Biomedical Engineering Reference
In-Depth Information
demonstrated as a critical component for healing processes in tissues and bones [ 26 ] .
Therefore, the ability to quantify these circulating VitD metabolites would be highly
valuable for the clinical studies of diseases that are associated with the deficiency
and/or metabolic dysfunction of VitD.
A wide variety of methodologies have been developed for the quantification of
VitD metabolites in serum/plasma samples. Among them, immunoassays including
the radioimmunoassay (RIA) and enzyme immunoassay (EIA) are the most preva-
lent [ 24 ]. Nevertheless, the limitation of most of the RIA and EIA methods is that
only one metabolite can be measured per assay therefore the selectivity, accuracy,
and interbatch/lab reproducibility could be problematic [ 24, 27, 28 ] . Other methods
include competitive protein binding assay, automated chemiluminescent protein-
binding assay [ 29 ] and HPLC-UV [ 30 ]. More recently, liquid chromatography tan-
dem mass spectrometry (LC-MS/MS) has emerged as a promising alternative.
Although LC-MS/MS-based methods could readily measure 25(OH)VitD, which
presents at relatively high levels in serum, quantification of the dihydroxyl metabo-
lites can be highly challenging. For example, the 1,25(OH)2VitD3, which is highly
active and of primary interest for the research of many diseases [ 14, 24 ] , present at
extremely low levels (low pg/mL) in human serum that are significantly lower than
the detection limits of conventional LC-MS/MS methods. The relatively poor ion-
ization efficiency of this metabolite, further compounds the problem. Although a
recent study employed a derivatization procedure with conventional LC-MS/MS
achieved a lower limit of quantification (LLOQ) of 25 pg/mL using 0.5 mL of
human serum, the LLOQ achieved is not sufficiently sensitive for the measurement
of 1,25(OH)2VitD3 in human serum, especially for those patients (e.g., multiple
sclerosis) whose serum levels of VitD metabolites are expected to be lower than
healthy subjects.
Multiple sclerosis is a neurodegenerative, chronic inflammatory disease, and
has become the most prevalent neurological disorder affecting young adults in the
USA [ 31 ]. Much evidence suggests that low levels of the VitD metabolites are
potential risk factors for developing multiple sclerosis, probably owing to their
immune modulating effects [ 22 ]. For instance, some epidemiological studies
showed that low serum concentrations of 25(OH)VitD in adolescence are associ-
ated with an increased risk of developing multiple sclerosis and VitD supplemen-
tation and high serum concentrations of 25(OH)VitD are protective [ 32 ] .
Nevertheless, the underlying mechanisms for the association of VitD metabolites
and the development of multiple sclerosis remain unclear. In order to study this
association, an ultrasensitive, selective and robust analytical method for
quantification of the key circulating VitD metabolites in clinical samples is essen-
tial. In this study, four clinically related VitD metabolites were selected. They are
25(OH)VitD2 and 25(OH)VitD3, which are the indicators of VitD repletion;
1,25(OH)2VitD3 and 24,25(OH)2VitD3, which are biologically active metabo-
lites. In this study, an ultrasensitive and selective-SPE/m LC-MS/MS analytical
method was developed in order to quantify ultralow concentration of VitD metab-
olites in complex biological matrices and evaluated the method's applicability in
a large-scale clinical analysis.
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