토코페롤 검사를 위한 순상 고압액체크로마토그래피법 평가
Evaluation of Normal Phase High-Performance Liquid Chromatography for Analysis of Tocopherols
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배경 : 항산화 물질로 잘 알려져 있는 토코페롤의 항염증 작용및 항암 효과 등 여러 다른 역할 등이 최근에 밝혀짐에 따라, 토코페롤을 신속 정확하며 신뢰할 수 있게 검사할 수 있는 방법이요구되고 있다. 저자들은 순상 고압액체크로마코그래피(normalphase high performance liquid chromatography, NP-HPLC)이용하여 토코페롤 검사에 대한 타당성을 평가하고자 하였다. 방법 : 분석 컬럼으로 hypersil silica (4.6 mm×200 mm×5m, Thermo electron corporation, Keystone, PA, USA)를 장착한 HP1100 시리즈(Hewlett-Packard company, Wilmington, DE, USA) 시스템을 이용하였다. 이동상은 n-헥산 및 이소프로필 알코올이 98:2 용적비로 혼합된 것이었으며, 주입량은 10L, 속도는 0.7 mL/min, 검출파장은 295 nm에서 측정하였다. 토코페롤에 대한 분리인자, 직선성, 정확성, 정밀도, 검출한계 및 임상 검체의 농도 범위 평가를 시행하였다. 결과 : -, -, - 및 -토코페롤 검출시간은 15분 이내로 이들의 머무름 인자는 0.61-2.13이며 위치이성질체를 포함한 분리도 역시 탁월하였다. 각각의 토코페롤 일정 농도에서 상관계수는 0.999 이상으로 우수한 직선성을 보였다. 중간 및 고농도 표준용액의 정밀도 변이계수는 11% 이내, 저농도 표준용액의 정밀도 변이계수는 18% 이내에 들었다. 평균 회수율은 91.4-107.8%이었으며 bias 는 모두 15% 이내로 만족스러운 결과를 보였다. 또한 -, -, -및 -토코페롤의 최소검출한계값은 각각 0.038, 0.064, 0.078, 0.049 이었다. 결론 : NP-HPLC를 이용한 토코페롤 검사는 신속하고 정확하며 신뢰할 수 있는 분석법으로 향후 임상검사실에서 유용하게 쓰일 수 있을 것으로 사료된다.
Abstract
Background : Recently the tocopherol, which has been proven as an antioxidant, has also been found to possess non-antioxidant roles such as anti-inflammatory and chemopreventive effects, which has heightened the importance of rapid, accurate and reliable testing methods. We evaluated the performance of normal phase high-pressure liquid chromatography (NP-HPLC) for analysis of tocopherol in serum. Methods : The chromatographic separation parameters, linearity, precision, accuracy and detection limit were evaluated. The analytical column was hypersil silica (4.6 mm×200 mm×5 m, Thermo Electron Corporation, Keystone, PA, USA) and mobile phase comprised 2% (v/v) of isopropanol in n-hexane. Analyses were run at a flow rate of 0.7 mL/min at 295 nm. Results : The run time of separation of tocopherol was completed in less than 15 minutes and its retention factor (0.61-2.13) and resolution including positional isomers ( - and -tocopherol) were excellent. Their linearity was good (r >0.999). The coefficient of variation (CV) at medium and high concentrations did not exceed 15% and those at low concentrations did not exceed 20%. The range of its recovery was 91.4-107.8% and bias was within 15%. The detection limits of -, -, - and - tocopherol were 0.038, 0.064, 0.078 and 0.049, respectively. Conclusions : NP-HPLC provided a rapid, accurate and reliable performance for the separation of tocopherol and would be helpful as a routine method in the clinical laboratory.
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