Woźniak Barbara, Matraszek-Żuchowska Iwona, Witek Sebastian, Posyniak Andrzej
Department of Pharmacology and Toxicology National Veterinary Research Institute, 24-100 Pulawy, Poland.
J Vet Res. 2017 Apr 4;61(1):81-89. doi: 10.1515/jvetres-2017-0010. eCollection 2017 Mar.
In the European Union the use of steroid growth promoters is prohibited under Council Directive 96/22/EC. For effective control of illegal use of natural steroids, highly sensitive analytical methods are required, because sex hormones can be present in very low concentrations in biological samples. The aim of the study was to develop a confirmatory method for the detection of testosterone in bovine serum at ppt level.
17β-testosterone and internal standards of 17%-testosterone-d2 were extracted from serum samples with a mixture of tert-butyl methyl ether/petroleum ether and were directly analysed by an LC/MS/MS on QTRAP 5500 instrument with a TurboIon-Spray source operating in a positive ionisation mode. Chromatographic separation was achieved on the analytical column Inertsil® ODS-3 with an isocratic elution using mobile phase consisting of acetonitrile, methanol, and water. Method validation has been carried out in accordance with the Commission Decision 2002/657/EC.
The method was characterised by good recovery (82%) and precision (R.S.D 17 %). Decision limit (CCα) and detection capability (CCβ) was 0.05 μg L and 0.09 μg L respectively. The method met the criteria set out in Commission Decision 2002/657/EC for the purpose of confirmation in terms of retention time and ion ratio in the whole range of its application.
The developed method is specific and sensitive, suitable for measuring the natural level of testosterone in blood of cattle and for use in routine control programme for the detection of this hormone in bovine serum.
在欧盟，根据理事会指令96/22/EC，禁止使用类固醇生长促进剂。为有效控制天然类固醇的非法使用，需要高灵敏度的分析方法，因为生物样品中的性激素浓度可能非常低。本研究的目的是开发一种用于检测牛血清中ppt水平睾酮的确证方法。
用叔丁基甲醚/石油醚混合物从血清样品中提取17β-睾酮和17%-睾酮-d2内标，然后在配备TurboIon-Spray源且以正离子模式运行的QTRAP 5500仪器上通过液相色谱-串联质谱直接分析。在Inertsil® ODS-3分析柱上采用等度洗脱进行色谱分离，流动相由乙腈、甲醇和水组成。方法验证按照委员会决定2002/657/EC进行。
该方法具有良好的回收率（82%）和精密度（相对标准偏差17%）。决策限（CCα）和检测能力（CCβ）分别为0.05 μg/L和0.09 μg/L。该方法在其整个应用范围内，在保留时间和离子比方面符合委员会决定2002/657/EC规定的确证标准。
所开发的方法具有特异性和灵敏性，适用于测量牛血液中睾酮的天然水平，并用于牛血清中该激素检测的常规控制程序。
