Claudio De Nardi (Presenter)
Thermo Fisher Scientific
Authorship: Claudio De Nardi1, Johannes Engl2, Radouane Nagim2
(1) Thermo Fisher Scientific GmbH, Dreieich, Germany (2) RECIPE Chemicals+Instruments GmbH, Munich, Germany
| Short Abstract A clinical research analytical method for the quantification of 25-hydroxyvitamin D2 and D3 in human plasma or serum with chromatographic resolution of 3-epi-25-OH-vitamin D3 is reported. The method involves a simple protein precipitation step followed by online SPE using a Thermo Scientific™ Transcend™ II system; a Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer with atmospheric pressure chemical ionization is used for detection by single reaction monitoring (SRM) using 25-hydroxyvitamin D3-d6 as the internal standard. The method was analytically validated using the MS MS7000 ClinMass® LC-MS/MS Complete Kit 25-OH-Vitamin D2/D3 in Plasma and Serum – On-Line Analysis from RECIPE and limit of quantification, linearity range, accuracy and intra- and inter-assay precision were evaluated for both analytes. |
Long Abstract
Introduction
An analytical method for clinical research for the quantification of 25-hydroxyvitamin D2 and D3 in human plasma or serum is reported. The method, based on the MS7000 ClinMass® LC-MS/MS Complete Kit 25-OH-Vitamin D2/D3 in Plasma and Serum – On-Line Analysis from RECIPE, allows for the chromatographic separation of the C-3 epimeric form of 25-hydroxyvitamin D3 (3-epi-25-OH-vitamin D3), avoiding its overestimation especially in samples from newborns and children. Sample extraction is performed by protein precipitation followed by online SPE using a Thermo Scientific™ Transcend™ II system; a Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer with atmospheric pressure chemical ionization is used for detection by single reaction monitoring (SRM) using 25-hydroxyvitamin D3-d6 as the internal standard. Implementation of the kit included the evaluation of the limit of quantification, linearity ranges, accuracy and intra- and inter-assay precision for each analyte.
Methods
The reported analytical method for clinical research is based on the MS7000 ClinMass® LC-MS/MS Complete Kit 25-OH-Vitamin D2/D3 in Plasma and Serum – On-Line Analysis from RECIPE and allows for the quantification of 25-hydroxyvitamin D2 and D3 in human plasma or serum. In this method, the C-3 epimeric form of 25-hydroxyvitamin D3 (3-epi-25-OH-vitamin D3), which can have significant concentrations in samples of newborns and children, is separated from the analyte peak with a sufficient degree to avoid false high analytical values. The kit includes calibrators and controls at four and two different levels, respectively, covering a concentration range between 8.2 and 68.5 µg/L for 25-hydroxyvitamin D2 and between 9.4 and 77.3 µg/L for 25-hydroxyvitamin D3. 25-hydroxyvitamin D3-d6 is used as the internal standard for the quantification of both analytes. Sample clean-up is performed by a preliminary protein precipitation with internal standard addition followed by on-line solid phase extraction (SPE) on a Transcend II system using mobile phases, SPE cartridge and analytical column provided with the kit. Detection was performed by SRM on a TSQ Endura triple quadrupole mass spectrometer with atmospheric pressure chemical ionization operated in positive mode. Two SRM transitions for each analyte were included in the acquisition method for quantification and confirmation, respectively.
Results
The method was implemented evaluating limit of quantification, linearity ranges, accuracy and intra- and inter-assay precision for each analyte; the assay proved to be linear in the calibration range covered by the kit, with limits of quantification (LOQ) of 2.1 µg/L for both analytes, an upper limit (ULOQ) of 154.7 and 157.0 µg/L for 25-hydroxyvitamin D2 and D3, respectively and a correlation factor (R2) always above 0.997. Accuracy for the assay was evaluated in terms of trueness of measurement using the DEQAS Proficiency Test Sample #466 and #467 from DEQAS prepared and analyzed on 4 different days in single runs each day; the percentage bias between nominal and average back-calculated concentration for these control samples was 2.4 and 2.5% for 25-hydroxyvitamin D3 in sample #466 and #467, respectively. Intra-assay precision was evaluated in terms of percentage coefficient of variation (%CV) using the kit controls at both levels in replicates of eight (n=8) prepared and analyzed in one batch; the %CV for intra-assay precision was 4.5% and 3.1% for the lower and upper control for 25-hydroxyvitamin D2, respectively and 2.2% and 2.8% for the lower and upper control for 25-hydroxyvitamin D3, respectively. Inter-assay precision was evaluated on the same controls in replicates of three (n=3) prepared and analyzed on five different days; the %CV for inter-assay precision was 10.0% and 8.3% for the lower and upper control for 25-hydroxyvitamin D2, respectively and 4.3% and 3.0% for the lower and upper control for 25-hydroxyvitamin D3, respectively.
Conclusions
A liquid chromatography tandem mass spectrometry method for clinical research for the quantification of 25-hydroxyvitamin D2 and D3 in human plasma or serum using the MS7000 ClinMass® LC-MS/MS Complete Kit 25-OH-Vitamin D2/D3 in Plasma and Serum – On-Line Analysis from RECIPE was implemented and analytically validated on a Transcend II system connected to a TSQ Endura triple quadrupole mass spectrometer with atmospheric pressure chemical ionisation. This analytical method allows for the chromatographic resolution between 25-hydroxyvitamin D3 and its C-3 epimeric form, avoiding its overestimation especially in samples from newborns and children. The method meets research laboratory requirements, sensitivity and linearity of response covering analyte concentration ranges provided by the kit.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Thermo Fisher Scientific |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | Thermo Fisher Scientific |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |