同位素稀释液相色谱-串联质谱法检测血清睾酮候选参考方法的建立及应用

doi:10.3969/j.issn.1673-8640.2021.010.016

摘要/Abstract

摘要：

目的建立基于液相色谱串联质谱（LC-MS/MS）技术的血清睾酮候选参考方法,并采用该方法对临床常规检测方法（微粒子化学发光法）的正确性进行评价。方法采用睾酮-2,3,4-¹³C₃作为内标,以等体积比的正己烷-乙酸乙酯溶液对样本进行液液萃取,上清液采用氮气吹干后用流动相复溶,进行LC-MS/MS分析。采用五点包括法计算血清睾酮浓度。参考美国临床实验室标准化协会（CLSI）C62-A文件和EP15-A3文件对建立的同位素稀释液相色谱-串联质谱（ID-LC-MS/MS）方法进行性能评价（方法残留、特异性、线性范围、精密度、准确度）,参考CLSI EP9-A3文件对微粒子化学发光法的正确性进行评价。结果建立的ID-LC-MS/MS方法检测血清睾酮的分析时间为5 min,特异性好,无残留,线性范围为0.034~22.00 ng/mL,批内不精密度≤2.3%,批间不精密度≤2.2%,测定有证参考物质SRM971（Level male）的相对偏移为0.2%,测定2017 RELA-A、2017 RELA-B样本的相对偏移分别为-2.9%、-3.3%,测定2017 RELA-A样本的不确定度为 0.11 ng/mL。微粒子化学发光法与ID-LC-MS/MS的相关性较好（r=0.963）,但偏差较大[总平均偏差为-24.4%,低浓度样本（≤1 ng/mL）平均偏差为-62.4%,高浓度样本（>1 ng/mL）平均偏差为6.3%]。结论成功建立了检测血清睾酮的ID-LC-MS/MS候选参考方法。该方法的精密度、准确度均较好,且操作简单,分析时间短。

关键词: 睾酮, 同位素稀释液相色谱-串联质谱, 方法学评价, 候选参考方法

Abstract:

Objective To establish a candidate reference method for the determination of human serum testosterone based on liquid chromatography tandem mass spectrometry（LC-MS/MS）,and to use it into assessing the trueness of clinical method（microparticle chemiluminescence immunoassay）. Methods Testosterone-2,3,4-¹³C₃ was added as internal standard. Serum samples were treated with hexane-ethyl acetate（50∶50,volume fraction）. The upper layer was dried with nitrogen flow. The residual were reconstituted with mobile phase and analyzed by LC-MS/MS. Serum testosterone concentration was calculated by 5-point bracketing method. The isotope dilution liquid chromatography tandem mass spectrometry（ID-LC-MS/MS）（carryover,specificity,linearity,imprecision,accuracy） was validated according to the Clinical and Laboratory Standards Institute（CLSI） C62-A and EP15-A3. The trueness of microparticle chemiluminescence immunoassay was evaluated according to CLSI EP9-A3. Results The ID-LC-MS/MS with 5 min total run time and without carryover was validated over a concentration range of 0.034-22.000 ng/mL,and the specificity was good. The within-run imprecision was ≤2.3%,and the between-run imprecision was ≤2.2%. The bias of analyzing the candidate reference material SRM971（Level male）was 0.2%. The relative biases of analyzing 2017 RELA-A and 2017 RELA-B were -2.9% and -3.3%,respectively. The uncertainty for 2017 RELA-A was 0.11 ng/mL. The linearity between ID-LC-MS/MS and microparticle chemiluminescence immunoassay was good（r=0.963）. However,its average relative bias was large,which total average relative bias was -24.4%,the average relative bias of low concentration group（≤1 ng/mL） was -62.4%,and the average relative bias of high concentration group（>1 ng/mL） was 6.3%. Conclusions The ID-LC-MS/MS established successfully for serum testosterone has good precision and accuracy,with simple preparation and short analysis time and may be used as a candidate reference method.

Key words: Testosterone, Isotope dilution liquid chromatography tandem mass spectrometry, Methodological assessment, Candidate reference method

中图分类号:

R446.1

孙贺伟, 居漪, 朱岭峰, 李卿, 金中凎, 张素洁. 同位素稀释液相色谱-串联质谱法检测血清睾酮候选参考方法的建立及应用[J]. 检验医学, 2021, 36(10): 1064-1069.

SUN Hewei, JU Yi, ZHU Lingfeng, LI Qing, JIN Zhonggan, ZHANG Sujie. Isotope dilution liquid chromatography tandem mass spectrometry candidate reference method for serum testosterone[J]. Laboratory Medicine, 2021, 36(10): 1064-1069.

图/表 11

参考文献 14

[1]	BHASIN S, BRITO J P, CUNNINGHAM G R, et al. Testosterone therapy in men with hypogonadism:an endocrine society clinical practice guideline[J]. J Clin Endocrinol Metab, 2018, 103(5):1715-1744. DOI URL
[2]	LOBLAW D A, VIRGO K S, NAM R, et al. Initial hormonal management of androgen-sensitive metastatic,recurrent,or progressive prostate cancer:2006 update of an American Society of Clinical Oncology practice guideline[J]. J Clin Oncol, 2007, 25(12):1596-1605. DOI URL
[3]	WIERMAN M E, ARLT W, BASSON R, et al. Androgen therapy in women:a reappraisal:an endocrine society clinical practice guideline[J]. J Clin Endocrinol Metab, 2014, 99(10):3489-3510. DOI URL
[4]	HEROLD D A, FITZGERALD R L. Immunoassays for testosterone in women:better than a guess?[J]. Clin Chem, 2003, 49(8):1250-1251. DOI URL
[5]	SIEKMANN L. Determination of steroid hormones by the use of isotope dilution-mass spectrometry:a definitive method in clinical chemistry[J]. J Steroid Biochem, 1979, 11(1A):117-123. DOI URL
[6]	THIENPNT L M, DE BRABANDERE V I, STÖCKL D, et al. Use of cyclodextrins for prepurification of progesterone and testosterone from human serum prior to determination with isotope dilution gas chromatography/mass spectrometry[J]. Anal Chem, 1994, 66(22):4116-4119. DOI URL
[7]	TAI S S, XU B, WELCH M J, et al. Development and evaluation of a candidate reference measurement procedure for the determination of testosterone in human serum using isotope dilution liquid chromatography/tandem mass spectrometry[J]. Anal Bioanal Chem, 2007, 388(5-6):1087-1094. DOI URL
[8]	BOTELHO J C, SHACKLADY C, COOPER H C, et al. Isotope-dilution liquid chromatography-tandem mass spectrometry candidate reference method for total testosterone in human serum[J]. Clin Chem, 2013, 59(2):372-380. DOI URL
[9]	Clinical and Laboratory Standards Institute. Liquid chromatography-mass spectrometry methods[S]. C62-A,CLSI, 2014.
[10]	Clinical and Laboratory Standards Institute. User verification of precision and estimation of bias[S]. EP15-A3,CLSI, 2014.
[11]	Clinical and Laboratory Standards Institute. Measurement procedure comparison and bias estimation using patient samples[S]. EP9-A3,CLSI, 2013.
[12]	中国合格评定国家认可委员会. CNAS-GL006:2109化学分析中不确定度的评估指南[S]. 北京:中国合格评定国家认可委员会, 2002.
[13]	张天娇, 王玉飞, 曾洁, 等. 同位素稀释液相色谱串联质谱法测定血清睾酮[J]. 临床检验杂志, 2016, 34(12):892-896.
[14]	MOAL V, MATHIEU E, REYNIER P, et al. Low serum testosterone assayed by liquid chromatography-tandem mass spectrometry. Comparison with five immunoassay techniques[J]. Clin Chim Acta, 2007, 386(1-2):12-19. DOI URL

样本	批内精密度			批间精密度
样本	x/（ng/mL）	s/（ng/mL）	CV/%	x/（ng/mL）	s/（ng/mL）	CV/%
2017 RELA-A	4.35	0.10	2.3	4.32	0.09	2.2
2017 RELA-B	7.02	0.14	2.0	6.95	0.12	1.7

样本	批内精密度			批间精密度
样本	x/（ng/mL）	s/（ng/mL）	CV/%	x/（ng/mL）	s/（ng/mL）	CV/%
2017 RELA-A	4.35	0.10	2.3	4.32	0.09	2.2
2017 RELA-B	7.02	0.14	2.0	6.95	0.12	1.7

样本	测定均值/（ng/mL）	参考值/ （ng/mL）	偏移/%
2017 RELA-A	4.32	4.45	-2.9
2017 RELA-B	6.95	7.19	-3.3
SRM971	6.46	6.44	0.2

样本	测定均值/（ng/mL）	参考值/ （ng/mL）	偏移/%
2017 RELA-A	4.32	4.45	-2.9
2017 RELA-B	6.95	7.19	-3.3
SRM971	6.46	6.44	0.2

样本编号	测定均值/（pg/mL）	理论值/（pg/mL）	平均R_SN	CV/%	偏移/%
1	12.78	10	6	8.6	13.6
2	21.73	20	12	3.7	-4.2
3	49.11	50	29	3.3	-5.3