Turpeinen U, Linko S, Itkonen O, Hämäläinen E
HUSLAB, Laboratory of Women's Clinic, Helsinki and Uusimaa Hospital District, Finland.
Scand J Clin Lab Invest. 2008;68(1):50-7. doi: 10.1080/00365510701496496. Epub 2007 Jun 24.
Commercial direct immunoassays for serum testosterone sometimes result in inaccuracies in samples from women and children, leading to misdiagnosis and inappropriate treatment. The diagnosis of male hypogonadism also requires an accurate testosterone assay method. We therefore developed a sensitive and specific stable-isotope dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum testosterone at the concentrations encountered in women and children. Testosterone was extracted with ether-ethyl acetate from 250 microL or 500 microL of serum. Instrumental analysis was performed on an API 2000 tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode after separation on a reversed-phase column. The MRM transitions (m/z) were 289/97 for testosterone and 291/99 for d(2) testosterone. The calibration curves exhibited consistent linearity and repeatability in the range 0.2-100 nmol/L. Interassay CVs were 4.2-7.6 % at mean concentrations of testosterone of 3.3-45 nmol/L. Total measurement uncertainty (U, k = 2) was 12.9 % and 13.4 % at testosterone levels of 2.0 nmol/L and 20 nmol/L, respectively. The limit of detection was 0.05 nmol/L (signal-to-noise ratio = 3) and the overall method recovery of testosterone was 95 %. Correlation (r) with our in-house extraction RIA was 0.98 and with a commercial RIA 0.92. Reference intervals for adult males and females in age groups 18-30, 31-50, 51-70 and over 70 years were established. Sensitivity and specificity of the LC-MS/MS method offer advantages over immunoassay and make it suitable for use as a high-throughput assay in routine clinical laboratories. The high equipment costs are balanced by higher throughput together with shorter chromatographic run times.
用于血清睾酮检测的商业直接免疫分析方法有时会导致女性和儿童样本检测结果不准确，从而导致误诊和不恰当的治疗。男性性腺功能减退的诊断也需要一种准确的睾酮检测方法。因此，我们开发了一种灵敏且特异的稳定同位素稀释液相色谱 - 串联质谱（LC-MS/MS）方法，用于检测女性和儿童血清中常见浓度的睾酮。用乙醚 - 乙酸乙酯从250微升或500微升血清中提取睾酮。在反相柱上分离后，使用API 2000串联质谱仪在多反应监测（MRM）模式下进行仪器分析。睾酮的MRM跃迁（m/z）为289/97，氘代睾酮的MRM跃迁为291/99。校准曲线在0.2 - 100 nmol/L范围内呈现出一致的线性和重复性。在睾酮平均浓度为3.3 - 45 nmol/L时，批间变异系数为4.2 - 7.6%。在睾酮水平为2.0 nmol/L和20 nmol/L时，总测量不确定度（U，k = 2）分别为12.9%和13.4%。检测限为0.05 nmol/L（信噪比 = 3），睾酮的整体方法回收率为95%。与我们内部提取的放射免疫分析（RIA）的相关性（r）为0.98，与商业RIA的相关性为0.92。建立了18 - 30岁、31 - 50岁、51 - 70岁和70岁以上成年男性和女性的参考区间。LC-MS/MS方法的灵敏度和特异性优于免疫分析，使其适用于常规临床实验室的高通量检测。较高的设备成本通过更高的通量和更短的色谱运行时间得到平衡。
