On the other hand, it was observed an inefficient phase separation evidenced by on small volume of the surfactant rich phase (<50 μL), which affected the extraction efficiency and consequently their analytical response.At pH 5 the resulting coacervate phase volume was ca. 450 μL and showed turbid, which clear up after a protein precipitation step. At this pH, 3 TC and ABC prevailed in ionic form, while NFV and EFV prevailed in their nonionic form according to their pKa. On this basis, it was expected to see lower relative responses for the ionic analytes since they would have lower affinity for the nonionic micelle, and therefore, lower extraction efficiency for these ARV. However, the analytical response of ARV reached their highest values at pH 5 (>95%), except for NFV (ca. 70%), and remained invariant only for EFV at higher pH values. These results could be due to the tradeoff between the effect of affinity of the analyte by the micelles and their efficiency in forming, resulting in an improvement of the analytical signal. NFV analytical responses reached the highest values (>95%) at pH 9 and remained invariant at pH 11.5. The analytical responses of 3 TC and ABC decreased from pH 7 and pH 9, respectively. Phases separation was efficient from pH 7 ahead, obtaining a coacervate-phase volume of ca. 350 μL. Coacervate phase showed the highest dehydration and the smaller volume (ca. 300 μL) at pH of 11.5; resulting in a better phases separation performance and reducing of the loss of coacervate phase when the supernatant was discharged. The decrease in the analytical signal of ABC and 3 TC at pH 11.5 could be due to a signal suppression effect in the mass spectrometer because of a high matrix load in the solvent front. Considering the short retention time of ABC and 3 TC (1.30 and 1.34 min−1, respectively) it was expected that their analytical signal were more affected by this phenomenon than for NFV and EFV, whose retention time were higher (1.61 and 2.06 min−1, respectively).
Based on these results, pH 7.5 was adopted as working condition considering it as the best compromise pH that lead to successful analytical response for the studied ARV. Considering that human plasma pH is 7.4 [27], the extraction pH was not adjusted for CPE of ARV from plasma. This fact is relevant for the development of an analytical methodology for a multi-component analysis since it simplify the procedure.
3.4. Optimization of extraction temperature and time
Reported cloud point temperatures for TritonX-114 are within the range of 15–20 °C [19]. It refers to the temperature at which the surfactant coacervation results notorious in the extraction system, because of its turbid aspect. As it is well known, extraction temperatures above the cloud point, and/or the extended extraction time (>2 min), diminishes the water content of the resulting surfactant reach phase, and thus, its final volume [25]. This argument is based on the fact that as the extraction temperature increases, the hydrogen bonds are disrupted leading, thus, to dehydration of micelles [25]. On the other hand, it is important to take into account that the use of excessive high extraction temperature can reduce the CPE efficiency, because of the thermal stability of surfactant aggregates [30] as well as the analytes lability. Therefore, it was of interest to study the effect of the extraction temperature on the extraction system performance, as well as on the analytes analytical response along with the extraction time within the range 45–75 °C and 10–30 min, respectively. The extraction temperature assays were carried out considering a 20 min extraction time; while the time assays, were carried out at the optimum temperature resulting from the former. As the temperature increased from 45 to 65 °C a better surfactant-rich phase separation as well as an increased response of the analytes was observed. The results showed no significant change neither on the CPE performance nor on the analytical response of the studied ARV at extraction temperatures above 65 °C. Therefore, 65 °C were chosen for further assays. For the extraction time, it was observed that by increasing it, the relative response is subsequently increased, reaching the maximum analytical responses of the studied ARV at 20 min; after which, and remained invariant. Thus, in the following experiments, an extraction time of 20 min was selected for the extraction.
3.5. Analytical performance and method validation
The proposed analytical multi-component method was evaluated in terms of linearity, recovery, accuracy and precision intra- and inter-day, selectivity and sensitivity. Blank plasma was assessed in six different batches of plasma samples by analyzing blanks and spiked samples at LOQ levels. The calibration curves were made under optimized conditions following the proposed methodology, which was described above. Blank plasma were spiked with ARV standards achieving different concentration levels [31
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