Background

Highly accurate and sensitive method to measure testosterone in hypogonadal male, female and children is vital for proper diagnosis of hormone-related conditions and their treatment.

Objective

To develop an accurate and robust total testosterone ESI-LC-MS/MS quantification method with a simple sample preparation workflow and sufficient sensitivity for serum or plasma samples of all gender and age groups, via ketone functional group derivatization (using Amplifex™ Keto Reagent).

Method

A simple sample preparation method to accommodate both low and high numbers of samples was developed using simultaneous protein precipitation and derivatization with Amplifex™ Keto reagent, followed by centrifugation and direct injection of supernatant into an LC-MS/MS system (SCIEX Topaz™ IVD LC-MS/MS, in which MS is equivalent to a SCIEX 4500MD Mass Spectrometer). Total testosterone in human serum or plasma samples was quantified using an external calibration curve generated by calibrators spanning a broad concentration range of ∼1–2000 ng/dL (10–20,000 pg/mL), traceable to NIST 971 SRM. ¹³C₃-enriched testosterone was used as an internal standard to correct for both analyte loss during sample preparation and matrix effect during analysis (Supplementary Information: SI Fig. 4C). Two methods, one using a 96-well filter plate and another using Eppendorf tubes, were developed. Both methods were certified by the Centers for Disease Control (CDC) hormone standardization (HoSt) program for total serum testosterone. The feasibility of implementing the method for plasma and serum samples was tested via a small-scale method comparison study between matched pediatric serum and plasma samples derived from the same donor. In addition, plasma samples originating from the same donor collected in two different anticoagulant tube types (Li-heparin and K₂EDTA) were compared.

Results

Using in-house formulated NIST 971-traceable calibrators, the method was linear (r² > 0.999) between 1 and 2000 ng/dL (10 and 20,000 pg/mL) with a limit of detection of approximately 1 ng/dL (10 pg/mL). The testosterone concentration bias against 40 reference samples from the HoSt certification program was absolute <3% with an average %CV of ∼3–4%. More than 78% of samples passed the CDC bias criterion of ±6.4%. Comparison between pediatric matched serum and plasma samples resulted in high correlation (r² = 0.997) and bias of <5%. The calculated % difference between matched adult serum and plasma samples was ∼1%.

Conclusions

Feasibility for an accurate and streamlined method suitable for measuring total testosterone in all human samples was demonstrated with a choice of sample preparation workflow to suit low or high number of samples. The method can potentially be used for plasma matrix from different blood collection tubes (Li-Heparin and K₂EDTA).

中文翻译：

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使用 LC-MS/MS 开发经 CDC 认证的成人和儿童样本总睾酮测定

背景

测量性腺功能减退男性、女性和儿童睾酮的高度准确和灵敏的方法对于正确诊断激素相关疾病及其治疗至关重要。

客观的

通过酮官能团衍生化（使用 Amplifex™ Keto 试剂），开发一种准确且稳健的总睾酮 ESI-LC-MS/MS 定量方法，该方法具有简单的样品制备工作流程和足够的灵敏度，适用于所有性别和年龄组的血清或血浆样品.

方法

使用 Amplifex™ Keto 试剂同时进行蛋白质沉淀和衍生化，然后离心并将上清液直接注入 LC-MS/MS 系统（SCIEX Topaz™ IVD LC -MS/MS，其中 MS 相当于 SCIEX 4500MD 质谱仪）。使用校准器生成的外部校准曲线对人血清或血浆样品中的总睾酮进行量化，该曲线跨越 ~1–2000 ng/dL (10–20,000 pg/mL) 的广泛浓度范围，可追溯至 NIST 971 SRM。¹³ C ₃富集睾酮用作内标，以校正样品制备过程中的分析物损失和分析过程中的基质效应（补充信息：SI 图 4C）。开发了两种方法，一种使用 96 孔滤板，另一种使用 Eppendorf 管。这两种方法都通过了疾病控制中心 (CDC) 激素标准化 (HoSt) 计划的总血清睾酮认证。通过对来自同一供体的匹配的儿科血清和血浆样本之间的小规模方法比较研究，测试了实施血浆和血清样本方法的可行性。此外，比较了在两种不同抗凝管类型（Li-肝素和 K ₂ EDTA）中采集的来自同一供体的血浆样品。

结果

使用内部配制的 NIST 971 可溯源校准品，该方法在 1 和 2000 ng/dL（10 和 20,000 pg/mL）之间呈线性 (r ²  > 0.999)，检测限约为 1 ng/dL (10 pg /毫升）。来自 HoSt 认证计划的 40 个参考样本的睾酮浓度偏差绝对 <3%，平均 %CV 为 ~3-4%。超过 78% 的样本通过了 ±6.4% 的 CDC 偏差标准。儿科匹配的血清和血浆样本之间的比较导致高度相关（r ²  = 0.997）和<5%的偏差。匹配的成人血清和血浆样本之间的计算百分比差异为~1%。

结论

通过选择适合少量或大量样品的样品制备工作流程，证明了适用于测量所有人体样品中总睾酮的准确和简化方法的可行性。该方法可以潜在地用于来自不同采血管（Li-Heparin 和 K ₂ EDTA）的血浆基质。