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Method Validation and Quantification of Lutein and Zeaxanthin from Green Leafy Vegetables using the UPLC System

UPLC를 이용한 lutein과 zeaxanthin의 분석법 검증 및 엽채류에서의 정량적 평가

  • Kim, Suna (Department of Home Economics, College of Natural Science, Korea National Open University) ;
  • Kim, Ji-Sun (Department of Home Economics, College of Natural Science, Korea National Open University)
  • 김선아 (한국방송통신대학교 자연과학대학 가정학과) ;
  • 김지선 (한국방송통신대학교 자연과학대학 가정학과)
  • Received : 2012.07.25
  • Accepted : 2012.10.30
  • Published : 2012.12.31

Abstract

The objective of this research is to present method development and validation for the simultaneous determination of lutein and zeaxanthin using ultra performance liquid chromatography (UPLC). Also, rapid quantification was performed on six green leafy vegetables (Allium tuberosum, Aster scaber, Hemerocallis fulva, Pimpinella brachycarpa, Sedum sarmentosum and Spinacia oleracea) that are commonly consumed in Korea. Separation and quantification were successfully achieved with a Waters Acquity BEH C18 ($50{\times}2.1mm$, $1.7{\mu}m$) column by 85% methanol within 5 min. Two compounds showed good linearity ($r^2$ > 0.9968) in $1-150{\mu}g/mL$. Limit of detection (LOD) and quantification (LOQ) for lutein and zeaxanthin were 1.7 and 5.1 g/mL and 2.1 and 6.3 g/mL, respectively. The RSD for intra- and inter-day precision of each compound was less than 10.69%. The recovery of each compound was in the range of 91.75-105.13%. Aster scaber and Spinacia oleracea contained significantly higher amounts of lutein ($4.06{\pm}0.24$ and $3.97{\pm}0.10mg$/100 g of fresh weight), respectively.

본 연구에서는 UPLC를 이용하여 lutein과 zeaxanthin을 단시간에 동시 분석할 수 있는 새로운 방법을 개발한 후 분석법에 대한 평가를 수행하였으며, 확립된 방법으로 6종의 엽채류에서 lutein과 zeaxanthin을 정량 분석하였다. 분석 컬럼은 Acquity UPLC BEH C18($1.7{\mu}m$, $2.1{\times}50mm$), 이동상 용매는 85% methanol을 사용하였으며, 검출파장은 450 nm, 이동상의 유속은 0.5 mL/min, 분석온도는 $40^{\circ}C$, 시료주입량은 $1.0{\mu}L$로 설정하여 분석하였다. 확립된 분석조건에서 lutein과 zeaxanthin의 피크머무름시간(RT)은 각각 4.35분과 4.56분이며, 표준용액의 피크머무름시간과 엽채류 시료들의 피크머무름시간은 일치하였다. lutein과 zeaxanthin에 대한 표준검정곡선은 $1-150{\mu}g/mL$ 농도범위에서 상관계수($r^2$)는 0.9968 이상의 양호한 직선성을 나타내어 분석에 적합함을 알 수 있었으며, 검출한계는 lutein과 zeaxanthin이 각각 $1.7{\mu}g/mL$$2.1{\mu}g/mL$, 정량한계는 lutein과 zeaxanthin이 각각 $5.1{\mu}g/mL$$6.3{\mu}g/mL$로 설정되었다. Lutein과 zeaxanthin의 회수율은 각각 95.76-105.13%와 91.75-103.24% 범위를 보였다. Lutein의 일내 RSD값은 4.65-7.33%, 일간 RSD값은 4.04-10.69%이었으며, zeaxanthin의 일내 RSD값은 5.41-7.31%, 일간 RSD값은 3.79-9.82%이었다. 확립된 분석법으로 한국인이 많이 섭취하는 녹색과 엽채류인 부추, 취나물, 원추리, 참나물, 돌나물 시금치에서 lutein과 zeaxanthin을 분석한 결과 lutein 함량은 시금치와 취나물이 $3.97{\pm}0.10mg$/100 g fw, $4.06{\pm}0.24mg$/100 g fw로 가장 높았으며, 참나물과 원추리가 $3.39{\pm}0.43mg$/100 g fw, $3.39{\pm}0.15$ mg/100 g fw로 높았다. Zeaxanthin의 경우 취나물과 원추리가 각각 $0.95{\pm}0.00mg$/100 g fw와 $0.96{\pm}0.01mg$/100 g fw로 가장 높았다. 본 연구에서 확립된 UPLC 분석법은 lutein과 zeaxanthin을 신속하고 효과적으로 동시 분석하는데 이용될 수 있을 것이며, 한국인이 많이 섭취하는 엽채류 중 시금치와 취나물은 lutein과 zeaxanthin의 좋은 식이 급원일 뿐 아니라, 꾸준히 섭취하면 눈건강에 도움을 줄 수 있을 것으로 사료된다.

Keywords

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