Biomedical Engineering Reference
In-Depth Information
One relatively common type is consistently higher IS response for one whole
subject, including the predose sample. If a particular matrix component is absent or
exists in less amount in one subject and this component happens to be one of the
those that cause ion suppression, then the IS responses for all the samples from this
subject will be higher than those of other subjects. For example, consistently high
IS responses (higher than those of CS and QC samples) were observed for all the
samples of a subject (including the predose sample) during the analysis of repaglin-
ide in a study (Fig.
12a
). Postcolumn (postelution) infusion tests demonstrated the
presence of ion suppression in the pooled control human plasma used for the prepa-
ration of CS and QC samples (Fig.
12b
). However, this ion suppression was absent
for the predose sample from the subject that had high IS responses (Fig.
12c
). Since
the number of lots tested in method development and validation (e.g., 6) is usually
much smaller than that of a study (e.g., 40), this interlot difference might not be
observed during method development and validation. In addition, as the component(s)
that can cause this ion suppression is (are) present in most of the blank sources and
several lots are usually pooled to prepare CS and QC samples, the ion suppression
would be always present for CS and QC samples. For incurred samples, the matrix
is from individual source. Therefore, this type of IS response variations could occur
to some subjects.
The interlot or intersample differences can also cause variations in the recovery
for an analyte and its internal standard. For example, in a method based on liquid-
liquid extraction for
p
-hydroxy-atorvastatin, the recovery of internal standard varied
from 67.19 to 89.99 % (1.5-fold) for the four subjects tested despite the fact that the
ratios of analyte to IS were relatively independent of subject sources, i.e., no impact
on the quantitation [
36
]. It should be borne in mind that a method is usually opti-
mized aiming the maximum recovery for an analyte, i.e., not for any matrix compo-
nents. In case where a matrix component causes matrix effect and the optimal
extraction conditions happen to be an unreliable extraction condition for the matrix
component, variable IS response is very likely.
Moreover, this inter matrix lot difference can result in IS variation through other
routes. For example, rosuvastatin in plasma samples was extracted by LLE with the
automatic transfer of organic layers by a liquid handling system [
41
] . During
incurred sample analysis, consistently low IS responses were observed for a few
subjects in the study, including the predose samples (Fig.
13
). Through careful
examination of the extraction procedure, it was found that the intermediate layers
between organic and aqueous layers in LLE were thicker for some subjects than
those of CS and QC samples, based on which the aspirating height had been set dur-
ing the method development. As this preset aspirating height was not appropriate
for some subjects, a little bit of the intermediate layer between the organic and aque-
ous layers during LLE, which contains salts, was transferred, leading to subsequent
ion suppression during MS detection. The problem was successfully solved by read-
justing the aspirating height. It should be noted that this type of IS response varia-
tions can also happen to other type of LLE with decanting of organic phases, such
as flash-freeze LLE or normal LLE.
