Measurement of Serum Levels of 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 Using Diels-Alder Derivatization and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

Hyungsuk Kim; Sun-Hee Jun; Taeksoo Kim; Sang Hoon Song; Kyoung Un Park; Junghan Song

doi:10.3343/lmo.2012.2.4.188

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Kim, Jun, Kim, Song, Park, and Song: Measurement of Serum Levels of 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 Using Diels-Alder Derivatization and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

Original Article

Clinical Chemistry

Laboratory Medicine Online

Laboratory Medicine Online 2012; 2(4): 188-196.

Published online: 1 October 2012

DOI: https://doi.org/10.3343/lmo.2012.2.4.188

Measurement of Serum Levels of 25-Hydroxyvitamin D₃ and 25-Hydroxyvitamin D₂ Using Diels-Alder Derivatization and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

Hyungsuk Kim, M.D.¹, Sun-Hee Jun, M.T.², Taeksoo Kim, M.D.^1,², Sang Hoon Song, M.D.^1,², Kyoung Un Park, M.D.^1,², Junghan Song, M.D.^1,²

¹Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea.

²Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Korea.

Corresponding author: Junghan Song, M.D. Department of Laboratory Medicine, Seoul National University Bundang Hospital, 166 Gumi-ro, Bundang-gu, Seongnam 463-707, Korea. Tel: +82-31-787-7691, Fax: +82-31-787-4015, songjhcp@snu.ac.kr

Received 14 December 2011 Revised 23 April 2012 Accepted 30 April 2012

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Vitamin D has been recently shown to play important roles in the functioning of various systems. Most of the current analytical methods for measuring vitamin D levels are based on immunoassays. We simultaneously measured the levels of 25-hydroxyvitamin D₃ [ 25(OH)D₃ ] and 25-hydroxyvitamin D₂ [ 25(OH)D₂ ] in human serum by performing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) after Diels-Alder derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and evaluated the performance of our method.

Methods

After liquid-liquid extraction, samples were dried under N₂ at 50℃ for 1 hr followed by Diels-Alder derivatization with ethyl acetate containing 0.1 mg/mL PTAD. The samples were resuspended in 60 µL of methanol:10 mM ammonium formate solution (1:1, V/V). C18 UPLC column and positive ion multiple reaction monitoring transitions such as m/z 558.35→298.1, 25(OH)D₃; m/z 570.35→298.1, 25(OH)D₂; and m/z 564.35→298.1, hexadeuterated-25(OH)D₃ were used for UPLC-MS/MS.

Results

The within-run imprecision (CVs) for 25(OH)D₃ and 25(OH)D₂ were 3.5-4.0% and 3.8-4.2%, respectively, and the corresponding between-run CVs were 3.3-5.5% and 4.7-5.8%. The lower limit of quantification for 25(OH)D₃ and 25(OH)D₂ were 0.5 and 1.0 ng/mL, respectively. The curve for interassay calibration variability data obtained over concentrations of 0-120 ng/mL for 25(OH)D₃ and 0-90 ng/mL for 25(OH)D₂ was linear and reproducible [ 25(OH)D₃, R²=0.993; 25(OH)D₂, R²=0.998]. The total 25(OH)D levels in Koreans (average, 18.7 ng/mL) were lower than those in American Caucasians, and the percentage of people with total 25(OH)D levels under 10 ng/mL was 8.1%.

Conclusions

Our method to measure 25(OH)D₃ and 25(OH)D₂ levels by performing UPLC-MS/MS after PTAD derivatization showed good performance as a sensitive and reproducible method for routine analysis of vitamin D status.

Keywords: Vitamin D, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), Tandem mass spectrometry, Ultra performance liquid chromatography, Immunoassay

Figures and Tables

Fig. 1

Representative chromatogram for ultra-performance liquid chromatography-multiple reaction monitoring of 25-hydroxyvitamin D₂ [25(OH)D₂], hexadeuterated 25-hydroxyvitamin D₃ [d6-25(OH)D₃], and 25-hydroxyvitamin D₃ [25(OH)D₃].

Fig. 2

Chromatograms obtained using a post-column infusion system showing identifiable ion suppression at around 0.4 min and retention of target metabolites at 1.9 min. (A) 25-hydroxyvitamin D₂ [25(OH)D₂], serum; (B) hexadeuterated 25-hydroxyvitamin D₃ [d6-25(OH)D₃], serum; (C) 25(OH)D₂, mobile phase; (D) d6-25(OH)D₃, mobile phase.

Fig. 3

Passing-Bablok Regression Analysis to compare ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with (A) DiaSorin radioimmunoassay (RIA), 25-hydroxyvitamin D [25(OH)D] and (B) BioSource RIA, 25-hydroxyvitamin D₃ [25(OH)D₃].

Table 1

Imprecision of 25-hydroxyvitamin D analysis

^*Unit of mean, ng/mL.

Abbreviations: PTAD, 4-phenyl-1,2,4-triazoline-3,5-dione; UPLC-MS/MS, ultra-performance liquid chromatography-tandem mass spectrometry; 25(OH)D₃, 25-hydroxyvitamin D₃; 25(OH)D₂, 25-hydroxyvitamin D₂.

Table 2

Bias between 25-hydroxyvitamin D₃ [25(OH)D₃] and 25-hydroxyvitamin D₂ [25(OH)D₂] values measured by revised ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and the National Institute of Standards and Technology (NIST) target values provided in the NIST Standard Reference Material (SRM) 972

Table 3

Serum levels of 25-hydroxyvitamin D₃ [25(OH)D₃], 25-hydroxyvitamin D₂ [25(OH)D₂], and total 25-hydroxyvitamin D [25(OH)D] in a Korean population

^*Values shown as mean (range); ^†values shown as percentage of persons.

Table 4

Characteristic differences between various methods for measuring 25-hydroxyvitamin D [25(OH)D] levels

Abbreviations: LC-MS/MS, liquid chromatography-tandem mass spectrometry; NIST, National Institute of Standards and Technology; SRM, Standard Reference Material; 25(OH)D, 25-hydroxyvitamin D.

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