Determination of free cortisol and free cortisone in human urine by on-line turbulent flow chromatography coupled to fused-core chromatography–tandem mass spectrometry (TFC–HPLC–MS/MS)
Determination of free cortisol and free cortisone in human urine by on-line turbulent flow chromatography coupled to fused-core chromatography–tandem mass spectrometry (TFC–HPLC–MS/MS)
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DOI：
10.1007/s00216-013-7505-x
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发表时间：
2013
影响因子：
4.3
通讯作者：
A. Sánchez-Guijo;M. Hartmann;Lijie Shi;T. Remer;S. Wudy
中科院分区：
文献类型：
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作者：
A. Sánchez-Guijo;M. Hartmann;Lijie Shi;T. Remer;S. Wudy
尿游离皮质醇和尿游离可的松是诊断肾上腺功能障碍相关综合征或评估某些酶紊乱的决定性标志物。在这里，我们提出了一种新的方法，设计用于常规实验室使用，它可以快速测定这些分析物与少量的样品后处理。湍流色谱法缩短了样品制备时间，并连接到一个融合的核心颗粒填充柱（坚固的酰胺嵌入C18相）允许分析物的快速有效的分离，以及从尿液中存在的其他相关和同量异位化合物的额外分离。成功鉴定了尿中的同量异位素化合物。该方法仅需要每份样品100 μl的尿液上清液。进样之间的总时间为9.5 min。湍流色谱和分析色谱所用的溶剂是水和甲醇，在该方法中所需的相对较低的流量导致了柱寿命的延长。线性显示aR 2> 0.994。可的松的检测限和定量限为0.5和1.0 ng/ml，皮质醇为1.0和2.0 ng/ml。可的松的回收率范围为99.7 - 109.1%，皮质醇的回收率范围为98.7 - 102.9%。批内和批间实验的准确度值（相对误差）始终低于8%，而精密度（CV百分比）范围为3.7 - 10.7%。验证过程中未检测到基质效应。各分析物保留时间的重现性极佳，变异系数始终低于0.2%。最后的验证步骤包括对健康儿童和既往诊断为皮质激素紊乱的儿童的尿样进行研究。实现的高选择性可实现快速数据处理。
Urinary free cortisol and urinary free cortisone are decisive markers for the diagnosis of syndromes related to the dysfunction of the adrenal gland or to evaluate certain enzymatic disorders. Here, we present a new method, designed for routine laboratory use, which enables quick determination of these analytes with minor sample workup. Turbulent flow chromatography shortens sample preparation, and connection to a fused-core particle-packed column (rugged amide-embedded C18 phase) permits a rapid and effective separation of the analytes, as well as additional separation from other related and isobaric compounds present in urine. Urinary isobaric compounds were successfully identified. The method requires only 100 μl of urine supernatant per sample. The total time between injections is 9.5 min. The solvents used for both turbulent and analytical chromatography are water and methanol, and the relatively low flows needed during the method resulted in an extended life of the columns. Linearity showed aR2> 0.994. Limit of detection and limit of quantification are 0.5 and 1.0 ng/ml for cortisone and 1.0 and 2.0 ng/ml for cortisol. Recoveries ranged from 99.7 to 109.1 % for cortisone and from 98.7 to 102.9 % for cortisol. Accuracy values (relative errors) for intra- and inter-assay experiments were always below 8 %, whereas precision (percent CV) ranged from 3.7 to 10.7 %. No matrix effects were detected during the validation process. The reproducibility for each analyte’s retention time was excellent, with a coefficient of variation always below 0.2 %. The final validation step included the study of urine samples from healthy children and from children previously diagnosed with corticoidal disorders. The high selectivity achieved enables quick data handling.