triterpene saponins, 28-O-{[ß-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)]-[ß-D-glucopyranosyl-(1-6)]-ß-D-glucopyranosyl}-medicagenic acid and 3-O-[α-L-rhamnopyranosyl-(1 → 3)-ß-D-glucuronopyranosyl]-28-O-{[ß-D-glucopyranosyl-(1 → 3)-ß-D-xylopyranosyl-(1 → 4)]-[ß-D-apiofuranosyl-(1 → 3)]-α-L-rhamnopyranosyl-(1 → 2)-ß-D-fucopyranosyl}-medicagenic acid and three known flavonoids, quercetin-3-O-(2″-O-α-L-rhamnopyranosyl)-ß-D-glucuronopyranoside, rutin, and narcissin (isorhamnetin-3-O-rutinoside), were isolated using flash chromatography and successive semi-preparative HPLC and were well characterized by MS and 1D and 2D NMR spectroscopic techniques. These findings could contribute to the development of a standardized extract that can be used in prophylaxis and treatment of gall and kidney stones.

Development of immunoprecipitation - two-dimensional liquid chromatography - mass spectrometry methodology as biomarker read-out to quantify phosphorylated tau in cerebrospinal fluid from Alzheimer disease patients.

In Alzheimer's disease (AD) brain, one of the histopathological hallmarks is the neurofibrillary tangles consisting of aggregated and hyperphosphorylated tau. Currently many tau binding antibodies are under development to target the extracellular species responsible for the spreading of the disease in the brain. As such, an in-house developed antibody JNJ-63733657 with picomolar affinity towards tau phosphorylated at both T212 and T217 (further named p217+tau) was recently tested in phase I clinical trial NCT03375697. Following multiple dose administration in healthy subjects and subjects with AD, there were dose dependant reductions in free p217+tau fragments in cerebrospinal fluid (CSF) following antibody administration, as measured with a novel single molecule ELISA assay (Simoa PT3 x PT82 assay), demonstrating epitope engagement of the therapeutic antibody [Galpern, Haeverans, Janssens, Triana-Baltzer, Kolb, Li, Nandy, Mercken, Van Kolen, Sun, Van Nueten, 2020]. Total p217+tau levels also were reduced in CSF as measured with the Simoa PT3 x PT82 assay. In this study we developed an orthogonal immunoprecipitation - liquid chromatography - triple quadrupole mass spectrometry (IP-LC-TQMS) assay to verify the observed reductions in total p217+ tau levels. In this assay, an excess of JNJ-63733657 is added to the clinical CSF to ensure all p217+tau is bound by the antibody instead of having a pool of bound and unbound antigen and to immunoprecipitate all p217+tau, which is followed by on-bead digestion with trypsin to release surrogate peptides. Tryptic peptides with missed cleavages were monitored when phosphorylation occurred close to the cleavage site as this induced miscleavages. Compared with acidified mobile phases typically used for peptide analysis, reversed phase LC with mobile phase at basic pH resulted in sharper peaks and improved selectivity and sensitivity for the target peptides. With this setup a diphospho-tau tryptic peptide SRTPSLPTPPTREPK*2 could be measured with pT217 accounting for at least one of the phospho-sites. This is the first time that the presence of a diphopsho-tau peptide is reported to be present in human CSF. A two-dimensional LC-TQMS method was developed to remove matrix interferences. Selective trapping of diphospho-peptides via a metal oxide chromatography mechanism was achieved in a first dimension with a conventional reversed phase stationary phase and acidified mobile phase. Subsequent elution at basic pH enabled detection of low picomolar p217+tau levels in human CSF (lower limit of quantification: 2 pM), resulting in an approximate 5-fold increase in sensitivity. This enabled the quantification of total p217+tau in CSF leading to the confirmation that in addition to reductions in free p217+tau levels total p217+tau levels were also reduced following administration of the tau mAb JNJ-63733657, correlating with the previous measurement with the PT3 x PT82 Simoa assay. An orthogonal sample clean-up using offline TiO2/ZrO2 combined with 1DLC-TQMS was developed to confirm the presence of mono-ptau (pT217) tryptic peptides in CSF.

Compound Characterization and Metabolic Profile Elucidation after In Vitro Gastrointestinal and Hepatic Biotransformation of an Herniaria hirsuta Extract Using Unbiased Dynamic Metabolomic Data Analysis.

Herniaria hirsuta L. (Caryophyllaceae) is used for treatment of urinary stones and as a diuretic. Little is known about the active compounds and the mechanism of action. The phytochemical composition of H. hirsuta was comprehensively characterized using UHPLC-UV-HRMS (Ultrahigh-Performance Liquid Chromatography-Ultraviolet-High Resolution Mass Spectrometry) data. An in vitro gastrointestinal model was used to simulate biotransformation, which allowed the monitoring of the relative abundances of individual compounds over time. To analyze the longitudinal multiclass LC-MS data, XCMS, a platform that enables online metabolomics data processing and interpretation, and EDGE, a statistical method for time series data, were used to extract significant differential profiles from the raw data. An interactive Shiny app in R was used to rate the quality of the resulting features. These ratings were used to train a random forest model. The most abundant aglycone after gastrointestinal biotransformation was subjected to hepatic biotransformation using human S9 fractions. A diversity of compounds was detected, mainly saponins and flavonoids. Besides the known saponins, 15 new saponins were tentatively identified as glycosides of medicagenic acid, acetylated medicagenic acid and zanhic acid. It is suggested that metabolites of phytochemicals present in H. hirsuta, most likely saponins, are responsible for the pharmaceutical effects. It was observed that the relative abundance of saponin aglycones increased, indicating loss of sugar moieties during colonic biotransformation, with medicagenic acid as the most abundant aglycone. Hepatic biotransformation of this aglycone resulted in different metabolites formed by phase I and II reactions.

Non-volatile and volatile composition of West African bulk and Ecuadorian fine-flavor cocoa liquor and chocolate.

Cocoa products are obtained from the seeds of Theobroma cacao L. In this research, cocoa liquor and chocolate produced from cocoa beans from West Africa (Forastero, "bulk" cacao) and Ecuador (Nacional variety, "fine-flavor" cacao), were investigated, using a novel approach in which various analytical techniques are combined in order to obtain in-depth knowledge of the studied cocoa samples. The levels of various classes of primary metabolites were determined and a wide range of secondary metabolites, including volatile organic acids, aldehydes, esters, pyrazines, polyphenols, methylxanthines and biogenic amines, were identified and/or quantified by HS-SPME GC-MS (headspace-solid phase microextraction gas chromatography - mass spectrometry). and UPLC-HRMS (ultra-performance liquid chromatography - high resolution mass spectrometry). Odor Activity Values (OAV) were calculated to assess the contribution of individual volatiles on the final aroma. Various volatile aroma compounds were more abundant in the West African cocoa liquor and chocolate, while the Ecuadorian samples were richer in most quantified non-volatile metabolites. Principal component analysis (PCA) confirmed that the four samples can be clearly distinguished. Alcohols, pyrazines, amino acids and biogenic amines were found to be highly influential in causing this differentiation. The proposed approach can be useful in future studies on more extensive cocoa sample collections, in order to highlight similarities and pinpoint typical differences in chemical composition among these samples.

Revelation of the metabolic pathway of hederacoside C using an innovative data analysis strategy for dynamic multiclass biotransformation experiments.

Although some herbal remedies have been used for decades, little is known about the active compounds and the mechanism of action. Many natural products, such as glycosides, can be considered as prodrugs, which become active after biotransformation. To optimize the workflow of in vitro biotransformation followed by automated data analysis, hederacoside C was used as a model compound for saponins. Hederacoside C was subjected to gastrointestinal enzymes and fecal microflora. Samples were analyzed with UHPLC-PDA-HRMS before, during and after in vitro biotransformation, which allowed the monitoring of the relative abundances of the compound and its metabolites. The data-analysis workflow was optimized to render as much information as possible from the longitudinal LCMS data. XCMS was used to convert the raw data into features via peak-picking, followed by grouping, and EDGE was used for the extraction of significant differential profiles. To evaluate if the workflow was suitable for dynamic multiclass metabolomics data, an interactive Shiny web app was developed in R to rate the quality of the resulting features. These ratings were used to train a random forest model for predicting experts response. A performance analysis revealed that the random forest model was capable of correctly predicting the reviewers response in most cases (AUC 0.926 with 10 fold cross validation). The automated data analysis workflow was used for unbiased screening for metabolites and revealed the biotransformation of hederacoside C. As expected, a decrease in relative abundance of hederacoside C was observed over time. Additionally, the relative abundance of metabolites increased, illustrating the biotransformation of hederacoside C, especially in the colon phase, where microbial fermentation takes place. Stepwise progressive elimination of sugar moieties was the major metabolic pathway.

Phytochemical characterization and comparative studies of four Cecropia species collected in Panama using multivariate data analysis.

Plant species of the genus Cecropia (Urticaceae) are used as traditional medicine in Latin-America, and are commercially available as food supplements. The aim of this study was to characterize and compare the phytochemical constituents of four Cecropia species collected in Panama. The structures of 11 compounds isolated from leaves of C. obtusifolia were elucidated based on high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopic analysis; the polyphenolic constituents of leaves of all four Cecropia species and commercial products were characterized using high performance liquid chromatography-diode array detection-quadrupole time of flight-tandem high resolution mass spectrometry (HPLC-DAD-QTOF). Forty-seven compounds were fully identified or tentatively characterized. Thirty-nine of these have not been previously reported for the species under investigation. Multivariate analysis revelead that C. obtusifolia and C. insignis are the most related species, while C. hispidissima is the most segregated one. Considering the importance of the description of novel chemical entities and the increasing interest and use of natural products, this study may be of great help for chemotaxonomic purposes, the interpretation of medicinal properties and for quality assessment of herbal supplements containing Cecropia leaves.

Ultrasound-assisted extraction optimization and validation of an HPLC-DAD method for the quantification of polyphenols in leaf extracts of Cecropia species.

Cecropia species are traditionally used in Latin American folk medicine and are available as food supplements with little information warranting their quality. The optimum conditions for the extraction of chlorogenic acid (CA), total flavonoids (TF) and flavonolignans (FL) from leaves of Cecropia species were determined using a fractional factorial design (FFD) and a central composite design (CCD). A reversed-phase high-performance liquid chromatographic method coupled to a diode array detector (HPLC-DAD) was validated for the quantification of CA, TF and FL, following the ICH guidelines. Quantitative and Principal Component Analysis (PCA) was also performed. The extraction-optimization methodology enabled us developing an appropriate extraction process with a time-efficient execution of experiments. The experimental values agreed with those predicted, thus indicating suitability of the proposed model. The validation parameters for all chemical markers of the quantification method were satisfactory. The results revealed that the method had excellent selectivity, linearity, precision (repeatability and intermediate precision were below than 2 and 5%, respectively) and accuracy (98-102%). The limits of detection and quantification were at nanogram per milliliter (ng/mL) level. In conclusion, the simultaneous quantification of chemical markers using the proposed method is an appropriate approach for species discrimination and quality evaluation of Cecropia sp.

Health promoting potential of herbal teas and tinctures from Artemisia campestris subsp. maritima: from traditional remedies to prospective products.

This work explored the biotechnological potential of the medicinal halophyte Artemisia campestris subsp. maritima (dune wormwood) as a source of health promoting commodities. For that purpose, infusions, decoctions and tinctures were prepared from roots and aerial-organs and evaluated for in vitro antioxidant, anti-diabetic and tyrosinase-inhibitory potential, and also for polyphenolic and mineral contents and toxicity. The dune wormwood extracts had high polyphenolic content and several phenolics were identified by ultra-high performance liquid chromatography-photodiode array-mass-spectrometry (UHPLC-PDA-MS). The main compounds were quinic, chlorogenic and caffeic acids, coumarin sulfates and dicaffeoylquinic acids; several of the identified phytoconstituents are here firstly reported in this A. campestris subspecies. Results obtained with this plant's extracts point to nutritional applications as mineral supplementary source, safe for human consumption, as suggested by the moderate to low toxicity of the extracts towards mammalian cell lines. The dune wormwood extracts had in general high antioxidant activity and also the capacity to inhibit α-glucosidase and tyrosinase. In summary, dune wormwood extracts are a significant source of polyphenolic and mineral constituents, antioxidants and α-glucosidase and tyrosinase inhibitors, and thus, relevant for different commercial segments like the pharmaceutical, cosmetic and/or food industries.

UPLC/MS MS data of testosterone metabolites in human and zebrafish liver microsomes and whole zebrafish larval microsomes.

This article represents data regarding a study published in Toxicology in vitro entitled " in vitro CYP-mediated drug metabolism in the zebrafish (embryo) using human reference compounds" (Saad et al., 2017) [1]. Data were acquired with ultra-performance liquid chromatography - accurate mass mass spectrometry (UPLC-amMS). A full spectrum scan was conducted for the testosterone (TST) metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM) and female (FLM) livers, whole body homogenates of 96 h post fertilization larvae (EM) and a pool of human liver microsomes from 50 donors (HLM). Data are expressed as the abundance from the extracted ion chromatogram of the metabolites.

In vitro gastrointestinal biotransformation and characterization of a Desmodium adscendens decoction: the first step in unravelling its behaviour in the human body.

OBJECTIVES: The isolation and identification of the flavonoids present in a decoction of Desmodium adscendens was performed. In view of the oral use of the decoction, this work focused on the stability in gastrointestinal conditions and biotransformation by intestinal microflora in the colon of D-pinitol, vitexin and the flavonoid fraction of the decoction, as a first step in unravelling its behaviour in the human body. METHODS: The freeze-dried decoction was first subjected to column chromatography. Subsequently an enriched flavonoid fraction, was separated by repeated semi-preparative high-performance liquid chromatography (HPLC) or by HPLC-SPE. The isolated compounds were elucidated by NMR. Biotransformation experiments were carried in an in vitro gastrointestinal dialysis model. KEY FINDINGS: The major flavonoids of a decoction of D. adscendens were characterized as vicenin-2, isoschaftoside, schaftoside, 2″-O-xylosylvitexin, 2″-O-pentosyl-C-hexosyl apigenin and a O-hexosyl-C-hexosyl apigenin, tentatively identified as 2″-O-glucosyl-vitexin. During their passage in the gastrointestinal dialysis model, vitexin and C-glycosides thereof were found to be stable. Only the O-glycosidic bonds of O-glycosides of vitexin or isovitexin were hydrolysed during the colonic phase. CONCLUSIONS: A D. adscendens decoction was found to be rich in vitexin and isovitexin glycosides from which vitexin and the C-glycosides thereof were found to be stable in the simulated gastrointestinal tract.

Chemical profiling of infusions and decoctions of Helichrysum italicum subsp. picardii by UHPLC-PDA-MS and in vitro biological activities comparatively with green tea (Camellia sinensis) and rooibos tisane (Aspalathus linearis).

Several medicinal plants are currently used by the food industry as functional additives, for example botanical extracts in herbal drinks. Moreover, the scientific community has recently begun focusing on halophytes as sources of functional beverages. Helichrysum italicum subsp. picardii (everlasting) is an aromatic halophyte common in southern Europe frequently used as spice and in traditional medicine. In this context, this work explored for the first time H. italicum subsp. picardii as a potential source of innovative herbal beverages with potential health promoting properties. For that purpose, infusions and decoctions were prepared from roots, vegetative aerial-organs (stems and leaves) and flowers and evaluated for in vitro antioxidant and anti-diabetic activities. Samples were also assessed for toxicity in different mammalian cell lines and chemically characterized by spectrophotometric methods and ultra-high performance liquid chromatography-photodiode array-mass-spectrometry (UHPLC-PDA-MS). Results were expressed relating to 'a cup-of-tea' and compared with those obtained with green tea (Camellia sinensis) and rooibos tisane (Aspalathus linearis). Tisanes from the everlasting's above-ground organs, particularly flowers, have high polyphenolic content and several phenolics were identified; the main compounds were chlorogenic and quinic acids, dicaffeoylquinic-acid isomers and gnaphaliin-A. The antioxidant activity of beverages from the everlasting's above-ground organs matched or surpassed that of green tea and rooibos. Its anti-diabetic activity was moderate and toxicity low. Overall, our results suggest that the everlasting is a potential source of innovative and functional herbal beverages.

In vitro CYP-mediated drug metabolism in the zebrafish (embryo) using human reference compounds.

The increasing use of zebrafish embryos as an alternative model for toxicological and pharmacological studies necessitates a better understanding of xenobiotic biotransformation in this species. As cytochrome P450 enzymes (CYPs) play an essential role in this process, in vitro drug metabolism of four human CYP-specific substrates, i.e. dextromethorphan (DXM), diclofenac (DIC), testosterone (TST) and midazolam (MDZ) was investigated in adult male and female zebrafish, and in zebrafish embryos and larvae up to 120hours post-fertilization. Substrate depletion and production of their respective metabolites were measured using tandem quadrupole UPLC-MS/MS. Human liver microsomes were used as positive control. Adult zebrafish produced the two major human metabolites of DIC and DXM. For DIC the metabolite ratio was similar to that in man, whereas it was different for DXM. For TST, the major human metabolite could not be detected and MDZ was not metabolized. No sex-related differences were detected, except for the higher TST depletion rate in adult females. Zebrafish embryos and larvae showed no or only low biotransformation capacity. In conclusion, in vitro CYP-mediated drug metabolism in adult zebrafish shows differences compared to man and appears to be lacking in the early zebrafish life stages. As CYP-mediated drug metabolism in zebrafish may not be predictive for the one in man, we recommend including the zebrafish in metabolic stability testing of new compounds when considering non-clinical species for human risk assessment.

Bridging the gap between comprehensive extraction protocols in plant metabolomics studies and method validation.

It is vital to pay much attention to the design of extraction methods developed for plant metabolomics, as any non-extracted or converted metabolites will greatly affect the overall quality of the metabolomics study. Method validation is however often omitted in plant metabolome studies, as the well-established methodologies for classical targeted analyses such as recovery optimization cannot be strictly applied. The aim of the present study is to thoroughly evaluate state-of-the-art comprehensive extraction protocols for plant metabolomics with liquid chromatography-photodiode array-accurate mass mass spectrometry (LC-PDA-amMS) by bridging the gap with method validation. Validation of an extraction protocol in untargeted plant metabolomics should ideally be accomplished by validating the protocol for all possible outcomes, i.e. for all secondary metabolites potentially present in the plant. In an effort to approach this ideal validation scenario, two plant matrices were selected based on their wide versatility of phytochemicals: meadowsweet (Filipendula ulmaria) for its polyphenols content, and spicy paprika powder (from the genus Capsicum) for its apolar phytochemicals content (carotenoids, phytosterols, capsaicinoids). These matrices were extracted with comprehensive extraction protocols adapted from literature and analysed with a generic LC-PDA-amMS characterization platform that was previously validated for broad range phytochemical analysis. The performance of the comprehensive sample preparation protocols was assessed based on extraction efficiency, repeatability and intermediate precision and on ionization suppression/enhancement evaluation. The manuscript elaborates on the finding that none of the extraction methods allowed to exhaustively extract the metabolites. Furthermore, it is shown that depending on the extraction conditions enzymatic degradation mechanisms can occur. Investigation of the fractions obtained with the different extraction methods revealed a low resolving power for phytochemicals for all methods. Nevertheless, an overall good repeatability was observed for all extraction methods, which is essential to allow direct comparison between samples. In summary, no single procedure outperforms the others and compromises will have to be made during method selection.

Tackling the challenge of selective analytical clean-up of complex natural extracts: the curious case of chlorophyll removal.

Alkaline saponification is often used to remove interfering chlorophylls and lipids during carotenoids analysis. However, saponification also hydrolyses esterified carotenoids and is known to induce artifacts. To avoid carotenoid artifact formation during saponification, Larsen and Christensen (2005) developed a gentler and simpler analytical clean-up procedure involving the use of a strong basic resin (Ambersep 900 OH). They hypothesised a saponification mechanism based on their Liquid Chromatography-Photodiode Array (LC-PDA) data. In the present study, we show with LC-PDA-accurate mass-Mass Spectrometry that the main chlorophyll removal mechanism is not based on saponification, apolar adsorption or anion exchange, but most probably an adsorption mechanism caused by H-bonds and dipole-dipole interactions. We showed experimentally that esterified carotenoids and glycerolipids were not removed, indicating a much more selective mechanism than initially hypothesised. This opens new research opportunities towards a much wider scope of applications (e.g. the refinement of oils rich in phytochemical content).

Ultra high performance liquid chromatography versus high performance liquid chromatography: stationary phase selectivity for generic carotenoid screening.

Aim of study was to find the most suitable LC column for generic carotenoid screening. To represent the diversity of carotenoids in nature and to optimize chromatographic separation, a set of carotenoid standards was carefully chosen to account for the various classes of carotenoids. The HPLC C30 column has since long been the 'golden standard' in the chromatographic separation of carotenoids. Since approximately one decade, new UHPLC technology has led to much shorter analysis times, smaller peak widths and higher chromatographic resolution. However, there are currently no UHPLC columns on the market containing the specific stationary phase chemistry of the HPLC C30 column. Therefore during this study, we investigated the separation of carotenoids on a set of UHPLC columns and compared it to their separation on the HPLC C30 column. Comparison of carotenoids separations on the different stationary phases with objective column comparison parameters clearly indicated that the HPLC C30 column is an overall better performer in the separation of carotenoids. This is due to the lack of UHPLC column chemistries that are adapted for carotenoid analysis. However, analysis time on the HPLC C30 column takes about four times longer compared to UHPLC analysis. Therefore, with the range of columns that are commercially available nowadays, a choice has to be made between very high selectivity (HPLC C30 column) and analysis times that are adapted to modern laboratory requirements (UHPLC technology). Therefore, carotenoid separations would be even more performing if an appropriate UHPLC C30 column would be available.