Biomedical Engineering Reference
In-Depth Information
2.4
Selection of Sample Cleanup Procedure
Many factors need to be taken into consideration while choosing a particular sample
cleanup procedure for a LC-MS/MS assay. It is very beneficial to obtain critical
information prior to initializing laboratory work, such as the scope of the project,
the timeline of the project, the target Lower Limit of Quantitation (LLOQ) level, the
maximal aliquot size, the polarity and stability of the analyte of interest. It is also
important to recognize the benefit and pitfall of each sample cleanup technique.
However, automation should be always considered in order to improve throughput
and eliminate human error. In general, PPE extraction should be the first choice for
an assay if the target LLOQ is at ng/mL or sub ng/mL level and the method is
intended to support early drug screening and discovery study. Because PPE is the
most nonselective extraction technique and tends to have severe matrix effect, it is
recommended that the maximal sample aliquot volume should not be more than
100 mL. If the target LLOQ is at pg/mL or sub pg/mL level and it is used to support
GLP study or clinical study for long period, LLE or SLE or SPE at 96-well plate
format should be explored. LLE or SLE extraction can remove most matrix effect
components and produce clean extracts. Based on the requirement of sample vol-
ume needed for desired LLOQ, automated SLE extraction can be used for sample
aliquot volume less than 400 mL and larger sample aliquot volume for manual LLE
extraction. While SLE/LLE is only suitable for nonpolar analytes, SPE is the best
extraction option for an assay containing prodrug, active drug, and metabolite with
vastly diversi fi ed polarity.
When developing sample cleanup procedure, the traditional approach is to start
from the simplest extraction such as PPE, then move into cleaner extraction such as
SLE or SPE. This linear and trial and error approach is time-consuming and
inefficient. In our laboratory, a well-thought and systematic approach, nicknamed as
“Amoeba Method Development Program,” is implemented. Using Amoeba Sample
Preparation Screening Protocol, three extraction techniques, i.e., PPE, SLE and SPE
under various pH and solvent conditions were evaluated simultaneously. The vari-
ous conditions tested in this protocol are: PPE with acidic, or neutral or basic ace-
tonrile (MeCN); SLE with three different pH and three different organic solvents
such as EtOAc, MtBE, and 1:1 hexane-EtOAc using Isolute
®
200 mg SLE + plate;
SPE under various combination of wash and elution solvent with acidic or neutral
or basic conditions using Oasis MD SPE plate (Waters, Milford, MA, USA). With
replicate of three for each condition, one test batch contains 26 different extraction
conditions and a total of 81 samples including three neat control samples.
An example of the above extraction screening protocol is shown in Fig.
3
using
guanfacine as model compound. In PPE and SLE groups, the letter of A or B or N
represents acidic or basic or neutral condition. In SPE group, the first letter “A” in
A/B represents acidic wash solvent and the second letter “B” means basic elution
solvent. As shown in Fig.
3
, the highest recovery was obtained using SLE and MtBE
under basic condition. It was also noticed that there was relatively high recovery
using various SPE conditions and the most interesting results were from MCX
