Biomedical Engineering Reference
In-Depth Information
and 96 % of the patients, the serum levels were above a LOQ of 5 pg/mL. Apparently,
conventional LC-MS/MS is not sufficiently sensitive for this task. The quantitative
results revealed that the serum levels of both 1,25(OH)2VitD3 and 24,25(OH)2VitD3
are significantly lower in multiple sclerosis patients than in healthy subjects.
However, with a cognizance that this study may be limited in the sample sizes, this
finding may need to be examined in further clinical investigation. More mechanism-
related studies are desired to elucidate how this altered VitD metabolism relates to
multiple sclerosis development. The method developed here permitted, for the first
time, the consistent and robust quantification of low-abundance dihydroxyl VitD
metabolites such as the 1,25(OH)2VitD3 in clinical samples by LC-MS/MS-based
methods. This work also demonstrated the applicability of the selective-SPE-m LC-
MS/MS strategy in large-scale clinical analysis. Therefore, the method is well suited
for the analysis of VitD metabolites in large-scale clinical studies, especially for
these involving pathological conditions where the levels of the dihydroxyl VitD
metabolites could be low.
5
Conclusion
In the above paradigms, selective SPE strategies were optimized and applied for
preparations of highly complex biological samples. Because of this selective
extraction procedure, the acceptable
V
inj
of biological samples on the m LC column
was increased significantly. An additional benefit of this selective SPE strategy
was an improved S/N resulting from decreased ion suppression. A two-segment
gradient which included on-column focusing in first segment was used to main-
tain an optimal chromatographic separation. As a result, a high injection volume
is feasible for mLC-MS analysis and sensitivity was increased, without compro-
mising chromatographic performance or operational robustness. In injection vol-
ume optimization, the retention times of target compounds increased only slightly
as
V
inj
was increased, but peak broadening or peak shape deterioration was not
observed over a broad range of
V
inj
up to 9.5 mL. Furthermore, the S/N for all tar-
gets increased in proportion with
V
inj
. This indicates that the selective SPE proce-
dure reduced the complexity of the plasma samples significantly, to a degree that
would permit a high
V
inj
on the mLC column without causing column overcapacity.
Furthermore, the utilization of separate “trapping” and “separation” gradient seg-
ments (described above) counteracted the tendency of increased
V
inj
to increase
peak widths and thus rendered peak widths for the target compounds independent
of loading times.
Compared with current LC-MS/MS methods, this approach increased assay
sensitivity greatly (LOQ at low-pg/mL in plasma). The developed methods are
also superior to some highly sensitive RIA methods, in that it offers significantly
better selectivity and quantitative accuracy.
Collectively, the method has potentially widespread applications given the per-
vasive clinical investigation of diverse and highly potent pharmaceutical agents
