Detection of physiological concentrations of cortisol and cortisone in human hair
Detection of physiological concentrations of cortisol and cortisone in human hair
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DOI：
10.1016/j.clinbiochem.2004.02.010
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发表时间：
2004-12-01
影响因子：
2.8
通讯作者：
Kintz, P
中科院分区：
文献类型：
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作者：
Raul, JS;Cirimele, V;Kintz, P
目的：自20世纪60年代以来，糖皮质激素被运动员用于提高运动成绩。它们的使用仅限于体育运动。头发可以证明长期滥用，因此可以作为兴奋剂控制的补充材料。我们已经开发了一种新的提取，纯化和分离技术，利用液相色谱和质谱鉴定和定量的两种内源性糖皮质激素：皮质醇和cortisone.Methods：定性和定量调查，实现了44头发样本（17男，27女，年龄从2到90岁）。将发束在二氯甲烷中洗涤，将前两厘米的发束切割并在球磨机中粉碎。将粉末状毛发在2 mL Soerensen缓冲液（pH 7.6）中于40 ℃孵育16 h，以皮质醇-d（3）作为内标。在SPE C18 Isolute萃取柱上纯化孵育培养基，然后用乙醚进行碱性液-液萃取。将沉淀物蒸发至干并重悬于25 μ L穆尔乙腈/甲酸铵（1：1，v/v）中。色谱在LC Packings Superba Nucleosil C18柱上进行，使用乙腈线性梯度（10 min内从30%至70%）。检测器为Perkin Elmer Sciex API 100质谱仪。检测器的响应是线性的皮质醇和可的松浓度范围从1到500 pg/mg。在50 pg/mg下，皮质醇的提取回收率为74%，可的松为32%。在两种情况下，7 pg/mg皮质醇和50 pg/mg可的松的重复性（CV值n = 8）均为11%。两种化合物的检测限和定量限分别为1和5 pg/mg。结果：头发中皮质醇浓度范围为5 ~ 91 pg/mg（平均18 pg/mg）。毛发中的可的松浓度范围为12至163 pg/mg（平均70 pg/mg）。没有发现头发颜色的影响，性别对可的松浓度的影响似乎是可能的，但无法在统计学上证明。最后，头发中的可的松浓度在20岁之前明显更高。皮质醇和可的松在头发中的结合可以在汗腺中的2型11-β-羟基类固醇脱氢酶将部分皮质醇转化为可的松后通过汗液被动扩散。这些分析记录了该问题。(C)2004年加拿大临床化学家协会。All rights reserved.
Objective: Since the 1960s, glucocorticoids are used by athletes to improve their performances. Their use is restricted in sports. Hair can document chronic abuse and can be therefore a complementary matrix for doping control. We have developed a new extraction, purification, and separation technique using liquid chromatography and mass spectrometry for the identification and quantification of two endogenous glucocorticoids: cortisol and cortisone.Methods: Qualitative and quantitative investigations were achieved with 44 hair samples (17 males, 27 females; age ranging from 2 to 90 years). Hair strands were washed in methylene chloride, the first two centimeters of the strand were cut and pulverized in a ball mill. The powdered hair was incubated in 2 mL Soerensen buffer, pH 7.6, for 16 h at 40degreesC, in the presence of cortisol-d(3) as an internal standard. Purification of the incubation medium was achieved on SPE C18 Isolute extraction columns followed by an alkaline liquid-liquid extraction with diethylether. The eluate was evaporated to dryness and resuspended in 25 muL of acetonitrile/ammonium formiate (1:1,v/v). The chromatography was operated on a LC Packings Superba Nucleosil C18 column using a linear gradient of acetonitrile from 30% to 70% in 10 min. The detector was a Perkin Elmer Sciex API 100 mass spectrometer. The detector's response was linear for cortisol and cortisone concentrations ranging from I to 500 pg/mg. Extraction recovery at 50 pg/mg was 74% for cortisol and 32% for cortisone. Repeatability (CV values n = 8) at 7 pg/mg cortisol and at 50 pg/mg cortisone were 11% in both cases. Limit of detection and limit of quantification were 1 and 5 pg/mg, for both compounds, respectively.Results: Cortisol concentrations in hair ranged from 5 to 91 pg/mg (mean 18 pg/mg). Cortisone concentrations in hair ranged from 12 to 163 pg/mg (mean 70 pg/mg). No influence of hair colour could be found. Influence of sex on cortisone concentrations seems possible but could not be statistically demonstrated. Finally, cortisone concentrations in hair are significantly higher before the age of 20. Incorporation of cortisol and cortisone in hair could follow a passive diffusion through sweat after conversion of part of cortisol to cortisone by Type 2 11-beta-Hydroxysteroid-dehydrogenase in sweat glands. This issue was documented by these analyses. (C) 2004 The Canadian Society of Clinical Chemists. All rights reserved.