Zhou Hui, Wang Yuesong, Gatcombe Matthew, Farris Jacob, Botelho Julianne C, Caudill Samuel P, Vesper Hubert W
Centers For Disease Control and Prevention, National Center For Environmental Health, Division of Laboratory Sciences, Clinical Chemistry Branch, 4770 Buford Hwy NE, Atlanta, GA, 30341, USA.
Anal Bioanal Chem. 2017 Oct;409(25):5943-5954. doi: 10.1007/s00216-017-0529-x. Epub 2017 Aug 11.
Reliable measurement of total testosterone and estradiol is critical for their use as biomarkers of hormone-related disorders in patient care and translational research. We developed and validated a mass spectrometry method to simultaneously quantify these analytes in human serum without chemical derivatization. Serum is equilibrated with isotopic internal standards and treated with acidic buffer to release hormones from their binding proteins. Lipids are isolated and polar impurities are removed by two serial liquid-liquid extraction steps. Total testosterone and estradiol are measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) in combination of positive and negative electrospray ionization modes. The method shows broad analytical measurement range for both testosterone 0.03-48.5 nM (0.75-1400 ng/dL) and estradiol 11.0-5138 pM (2.99-1400 pg/mL) and excellent agreement with certified reference materials (mean bias less than 2.1% to SRM 971, BCR 576, 577, and 578) and a high order reference method (mean bias 1.25% for testosterone and -0.84% for estradiol). The high accuracy of the method was monitored and certified by CDC Hormone Standardization (HoSt) Program for 2 years with mean bias -0.7% (95% CI -1.6% to 0.2%) for testosterone and 0.1% (95% CI -2.2% to 2.3%) for estradiol. The method precision over a 2-year period for quality control pools at low, medium, and high concentrations was 2.7-2.9% for testosterone and 3.3-5.3% for estradiol. With the consistently excellent accuracy and precision, this method is readily applicable for high-throughput clinical and epidemiological studies.
可靠测量总睾酮和雌二醇对于将其用作患者护理和转化研究中激素相关疾病的生物标志物至关重要。我们开发并验证了一种质谱方法，可在不进行化学衍生化的情况下同时定量人血清中的这些分析物。血清与同位素内标平衡，并用酸性缓冲液处理以从其结合蛋白中释放激素。通过两个连续的液-液萃取步骤分离脂质并去除极性杂质。使用液相色谱串联质谱法（LC-MS/MS）结合正电喷雾电离和负电喷雾电离模式测量总睾酮和雌二醇。该方法对睾酮的分析测量范围为0.03-48.5 nM（0.75-1400 ng/dL），对雌二醇的分析测量范围为11.0-5138 pM（2.99-1400 pg/mL），与认证参考物质（与SRM 971、BCR 576、577和578的平均偏差小于2.1%）和高阶参考方法（睾酮平均偏差为1.25%，雌二醇平均偏差为-0.84%）具有良好的一致性。该方法的高精度由美国疾病控制与预防中心激素标准化（HoSt）计划监测和认证了两年，睾酮的平均偏差为-0.7%（95%可信区间为-1.6%至0.2%），雌二醇的平均偏差为0.1%（95%可信区间为-2.2%至2.3%）。在两年期间，低、中、高浓度质量控制池的方法精密度对于睾酮为2.7-2.9%，对于雌二醇为3.3-5.3%。凭借始终如一的出色准确性和精密度，该方法易于应用于高通量临床和流行病学研究。
