Struck-Lewicka Wiktoria, Karpińska Beata, Rodzaj Wojciech, Nasal Antoni, Wielgomas Bartosz, Markuszewski Michał Jan, Siluk Danuta
Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk, Gdańsk, Poland.
Department of Toxicology, Medical University of Gdańsk, Gdańsk, Poland.
Front Mol Biosci. 2023 Mar 6;10:1074263. doi: 10.3389/fmolb.2023.1074263. eCollection 2023.
In the present study, the development and optimization of a thin film solid phase microextraction method (TF-SPME) was conducted for metabolomics profiling of eight steroid compounds (androsterone, dihydrotestosterone, dihydroepiandrosterone, estradiol, hydroxyprogesterone, pregnenolone, progesterone and testosterone) from urine samples. For optimization of extraction method, two extraction sorbents (PAN-C18 and PS-DVB) were used as they are known to be effective for isolation of low-polarity analytes. The stages of sample extraction and analyte desorption were considered as the most crucial steps in the process. Regarding the selection of the most suitable desorption solution, six different mixtures were analyzed. As a result, the mixture of ACN: MeOH (1:1, ) was chosen in terms of the highest analytes' abundances that were achieved using the chosen solvent. Besides other factors were examined such as the volume of desorption solvent and the time of both extraction and desorption processes. The analytical determination was carried out using the ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry detection in electrospray ionization and positive polarity in a scan mode (UHPLC-ESI-QTOF/MS). The developed and optimized TF-SPME method was validated in terms of such parameters as extraction efficiency, recovery as well as matrix effect. As a result, the extraction efficiency and recovery were in a range from 79.3% to 99.2% and from 88.8% to 111.8%, respectively. Matrix effect, calculated as coefficient of variation was less than 15% and was in a range from 1.4% to 11.1%. The values of both validation parameters (recovery and matrix effect) were acceptable in terms of EMA criteria. The proposed TF-SPME method was used successfully for isolation of steroids hormones from pooled urine samples before and after enzymatic hydrolysis of analytes.
在本研究中，开展了一种薄膜固相微萃取方法（TF-SPME）的开发与优化，用于对尿液样本中的八种甾体化合物（雄甾酮、二氢睾酮、脱氢表雄酮、雌二醇、羟孕酮、孕烯醇酮、孕酮和睾酮）进行代谢组学分析。为优化萃取方法，使用了两种萃取吸附剂（聚丙烯腈-十八烷基硅烷键合硅胶和聚苯乙烯-二乙烯基苯），因为已知它们对低极性分析物的分离有效。样品萃取和分析物解吸阶段被视为该过程中最关键的步骤。关于最合适解吸溶液的选择，分析了六种不同的混合物。结果，就使用所选溶剂实现的最高分析物丰度而言，选择了乙腈：甲醇（1:1）的混合物。此外，还考察了其他因素，如解吸溶剂的体积以及萃取和解吸过程的时间。采用超高效液相色谱结合电喷雾电离和正离子模式下的高分辨率串联质谱检测（UHPLC-ESI-QTOF/MS）进行分析测定。所开发和优化的TF-SPME方法在萃取效率、回收率以及基质效应等参数方面进行了验证。结果，萃取效率和回收率分别在79.3%至99.2%以及88.8%至111.8%的范围内。以变异系数计算的基质效应小于15%，在1.4%至11.1%的范围内。根据欧洲药品管理局（EMA）标准，两个验证参数（回收率和基质效应）的值都是可接受的。所提出的TF-SPME方法成功用于在分析物酶解前后从混合尿液样本中分离甾体激素。
