Introduction
This poster compares sample preparation options for the extraction of a panel of endogenous steroids from serum. LC-MS/MS parameters were investigated for increased sensitivity: MRM transitions, chromatography and mobile phase additives for use with positive and negative ionisation modes. Particular emphasis was placed on the sample preparation to provide high reproducible recoveries whilst minimizing matrix effects and co-extracted materials such as proteins and phospholipids. Solid phase extraction was compared to supported liquid extraction in terms of recoveries, ion suppression, phospholipid content, calibration curve performance and overall sensitivity.
Experimental
Reagents Standards, ammonium acetate and ammonium fluoride were purchased from Sigma-Aldrich Company ltd. (Gillingham, UK). LC/MS grade solvents were from Honeywell Research Chemicals (Bucharest, Romania). Water (18.2 MΩ.cm) was drawn fresh daily from a Direct-Q 5 water purifier (Merck Millipore, Watford, UK). Pooled human plasma was from The Welsh Blood Service (Pontyclun, UK) or Golden West Biologicals, Inc. (Temecula CA). Sample Preparation Extractions were developed using supported liquid extraction or polymer-based SPE in 96 fixed well plate format. ISOLUTE® SLE+ was used in the 400 μL capacity 96-well plate format (P/N 820-0400-P01) following a load-wait-elute procedure (Figure 2).
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