Vincent Bessonneau

Analysis of human saliva metabolome by direct immersion solid-phase microextraction LC and benchtop orbitrap MS

BACKGROUND Saliva samples collected from one 58 year old male and one 35 year old female during 7 days of fasting were analyzed by direct immersion of both C18 and mixed-mode biocompatible solid-phase microextraction fibers, in combination with a LC-MS method using a benchtop orbitrap instrument in both positive and negative ionization modes, in order to evaluate the difference in terms of metabolite coverage. RESULTS The mixed-mode coating provided better results, with the simultaneous extraction of a higher number of both hydrophilic and hydrophobic metabolites. The ability of detected features to distinguish differences between the individuals and changes in saliva metabolome induced by diet was also demonstrated. CONCLUSION Saliva may be useful for medical diagnostics as it is non-invasive. The use of biocompatible solid-phase microextraction fibers can play an important role as an alternative sample preparation method for untargeted LC-MS metabolomics studies on human saliva because it provides simultaneous extraction of metabolites with a wide range of polarity, thus allowing the detection of changes in metabolic pathways with unsupervised statistical analyses.

In vivo solid phase microextraction sampling of human saliva for non-invasive and on-site monitoring.

On-site sample preparation is an analytical approach based on direct sampling from the system under investigation. It has the advantage of combining sampling and sample preparation into a single step, thus generally is fast, minimizes the potential sources of error and eliminates the risks for analytes instability. For such analysis solid phase microextraction in thin film geometry (TF-SPME) can provide robust and convenient in vivo sampling, offering in the same time faster analysis and higher extraction recovery (i.e., better sensitivity) due to large surface to volume ratio. In this study, TF-SPME in coated blade and membrane formats with a single extraction phase were used for in vivo and ex vivo saliva extraction and separation by LC and GC, respectively. Due to applicability for wide range of polarity of analytes as well as thermal and solvent stability during the desorption, hydrophilic lipophilic balanced particles (HLB) were chosen as extraction phase and used for fast (5 min) in vivo and ex vivo sampling. The results of metabolomic profiling of the saliva are indicating that even 5 min in vivo sampling using TF-SPME followed by GC and LC analyses provides complementary coverage of wide range of analytes with different physical and chemical properties. To demonstrate the applicability of the method for doping analyses, the SPME-LC-MS/MS method was validated for simultaneous quantification of 49 prohibited substances with limit of quantification (LOQ) ranging between 0.004 and 0.98 ng mL(-1). Moreover, the method was also validated and successfully applied for determination of endogenous steroids in saliva where the concentrations of the analytes are substantially low. The developed assay offers fast and reliable multiresidue analysis of saliva as an attractive alternative to the standard analysis methods.

In vivo solid-phase microextraction liquid chromatography-tandem mass spectrometry for monitoring blood eicosanoids time profile after lipopolysaccharide-induced inflammation in Sprague-Dawley rats.

A fast and non-lethal in vivo solid-phase microextraction (SPME) sampling method for rat blood coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) was developed for monitoring rapid changes in concentrations of eicosanoids - lipid mediators involved in the development of inflammatory conditions - using diffusion-based calibration. Sampling rates of target eicosanoids were pre-determined under laboratory conditions with a precision of ≤10%, and directly used for quantification of analyte concentrations in blood after lipopolysaccharide-induced inflammation in Sprague-Dawley rats. Results showed significant changes in unbound plasma concentrations of arachidonic acid (AA) and 12-hydroxyeicosatetraenoic acid (12-HETE) in response to the treatment. Next, performance of the proposed method was compared with protein precipitation (PP) of plasma, a conventional sample preparation technique. Finally, percentages of plasma protein binding (PPB) of specific eicosanoids were determined. PPB of target eicosanoids was in agreement with literature values, ranging from 99.3 to 99.9% for 12-HETE and DHA, respectively. We envision that the proposed method is a particularly suitable alternative to lethal sampling and current methods based on sample depletion in animal studies for accurate monitoring of rapid changes in blood concentrations of small molecules.

Cinnamaldehyde Characterization as an Antibacterial Agent toward E. coli Metabolic Profile Using 96-Blade Solid-Phase Microextraction Coupled to Liquid Chromatography–Mass Spectrometry

Sampling and sample preparation plays an important role in untargeted analysis as it influences final composition of the analyzed extract and consequently reflection of the metabolome. In the current work, mechanism of bactericidal action of cinnamaldehyde (CA) against Escherichia coli (E. coli) during bacteria growth applying high-throughput solid-phase microextraction in direct immersion mode coupled to a high-performance liquid chromatography-mass spectrometry system was investigated. Numerous discriminant metabolites due to CA addition to the bacteria culture were mapped in the E. coli metabolic pathways. We propose new metabolic pathways confirming that CA acts as an oxidative stress agent against E. coli. The results of the current research have successfully demonstrated that CA changes the bacterial metabolism through interactions with different biochemical families such as proteins, nucleic acids, lipids, and carbohydrates, which needs further validation by proteomics and transcriptomics studies. The results presented here show the great potential of the novel approach in drug discovery and food safety.

Determination of bronchoalveolar lavage bile acids by solid phase microextraction liquid chromatography-tandem mass spectrometry in combination with metabolite profiling: comparison with enzymatic assay.

A thin-film solid-phase microextraction (SPME) method coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was developed for high-throughput determination of bronchoalveolar lavage bile acids. The proposed method was validated according to the bioanalytical method validation guidelines. LOQ and LLOQ were 0.007 and 0.02 μmol/L, respectively. The accuracy and the precision were <7 and <4%, respectively. The performance of the proposed method was also compared with an optimized enzymatic cycling assay. Results showed a weak correlation between total concentration of bile acid (BAs) obtained with enzymatic assay and cumulative concentration of specific BAs determined with SPME-LC-MS/MS. This discrepancy was probably due to the presence of other BAs in bronchoalveolar lavage fluid (BALF) samples. Metabolites profiling of BALF samples extracts using a high-resolution mass spectrometer (HRMS) revealed the presence of additional BAs, which were not included in the proposed method. After considering these additional BAs, a strong correlation was found between the LC-MS method and the enzymatic assay. Unsupervised statistical analysis conducted on HRMS data also showed clear separation within BALF samples, depending on the presence of BAs and other lipids. SPME-LC-MS-based metabolites profiling may provide additional information for diagnosis occurrence and severity of gastric reflux/aspiration in lung transplant patients.

In vivo microsampling to capture the elusive exposome

Loss and/or degradation of small molecules during sampling, sample transportation and storage can adversely impact biological interpretation of metabolomics data. In this study, we performed in vivo sampling using solid-phase microextraction (SPME) in combination with non-targeted liquid chromatography and high-resolution tandem mass spectrometry (LC-MS/MS) to capture the fish tissue exposome using molecular networking analysis, and the results were contrasted with molecular differences obtained with ex vivo SPME sampling. Based on 494 MS/MS spectra comparisons, we demonstrated that in vivo SPME sampling provided better extraction and stabilization of highly reactive molecules, such as 1-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoleoyl-glycero-3-phosphocholine, from fish tissue samples. This sampling approach, that minimizes sample handling and preparation, offers the opportunity to perform longitudinal monitoring of the exposome in biological systems and improve the reliability of exposure-measurement in exposome-wide association studies.

The Saliva Exposome for Monitoring of Individuals’ Health Trajectories

Background: There is increasing evidence that environmental, rather than genetic, factors are the major causes of most chronic diseases. By measuring entire classes of chemicals in archived biospecimens, exposome-wide association studies (EWAS) are being conducted to investigate associations between a myriad of exposures received during life and chronic diseases. Objectives: Because the intraindividual variability in biomarker levels, arising from changes in environmental exposures from conception onwards, leads to attenuation of exposure–disease associations, we posit that saliva can be collected repeatedly in longitudinal studies to reduce exposure–measurement errors in EWAS. Methods: From the literature and an open-source saliva–metabolome database, we obtained concentrations of 1,233 chemicals that had been detected in saliva. We connected salivary metabolites with human metabolic pathways and PubMed Medical Subject Heading (MeSH) terms, and performed pathway enrichment and pathway topology analyses. Results: One hundred ninety-six salivary metabolites were mapped into 49 metabolic pathways and connected with human metabolic diseases, central nervous system diseases, and neoplasms. We found that the saliva exposome represents at least 14 metabolic pathways, including amino acid metabolism, TCA cycle, gluconeogenesis, glutathione metabolism, pantothenate and CoA biosynthesis, and butanoate metabolism. Conclusions: Saliva contains molecular information worthy of interrogation via EWAS. The simplicity of specimen collection suggests that saliva offers a practical alternative to blood for measurements that can be used to characterize individual exposomes. https://doi.org/10.1289/EHP1011

Tissue storage affects lipidome profiling in comparison to in vivo microsampling approach

Low-invasive in vivo solid-phase microextraction (SPME) was used to investigate the lipid profiles of muscle tissue of living fish. Briefly, mixed mode SPME fibers were inserted into the muscle for 20 min extraction, and then the fibers were desorbed in an optimal mixture of solvents. The obtained lipid profile was then compared and contrasted to that obtained with employment of ex vivo SPME and solid-liquid extraction (SLE) from fish muscle tissue belonging to the same group of fish, following a one-year storage period. Ex vivo SPME analysis of stored muscle samples revealed 10-fold decrease in the number of detected molecular features in comparison to in vivo study. Moreover, in vivo microsampling enabled the identification of different classes of bioactive lipids, including fatty acyls, not present in the lipid profile obtained through ex vivo SPME and SLE, suggesting the alterations occurring in the unbound lipid fraction of the system under study during the storage and also indicating the advantage of the in vivo extraction approach.

In situ chemical exploration of underwater ecosystems with microsampling/enrichment device

The study of organic compounds present in water surrounding marine organisms can be useful for a better understanding of ecosystem dynamics, since these compounds may be important in signaling. However, the detection of organic compounds underwater is challenging, as they are present at very low levels. We introduce in situ solid-phase microextraction (SPME) as a non-invasive and non-polluting technique for simultaneous underwater sampling and extraction of small molecules. Analyses were conducted with a LC-MS method using a benchtop orbitrap instrument in both positive and negative modes. We provided preliminary results on the ability of SPME fibers to extract simultaneously around 280 compounds with a wide range of polarities (logP from -2.6 to 15.3), including short-lived intermediates, directly from underwater. We envision that this approach can offer new opportunities to explore underwater ecosystems dynamics.