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Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of ammonium fluoride
Journal of Chromatography B ( IF 2.8 ) Pub Date : 2022-08-09 , DOI: 10.1016/j.jchromb.2022.123413
Lina Schiffer 1 , Fozia Shaheen 1 , Lorna C Gilligan 1 , Karl-Heinz Storbeck 2 , James M Hawley 3 , Brian G Keevil 4 , Wiebke Arlt 1 , Angela E Taylor 1
Affiliation  

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:

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。

更新日期:2022-08-09
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