Simultaneous determination of 5 steroid hormones in human serum by ultra-performance liquid chromatography-tandem mass spectrometry and establishment of reference intervals in healthy adults

doi:10.3969/j.issn.1673-8640.2022.02.015

Abstract

Abstract:

Objective To establish and validate a method for the simultaneous determination of testosterone（T）,dihydrotestosterone（DHT）,androstenedione（AD）,dehydroepiandrosterone（DHEA） and 17alpha-hydroxyprogesterone（17OHP） in human serum by ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）,and to establish the reference intervals of 5 steroid hormones in healthy adults. Methods The serum samples were purified by SOLAμ HRP solid-phase extraction,and they were separated by Poroshell 120 EC-C18 analytical column. Mobile phase was constituted of 30% acetonitrile solution and 90% acetonitrile solution containing 0.05% formic acid with gradient elution. The electrospray positive ion multi-reaction monitoring mode was used for determination,and the isotope internal standard method was used to quantify the concentration of steroid hormones. The performance of the method was verified comprehensively,and the reference intervals suitable for the local population in Shanghai were established. Results The minimum limits of quantification of the 5 steroid hormones in human serum were 0.01-0.10 ng/mL. The standard curve range covered 1 000 times （r²≥0.999 5） and showed good linearity,and the sample after dilution can be reported to cover the range of 5 000 times. The average accuracy of 5 steroid hormones ranged from 95.0% to 105.6%,with the precision of <8%. None of the 8 structural analogs interfered with the target analytes,high triglyceride blood did not affect the determination results,and hemolysis resulted in low DHEA concentration. The extraction recoveries of the analytes were 60.5%-93.3%,and there was no obvious matrix effect after calibration of internal standard. Analytes demonstrated good stability at room temperature for 4 h,at -25--15 ℃ for 30 d or at -90--70 ℃ for 100 d. The method was used and participated in a survey of endocrine accuracy verification supplied by the National Center for Clinical Laboratories in 2019. The determination deviations of T and 17OHP were -5.00%-0.38%. The results of 367 physical examination subjects were analyzed and showed that there was statistical significance in the concentration of 5 steroid hormones between females and males（P<0.05）. There was statistical significance between the 2 age subgroups（aged 16-50 years and aged>50 years） of female groups（P<0.05）. Conclusions A UPLC-MS/MS has been established for the determination of 5 steroid hormones in human serum,and the reference intervals suitable for the Central Laboratory of Shanghai Xuhui Central Hospital have been verified.

Key words: Ultra-performance liquid chromatography, Mass spectrometry, Steroid hormone, Human serum, Reference interval

CLC Number:

R446.1

LU Youli, YANG Shuangshuang, ZHANG Meiwei, OU Meixian, DONG Chunxia, SHEN Weiwei, JIANG Fengli, LI Shuijun. Simultaneous determination of 5 steroid hormones in human serum by ultra-performance liquid chromatography-tandem mass spectrometry and establishment of reference intervals in healthy adults[J]. Laboratory Medicine, 2022, 37(2): 165-173.

Figures/Tables 9

References 18

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分析物	标准曲线添加液						质控添加液
分析物	S1	S2	S3	S4	S5	S6	低浓度	中浓度	高浓度
T、AD、17OHP	1	2	10	50	250	1 000	3	25	800
DHT	2	4	20	100	500	2 000	6	50	1 600
DHEA	10	20	100	500	2 500	10 000	30	250	8 000

分析物	标准曲线添加液						质控添加液
分析物	S1	S2	S3	S4	S5	S6	低浓度	中浓度	高浓度
T、AD、17OHP	1	2	10	50	250	1 000	3	25	800
DHT	2	4	20	100	500	2 000	6	50	1 600
DHEA	10	20	100	500	2 500	10 000	30	250	8 000

分析物名称	监测离子对/（m/z）	碰撞能量/eV	监测方式	内标
T	289.2^①/97.0^②/109.0^③	31/35	+	T-d₃
DHT	291.2^①/255.2^②/159.0^③	21/31	+	DHT-d₃
AD	287.2^①/97.0^②/109.0^③	30/33	+	AD-¹³C₃
DHEA	289.2^①/253.2^②/213.1^③	16/26	+	DHEA-d₅
17OHP	331.2^①/97.0^②/109.0^③	33/33	+	17OHP-d₈
T-d₃	292.2^①/97.0^②	31	+	无
DHT-d₃	294.2^①/258.2^②	21	+	无
AD-¹³C₃	290.2^①/100.0^②	30	+	无
DHEA-d₅	294.3^①/258.2^②	16	+	无
17OHP-d₈	339.2^①/100.0^②	33	+	无

分析物名称	监测离子对/（m/z）	碰撞能量/eV	监测方式	内标
T	289.2^①/97.0^②/109.0^③	31/35	+	T-d₃
DHT	291.2^①/255.2^②/159.0^③	21/31	+	DHT-d₃
AD	287.2^①/97.0^②/109.0^③	30/33	+	AD-¹³C₃
DHEA	289.2^①/253.2^②/213.1^③	16/26	+	DHEA-d₅
17OHP	331.2^①/97.0^②/109.0^③	33/33	+	17OHP-d₈
T-d₃	292.2^①/97.0^②	31	+	无
DHT-d₃	294.2^①/258.2^②	21	+	无
AD-¹³C₃	290.2^①/100.0^②	30	+	无
DHEA-d₅	294.3^①/258.2^②	16	+	无
17OHP-d₈	339.2^①/100.0^②	33	+	无

添加浓度/（ng/mL）	批内		批间
添加浓度/（ng/mL）	精密度（CV）/%	准确度/%	精密度（CV）/%	准确度/%
T
0.03	3.43	102.8	2.91	100.9
0.25	4.12	101.1	3.64	100.7
8.00	2.30	97.4	3.01	97.1
DHT
0.06	2.92	101.9	3.56	100.5
0.50	3.22	104.8	3.49	105.2
16.00	2.53	100.4	2.87	102.4
AD
0.03	2.62	99.2	4.47	99.6
0.25	3.54	99.8	3.57	101.4
8.00	2.59	104.2	2.97	102.3
DHEA
0.30	4.75	101.8	4.75	98.2
2.50	3.08	101.7	5.33	101.8
80.00	3.74	100.5	5.66	102.6
17OHP
0.03	2.83	96.9	4.68	95.0
0.25	3.51	95.2	4.80	96.3
8.00	2.85	99.1	4.35	101.2