In the early 19th century a ground-breaking discovery was made that linked a dietary
deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin
was named vitamin D and extensive research regarding the physiological importance of this
vitamin followed ever since. It is currently known that vitamin D plays an important role in
maintaining the calcium and phosphate homeostasis in the human body. Less clear
evidence states the medical importance of vitamin D in the prevention and cancer,
autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct
forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term
“vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D
status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive
form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a
continuously growing field both on individual and epidemiological level. For decades
laboratories have struggled to produce a robust method capable of quantifying these
different vitamin D analogues and uncovered a new form of complexity regarding the
analysis of these analogues. The identification of the C3-epimeric forms of vitamin D
metabolites has forced laboratories to rethink their analytical methods and several concerns
were raised regarding the overestimation of the true vitamin D status by current analytical
methods. The quantification of the biologically active and inactive forms of vitamin D is
reported to be difficult and to date very few LC-MS/MS methods reported in the literature are
able to quantify various vitamin D analogues. However, to our knowledge none of these
methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the
biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single
run.
Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method
for the quantification of twelve vitamin D analogues in a single run. This was done by
optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in
positive ESI mode and sufficient chromatographic separation between analytes with similar
chemical properties. Furthermore, the optimised method was validated to ensure the
accuracy and precision of the method before implementation into a clinical environment. The
vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2,
25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi-
25(OH)D3, 7(OH)4C3 and 1α(OH)D3.
A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most
efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes.
It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin
D analogue can produce a more abundant MS1 signal and that the ESI source parameters
vary between analytes with different chemical properties and should therefore be optimised
individually for each analyte. Various columns were assessed and sufficient
chromatographic separation between the relevant analytes was achieved with an Agilent
Technologies Pentafluorophenyl column. Baseline separation was achieved between
25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a
requirement for this method to be viable. The method was subjected to a series of validation
steps to ensure the accuracy and precision of the method. These included the assessment
of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and
recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable
repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method
accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for
48 hours after sample preparation with no interferences found for co-eluting analytes.
Finally, based on the sigma metric scale specifications, it was calculated that this method
proved to be “world class” and very little QC is needed to ensure the quality of the data
derived from this method.
Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the
quantification of twelve vitamin D analogues in a single run was successfully developed. All
the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each
analyte and the method was validated based on a series of method validation steps required
for implementation into a clinical laboratory. This validation proved this method to be ready
for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015
| Identifer | oai:union.ndltd.org:NWUBOLOKA1/oai:dspace.nwu.ac.za:10394/14893 |
| Date | January 2014 |
| Creators | Van der Westhuizen, Jacobus Cornelius |
| Source Sets | North-West University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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