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13


Results and Discussion SLE Method Optimisation


The optimisation of SLE methodology involved the evaluation of analyte recovery, average reproducibility, and matrix effect as a function of elution solvent, solvent volume and sample soaking time. Elution solvents investigated included: MTBE, 1-Chlorobutane, DCM:EtOAc (1:1), MTBE:Chloroform (4:1) and MTBE:Hexane (4:1). A total volume of 1.2 mL was used for each elution solvent, applied in different aliquots: 3 x 400 µL, as two aliquots of 600 µL, 2 x 600 µL and 1 x 1200 µL. Results demonstrated in Figure 5 indicate that (a) the synthetic SLE sorbent provides higher analyte recovery, more consistent reproducibility, and reduced matrix effect when compared to DE, and (b) DCM/EtOAc (1:1) and EtOAc provide optimal recovery for each analyte, however elution with EtOAc exhibited higher matrix effect for NFNN. Consequently, DCM/ EtOAc (1:1) was selected as the preferred elution solvent. Notably, when using the synthetic SLE sorbent, recovery was largely independent of the number of elution aliquots; 2 x 600 µL provided marginally improved recovery and was therefore selected as the final elution scheme. However, when using DE SLE, changes in recovery were more significant with different elution steps.


Equilibration time following sample loading on the SLE sorbent was investigated for up


to 40 min. Results indicate no significant variation in recovery at different equilibrium time, and therefore 5 min was used to optimise sample throughput. Fact that recovery was not impacted with additional equilibration time ensures a level of robustness in the methodology, since analytes do not irreversibly bind to the sorbent substrate with extended soaking.


Phospholipid Depletion


Figure 4: SLE procedure for mouse brain homogenate using synthetic SLE sorbent.


Phospholipids (PPLs) have been identified as a major source of matrix effect in LC-MS/ MS assays, leading to signal supression under ESI conditions. As PPLs elute over a wide range of retention times, their removal via sample preparation is critical to minimise the likelihood of coelution with analyte, or accumulation within the LC/MS system. Within many different classes of phospholipids in biological matrix, phosphatidylcholine (PC) and lysophosphatidylcholine (Lyso-PC) are the two most abundant classes. Therefore, eight PC and four Lyso-PC compounds, together with total PPLs (184 > 184), were monitored for depletion following SLE on both synthetic


SLE sorbent and diatomaceous earth substrates. Results reported in Figure 6 demonstrate significant improvement for PPL depletion using the synthetic SLE sorbent, with > 50% of total PPL retained on the sorbent compared to diatomaceous earth.


With greater PPL depletion efficiency using the synthetic SLE sorbent, many benefits are conferred to the bio-analytical scientist, including (a) reduced likelihood for ionisation suppression, (b) the ability to develop faster chromatography without fear of PPL co-elution, and (c) increased assay robustness by elimination of PPL accumulation on-column and in the ion source, leading to extended column lifetime and reduced instrument downtime.


Figure 5: SLE elution solvent optimisation.


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