Urinary and plasma metabolite differences detected by HPLC-ESI-QTOF-MS in systemic sclerosis patients.

Systemic Sclerosis (SSc) is a chronic autoimmune disease whose origin and pathogenesis are not yet well known. Recent studies are allowing a better definition of the disease. However, few studies have been performed based on metabolomics. In this way, this study aims to find altered metabolites in SSc patients in order to improve their diagnosis, prognosis and treatment. For that, 59 SSc patients and 28 healthy volunteers participated in this study. Urine and plasma samples were analysed by a fingerprinting metabolomic approach based on HPLC-ESI-QTOF-MS. We observed larger differences in urine than plasma metabolites. The main deregulated metabolic families in urine were acylcarnitines, acylglycines and metabolites derived from amino acids, specifically from proline, histidine and glutamine. These results indicate perturbations in fatty acid beta oxidation and amino acid pathways in scleroderma patients. On the other hand, the main plasma biomarker candidate was 2-arachidonoylglycerol, which is involved in the endocannabinoid system with potential implications in the induction and propagation of systemic sclerosis and autoimmunity.

Simultaneous ultra-high performance liquid chromathograpy-electrospray ionization-quadrupole-time of flight mass spectrometry quantification of endogenous anandamide and related N-acylethanolamides in bio-matrices.

We describe and validate a sensitive UHPLC-ESI-QTOF-MS method for the simultaneous quantification of seven endocannabinoids and non-endocannabinoids related N-acylethanolamides: N-arachidonoylethanolamide, N-palmitoylethanolamide, N-stearoylethanolamide, N-oleoylethanolamide, N-linoleoylethanolamide, N-α-linolenoylethanolamide and N-eicosapentaenoylethanolamide in several bio-matrices for the purpose of research and clinical application. We examined effects of different liquid-liquid and solid phase extraction on the recovery of endocannabinoids and N-acylethanolamides. Protein precipitation with cooled acetone and extraction with acetonitrile (1% v/v formic acid) using OASIS HLB cartridge gave better results. Separation was performed on a Waters Acquity UPLC HSST3 column using a 9min elution gradient coupled with high resolution mass spectrometry (QTOF/MS). The high sensitivity of the developed method allow its application on sample with low volumes or low levels of endocannabinoids and N-acylethanolamides and make the method suitable for routine measurement in human bio-matrices, such as plasma, serum (500µL), urine (1mL) and tissues (10-30mg). Its application in clinical research could contribute to unravel pathophysiological roles of these family of lipid mediators and disclose novel diagnostic and prognostic markers.

Analytical approaches for the determination of phytocannabinoids and endocannabinoids in human matrices.

Over the last two decades, the role played by phytocannabinoids and endocannabinoids in medicine has gained increasing interest in the scientific community. Upon identification of the plant compound Δ(9)-tetrahydrocannabinol (THC) and of the endogenous substance anandamide (AEA), different methodological approaches and innovative techniques have been developed, in order to evaluate the content of these molecules in various human matrices. In this review, we discuss the analytical methods that are currently used for the identification of phytocannabinoids and endocannabinoids, and we summarize the benefits and limitations of these procedures. Moreover, we provide an overview of the main biological matrices that have been analyzed to date for qualitative detection and quantitative determination of these compounds.