Biomedical Engineering Reference
In-Depth Information
which typically includes dilution, extraction, evaporation, and reconstitution.
Variable losses of the analytes may occur during these sample treatment steps. In
addition, there might be variations during the LC-MS analysis, such as variations in
injection volume and particularly in ionization (ion suppression or enhancement
caused by coeluted matrix components; refer to [
1
] ).
To reduce the impact of analyte losses and instrumental variations on the quanti-
tation of an analyte, an internal standard (IS), which has the same or similar physical
and chemical properties as the analyte, is added in equal amount to both concentra-
tion-known (calibration standards and quality controls) and unknown samples prior
to sample treatment. By using signal ratios of the analyte to its IS (instead of using
analyte absolute signal) for quantitation, the losses and variations can be corrected,
which improves the precision and accuracy of final analytical results for unknown
samples.
Internal standards could be used in external calibration, matrix-matched external
calibration, and standard addition calibration [
2
]. However, the use of internal stan-
dards in LC-MS quantitative methods should not be confused with internal calibra-
tion in which an internal standard is employed as a calibrant and the concentration
of a unknown sample is calculated from the concentration of this internal standard
and its analyte/IS signal ratio, i.e., the concentration of the unknown sample is
calculated without the need for a calibration curve [
3
]. The use of internal standards
in most LC-MS quantitative methods belongs to “signal-ratio calibration” or inter-
nal standardization [
2,
4
]. In fact, the majority of bioanalytical LC-MS methods use
matrix-matched signal-ratio external calibration.
The analytes of interest in quantitative bioanalysis vary, from small molecules
with molecular weights usually less than 1,000 Da (e.g., drugs and their metabo-
lites) to large biopolymers, such as proteins. The focus of this chapter is on the
quantitation of small molecules in biological samples by LC-MS, though some of
the principles presented in this chapter are also applicable to the quantitative analysis
of large molecules in biological samples.
2
Selection and Use of Internal Standards for Quantitative
LC-MS Bioanalysis
2.1
Requirements on Internal Standards
An internal standard should meet the following three requirements. First, it should
have the same or very similar physical-chemical properties as the analyte, particu-
larly hydrophobicity and ionization characteristics, so that it can mimic closely the
performance of the analyte in every stage of analysis, i.e., from sample preparation,
chromatographic separation, to mass spectrometric detection. In this way, any losses
during sample preparation or variations in the mass spectrometry detection can be
corrected.
