可溯源干血片样本类固醇激素标准曲线的建立

doi:10.3969/j.issn.1673-8640.2023.05.015

摘要/Abstract

摘要：

目的建立可溯源的干血片样本19种类固醇激素液相色谱-串联质谱（LC-MS/MS）标准曲线。方法收集乙二胺四乙酸抗凝新鲜全血，用0.9% NaCl溶液清洗血细胞，将清洗后的血细胞和无类固醇激素的血浆按0.55∶0.45比例混合，制备模拟全血。根据拟制备标准曲线浓度添加不同量类固醇激素至模拟全血样本中，制备不同浓度的类固醇激素标准曲线干血片。采用液相色谱-串联质谱（LC-MS/MS）检测各标准曲线干血片类固醇激素含量，分析其线性范围，并对类固醇激素标准曲线干血片进行性能验证。结果类固醇激素标准曲线干血片中孕烯醇酮、孕酮、可的松、脱氢表雄酮的线性范围为0.25~100 ng/mL，11-脱氧皮质酮、21-脱氧皮质醇、18-羟皮质醇、11-酮睾酮的线性范围为0.05~20 ng/mL，皮质酮、11-羟睾酮的线性范围为0.025~10 ng/mL，17-羟孕酮、18-羟皮质酮、11-脱氧皮质醇、雄烯二酮、11-羟雄烯二酮、睾酮的线性范围为0.1~40 ng/mL，皮质醇的线性范围为1.5~600 ng/mL，17-羟孕烯醇酮的线性范围为0.2~80 ng/mL，硫酸脱氢表雄酮的线性范围为25~10 000 ng/mL；相关系数（r²）≥0.997。结论建立的干血片样本类固醇激素标准曲线具有良好的线性，可实现相关项目测量结果的溯源。

关键词: 类固醇激素, 标准曲线, 干血片, 液相质谱-串联质谱, 溯源性

Abstract:

Objective To establish a traceable standard curve for 19 steroid hormones in dried blood spot samples by liquid chromatography tandem mass spectrometry（LC-MS/MS）. Methods Ethylenediaminetetraacetic acid fresh anticoagulated whole blood was collected，blood cells were washed with 0.9 NaCl saline，and the washed blood cells and steroid hormone-free plasma were mixed in the ratio of 0.55∶0.45. Simulated whole blood samples was prepared. Different amounts of steroid hormones were added to simulated whole blood samples according to the prepared standard curve concentration in order to prepare different concentrations of steroid hormone standard curve dried blood spot samples. Steroid hormone concentrations in standard curve dried blood spot samples were determined by LC-MS/MS，and its linear range was analyzed. The performance of dried blood spot steroid hormone standard curve was verified. Results The linear ranges of dried blood spot steroid hormone standard curve for pregnenolone，progesterone，cortisone and dehydroepiandrosterone were 0.25-100 ng/mL. 11-Deoxycorticosterone，21-deoxycortisol，18-hydroxycortisol and 11-keto testosterone were in the linear range of 0.05-20 ng/mL. Corticosterone and 11-hydroxytestosterone were in the linear range of 0.025-10 ng/mL. 17-Hydroxyprogesterone，18-hydroxycorticosterone，11-deoxycortisol，androstenedione，11-hydroxyandrostenedione and testosterone were in the linear range of 0.1-40 ng/mL. Cortisol was in the linear range of 1.5-600 ng/mL，17-hydroxypregnenolone was in the linear range of 0.2-80 ng/mL，and dehydroepiandrosterone sulfate was in the linear range of 25-10 000 ng/mL with a correlation coefficient of squared（r²）≥0.997. Conclusions The established steroid hormone standard curves for dried blood spot samples have good linearities and can achieve the traceability of steroid hormone determination results in dried blood spots samples.

Key words: Steroid hormone, Standard curve, Dried blood spot, Liquid chromatography tandem mass spectrometry, Traceability

中图分类号:

R446.62

吴洁, 李增和, 陈宝荣, 孔许净. 可溯源干血片样本类固醇激素标准曲线的建立[J]. 检验医学, 2023, 38(5): 489-493.

WU Jie, LI Zenghe, CHEN Baorong, KONG Xujing. Establishment of traceable steroid hormone standard curve for dried blood spot samples[J]. Laboratory Medicine, 2023, 38(5): 489-493.

图/表 3

参考文献 9

[1]	KOAL T, SCHMIEDERER D, PHAM-TUAN H, et al. Standardized LC-MS/MS based steroid hormone profile-analysis[J]. J Steroid Biochem Mol Biol, 2012, 129(3-5):129-138. DOI URL
[2]	HOU L, LIANG L, LIN S, et al. Analysis of phenotypes and genotypes in 84 patients with 21-hydroxylase deficiency in southern China[J]. Steroids, 2019, 151:108474. DOI URL
[3]	GU C, TAN H, YANG J, et al. Congenital adrenal hyperplasia due to 11-hydroxylase deficiency-compound heterozygous mutations of a prevalent and two novel CYP11B1 mutations[J]. Gene, 2017, 626:89-94. DOI PMID
[4]	PALMIERI S, MORELLI V, POLLEDRI E, et al. The role of salivary cortisol measured by liquid chromatography-tandem mass spectrometry in the diagnosis of subclinical hypercortisolism[J]. Eur J Endocrinol, 2013, 168(3):289-296. DOI PMID
[5]	QASRAWI D O, BOYD J M, SADRZADEH S M H. Measuring steroids from dried blood spots using tandem mass spectrometry to diagnose congenital adrenal hyperplasia[J]. Clin Chim Acta, 2021, 520:202-207. DOI PMID
[6]	JANZEN N, SANDER S, TERHARDT M, et al. Rapid steroid hormone quantification for congenital adrenal hyperplasia(CAH) in dried blood spots using UPLC liquid chromatography-tandem mass spectrometry[J]. Steroids, 2011, 76(13):1437-1442. DOI URL
[7]	KIM B, LEE M N, PARK H D, et al. Dried blood spot testing for seven steroids using liquid chromatography-tandem mass spectrometry with reference interval determination in the Korean population[J]. Ann Lab Med, 2015, 35(6):578-585. DOI PMID
[8]	孙萌, 李育霖, 邹卉. 液相色谱-串联质谱技术在新生儿先天性肾上腺皮质增生症筛查中的应用进展[J]. 中国妇幼保健, 2021, 36(13):3168-3169.
[9]	STANCZYK F Z, CLARKE N J. Advantages and challenges of mass spectrometry assays for steroid hormones[J]. J Steroid Biochem Mol Biol, 2010, 121(3-5):491-495. DOI URL

激素	线性范围/ （ng/mL）	斜率（a）	r²值
孕烯醇酮	0.250~100	0.030 5	0.998
17-羟孕烯醇酮	0.200~80	0.022 0	0.998
孕酮	0.250~100	0.222 1	0.998
17-羟孕酮	0.100~40	0.162 7	0.998
皮质酮	0.250~100	0.087 3	0.997
11-脱氧皮质酮	0.050~20	0.323 0	0.998
18-羟皮质酮	0.100~40	0.144 8	0.999
皮质醇	1.500~600	0.020 2	0.998
可的松	0.250~100	0.058 4	0.998
11-脱氧皮质醇	0.100~40	0.180 8	0.998
21-脱氧皮质醇	0.050~20	0.037 0	0.999
18-羟皮质醇	0.050~20	0.386 0	0.999
脱氢表雄酮	0.250~100	0.015 0	0.997
硫酸脱氢表雄酮	25.000~10 000	0.000 1	0.998
雄烯二酮	0.100~40	0.192 8	0.997
11-羟雄烯二酮	0.100~40	0.139 1	0.999
睾酮	0.100~40	0.270 3	0.998
11-羟睾酮	0.025~10	0.220 4	0.999
11-酮睾酮	0.050~20	0.753 8	0.998

激素	线性范围/ （ng/mL）	斜率（a）	r²值
孕烯醇酮	0.250~100	0.030 5	0.998
17-羟孕烯醇酮	0.200~80	0.022 0	0.998
孕酮	0.250~100	0.222 1	0.998
17-羟孕酮	0.100~40	0.162 7	0.998
皮质酮	0.250~100	0.087 3	0.997
11-脱氧皮质酮	0.050~20	0.323 0	0.998
18-羟皮质酮	0.100~40	0.144 8	0.999
皮质醇	1.500~600	0.020 2	0.998
可的松	0.250~100	0.058 4	0.998
11-脱氧皮质醇	0.100~40	0.180 8	0.998
21-脱氧皮质醇	0.050~20	0.037 0	0.999
18-羟皮质醇	0.050~20	0.386 0	0.999
脱氢表雄酮	0.250~100	0.015 0	0.997
硫酸脱氢表雄酮	25.000~10 000	0.000 1	0.998
雄烯二酮	0.100~40	0.192 8	0.997
11-羟雄烯二酮	0.100~40	0.139 1	0.999
睾酮	0.100~40	0.270 3	0.998
11-羟睾酮	0.025~10	0.220 4	0.999
11-酮睾酮	0.050~20	0.753 8	0.998

[1]	李西就, 周健伟, 张芹, 龙允麟, 吴英松. 滤纸干血片6种标志物UPLC-MS/MS方法的建立和性能评价[J]. 检验医学, 2023, 38(5): 446-453.
[2]	孟舒婷, 周卫平, 戴绘芬, 董德平, 董业峰, 葛仁美, 王惠民. 蒙特卡洛法对校准曲线法测定总胆红素浓度的不确定度评定[J]. 检验医学, 2022, 37(7): 674-679.
[3]	谌玉佳, 孙智勇, 王少静, 杨江涛, 吴本清. 自制吹干装置在滤纸干血片样本制备中的应用[J]. 检验医学, 2021, 36(8): 857-863.
[4]	王燕敏, 田国力, 纪伟, 张潇分. 肌酸激酶MM同工酶在新生儿杜氏肌营养不良症筛查中的应用[J]. 检验医学, 2020, 35(11): 1115-1119.
[5]	虞啸炫, 欧元祝, 唐立萍, 王美娟, 刘文彬, 葛丹红, 王茗. 上海地区γ-谷氨酰转移酶正确度验证计划调查结果分析[J]. 检验医学, 2018, 33(11): 1032-1035.
[6]	欧元祝, 虞啸炫, 唐立萍, 居漪, 王美娟, 刘文彬, 金中淦. ALT正确度控制品稳定性研究及调查结果分析[J]. 检验医学, 2017, 32(8): 713-717.
[7]	冯仁丰. 脂蛋白（a）检测的标准化[J]. 检验医学, 2017, 32(7): 555-560.
[8]	万志丹, 黄湘, 钟裕恒, 吴学威, 李冬秀. 滤纸干血片毛细管电泳技术检测HbA在筛查新生儿β-地中海贫血中的应用价值[J]. 检验医学, 2017, 32(12): 1128-1131.
[9]	梅四清, 缪希莉. γ-谷氨酰氨基转移酶检测结果一致性及正确性分析[J]. 检验医学, 2017, 32(11): 1051-1054.
[10]	田国力, 王燕敏, 许洪平, 郭静, 周卓, 姚静. 串联质谱法和荧光分析法检测滤纸干血片苯丙氨酸的比较[J]. 检验医学, 2016, 31(9): 814-819.
[11]	周亚飞, 王月婷, 于嘉屏. HPLC-MS/MS同位素稀释法测定人体血清中类固醇激素的研究[J]. 检验医学, 2015, 30(5): 427-432.
[12]	冯仁丰. 互换性依然是问题[J]. 检验医学, 2013, 28(12): 1063-1068.
[13]	彭凤综述;于嘉屏;应春妹　审校. 免疫透射比浊测定技术的进展和分析特性[J]. 检验医学, 2012, 27(4): 252-256.
[14]	乐爱平;胡国信;李太原;罗忠勤;黄清水. 血清乙型肝炎病毒大蛋白浓度标准曲线的拟合与优选[J]. 检验医学, 2009, 24(06): 463-465.
[15]	陈化禹;耿洁;焦连亭. 不同溯源的两台血凝仪测定纤维蛋白原方法学比较[J]. 检验医学, 2009, 24(04): 278-280.