Biomedical Engineering Reference
In-Depth Information
Table 1
Major fragments, MRM transitions, and optimal collision energies of the four corticosteroids
Compound
(
m/z
)
(Precursor/product)
Energy (eV)
BUD
413, 147, 173, 225, 323
431/413
17
DEX
373, 355, 147, 237, 171,
337
393/373
16
TACA
415, 339, 213, 397, 147,
357
435/415
15
DEX-AC 415, 397, 337, 147, 319 435/415 15
The fragments are listed in order of relative abundance, from high to low (Reproduced with
permission from American Chemical Society)
result in compromised ruggedness of analytical methods compared with larger I.D.
columns. Suf fi cient mLC resolution was desirable for ultrasensitive quantification
for the following reasons: first, plasma samples are highly complex and the concen-
tration of target compounds is extremely low; interferences from the sample matrix
may present a significant problem. Therefore, chromatographic separation of those
interfering components from the target corticosteroids was necessary to ensure
quantitative accuracy at low target concentrations. Second, the formation of both
[M + Na]
+
and [M + K]
+
ions was relatively high when minimal mLC separation was
used, even when selective SPE cleanup was employed. However, with sufficient
chromatographic separation, undesirable adduct ions were much reduced in inten-
sity relative to [M + H]
+
in this study. This observation probably results from the fact
that K
+
and Na
+
salts do not co-elute with the target compounds when sufficient
chromatographic separation is obtained. Third, when using the minimal m LC sepa-
ration, a severe ion suppression effect arising from the complex sample matrix was
observed. This effect, which depresses the ionization of all target compounds, was
demonstrated using an evaluation approach we established previously [
7
] . By
achieving suf fi cient mLC separation, it was possible to minimize this effect and thus
improve sensitivity.
In order to maximize the acceptable
V
inj
without causing peak broadening, a two-
segment gradient was designed in this study: the first gradient step is the “trapping
segment,” and it employs a high percent of the aqueous mobile phase A. As a result,
relatively hydrophobic compounds such as the corticosteroids are focused on the
front end of the column during sample loading. The subsequent gradient steps
constitute the “separation segment,” in which a gradually increasing percentage of
mobile phases B separate the compounds that were focused on the column during
the trapping segment of the gradient. To determine the optimal gradient conditions
in the separation segment, we investigated the chromatographic retention vs. S/N.
Different partition ratios (
k
) of corticosteroids were achieved with five different
gradient elution conditions, using an injection volume of 5 mL; Fig.
4
[
4
] shows the
S/N as a function of mLC partition ratio (
k
). From this data, it is clear that the S/N
of the target analytes improved considerably when their chromatographic retention
increased, and for that beyond a certain extent of retention, the S/N approaches a
maximum. Column-overloading was overcome to a great extent by applying this
on-column focusing strategy.
