Biomedical Engineering Reference
In-Depth Information
2
1
0
0
20
40
60
80
100
120
Sample Sequence No.
System Suitability Sample
CS and QC
Incurred Sample
Fig. 17
Low internal standard responses caused by using rubber-line caps, instead of PTFE-lined
caps during liquid-liquid extraction. Analyte: tamsulosin; internal standard: tamsulosin-d
4
.
Reproduced from ref. [
36
] with permission from Elsevier
issue with the LC-MS/MS system or reagents used. Furthermore, when those
incurred samples with unaccepted low internal standard responses were reanalyzed,
their internal standard responses were back to normal. The reassay results matched
those of the first analyses if low internal standard responses were not considered.
Based on the above, it was deemed necessary to use the incurred samples for
further troubleshooting. Unfortunately, this was not granted by the client, which left
the root cause unidentified. The speculation is that the randomly low IS responses
were related to the ascorbic acid added to the incurred samples at the clinic for the
stabilization of the analyte. Although ascorbic acid was also added in the pooled
control plasma used for the preparation of the CS and QC samples, there exist dif-
ferences as outlined in Table
1
. For example, the incurred samples had gone through
only one thawing during the first analysis while the plasma matrix for CS and QC
samples had gone through thawing twice when they were extracted (the first thaw-
ing occurred during the preparation of the CS and QC samples, which were usually
aliquoted and stored in a freezer after the preparation, while the second thawing
took place when the aliquoted CS and QC samples were retrieved from the freezer
prior to being used for sample analysis).
3.3.4
The Impact of Internal Standard Response Variations on Quantitation
The main purpose of troubleshooting IS response variations is to make sure that the
quantitation of unknown samples has not been impacted despite the variable or abnor-
mal IS responses observed. Unfortunately, there is no clear-cut “yes or no” answer to
