Abstract
A selective and sensitive method for the derivatization of total homocysteine (Hcy) and the related compounds in plasma by gas chromatograph (GC)-electron capture detector (ECD) has been developed. To determine total homocysteine, cysteine (Cys), and methionine (Met) in human plasma using GC-ECD, analytes were reduced and converted into their N(O,S)-ethylearbonyl pentafluoropropyl (PFP) ester by derivatization with ethyl chloroformate and pentafluoropropyl alcohol (PFP-OH) in plasma. The best derivatizing agent N(O,S)-ethyl carbonyl PFP ester, was chosen by comparing the sensitivity of derivatized analysis in GC-ECD. The derivatized analytes in plasma were extracted by chloroform, and subsequently back-extracted with hexane and analyzed by GC-ECD. The calibration carves ($R^2$ > 0.990) were linear over the range $5-50{\mu}mol/L$ of Hcy and Met, $40-400{\mu}mol/L$ of Cys spiked in plasma. The detection limit observed by the established method was below $0.5{\mu}mol/L$. This method is highly sensitive and specific in the analysis of Hcy, Cys, and Met. Therefore, we suggest that this method is appropriate in the analysis of trace concentration of Hcy, Cys, and Met in biological fluids.
기체 크로마토그래피-전자포획검출기 (gas chromatograph-electron capture detector : GC-ECD)를 이용하여 사람의 혈액 내에 존재하는 homocysteine (Hcy), 관련 화합물인 cysteine (Cys)과 methionine (Met)에 대해 선택적이고 감도 좋은 분석방법에 대하여 연구하였다. 시료는 혈장 내에서 환원과정을 거친 후 ethyl chloroformate (ECF)와 pentafluoropropyl alcohol (PEP-OH) 유도체화 시약을 사용하여 N(O,S)-ethylearhonyl pentafluoropropyl (PEP) ester 유도체를 만들어 추출의 효율을 높이면서 동시에 GC-ECD에 대한 크로마토그래피 성질을 향상시켰다. 생성된 유도체들은 chloroform으로 추출한 후 잔류물을 완전히 건조시키고 hexane으로 추출하여 GC-ECD로 분석하였다. Hcy와 Met는 $5-50{\mu}mol/L$, Cys는 $40-400{\mu}mol/L$ 범위에서 $R^2=0.990$ 이상의 직선상의 상관관계를 나타내었다. 이 작성한 질량곡선을 통해서 5명의 자원자들의 혈액 중에 존재하는 Hcy, Cys, met의 함량을 분석하였다.
