Novel Cookson-type reagent for LC/MS/MS of vitamin D compounds
[2] E. Hyppönen, E. Läärä, A. Reunanen, M. R. Järvelin,
a CN column [YMC-Pack CN (5 μm, 150 × 2.0 mm i.d.; YMC)],
combined with different mobile phases, the satisfactory
separation of 25(OH)D3 and 3-epi-25(OH)D3 could not be
achieved as the PTAD-derivatives; the tRs were 7.3 and 8.1 min
(twin peaks) for the 25(OH)D3-PTAD and 7.6 and 7.8 min (twin
peaks) for 3-epi-25(OH)D3-PTAD, when a YMC-Pack Pro C18
RS column was used with the mobile phase of methanol/
10 mM ammonium formate (4:1, v/v). The peak derived from
3-epi-25(OH)D3 partly overlapped with the major peak of
25(OH)D3-PTAD (Fig. 4(c)). Based on these results, the PTAD-
derivatization-based assay may lead to overestimation of the 25
(OH)D3 levels, i.e., false diagnosis of vitamin D deficiency in
infants. On the contrary, our new method with the DAPTAD-
derivatization can avoid the overestimation of the 25(OH)D3
levels, which is the greatest advantage of our method over the
method employing the PTAD-derivatization. To separate
25(OH)D3-PTAD from 3-epi-25(OH)D3-PTAD, additional acetyl-
ation of the hydroxy group at the 3-position was needed.[19]
S. M. Virtanen. Intake of vitamin D and risk of type 1
diabetes: a birth-cohort study. Lancet 2001, 358,1500.
[3] C. J. Willer, D. A. Dyment, A. D. Sadovnick, P. M. Rothwell,
T. J. Murray, G. C. Ebers. Canadian Collaborative Study
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maternal vitamin D as a risk factor for schizophrenia: a pilot
study using banked sera. Schizophr. Res. 2003, 63, 73.
[5] T. Higashi, K. Shimada, T. Toyo’oka. Advances in
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[6] J. M. El-Khoury, E. Z. Reineks, S. Wang. Progress of liquid
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[7] M. Vogeser. Quantification of circulating 25-hydroxyvitamin
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[8] S. S.-C. Tai, M. Bedner, K. W. Phinney. Development of a
candidate reference measurement procedure for the determi-
nation of 25-hydroxyvitamin D3 and 25-hydroxyvitamin
D2 in human serum using isotope-dilution liquid
chromatography–tandem mass spectrometry. Anal. Chem.
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[9] J. M. W. van den Ouweland, A. M. Beijers, P. N. M. Demacker,
H. van Daal. Measurement of 25-OH-vitamin D in human
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CONCLUSIONS
We developed a novel Cookson-type reagent, DAPTAD, for
enhancing sensitivity and specificity in the LC/ESI-MS/MS
assay of 25(OH)D3. The detection response of the DAPTAD-
derivative was 30-fold and twice as high as that of the intact
25(OH)D3 and the PTAD-derivative, respectively, and the LOD
was 0.25 fmol. The derivatization procedure with DAPTAD
has a sufficient applicability for the assay of neonatal DBSs;
the DAPTAD-derivatization followed by LC/ESI-MS/MS
enabled the detection of trace amounts (in the low-ng/mL
range) of 25(OH)D3 with a simple pretreatment (only methanol
extraction), small sample volume (a disk with a 3-mm diameter,
equivalent to 2.65 μL of whole blood) and short chromatog-
raphic run time (10 min). Based on the DAPTAD-derivatization,
25(OH)D3 was well separated from a potent interfering
metabolite, 3-epi-25(OH)D3. Thus, our new method using
DAPTAD can reduce the overestimation of the 25(OH)D3 levels,
and will prove helpful in the diagnosis of vitamin D deficiency
in infants.
[12] T. Higashi, Y. Shibayama, M. Fuji, K. Shimada. Liquid
chromatography–tandem mass spectrometric method for
the determination of salivary 25-hydroxyvitamin D3: a
noninvasive tool for the assessment of vitamin D status.
Anal. Bioanal. Chem. 2008, 391, 229.
[13] P. A. Aronov, L. M. Hall, K. Dettmer, C. B. Stephensen,
B. D. Hammock. Metabolic profiling of major vitamin D
metabolites using Diels-Alder derivatization and ultra-
We are now studying the application of the DAPTAD-
derivatization for the quantitative analysis of 1α,25-
dihydroxyvitamin D3, an active form of vitamin D3, in plasma
(in the pg/mL range). Details of these results will be reported
in the future.
performance
liquid
chromatography–tandem
mass
spectrometry. Anal. Bioanal. Chem. 2008, 391, 1917.
[14] D. Eyles, C. Anderson, P. Ko, A. Jones, A. Thomas, T. Burne,
P. B. Mortensen, B. Nørgaard-Pedersen, D. M. Hougaard,
J. McGrath. A sensitive LC/MS/MS assay of 25OH vitamin
D3 and 25OH vitamin D2 in dried blood spots. Clin. Chim.
Acta 2009, 403, 145.
[15] X. Duan, B. Weinstock-Guttman, H. Wang, E. Bang, J. Li,
M. Ramanathan, J. Qu. Ultrasensitive quantification of serum
vitamin D metabolites using selective solid-phase extraction
coupled to microflow liquid chromatography and isotope-
dilution mass spectrometry. Anal. Chem. 2010, 82, 2488.
[16] S. Ding, I. Schoenmakers, K. Jones, A. Koulman, A. Prentice,
D. A. Volmer. Quantitative determination of vitamin D
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Acknowledgements
This study was supported in part by a Grant-in-Aid for
Scientific Research (C) for 2011–2013 (to T. H., Grant No.
23590046) from the Japan Society for the Promotion of Science.
Our thanks are due to Emeritus Professor Kazutake Shimada
(Kanazawa University) for his insightful comments and
suggestions.
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Copyright © 2013 John Wiley & Sons, Ltd.
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