Biomedical Engineering Reference
In-Depth Information
4.2
Experimental
The detailed experiment conditions are shown in a previous publication [
6
] .
4.3
Results and Discussion
In order to achieve highest sensitivity of VitD metabolites detection, the efforts for
method development were focused on three components of the analytical flow path
(1) the selective SPE extraction procedure, (2) the ionization and SRM method for
MS/MS, and (3) the mLC loading and separation method.
For the purpose of achieving a selective SPE extraction that eliminates matrix
components to the greatest extent possible without loss of analytes, an extensive
optimization of the wash/elute conditions was conducted, the procedure is similar as
described in previous examples. In order to achieve high efficiencies of extraction
and reproducibility of quantification, a protein precipitation with ACN/methanol
prior to SPE extraction is found important to assist releasing VitD metabolites from
serum protein and derivatization by PTAD plays a critical role to improve the stability
of VitD metabolites.
In this study, a 0.5-mm-I.D. capillary column was chosen; however, the manu-
facturer-recommended injection volume (I.V.) for this mLC column is 0.2-0.4 m L.
Nonetheless, the hypothesis is that the serum matrix has been reduced substantially
by the selective SPE, and therefore, a markedly higher I.V. of the extracted sample
can be tolerated without compromising chromatographic performance, provided an
efficient sample focusing strategy was employed. To test this hypothesis, the evalu-
ation of the mLC-MS/MS performance as a function of I.V. using serum samples
fortified with the four analytes was performed. Typical chromatograms are shown in
Fig.
11
[
6
]. The retention times of the analyte increased only slightly as the I.V.
increased, but no appreciable change in peak width and peak shape was observed
over the range of I.V. from 0.2 to 9.5 mL, while both the intensity and S/N increased
roughly in proportion with I.V. This indicated the selective SPE procedure, in con-
junction with the sample focusing approach, permits an I.V. that is more than 20-fold
higher than manufacturer's recommendation, without causing column overcapacity.
Therefore, an I.V. of 9.5 mL was selected for quantification of serum samples.
Sufficient chromatographic separation is critical in this work, for three reasons:
first, the peak of target compounds must be separated from isomeric peaks, interfer-
ing signals and endogenous compounds that cause ion suppression. Second, given
the close similarity in the structure and
m
/
z
among the analytes and I.S., it is neces-
sary to resolve these compounds chromatographically to avoid various “cross talk”
effects. Third, a sufficient mLC separation was found further helped to suppress the
sodium-adducted precursors, a phenomenon also observed previously. Other consid-
erations for developing the mLC method included the following: (1) upon complete
elution of all analytes, it is necessary to perform an extensive cleaning of the column
and spray tip, to minimize the memory effect and (2) a relatively high throughput is
