The BioPhorum protocol published 2014 recommends three extractions with dichloromethane (DCM) at 1:1 (volume/volume) ratio. It is allowed to alter volume ratios or the number of extraction cycles to reach sufficient spike recovery and LOD of the standard(s) used in method qualification.

The volume of the combined DCM fractions will depend on the volume of extract used for LLE Thus, the recommendation from 2014 of evaporating down to ~1mL may not be applicable, depending on the starting volume. It is crucial not to evaporate to dryness, as compounds may be lost or not dissolve again. There is no need to reconstitute the concentrated extract to the original sample aliquot volume.

The selection of the extraction solvents was intended to encompass the widest range of potential extractables relevant to our processes (following the 80/20 approach). The extractable profiles of the solvents are selected to overlap to ensure this coverage is thorough. Therefore, we acknowledge that there are some compounds whose extraction efficiency is improved by pH adjustments to the extraction solvents. However, since the goal is to gain a complete picture of the extractables via all of the solvents and time points, the loss of efficiency in one solvent condition is compensated with a more complete picture by the other solvents. If a laboratory prefers to pH adjust, then that is fine and should be specified within the report. If there are specific extractables that a company expects to be present and are known to require pH adjustment, then we support this approach as long as the adjustment and rationale are detailed in the report.