LIQUID–LIQUID EXTRACTION continued
Metabolomics The study of metabolic compounds provides insight into biological systems and improves knowledge in key areas of drug discovery and development. This ranges from understanding the basis of a disease to identifying a drug’s mechanism of action and determining any relevant biomarkers to inform treatment op- tions. Metabolites need to be extracted from a cell or tissue sample and separated from other,
undesired, compounds to be studied. This pro- cess can be extremely challenging, due to their diverse physicochemical properties and the broad spectrum of metabolic concentrations within a single sample.
The extraction of metabolic compounds is most often performed by liquid–liquid extrac- tion (LLE). Bligh and Dyer LLE techniques, such as Folch extraction, are favored due to their ability to efficiently extract lipids and polar
endogenous metabolites. Typically, these
extraction techniques are performed manu- ally, a clear bottleneck in many pharmaceutical laboratories. Addressing this and identifying a viable solution is paramount, as the industry moves to more automated analyses for han- dling larger sets of samples.
Automating separation
Table 1 – Calculated logP, logD and pKa for the compounds analyzed (calculated with Percepta software release 2012 [ACD/Labs, Toronto, Canada])
and extraction In this study, a PAL RTC robot was used to complete the necessary Bligh and Dyer sample preparation protocol and man- age sample injection as seen in Figure 1. A fully automated protocol was developed using this setup. It partners Bligh and Dyer extraction with dual-column ultrahigh- performance liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/ MS) separation for the metabolic analysis of algae cell culture. The instrumentation and software are detailed in Figure 2.
Following preliminary reagent addition (Figure 3), the RTC platform performed all the neces- sary extractions and sample injections. This included adding the set reagents and splitting the aqueous and organic fractions prior to injection into the columns (Figure 4). Even with the preliminary manual step, this automated method was less labor-intensive than the com- plex manual method, and significantly reduced the overall sample preparation time.
Figure 5 – Column diagrams showing the peak areas of selected variables (by m/z and retention time, RT) as well as variation obtained after analysis of the aqueous (AQ) and organic (ORG) B&D fractions from the automated or the manual extraction procedure (n = 5). a) AQ fractions at pH 3—C18 column, b) AQ fractions at pH 8—C18 column, c) ORG fractions (pH 4)—C8 column.
AMERICAN LABORATORY 30 MARCH 2016
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