Lab Med Online.  2012 Oct;2(4):188-196.

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

• Kim H
• Jun SH
• Kim T
• Song SH
• Park KU
• Song J

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 50degrees C for 1 hr followed by Diels-Alder derivatization with ethyl acetate containing 0.1 mg/mL PTAD. The samples were resuspended in 60 microL 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.

Keyword

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

MeSH Terms

25-Hydroxyvitamin D 2
Acetates
Calcifediol
Calibration
Formates
Humans
Immunoassay
Liquid-Liquid Extraction
Mass Spectrometry
Quaternary Ammonium Compounds
Tandem Mass Spectrometry
Triazoles
Vitamin D
25-Hydroxyvitamin D 2
Acetates
Calcifediol
Formates
Quaternary Ammonium Compounds
Triazoles
Vitamin D

Figure

• 

  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].

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