Electromembrane Extraction of Highly Polar Compounds: Analysis of Cardiovascular Biomarkers in Plasma

Abstract

Cardiovascular diseases (CVDs) represent a major concern in today's society, with more than 17.5 million deaths reported annually worldwide. Recently, five metabolites related to the gut metabolism of phospholipids were identified as promising predictive biomarker candidates for CVD. Validation of those biomarker candidates is crucial for applications to the clinic, showing the need for high-throughput analysis of large numbers of samples. These five compounds, trimethylamine N-oxide (TMAO), choline, betaine, l-carnitine, and deoxy-l-carnitine (4-trimethylammoniobutanoic acid), are highly polar compounds and show poor retention on conventional reversed phase chromatography, which can lead to strong matrix effects when using mass spectrometry detection, especially when high-throughput analysis approaches are used with limited separation of analytes from interferences. In order to reduce the potential matrix effects, we propose a novel fast parallel electromembrane extraction (Pa-EME) method for the analysis of these metabolites in plasma samples. The evaluation of Pa-EME parameters was performed using multi segment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Recoveries up to 100% were achieved, with variability as low as 2%. Overall, this study highlights the necessity of protein precipitation prior to EME for the extraction of highly polar compounds. The developed Pa-EME method was evaluated in terms of concentration range and response function, as well as matrix effects using fast-LC-MS/MS. Finally, the developed workflow was compared to conventional sample pre-treatment, i.e., protein precipitation using methanol, and fast-LC-MS/MS. Data show very strong correlations between both workflows, highlighting the great potential of Pa-EME for high-throughput biological applications.

An investigation on the effect of filtration and dilution of biological samples on electromembrane extraction efficiency: Determination of basic drugs in plasma and urine samples

“…Therefore, usually, high voltages cannot be employed, so in lower voltages, the extraction duration becomes very long. Applying high voltages to the extraction system can be very helpful specifically in the extraction of polar analytes by EME [25]. The results revealed that the proposed method brings a successful extraction of analytes from two complex biological samples in high voltages.…”

Metabolomics meets lipidomics: Assessing the small molecule component of metabolism

“…Among the technological platforms used to resolve chemical diversity, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) constitute two main, widely applied technologies for broad-range metabolite and lipid analysis. [66] While NMR has the advantage of being quantitative, non-destructive and highly reproducible-mainly due to the lack of direct interaction between the sample and the instrument, its important drawbacks are the lack of sensitivity and spectral resolution or overlapping signals making unambiguous metabolite identification and quantification difficult when analyzing complex biological matrices. [27][28][29] NMR is widely used for the analysis of urinary metabolites, present in high concentrations, in the context of large-scale population studies, in view of its high robustness over time.…”

“…Major improvements in TA B L E 1 Technological platforms used to resolve polar and lipid metabolite diversity. The most common and successful applications are described [31][32][33][34][35][36] Flow [37] Complex lipids: Glycerolipids, glycerophospholipids, sphingolipids [38][39][40] Surface [41][42][43] Fatty acids Complex lipids: Glycerolipids, glycerophospholipids, sphingolipids [44][45][46][47][48] Gas [49][50][51] Fatty acids, oxysterols, oxylipins, steroids, aldehydes [24,[52][53][54][55][56] Liquid [57][58][59][60][61] Fatty acids, oxysterols, oxylipins, steroids, acyl carnitines [56,[62][63][64] Complex lipids: Glycerolipids, glycerophospholipids, sphingolipids [65,66] Capillary electrophoresis mass spectrometry (CE-MS) [67][68][69][70]…”

Bifidobacterium adolescentis improves lifespan and healthspan by regulating catalase activity and oxidative stress-associated metabolites

“…Some other antioxidants also exhibited enrichment with B. adolescentis supplement, including ginsenoside Ia 28 , 2-hydroxycinnamic acid 29 , daidzin 30 and L-malic acid 31 , while ROS producer such as hypoxanthine 32 was reduced. Furthermore, several metabolites with proin ammatory property or associated with cardiovascular diseases were enriched in Terc −/− aged mice, including cholic acid 33 , 9,10-DHOME 34 , 3dehydroxycarnitine 35 and 4-trimethylammoniobutanoic acid 36 . Cosmosiin 37 with anti-aging potential and enterodiol 38 with tumor-suppression activity were enriched in B. adolescentis-gavaged Terc −/− aged mice ( Fig.…”