Evaluation of the suitability of ionic liquid-based liquid-liquid microextractions for blood protein removal

Abstract

The analysis of biological samples, such as whole blood, comes with several sample preparation challenges. Biological matrices often contain a variety of endogenous components that can interfere with the determination of xenobiotics. Especially blood plasma proteins (e.g. serum albumin) are known to interfere with electrospray ionization and result in analyte ion suppression. Sample preparation techniques should guarantee adequate removal of these biomolecules. The current study aims to determine to which extent proteins are removed from whole blood samples, using ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME). A qualitative comparison of the protein presence in extracts of IL-DLLME, solidphase extraction (SPE) and protein precipitation (PP) was performed, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, UV/VIS spectrophotometry was used to determine the protein content of a whole blood sample and IL-DLLME, SPE and PP extracts of the same sample. Finally, a quantitative comparison of matrix effects of benzodiazepines present in both whole blood and water samples. SDS-PAGE results showed that IL-DLLME extracts still contained proteins (i.e. albumin, hemoglobin); however, band intensities were comparable to SPE extracts. Spectrophotometric tests showed a total protein content of approximately 2 mg/mL in the final extracts. PP showed the highest protein extraction rate (19 mg/mL). Quantitative ME results showed no significant differences (α = 0.05) between blood and water IL-DLLME extracts. Overall, this is the first study to conclude that IL-DLLME is able to sufficiently remove blood proteins from whole blood samples, in order to avoid significant ion suppression.

The variation in the levels of 18 amino acids in the cortex and plasma of cerebral ischemia C57BL/6 mice

“…The pretreated samples were then investigated for the clearance of proteins and lipids according to the method reported previously (De Boeck et al, 2019; Visnovitz et al, 2019). The results showed that the MTBE + ACN method could significantly reduce the lipids of the sample compared with ACN alone.…”

“…The protein has a strong absorption at 280 nm. Protein absorption has been reported to be proportional to the concentration (De Boeck et al, 2019). The sulfo-phospho-vanillin lipids assay was introduced to determine the content of lipids in the pretreated samples (Visnovitz et al, 2019).…”

“…at 15,000g for 15 min at 4 C. MTBE + ACN group: 300 μL of cortex homogenate or plasma was added to 300 μL MTBE and the mixture vortexed for 30 s and then centrifuged at 15,000g for 15 min at 4 C. Then, 300 μL of subnatant was added to 600 μL of ACN and the mixture vortexed for 30 s and then centrifuged at 15,000g for 15 min at 4 C. 0.4 M PCA group: 300 μL of cortex homogenate or plasma was added to 600 μL of 0.4 molÁL −1 PCA and the mixture vortexed for 30 s and then centrifuged at 15,000g for 15 min at 4 C. MTBE + 0.4 M PCA group: 300 μL of cortex homogenate or plasma was added to 300 μL MTBE and the mixture vortexed for 30 s and then centrifuged at 15,000g for 15 min at 4 C. Then, 300 μL of subnatant was added to 600 μL of 0.4 molÁL −1 PCA and the mixture vortexed for 30 s and then centrifuged at 15,000g for 15 min at 4 C. The pretreated samples were then investigated for the clearance of proteins and lipids according to the method reported previously(De Boeck et al, 2019;Visnovitz et al, 2019). The results showed that the MTBE + ACN method could significantly reduce the lipids of the sample compared with ACN alone.…”

Development and validation of the UPLC-MS method for simultaneous determination of six new psychoactive substances

“…The currently common pretreatments are solid phase extraction (SPE), liquid liquid extraction (LLE) and protein precipitation (PP). [32][33][34][35] SPE bases on the difference in partition coefficient between the solid phase and the liquid phase of the target to separate the target from urine sample. And it requires long-time extraction and complex multi-step process.…”