• Sample preparation
Serum samples (0.5 mL) were mixed with equal volumes of
internal standard (in acetonitrile) to precipitate protein. After
centrifugation, vitamin D metabolites in the supernatant were
purified by solid phase extraction and dried under nitrogen.
• PTAD derivatization
• LC-MS/MS method
Analysis was performed on a Qtrap5500 mass spectrometer
coupled with a Shimadzu HPLC. In the current method (6 min),
analytes were separated on an Acquity BEH 1.7 µm C18
column 2.1X100 mm (Waters) using acetonitrile and water with
0.1% formic acid as mobile phase at a flow rate of 0.6 mL/min.
In a longer method (9 min), a Kinetex 1.7 µm Phenyl-hexyl
column, 2.1X100 mm (Phenomenex), was used. MRM
transitions and ion source parameters were set according to the
previous report (3). Quantitation was performed in MassHunter
with a six-point calibration based on the ratio of light vs. heavy
labeled internal standard (2H6-1α,25-(OH)2VitD).
CONCLUSIONS
Root Cause Analysis of 1α,25-Dihydroxyvitamin D Overestimation in
LC-MS/MS Assay
Zhicheng Jin, PhD; Ashley N. Siverly; and Xin Yi, PhDDepartment of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX 77030
RESULTS RESULTS continued
• We found that two diastereoisomers were produced in
the reaction between 1α,25-(OH)2VitD3 and PTAD.
• Separation of diastereoisomer peaks offers improved
quantification of DHVD.
• We found that two diastereoisomers were produced in the Diels-Alder
reaction of 1α,25-(OH)2VitD3 with PTAD.
• The presence of 4β,25-(OH)2VitD3 may cause positive bias when
using a short LC-MS/MS assay of 1α,25-(OH)2VitD3.
• Separation of two diastereoisomer peaks provides improved
quantification of DHVD.
• We developed a LC-MS/MS method to quantity 1α,25-(OH)2VitD3 and
1α,25-(OH)2VitD2 without using antibody enrichment.
REFERENCES
1. Eyles D, Anderson C, Ko P, Jones A, Thomas A, Burne T, Mortensen PB,
Norgaard-Pedersen B, Hougarrd DM, McGrath J. A sensitive LC/MS/MS
assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots. Clinica
Chimica Acta, (2009) 403:145-151.
2. Laha TJ, Strathmann FG, Wang Z, de Boer IH, Thummel KE, Hoofnagle AN.
Characterizing and harnessing antibody cross-reactivity for the immunoaffinity
purification of analytes prior to multiplexed liquid chromatography-tandem
mass spectrometry. Clin chem (2012), 58(12):1711-1716.
3. Wang Z, Senn T, Kalhorn T, Zheng XE, Zheng S, Davis CL, Hebert MF, Lin
YS, Thummel KE. Simultaneous measurement of plasma vitamin D3
metabolites including 4β,25-dihydroxyvitamin D3 using liquid chromatography-
tandem mass spectrometry. Anal Biochem, (2011), 418(1):126-133.
The 1α,25-dihydroxyvitamin D (DHVD) test is used to assess
the level of the biologically active form of vitamin D in
patients with chronic renal disease or for differential
diagnosis of hypercalcemia. The DHVD assay is challenging
because there are numerous isomers present in patient
specimens, and the active form has very low circulating
concentrations (picomolar). Our hospital is currently using a
Cookson-type triazolinedione derivatization protocol followed
by LC-MS/MS quantification to determine 1α,25-
dihydroxyvitamin D2 and 1α,25-dihydroxyvitamin D3 levels in
serum specimens (1). While investigating a positive
comparison bias for DHVD assay results, we discovered that
the reference laboratories had adopted a new methodology,
including an antibody purification step (2). We thus modified
our methodology to address these discrepancies.
INTRODUCTION
Positive bias in a DHVD method comparison study
Challenges in the quantification of DHVD: other vitamin D metabolites, such as
4β,25-(OH)2VitD3 and 3-epi-1α,25-(OH)2D3, may be co-eluted with 1α,25-
(OH)2VitD3 peak in a short separation method (2,3).
MATERIALS & METHODS
No.
Reference
method
Short LC-MS/MS
method
Total DHVD
bias
1 59.7 82.3 38%
2 74.6 93.3 25%
3 113 139.0 23%
4 34 51.1 50%
5 45 76.5 70% 4β,25-(OH)2VitD3
R S
Diels-Alder
reaction
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0Time, min
0
50
100 5.97
5.74
Inte
nsity (
%)
B
4β,25-(OH)2VitD3
3-epi-1,25-(OH)2VitD3
Ratio of A:B < 2.
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Time, min
0
50
1005.96
5.73
Inte
nsity (
%)
A
S
R
Reference method
with immunoextraction
LC-MS/MS method
(without using antibody)
No. DHVD3 DHVD2 Total DHVD3 DHVD2 Total Recovery
1 17 42.7 59.7 19.3 38.6 57.9 97%
2 61.5 13.1 74.6 62.6 9.7 72.3 97%
3 113 <5 113 119.4 1.2 120.6 107%
4 34 <5 34 36.9 1.1 38.0 112%
5 24 21 45 24.7 22.9 47.6 106%
0.0 1.0 2.0 3.0 4.0 5.0
Time, min
0
50
100 3.5In
ten
sity (
%)
1α,25-(OH)2VitD3
574.4>314.1
A
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0Time, min
0
50
1005.9
5.7
Neat 1α,25-(OH)2VitD3
574.4>314.1
Inte
nsity (
%)
B
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0Time, min
0
50
100
Inte
nsity (
%)
5.9
5.7
2H6-1α,25-(OH)2VitD3
580.4>314.1
Ratio of two peaks is 2.
C
Figure 1. A: 1α,25-(OH)2VitD3 peak in the
short method. B: Neat 1α,25-(OH)2VitD3
peaks in the longer LC-MS/MS method.
C: Internal standard 2H6-1α,25-(OH)2VitD3
also has two peaks in the longer method.
Ratio of the two peaks is 2. D: Neat
4β,25-(OH)2VitD3 has only one peak in
the longer method. E: Mixture of 1α,25-
(OH)2VitD3 and 4β,25-(OH)2VitD3 (ratio:
1:1) shows three peaks.
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0Time, min
0
50
100 5.74
Inte
nsity (
%)
Neat 4β,25-(OH)2VitD3
574.4>314.1
D
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0Time, min
0
50
1005.80
5.9
5.7
Inte
nsity (
%)
4β,25-(OH)2VitD3
574.4>314.1
E
Ratio of two peaks is 2.
Investigating possible DHVD interferences using an optimized
LC-MS/MS method
Figure 2. Extracted ion chromatogram of 1α,25-(OH)2VitD3 in serum
specimens without (A) and with (B) interference peaks.
Mixture of B and D