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Diels-Alder 유도체화와 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry를 이용한 혈청 25-Hydroxyvitamin D3 및 25-Hydroxyvitamin D2의 측정

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

김택수|전선희|송상훈|박경운|송정한|김형석
대한진단검사의학회 (Korean Society For Laboratory Medicine)|2012.10|Laboratory Medicine Online|제2권 제4호|pp. 188-196

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Vitamin D2Tandem mass spectrometryImmunoassay4-phenyl-14-triazoline-35-dione (PTAD)Ultra performance liquid chromatography

초록

배경: 최근 비타민 D가 체내에서 다양한 기능을 수행하는데 중요한 역할을 한다는 사실이 밝혀지고 있다. 체내 비타민 D 수준을평가하는데 현재 면역검사법이 많이 이용되고 있다. 본 연구에서는 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD)를 활용한 Diels-Alder 유도체화와 ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)를 이용하여 25-hydroxyvitamin D3 [25(OH)D3] 및 25-hydroxyvitamin D2 [25(OH)D2]를 동시에 검사하는 방법을 개발하였고, 그 성능을 평가하였다. 방법: 혈청에서 비타민 D를 유기용매로 추출하고 50˚C N2에서 1시간 동안 건조시킨 후, 0.1 mg/mL의 PTAD가 포함된 ethyl acetate 를 이용하여 Diels-Alder 유도체화를 시켰다. 이후 methanol-10mM ammonium formate (1:1 V/V) 60 μL에 검체를 재부유시켰다. C18 UPLC 컬럼을 이용하여 분리하였고, 25(OH)D3, m/z 558.35→298.1; 25(OH)D2, m/z 570.35→298.1; hexadeuterated-25(OH)D3,m/z 564.35→298.1의 질량분석기 조건에서 정량하였다. 결과: 검사차례내 및 검사차례간 정밀도는 25(OH)D3의 경우 3.5-4.0% 및 3.3-5.5%이었고, 25(OH)D2는 3.8-4.2% 및 4.7-5.8%이었다. 최저정량한계농도는 25(OH)D3 및 25(OH)D2 각각 0.5와 1 ng/mL 이었다. 25(OH)D3는 0-120 ng/mL, 25(OH)D2는 0-90 ng/mL 범위에 걸쳐 모두 직선성이 잘 유지되었다[25(OH)D3, R2 =0.993; 25(OH)D2, R2 =0.998]. 우리나라 성인 집단의 총 25(OH)D의 평균 농도는 18.7 ng/mL로 미국평균 보다는 약간 낮은 분포를 보였고, 10ng/mL 미만의 결핍 비율도 8.1%로 높은 비율을 보였다. 결론: PTAD 유도체화와 UPLC-MS/MS를 이용한 25(OH)D3 및25(OH)D2에 대한 본 검사법은 우수한 검사수행능을 보여 임상 검사실에서 비타민 D의 일상적인 검사에 유용하게 사용할 수 있을것으로 기대된다.

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 (UPLCMS/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˚C 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 betweenrun 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.

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