Biomedical Engineering Reference
In-Depth Information
4
3
2
1
0
0
40
80
120
Sample Sequence No.
System Suitability
CS and QC
Normal Samples
Problematic Samples
Fig. 8
Example of missed and double addition of the internal standard. Analyte: clopidogrel
carboxylic acid; extraction: evaporation-free protein precipitation; sample volume: 50 m L; IS
volume: 150 mL. Reproduced from ref. [
36
] with permission from Elsevier
an internal standard is added to all the samples including concentration-known cali-
bration standards and quality controls as well as concentration-unknown incurred
samples. Therefore, this type of variation or error directly affects the accuracy of
reported concentration, and it should be minimized or eliminated whenever
possible.
It is relatively easy to spot variation or an error in the addition of IS for CS and
QC samples. As their concentrations are known, large variations in internal standard
addition for CS and QC samples would result in the rejection of the CS or QC
samples, even a whole run. However, the variation or error in IS addition for
unknown incurred samples would be difficult to be ascertained unless the addition
of the internal standard is doubled or missed (Fig.
8
). Even when a doubled or near
zero IS response is observed, there could be other reasons than the addition of the
IS. For example, missed addition or incorrect amount of a derivatization reagent can
produce near zero IS response as well.
Though human error is usually the cause, proper method development can make
a difference in reducing this type of error or variations. First, a large volume of IS,
such as 200 mL should be used when it is added by a repeater pipette because small
volumes (such as 50 mL or less) are more prone to imprecision than large ones. In
addition, it would be extremely difficult to visually spot missed or doubled addition
for an internal standard when its volume is much smaller than that of samples and/
or other reagents (e.g., buffer). Second, it would be helpful to reduce errors by
adding the usually colorless IS solution first and then incurred samples, which are
usually colored, such as slight yellowish for plasma samples or dark red for whole
blood samples.
