Background

Multi-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride.

Methods

Following liquid–liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C₁₈ column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation.

Results

Post-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122–140 % for 3βOH-Δ5 steroids and 477–1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range −14 % to 11.9 % for 21/23, −21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from −29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured.

Conclusions

UHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.

中文翻译：

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通过柱后注入氟化铵的 UHPLC-MS/MS 进行多类固醇分析

背景

多类固醇分析是一种强大的分析工具，可同时量化来自不同生物合成途径的类固醇。在这里，我们提出了一种超高效液相色谱-串联质谱 (UHPLC-MS/MS) 测定法，用于使用柱后输注氟化铵对 23 种类固醇进行分析。

方法

液-液萃取后，使用 Phenomenex Luna Omega C ₁₈色谱柱和水（0.1% 甲酸）甲醇梯度在 5 分钟内对类固醇进行色谱分离。在 Waters Acquity UHPLC 和 Xevo® TQ-XS 质谱仪上进行定量。比较了氟化铵（6 mmol/L，柱后注入）和甲酸（0.1 % (vol/vol)，流动相添加剂）作为有助于离子化的添加剂。

结果

与甲酸相比（3βOH-Δ5 类固醇为 122-140%，3-keto-Δ4 类固醇为 477-1274%），氟化铵的柱后注入以类固醇结构依赖性方式增强了离子化。因此，我们分析验证了氟化铵的柱后注入。定量下限为 0.3 至 3 nmol/L；所有分析物均以可接受的准确度进行量化（21/23 的偏差范围为 -14 % 至 11.9 %，所有分析物的偏差范围为 -21 % 至 11.9 %）。平均回收率为 91.6 % 至 113.6 %，平均基质效应为 -29.9 % 至 19.9 %。所有分析物的不精密度范围为 2.3% 至 23%，18/23 分析物的不精密度 < 15%。测量了 10 名健康男性和 10 名健康女性的血清多类固醇谱。

结论

柱后注入氟化铵的 UHPLC-MS/MS 可通过增强电离实现全面的多类固醇分析，特别有利于 3-keto-Δ4 类固醇的检测。