高效液相色谱测定血清皮质醇

DETERMINATION OF SERUM CORTISOL BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

高效液相色谱测定血清皮质醇的方法，０．５ｍｌ血清加１５０ｎｇ地塞美松（内标剂）在５５℃孵育１５分钟后用５ｍｌ乙醚提取类固醇。移有机相于一尖底管、蒸干，残留物用１００μｌ动相（１，２─二氯乙烷、异丙醇、环已烷按６／１１／８３容积混合，每升加冰醋酸０．２ｍｌ）重溶，注２０μｌ于色谱系统。分析柱为Ｓｈｉｍ─ｐａｃｋＣＬＣ─ＣＮ柱，柱温４０℃，流速２．０ｍｌ／分。检测器波长２４２ｎｍ，０．００１Ａ满刻度。皮质醇和内标剂的相对保留时间分别为４．６８和５．１４分。分析回收率平均在９８．１～１００．０％之间，批内和批间ＣＶ＜４％。本法在６８．８９～１１０３．５４ｎｍｏｌ／Ｌ之间呈线性，回归方程ｙ＝０．０２４２＋０．００５２ｘ，ｒ＝０．９９９６，最低检出限１３．８ｎｍｏｌ／Ｌ。１５８名健康成人上午８～９时血清用本法测得皮质醇浓度为１９３．１２土６５．００ｎｍｏｌ／Ｌ（７０．０７＋２３．５６μｌ／Ｌ），男性（ｎ＝８１）平均２１３．６９土６１．３Ｏｎｍｏｌ／Ｌ（７７．３１±２２．２２μｇ／Ｌ）明显高于女性的（ｎ＝７７）１７２．２３±６２．４３ｎｍｏｌ／Ｌ（６２．４６±２２．６３μｇ／Ｌ），Ｐ＜０．００１。

In the present article,a metbod determining serum cortisol by highperformance liquid chromatography was described.After 0.5ml of serum together with150ng of internal standard,dexamethasone,was incubated in a 55℃ water both,steroids were extracted by diethyl ether and the extract was evaporated.The residuewas redissolved with 100μL mobile phase which consisted of 1,2─dichloroethane/iso─propanol/cyclohexane(6/11/83,by Vol.) containing,per litre,0.2mL of gliacial aceticand then injected 20μL into chromatograp.Steroids were separated by Shim─packCLC─CN column at a flow rate of 2.0mL/min and the eluates were detected at 242nm.The relative retention time of cortisol and dexamethasone was 4.68 and 5.14min,respectively,The average analytical recoveries were between 98.1%and 100.0%.Thewithim─run CVs were 1.1%─4.0%and between─run was 3.8%.The linearity of thismethod ranged from 68.89 to 1103.54nmol/L,with the lower detection limit of 13.8nmol/L(signal/noise=3).Serum cortisol concentration determined by this method in158 health adults was 193.12士65.00nmol/L,whose blood samples were obtainedbetween 8 to 9 in the morning.The value in males(213.29土61.3nmol/L,n=81)wassignificantly higher(P<.001)than that in femals(172.2土62.4nmol/L,n=77).