Department of Clinical Biochemistry, University Hospital of South Manchester, Southmoor Road, Wythenshawe, Manchester M23 9LT, United Kingdom.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Jan 15;881-882:42-8. doi: 10.1016/j.jchromb.2011.11.036. Epub 2011 Nov 30.
Salivary cortisol measurements are increasingly being used in the investigation of disorders of the hypothalamic-pituitary-adrenal axis. In the salivary gland, cortisol is metabolised to cortisone by the action of 11β-hydroxysteroid dehydrogenase type 2, and cortisone is partly responsible for the variable interference observed in current salivary cortisol immunoassays. The aim of this study was to validate an assay for the simultaneous analysis of salivary cortisol and cortisone using the Spark Holland Symbiosis™ in eXtraction liquid chromatography-tandem mass spectrometry (XLC-MS/MS) mode for fully automated online solid phase extraction (SPE). Saliva samples were diluted in water with the addition of internal standard (d4-cortisol and d7-cortisone). Online SPE was performed using the Spark Holland Symbiosis™ with HySphere™ C18 SPE cartridges and compounds were eluted onto a Phenomenex® C18 guard column attached to a Phenomenex® Onyx monolithic C18 column for chromatography. Mass spectrometry used the Waters® Xevo™ TQ MS in electrospray positive mode. Cortisol and cortisone eluted with their internal standards at 1.95 and 2.17 min, respectively, with a total run time of four minutes. No evidence of ion-suppression was observed. The assay was linear up to 3393 nmol/L for cortisol and 3676 nmol/L for cortisone, with lower limits of quantitation of 0.75 nmol/L and 0.50 nmol/L, respectively. Intra- and inter-assay imprecision was <8.9% for cortisol and <6.5% for cortisone across three levels of internal quality control, with accuracy and recovery within accepted limits. High specificity was demonstrated following interference studies which assessed 29 structurally-related steroids at supra-physiological concentrations. We have successfully validated an assay for the simultaneous analysis of salivary cortisol and cortisone using XLC-MS/MS and fully automated online SPE. The assay benefits from increased specificity compared to immunoassay and minimal sample preparation which allows high sample throughput and is thus suitable for use in a routine clinical laboratory.
唾液皮质醇测量在研究下丘脑-垂体-肾上腺轴紊乱方面的应用越来越多。在唾液腺中，皮质醇被 11β-羟类固醇脱氢酶 2 作用代谢为皮质酮，而皮质酮部分导致当前唾液皮质醇免疫测定中观察到的可变干扰。本研究的目的是使用 Spark Holland Symbiosis™在 XLC-MS/MS 模式下验证一种同时分析唾液皮质醇和皮质酮的测定方法，该方法用于全自动在线固相萃取 (SPE)。唾液样本用含内标 (d4-皮质醇和 d7-皮质酮) 的水稀释。在线 SPE 使用 Spark Holland Symbiosis™与 HySphere™ C18 SPE 小柱进行，化合物洗脱到连接 Phenomenex® Onyx 整体 C18 柱的 Phenomenex® C18 保护柱上进行色谱分离。质谱采用 Waters® Xevo™ TQ MS 在电喷雾正模式下进行。皮质醇和皮质酮分别在内标物 1.95 和 2.17 分钟洗脱，总运行时间为四分钟。未观察到离子抑制。该测定方法对皮质醇的线性范围为 0.75 nmol/L 至 3393 nmol/L，对皮质酮的线性范围为 0.50 nmol/L 至 3676 nmol/L。皮质醇和皮质酮的定量下限分别为 0.75 nmol/L 和 0.50 nmol/L。三个内部质控水平的皮质醇和皮质酮的批内和批间精密度均<8.9%，准确度和回收率均在可接受范围内。通过评估 29 种超生理浓度结构相关类固醇的干扰研究，证明了该方法具有高特异性。我们成功地使用 XLC-MS/MS 和全自动在线 SPE 验证了一种同时分析唾液皮质醇和皮质酮的测定方法。与免疫测定相比，该测定方法具有更高的特异性，且样品制备量最小，允许高通量处理样本，因此适用于常规临床实验室。
