Chromatography
Figure 5. Chromatogram of matrix match acidic analytes standard at 0.5 µg/mL. Method is shown in Table 2 and SIM ion used is shown in Table 5.
Figure 3. Schematic of all plasma samples including matrix-matched standards (Row G) loaded onto the MicroluteTM SLE 200 mg.
LLE
LLE was performed using 1.5 mL centrifuge tubes. 2 x 500 µL of solvent (see Table 1 for solvent used for each type of analyte) was added to 200 µL of diluted plasma (as with SLE, the same pre-treatment was applied). For each extraction, the samples were shaken for 5 minutes by hand and centrifuged for 5 minutes at 10,000 RCF. The organic layer was carefully transferred with a glass pasteur pipette from the tube into
a collection plate (Cat no: 219250), evaporated to dryness with N2 with the Porvair Sciences Ultravap® Levante (Cat no: 500226) at 35°C and reconstituted in 200 µL of starting mobile phase.
Results and Discussion
Chromatography Figures 4, 5 and 6 represent chromatograms of basic, acidic and neutral analytes respectively. A Raptor Biphenyl (30 mm x 2.1 mm, 1.8 µm) column was used to separate the three classes of compounds with three different chromatographic methods (Tables 1, 2, 3 and 4) all in under 10 minutes.
Figure 6. Chromatogram of matrix match neutral analytes standard at 0.5 µg/mL. Method is shown in Table 3 and SIM ion used is shown in Table 5.
Recovery and Reproducibility Recovery and Reproducibility Analyte recovery was calculated using the following equation:
Analyte reproducibility was measured using relative standard deviation which was calculated using the following equation:
Figure 4. Chromatogram of matrix match basic analytes standard at 0.5 µg/mL. Method is shown in Table 1 and SIM ion used is shown in Table 5.
Figure 7 shows the mean recoveries for SLE and LLE side by side. Microlute™ SLE shows improved reproducible recoveries across the analytes from each class (acidic, basic and neutral). The average recovery across all analytes tested was 94±6%. The recoveries ranged from 89 - 106%. For LLE recoveries were not as good with an average of 87±7% with recoveries ranging from 79-98%. So whilst it gave some high recoveries, on average it was lower than SLE and was less reproducible. For LLE recoveries can be improved by performing a third extraction on the sample. However, this would not provide a direct comparison to SLE and would further increase the sample processing time. It may also cause issues by introducing matrix effects, due to the further extraction of unwanted compounds which can affect the ionisation in the instrument’s source.
When comparing the differences between the two techniques, SLE recovery is equivalent
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