Nadarajah Nirosa, Skadberg Øyvind, Adaway Joanne, Brede Cato
Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences, PO Box 4, NO-0130 Oslo, Norway.
Department of Medical Biochemistry, Stavanger University Hospital, PO Box 8100, NO-4068 Stavanger, Norway.
Clin Mass Spectrom. 2017 Aug 3;4-5:1-10. doi: 10.1016/j.clinms.2017.08.001. eCollection 2017 Apr-Aug.
LC-MS/MS methods for multiplexed analysis of steroid hormones in saliva are useful research tools in endocrinology, but require sensitive detection.
To explore the use of hydrazide and hydrazine derivatization to improve sensitivity of detection for ketosteroids. On development and validation of a sample preparation method based on robot pipetting in the 96-well format we intended to then establish normal reference ranges for women and men, with saliva collected both in the early morning and late at night.
Four hydrazides and one hydrazine were evaluated for their effectiveness as derivatization reagents based on the comparative signal response and efficacy of LC separation of five ketosteroid hydrazones via a methanol gradient mixed with either 0.2% formic acid or 0.1% ammonium hydroxide. Processing of saliva via liquid-liquid extraction (LLE) was optimized by examining variations in both extraction solvent polarity and protein precipitation reagents intended to inhibit emulsion.
LLE using 10 % butanol in methyl -butyl ether (MTBE), with tannic acid as emulsion inhibitor, followed by 2-hydrazinopyridine (2-HP) derivatization enabled the multiplexed measurement of cortisol, cortisone, testosterone, dehydroepiandrosterone (DHEA), progesterone, and 17-alpha-hydroxyprogesterone (17-OHP) in the majority of saliva samples from women and men for this study.
Tannic acid is a novel and effective protein precipitation reagent in bioanalytical sample preparation. The validated method was used in the establishment of reference ranges, the success of which indicated that the method is suitable for Cushing's screening and has potential as a novel analytical research tool.
用于唾液中甾体激素多重分析的液相色谱-串联质谱（LC-MS/MS）方法是内分泌学中有用的研究工具，但需要灵敏的检测。
探索使用酰肼和肼衍生化来提高对酮甾体检测的灵敏度。在开发和验证基于96孔板自动移液的样品制备方法后，我们打算为男性和女性建立正常参考范围，分别在清晨和深夜采集唾液。
基于通过甲醇梯度与0.2%甲酸或0.1%氢氧化铵混合后对五种酮甾体腙的比较信号响应和LC分离效果，评估了四种酰肼和一种肼作为衍生化试剂的有效性。通过检查萃取溶剂极性和旨在抑制乳化的蛋白质沉淀试剂的变化，优化了通过液-液萃取（LLE）处理唾液的方法。
在本研究中，使用10%丁醇的甲基叔丁基醚（MTBE）进行LLE，以单宁酸作为乳化抑制剂，然后进行2-肼基吡啶（2-HP）衍生化，能够对大多数男性和女性唾液样本中的皮质醇、可的松、睾酮、脱氢表雄酮（DHEA）、孕酮和17-α-羟孕酮（17-OHP）进行多重测量。
单宁酸是生物分析样品制备中一种新型有效的蛋白质沉淀试剂。该经过验证的方法用于建立参考范围，其成功表明该方法适用于库欣综合征筛查，并有潜力作为一种新型分析研究工具。
