Biomedical Engineering Reference
In-Depth Information
development and can persist into routine method validation and sample analysis as
well. In general, carryover is assessed by injecting one or more control blanks (dou-
ble blanks) or QC0 (control blank spiked with internal standard only) immediately
after the Upper Limit of the Quantitation (ULOQ) sample. If carryover is observed
during method development, it is critical to determine the source of carryover and
eliminate or minimize it accordingly. In general, there are two types of carryover:
autosampler injector carryover and LC column carryover. The latter is caused by an
ill-suited combination of column type, mobile phase solvents, and LC gradient elu-
tion program. For example, a fast and steep gradient on a retentive C18 LC column
is more prone for column carryover. Fortunately, column carryover can be over-
come by simply changing to isocratic conditions or using a shallower gradient or
switching to a less retentive LC column type such as C4, C8, or phenyl. For autosam-
pler injector carryover, the selection and combination of appropriate wash solvents
must be evaluated. For a strong wash solvent, the strength of the organic solvent is
critical. However, the addition of acid or base modifier which can facilitate the solu-
bility of the analyte is very useful.
4.3
Internal Standard Response
LC-MS/MS assays typically rely on the use of an internal standard that mimics the
performance of the analyte to improve the precision, reproducibility and reliability
of the assay. An ideal internal standard candidate is a stable-isotope labeled (“stable
labeled”) form of the drug. Because synthesizing stable labeled chemicals can be
expensive and time-consuming, it is very common to use a chemically similar
“structural analog” of the analyte(s) as the internal standard, especially during the
early phases of drug development.
From a practical point of view, internal standard in a LC-MS/MS assay serves three
distinct purposes in the analytical process. The first purpose is to compensate
extraction recovery inconsistencies. The second purpose is to compensate injection
volume variation. The third purpose is to compensate possible “matrix effects” during
the MS ionization process as has already been discussed in detail above. In 2009, Tan
A. et al. reported 12 case studies from incurred sample analyses using a wide variety
of bioanalytical methods for the investigation of inconsistent internal standard response
[
23
]. For similar reasons, it has now become common for laboratory SOPs to contain
specific requirements for the acceptable internal standard response of each individual
sample within a sample batch during regulated bioanalysis. These requirements (e.g.,
60-140 %, 50-150 % of the average internal standard area for all samples in the
batch) ensure that the behavior of the internal standard, regardless of how well it
tracks the analyte, is under control, and is consistent in all samples.
Although these measures and criteria were necessary during sample analysis, it
is recommended to take proactive action steps to ensure appropriate IS behavior
during method development. In general, inconsistent internal standard response is
typically caused by either nonideal extraction conditions such as poor extraction
