Method Development, Validation, and Sample Analysis for Regulated Quantitative Bioanalysis Using LC-MS/MS - LC-MS in Drug Bioanalysis - page 35

analysis, biological samples need to be extracted from biological matrixes via
various cleanup procedures. Currently the widely used extraction techniques are
protein precipitation extraction (PPE), liquid-liquid extraction (LLE) and solid
phase extraction (SPE). Additionally, several novel extraction techniques, i.e., sup-
ported-liquid extraction (SLE), online solid phase extraction (online-SPE), phos-
pholipid removal SPE/PPE extraction are also available.

2.1 Protein Precipitation Extraction (PPE)

PPE is the most commonly used extraction method. In principle, the underlying
mechanism of protein precipitation is to lower the solubility of the analytes by add-
ing organic solvent such as methanol (MeOH) or acetonitrile (MeCN) or saturated
buffer salt such as 10 M zinc sulfate (ZnSO 4 ) or high concentration of strong acid or
base such as 5-10 % Trifluoroacetic acid (TFA) or hydroperchloric acid (HClO 4 ).
In LC-MS/MS application, due to incompatibility of high salt or strong acid or base
with MS source, the most common approach is to add organic solvent at minimal
ratio of 1:3 (v/v) of matrix-organic solvent. If the active drug is highly bound to
protein, a volatile acid or base such as formic acid (FA) or ammonium hydroxide
(NH 4 OH) respectively at 5-10 % is utilized to disrupt the binding thus increasing
the recovery of the drug.
Historically PPE has been conducted in tubes. In recent years, PPE in 96-well
format such as commercial PPE precipitation plate has gained popularity in order to
increase throughput. PPE in tubes is inexpensive and reliable but requires manual
transfer/pipetting, vortex mixing, and centrifugation. In PPE in 96-well format, all
96 samples can be transferred simultaneously which increases the throughput tre-
mendously. Because the transfer volume among samples is the same and pro-
grammed in advance, it ensures consistent recovery, thus improving the overall
performance of the assay.

2.2 Liquid-Liquid Extraction and Supported-Liquid Extraction

Although PPE is the most efficient and inexpensive extraction technique, it is also
the most nonspecific extraction procedure which is known to be susceptible to
matrix effect for LC-MS/MS assay. In contrast, LLE provides a much cleaner
extract. LLE, also known as solvent extraction and partitioning, separates analytes
based on their relative solubility in two different immiscible solvents, usually water
and an organic solvent. The most commonly used solvents for LLE are ethyl acetate
(EtOAc), methyl tert -butyl-ether (MtBE), methylene chloride (CH 2 Cl 2 ), and hexane
or the combination of the above solvents. In order to manipulate the polarity of the
analytes, often a volatile acid or base such as FA or NH 4 OH respectively at 5-10 %