Biomedical Engineering Reference
In-Depth Information
hydrophilic metabolites. Analyte specific tuning of ionization conditions favouring
the ionization of non-conjugated analyte may reduce the impact of in source trans-
formation, but cannot rule out its occurrence in individual samples. Hence, sufficient
chromatographic resolution is the most reliable approach for this aim, albeit requir-
ing additional analysis time.
In-source transformation effects can only be perceived during method develop-
ment if extracts of biological samples are analyzed under extended chromatographic
runs. Interference by in-source transformation will not become evident if only
spiked quality control materials are used which do not contain relevant metabolites.
If one or more additional peak in the SRM trace of the target analyte is observed in
comparison to analyte reference solutions, chromatography may only be “acceler-
ated” to such degree that these peaks still remain baseline separated from the
analyte.
3.3
Isobaric Mass Transitions and Isomers
In
single-stage
LC-MS, potential inaccuracy due to isobaric constituents present in
complex biological samples is well recognized, since isobaric compounds share one
nominal molecular mass and are in general indistinguishable by mere molecular
mass based detection. Isobars are either structural isomers of the target analyte shar-
ing the same elemental formula or are structurally not related compounds sharing
the same nominal molecular mass with or without sharing the same molecular
formula.
Using LC-MS/MS, the probability that an analyte (or its internal standard com-
pound) shares both precursor and product ion masses with other unrelated com-
pounds present in the sample is far smaller than the probability of isobar effects in
single stage LC-MS with mere molecular weight selective detection. Nevertheless,
due to the extreme complexity of the metabolome and proteome, isobaric mass
transitions are a ubiquitous potential source of analytical inaccuracy in clinical
LC-MS/MS applications [
54
]. It must be noted, that shared mass transition of ana-
lyte and potential disturber may not necessarily be one of the most favoured mass
transition of the latter: If the disturber is present in much higher concentrations
compared to the target analyte, substantial problems may result even if only a less
populated fragment ion of the disturber is shared by the target analyte.
With at least two different SRM-transitions available for the analysis of a com-
pound, one transition can be used for quantification (“quantifier”) and the other one
for result confirmation (“qualifier”). If the branching ratio of quantifier to qualifier
is found outside pre-defined ranges of acceptance, it is typically tried to apply a re-
analysis with a more extensive chromatographic separation in order to overcome
co-elution of interfering compound and analyte. This quantifier-qualifier-principle
is used extensively in GC-MS and is now often applied for quantitative LC-MS/MS
application too—especially in legally strictly regulated environments as forensic
toxicology or pesticide and mycotoxin analysis in feed and foodstuff [
55
] . In contrast,
