• 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