Biomedical Engineering Reference
In-Depth Information
monitoring several MS/MS transitions. Applications range from single compound
assay devoted to diverse analytes like parathyroid hormone (PTH) [
23
] , thyroglobulin
[
24
] , insulin [
25
] , haemoglobin A2 [
26
] , Zn- a 2 glycoprotein [
27
] , ghrelin [
28
] , to
multiplex approaches devoted to cardiac markers [
29,
30
] . Since the methodological
cornerstones of quantitative clinical proteomics—i.e. analyte enrichment, chro-
matographic separation, and MS/MS based quantification are following the same
physical principles as in small molecule LC-MS/MS, causes of assay inaccuracy
and imprecision are akin [
31
]. However, due to the different chemical nature of
drugs and metabolites on the one and peptides and proteins on the other hand, addi-
tional specific analyte related challenges are encountered for each of the platforms.
3
Methodological Limitations
As soon as LC-MS/MS became more and more utilized in routine clinical laborato-
ries, potential limitations in the analytical performance of this powerful technology
became evident [
32
]. The selectivity of MS/MS detection was one aspect of this
technology that was particularly overestimated during the first years of its applica-
tion to clinical chemistry [
33-
35
]. The recent debate about the inaccuracy of
25-OH-vitamin D results obtained by LC-MS/MS [
36-
38
] was widely noticed and
has highlighted the need for rigorous quality assurance in clinical mass spectrome-
try. Indeed, quality assurance is a particular challenge in clinical LC-MS/MS appli-
cations because the end users themselves implement and validate the methods. Only
a few commercial LC-MS/MS assay kits are presently available—additionally, the
instrument configurations used to run these assays are extremely heterogeneous.
3.1
Ionization Ef fi cacy Modulation
Physico-chemical processes involved in ion generation and transfer under atmo-
spheric conditions (atmospheric pressure ionization, API) are complex and modu-
lated by a plethora of factors [
39
]. Generally, API operating under conventional
chromatographic flow rates is a rather inefficient process. Even if sophisticated
pneumatically assisted ion-sources are employed, only a minute fraction of target
analytes become ionized and actually enter the high vacuum area of the mass ana-
lyzer [
40
]. If not operated at its optimum, a significant ion yield fluctuations on a
timescale from seconds to minutes can be observed [
41
]. Hence, compared to UV-
or FLD-detection the stability of LC-MS/MS signals has to be considered rather
poor. This high degree of variation makes internal standardization mandatory for
quantitative LC-MS/MS analyses.
The term “matrix effects” globally refers to the impact of constituents from the
evaporated liquid (i.e. originating from the solvents and the sample) on the processes
of de-clustering and ionization of analytes within the ion source region [
42,
43
] . If
