Impact of sample preparation upon intracellular metabolite measurements in 3D cell culture systems.

INTRODUCTION: Interest in cell culture metabolomics has increased greatly in recent years because of its many potential applications and advantages (e.g., in toxicology). The first critical step for exploring the cellular metabolome is sample preparation. For metabolomics studies, an ideal sample preparation would extract a maximum number of metabolites and would enable reproducible, accurate analysis of a large number of samples and replicates. In addition, it would provide consistent results across several studies over a relatively long time frame. OBJECTIVES: This study was conducted to evaluate the impact of sample preparation strategies on monitoring intracellular metabolite responses, highlighting the potential critical step(s) in order to finally improve the quality of metabolomics studies. METHODS: The sample preparation strategies were evaluated by calculating the sample preparation effect, matrix factor, and process efficiency (PE) for 16 tobacco exposition-related metabolites, including nicotine, nicotine-derived nitrosamine ketone, their major metabolites, and glutathione, using isotopically-labelled internal standards. Samples were analyzed by liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS). RESULTS: A sample drying step increased losses or variability for some selected metabolites. By avoiding evaporation, good sample preparation recovery was obtained for these compounds. For some metabolites, the cell or culture type impacted PE and matrix factor. CONCLUSION: In our sample preparation protocol, the drying-reconstitution step was identified as the main cause of metabolite losses or increased data variability during metabolomics analysis by LC-HRMS. Furthermore, PE was affected by the type of matrix. Isotopologue internal standards fully compensate losses or enhancements.

Quantification of punicalagins in commercial preparations and pomegranate cultivars, by liquid chromatography-mass spectrometry.

BACKGROUND: Pomegranate (Punica granatum L.) - a delicious fruit once used in Ayurvedic medicine - is now largely known for the antioxidant properties of its juice, which has also been considered to have health benefits against diseases such as cancer and cardiovascular diseases. These beneficial effects are associated with the fruit's high content of polyphenolic compounds. High demand and lower production levels drive pomegranate prices up, which leads to the possibility of pomegranate products being adulterated, diluted or substituted. To ensure the presence of pomegranate in various preparations labeled as containing pomegranate, a simple method was developed to screen and quantify the specific punicalagins by mass spectrometry. RESULTS: The present method was used to analyze several pure and mixed beverages from the US market, and also to quantify punicalagins in the juice of 14 pomegranate cultivars. Punicalagins were detected in all cultivars, with higher concentrations in whole fruit juices compared with aril juices. Amongst the 20 commercial beverages, punicalagins were not detected in four preparations. CONCLUSION: The liquid chromatographic-mass spectrometric method presented herein enables an easy and rapid quantification of the specific punicalagins. The latter was detected in all cultivar samples, thus supporting that punicalagin is a suitable marker of these 14 pomegranate cultivars in commercial juices. Absence of the specific marker in four commercial preparations shows the necessity of having simple and rapid methods to evaluate the presence of pomegranate in preparations. © 2019 Society of Chemical Industry.

Metabolic Profiling of Chloroacetanilide Herbicides in Earthworm Coelomic Fluid Using 1H NMR and GC-MS.

Earthworms ( Eisenia fetida) are vital members of the soil environment. Because of their sensitivity to many contaminants, monitoring earthworm metabolism may be a useful indicator of environmental stressors. Here, metabolic profiles of exposure to five chloroacetanilide herbicides and one enantiomer (acetochlor, alachlor, butachlor, racemic metolachlor, S-metolachlor, and propachlor) are observed in earthworm coelomic fluid using proton nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS). Multiblocked-orthogonal partial least-squares-discriminant analysis (MB-OPLS-DA) and univariate analysis were used to identify metabolic perturbations in carnitine biosynthesis, carbohydrate metabolism, lipid metabolism, nitrogen metabolism, and the tricarboxylic acid cycle. Intriguingly, stereospecific metabolic responses were observed between racemic metolachlor and S-metolachlor exposed worms. These findings support the utility of coelomic fluid in monitoring metabolic perturbations induced by chloroacetanilide herbicides in nontarget organisms and reveal specificity in the metabolic impacts of herbicide analogues in earthworms.

Development of a Liquid Chromatography-High Resolution Mass Spectrometry Metabolomics Method with High Specificity for Metabolite Identification Using All Ion Fragmentation Acquisition.

High-resolution mass spectrometry (HRMS)-based metabolomics approaches have made significant advances. However, metabolite identification is still a major challenge with significant bottleneck in translating metabolomics data into biological context. In the current study, a liquid chromatography (LC)-HRMS metabolomics method was developed using an all ion fragmentation (AIF) acquisition approach. To increase the specificity in metabolite annotation, four criteria were considered: (i) accurate mass (AM), (ii) retention time (RT), (iii) MS/MS spectrum, and (iv) product/precursor ion intensity ratios. We constructed an in-house mass spectral library of 408 metabolites containing AMRT and MS/MS spectra information at four collision energies. The percent relative standard deviations between ion ratios of a metabolite in an analytical standard vs sample matrix were used as an additional metric for establishing metabolite identity. A data processing method for targeted metabolite screening was then created, merging m/z, RT, MS/MS, and ion ratio information for each of the 413 metabolites. In the data processing method, the precursor ion and product ion were considered as the quantifier and qualifier ion, respectively. We also included a scheme to distinguish coeluting isobaric compounds by selecting a specific product ion as the quantifier ion instead of the precursor ion. An advantage of the current AIF approach is the concurrent collection of full scan data, enabling identification of metabolites not included in the database. Our data acquisition strategy enables a simultaneous mixture of database-dependent targeted and nontargeted metabolomics in combination with improved accuracy in metabolite identification, increasing the quality of the biological information acquired in a metabolomics experiment.

Separation of ten phosphorylated mono-and disaccharides using HILIC and ion-pairing interactions.

Phosphorylated carbohydrates are indispensable cogs in several key metabolic wheels for all forms of life. Here, a straightforward liquid chromatography method coupled to mass spectrometry detection was developed for phosphorylated sugars. For separation of the targeted compounds, hydrophilic interaction chromatography (HILIC) was used with a bridged-ethylene hybrid amide column under alkaline conditions using triethylamine as a mobile phase modifier. Methylphosphonic acid was added to the aqueous mobile phase to reduce the tailing of compounds containing phosphate groups, which are known to interact with stainless steel components of the separation system. Under alkaline conditions and addition of methylphosphonic acid, the retention behavior can be attributed to both conventional HILIC mechanisms as well as ion-pairing interactions in the mobile phase. This hypothesis is supported by comparing the retention behavior of phosphorylated sugars and unmodified sugars. The HILIC method resolved eight biologically important phosphorylated sugars and thereby enables simultaneous detection and quantification of these compounds: fructose-1,6-bisphosphate, glucose-1-phosphate, glucose-6-phosphate, lactose-1-phosphate, mannose-6-phosphate, ribose-5-phosphate, sucrose-6-phosphate, and threhalose-6-phosphate. Fructose-1-phosphate and fructose-6-phosphate were not resolved but quantification of total fructose-phosphate is possible.

Survey of pyrrolizidine alkaloids in teas and herbal teas on the Swiss market using HPLC-MS/MS.

Pyrrolizidine alkaloids (PAs) are a large class of natural compounds amongst which the esterified 1,2-unsaturated necine base is toxic for humans and livestock. In the present study, a method was developed and validated for the screening and quantification of nine PAs and one PA N-oxide in teas (Camellia sinensis (L.) O. Kuntze) and herbal teas (camomile, fennel, linden, mint, rooibos, verbena). Samples were analysed by HPLC on a RP-column, packed with sub-2 µm core-shell particles, and quantified using tandem mass spectrometry operating in the positive electrospray ionisation mode. These PAs and some of their isomers were detected in a majority of the analysed beverages (50/70 samples). In 24 samples, PA concentrations were above the limit of quantification and the sum of the nine targeted PAs was between 0.021 and 0.954 µg per cup of tea. Thus, in some cases, total concentrations exceed the maximum daily intake recommended by the German Federal Institute for Risk Assessment and the UK's Committee On Toxicity (i.e. 0.007 µg kg(-1) bw).

Screening and determination of sibutramine in adulterated herbal slimming supplements by HPTLC-UV densitometry.

The adulteration of herbal supplements is of growing importance, especially when they contain undeclared compounds like sibutramine that are unsafe drugs. Sibutramine was withdrawn from US and European markets in 2010. In this study, an HPTLC-UV densitometric method was developed for the quantification of sibutramine in herbal diet foods. Sample extracts were directly applied onto HPTLC silica gel plates and separated with a mobile phase made of a toluene-methanol mixture. Sibutramine was quantified at 225 nm and its unequivocal identification was confirmed by MS using a TLC-MS interface. During two surveys, 52 weight loss supplements obtained via the Internet were screened. Half of those were adulterated with sibutramine at amounts reaching up to 35 mg per capsule. The results of this validated HPTLC method were compared with those obtained by HPLC-UV and HPLC-MS/MS. The results were not significantly different with the three methods.

Analysis of the neurotoxin anisatin in star anise by LC-MS/MS.

The aim of this work was to develop an analytical method capable of determining the presence of anisatin in star anise. This neurotoxin may induce severe side effects such as epileptic convulsions. It is therefore of prime importance to have rapid and accurate analytical methods able to detect and quantify anisatin in samples that are purportedly edible star anise. The sample preparation combined an automated accelerated solvent extraction with a solid-supported liquid-liquid purification step on EXtrelut®. Samples were analysed on a porous graphitic carbon HPLC column and quantified by tandem mass spectrometry operating in the negative ionisation mode. The quantification range of anisatin was between 0.2 and 8 mg kg⁻¹. The applicability of this validated method was demonstrated by the analysis of several Illicium species and star anise samples purchased on the Swiss market. High levels of anisatin were measured in Illicium lanceolatum, I. majus and I. anisatum, which may cause health concerns if they are misidentified or mixed with edible Illicium verum.

Simultaneous determination of nicotine and PAH metabolites in human hair specimen: a potential methodology to assess tobacco smoke contribution in PAH exposure.

The present methodology was developed to simultaneously assess chronic exposure to PAHs and to tobacco from the analysis of one hair specimen per examined individual. The method is a two step extraction of twelve mono-hydroxy-PAHs and of nicotine, and their separate analysis by optimized methods using gas chromatography-negative chemical ionization-mass spectrometry. After method validation and assessment of the hair decontamination procedure, 105 hair specimens from smokers and non-smokers were analyzed. All the hair samples tested positive for nicotine. Median concentration was 10.7ng/mg for smokers and 0.5ng/mg for non-smokers. 70% of the samples tested positive for OH-PAHs. The most common one was 2-naphthol (61%) and its concentration was significantly higher in smokers than in non-smokers (median: 111 vs 70pmol/g, p=0.006). 2-OH-benzo(c)phenanthrene and 6-OH-chrysene were only detected once in a non-smoker's hair. The concentration of the sum of all PAH-metabolites ranged from 24 to 67190pmol/g (median: 118pmol/g). Only six samples tested positive for more than two different metabolites. The simultaneous detection of nicotine and OH-PAHs in hair is possible and provides reliable results. This represents a useful tool for the accurate biomonitoring of chronic exposure to PAH and correct identification of the sources of exposure.