TMAO, choline, betaine, l
-carnitine, and deoxy-l
-carnitine, known predictive biomarkers for CVD [2
], were used as model compounds. All these metabolites are very polar compounds with logP between −4.49 and −0.93 (Table 1
). Due to their very low lipophilicity, these highly polar molecules are difficult to extract using conventional sample preparation methods such as solid-phase extraction or reversed-phase SPE [9
], which are based on the partition coefficients of analytes in two-phase systems. Indeed, only PP has been reported as efficient sample pre-treatment for these class metabolites so far [31
]. PP is typically used in metabolomics, especially in large-scale studies where high throughput is essential. Fast analytical techniques are also required, such as fast LC-MS, but may lead to strong ME, typically for poorly-retained compounds and particularly in combination with straightforward sample pre-treatments [9
]. This ME issue highlights the needs for novel sample preparation approaches adapted to the extraction of highly polar compounds.
3.1. Optimization of the Parallel Electromembrane Extraction Set-Up
The EME experimental conditions, i.e., applied voltage and sample composition, were first optimized to reach the highest EY and PE while lowering ME. Because most of the compounds of interest are endogenously present in human plasma, dISTDs were used during the Pa-EME optimization step, except for bupivacaine, which is a xenobiotic compound. Based on previous work [20
], 2-nitrophenylpentyl ether (NPPE) as SLM and 1% acetic acid (pH 2.8) as both acceptor and donor solutions were used as starting conditions. NPPE was selected due the expected good extraction recovery and low extraction variability for the selected compounds, while 1% acetic acid allowed for both protonation of the basic moiety and neutralization of carboxylic group of l
-carnitine and betaine. Moreover, 1% acetic acid generated a relatively low current which allowed for the application of higher voltages without generating excessive Joule heating. The obtained extracts were then analyzed using MSI-CE-MS. MSI-CE-MS consists of consecutive injections of up to seven different samples within the same analytical run. This leads to a significant increase in analysis throughput as well as decrease of analytical variability for sample injected in the same run [34
First, the extraction voltage was investigated, since EME recoveries are known to be directly correlated to the electric field applied during the electroextraction process [14
]. Figure 2
and Figure 3
illustrate the results obtained for three compounds, i.e., choline (positively charged and polar), l
-carnitine (partially charged and polar), and bupivacaine (positively charged and non-polar). As shown in Figure 2
, the EY (calculated according to Equation (1)) increased for all compounds with an increased extraction voltage. The gain in EY was especially important for l
-carnitine, where the EY showed a 5-fold enhancement when increasing the voltage from 75 V to 100 V. At 100 V, EYs up to 92% were obtained, with relative standard deviations (RSDs) as low as 4%. Choline and l
-carnitine are close compounds with logD values in the same range, i.e., −4.6 and −4.8 at pH 2.8, respectively. The difference observed in EY between both compounds might be explained by the higher molecular charge of l
-carnitine and the partial deprotonation of its carboxylic group (pKa 4.2) at pH 2.8, leading to a decrease of its net charge [37
] and thereby its susceptibility to electromigration. As expected, bupivacaine was easily extracted with EY above 85% with all tested voltages, showing that the Pa-EME setup was functioning well.
The highest voltage tested, i.e., 120 V, led to the highest EYs for all compounds. However, at this voltage, a significant fluid leakage between the acceptor and donor plate was observed. This was explained by gas production caused by electrolysis in the acceptor compartment, leading to an overpressure in this closed compartment and, ultimately, to the loss of the acceptor phase. This supports the apparent higher EY that were observed due to an overestimation of the acceptor compartment volume according to Equation (1).
Therefore, an applied voltage value of 100 V was selected for further experiments, leading to the highest EYs without any volume loss observed.
The influence of the concentration of untreated plasma in the donor compartment on the extraction was then investigated. As shown in Figure 3
, a significant drop of PE was observed for highly polar compounds in the presence of 10% untreated plasma or higher. The strong decrease in PE for betaine, l
-carnitine, and deoxy-l
-carnitine might be explained by partial deprotonation of their carboxylic acid group due to a pH increase in the compartment caused by addition of plasma (up to pH 4.5 with 50% of plasma) and the poor buffer capacity of acetic acid 1%. Therefore, the pH increase of the donor compartment led to a decrease of metabolite net charge. In these conditions, TMAO and choline remained both fully ionized, irrespective of the pH.
The observed decrease in PE for the two polar compounds might be further explained by the drastic reduction of the electric field in the system due to the plasma ionic strength, leading to lower logD values of polar compounds and slower migration into the SLM [39
]. On the other hand, bupivacaine was more slightly affected by this phenomenon when using up to 20% of plasma content, due to the known high logD of non-polar compounds in the SLM. However, when using 50% of plasma content, the important increase of ionic strength decreased the electric field, leading to a significantly lower PE for this analyte.
Another hypothesis, i.e., the disturbance of the interface between the organic layer and the plasma sample due to a superficial protein precipitation in this region, seems unlikely since a vortex is created in the donor compartment thanks to the very high agitation rate (i.e., 1400 rpm). In addition, since bupivacaine is a drug known to be 95% linked to plasmatic proteins but showed high PE values, the protein-binding hypothesis was discarded. However, PVDF material is well-known for its very high affinity and protein binding capacity. Therefore, this might lead to perturbation of the organic layer by competition between the organic solvent and proteins.
Finally, as high sensitivity is essential in metabolomics, an untreated plasma content of 10% was selected for further experiments.
In order to test our two hypotheses, we evaluated two approaches to modify the sample composition, namely, (1) addition of an organic solvent to the sample (e.g., MeOH) to enhance the electric field, and (2) PP prior to extraction to remove proteins.
Various proportions of MeOH were added to the donor compartment, i.e., 10%, 20%, and 50%, but no significant difference in PE was observed (data not shown). Higher concentrations of organic modifier were not tested to avoid possible SLM dissolution [40
PP using TCA is known to allow for an efficient protein removal (i.e., above 99%) with a limited dilution factor [41
]. The PP-TCA method takes place in pure aqueous phase, showing the benefit of avoiding the evaporation step that is necessary when using organic solvents for PP, which is favorable for compatibility with the EME approach. Moreover, the low pH that occurs when using PP-TCA is suitable for the extraction of cationic compounds. Therefore, PP-TCA was selected for further investigations, in a 0.05:1 ratio (TCA:sample). After a 10-fold dilution of the precipitated plasma, the TCA concentration was close to 30 mM, which was sufficient to obtain a pH of 2.0 and ensure protonation of all the compounds of interest. With an applied voltage of 100 V, a high current (more than 1–2 mA/well) was observed. This relatively high current could be explained by higher conductivity of the solution of 30 mM TCA compared to 1% acetic acid. In order to avoid the potential issues generated by a high current, the extraction current was set to 400 µA/well to minimize electrolysis and gas production, while maximizing both EY and PE. As presented in Figure 4
, good EY (up to 75% for polar compounds) and low variability (as low as 7%) were obtained using the optimal conditions.
For all the selected compounds, similar or higher PE (up to 100%) and low variability (as low as 2%) were obtained on protein precipitated plasma samples compared to neat solutions, especially for betaine and l
-carnitine where PE were 3.3 and 2.7-fold higher, respectively, when PP-TCA was used. This increase in PE remains unexplained and requires further investigations. Nevertheless, a PP step appears essential prior to EME of highly polar compounds to reduce both ionic strength and buffer capacity of biological fluids. To assess the maximal precipitated plasma volume extractable with the developed approach, different plasma contents, i.e., up to 50%, were evaluated. Using the same PP-TCA method, an increase of the precipitated plasma content into the donor compartment involved an increase of TCA concentration, up to 150 mM. The results are summarized in Table 2
Except for L-carnitine, good PE (between 50% and 100%) values were obtained for all tested precipitated plasma amounts. No change in PE was observed for TMAO, choline, and bupivacaine. An increase of PE for betaine with an increased plasma content and TCA concentration was obtained, explained by the lower pH observed, i.e., a pH value of 1.0–1.5 with 50% of plasma and 150 mM of TCA versus pH of 2.0 with 30 mM of TCA and 10% of plasma content. This decrease of pH induces a higher positive net charge on betaine, leading to an increase of its electrophoretic mobility. Nevertheless, the decrease in PE observed for deoxy-l-carnitine and carnitine remain unexplained but might be the consequence of stability issues of these metabolites in highly acidic conditions (pH ~1).
Finally, the linear response function of the developed method was evaluated. For this purpose, a fast LC-MS/MS method was used to be in similar conditions as what is observed in the context of large cohort studies with thousands of samples. The calibration curve was plotted using ratios of non-deuterated compounds and dISTDs in a neat solution. Calibration samples with increasing concentrations of TMAO, choline, betaine, l
-carnitine and deoxy-l
-carnitine (Table 2
) were made and mixed to constant concentrations of their dISTD in 30 mM TCA, to mimic EME conditions of the donor compartment composition. These calibration samples were extracted using the optimized Pa-EME setup and the selected experimental parameters (i.e., 400 µA/well, 15 min, 1400 rpm). As shown in Table 2
, a linear response function (R2
> 0.995) was obtained on concentration ranges of two orders of magnitude for all compounds of interest (Figure S1