HPLC-MS/MS同位素稀释法测定人体血清中类固醇激素的研究

doi:10.3969/j.issn.1673-8640.2015.05.005

摘要/Abstract

摘要： 目的建立一种能同时测定血清中多种类固醇激素[包括脱氢表雄酮(DHEA)、17α-羟孕酮(17α-OHP4)、雄烯二酮(AD)、雌酮(E1)、皮质酮(CORT)、双氢睾酮(DHT)、孕烯醇酮(P5)和17-羟孕烯醇酮(17-OHP5)]的快速、灵敏的稳定同位素稀释高效液相色谱串联质谱(ID-HPLC-MS/MS)方法。方法血清样品经甲基叔丁基醚(MTBE)提取,上清液吹干后加入羟胺进行柱前衍生化反应后检测。采用Phenomenex C18反相色谱柱和Phenomenex C18预保护柱。流动相A为含0.1%甲酸的水,流动相B为含0.1%甲酸的甲醇。质谱采用电喷雾电离(ESI)模式进行离子化,正离子多反应监测模式(MRM)扫描,内标法定量分析。结果血清中8种类固醇激素最低定量限[LLOQ,以信噪比(S/N)≥10为标准]为0.03~0.625 ng/mL;在0.05~50.00 ng/mL范围内线性良好,相关系数(r)均≥0.998 0;批内精密度为1.93%~13.81%,批间精密度为4.90%~16.18%,加标回收率为80.0%~130.6%。结论建立了同时测定8种类固醇激素的ID-HPLC-MS/MS。该法灵敏度高、特异性强、准确且高效,可在10 min内完成分离和检测,适用于人体血清中类固醇激素的定量分析。

关键词: 类固醇激素, 血清, 稳定同位素稀释, 液相色谱, 串联质谱

Abstract: Objective To establish a rapid, sensitive and stable isotope dilution high performance liquid chromatography-tandem mass spectrometry(ID-HPLC-MS/MS) for simultaneous quantitative determination of dehydroepiandrosterone(DHEA), 17 alpha-hydroxyprogesterone(17α-OHP4), androstenedione(AD), estrone(E1), corticosterone(CORT), dihydrotestosterone(DHT), pregnenolone(P5)and 17-hydroxylpregnenolone(17-OHP5). Methods The steroid hormones were extracted from human serum by methyl tert-butyl ether(MTBE), and the supernatant was treated with hydroxylamine to produce post-column derivatives before detection. Steroid hormones were separated by Phenomenex reversed-phase C18 with precolumn. Water containing 0.1% formic acid and methanol containing 0.1% formic acid were used as mobile phase A and B,respectively. Multiple reaction monitoring(MRM)with the positive ion detection mode was applied to selectively detect these 8 steroid hormones ionized by electrospray ionization (ESI) interface, and the quantitative analysis for them was carried out by using deuterium isotope as internal standard. Results The lower limits of quantitation(LLOQ)of these 8 hormones can reach 0.03-0.625 ng/mL on the basis of signal-noise ratio (S/N) ≥10. The correlation coefficients(r)were ≥0.998 0 with a good linear at the concentration of 0.05-50.00 ng/mL. The within-run precision was 1.93%-13.81%, and the between-run precision was 4.90%-16.18%. The recoveries were 80.0%-130.6%. Conclusions The ID-HPLC-MS/MS is established for simultaneous quantitative determination of 8 kinds of steroid hormones. It has the advantages of sensitivity, specificity and accuracy, which is suitable for the separation and determination of steroid hormones for human serum samples within 10 min.

Key words: Steroid hormone, Serum, Stable isotope dilution, Liquid Chromatography, Tandem mass spectrometry

中图分类号:

R446.1

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.

ZHOU Yafei, WANG Yueting, YU Jiaping. Research on the determination of serum steroid hormones by isotope dilution HPLC-MS/MS[J]. Laboratory Medicine, 2015, 30(5): 427-432.

图/表 5

表1 类固醇激素的条件优化结果

类固醇激素	内标物	保留时间(min)	MRM监测离子对(Q1/Q3)	DP	CE	CXP
DHEA	d6-DHEA	4.73	304.1/253.2	95	24	15
17α-OHP4	d8-17OHP4	4.92	361.1/112.2	145	45	10
17-OHP5	d8-17OHP4	4.54	348.1/330.3	60	14	8
E1	d₂-E1	4.42	286.1/253.0	75	22	10
CORT	d9-P4	4.49	410.0/377.2	80	25	9
AD	d3-T	4.85	317.1/112.2	130	41	6
P5	d9-P4	5.87	332.1/86.2	110	41	4
DHT	d3-T	5.24	306.2/81.1	127	53	15

图1 DHEA及其内标d6-DHEA、17α-OHP4及其内标d8-17OHP4的XIC图

表2 类固醇激素的线性拟合、最低定量限和检出限结果

分析物	曲线浓度 (ng/mL)	斜率	r值	LLOQ
分析物	曲线浓度 (ng/mL)	斜率	r值	$x ?$ (ng/mL)	S/N	CV(%)	准确度(%)
DHEA	0.125~40	0.423	0.999 5	0.125 0	14.0	4.6	119.0
17α-OHP4	0.125~10	0.226	0.999 0	0.125 0	10.5	5.8	97.4
AD	0.0625~10	0.351	0.999 6	0.062 5	10.0	6.1	110.0
P5	0.0313~10	0.275	0.999 3	0.031 3	10.5	9.2	88.0
17-OHP5	0.078~25	1.570	0.999 2	0.078 0	10.7	12.7	97.2
DHT	0.125~2	0.034	0.998 8	0.250 0	24.5	6.3	130.0
CORT	0.625~50	0.443	0.999 0	0.625 0	11.0	5.7	132.0
E1	0.05~2	0.052	0.998 0	0.031 3	10.0	13.5	137.0

表2 类固醇激素的线性拟合、最低定量限和检出限结果

分析物	曲线浓度 (ng/mL)	斜率	r值	LLOQ
分析物	曲线浓度 (ng/mL)	斜率	r值	$x ?$ (ng/mL)	S/N	CV(%)	准确度(%)
DHEA	0.125~40	0.423	0.999 5	0.125 0	14.0	4.6	119.0
17α-OHP4	0.125~10	0.226	0.999 0	0.125 0	10.5	5.8	97.4
AD	0.0625~10	0.351	0.999 6	0.062 5	10.0	6.1	110.0
P5	0.0313~10	0.275	0.999 3	0.031 3	10.5	9.2	88.0
17-OHP5	0.078~25	1.570	0.999 2	0.078 0	10.7	12.7	97.2
DHT	0.125~2	0.034	0.998 8	0.250 0	24.5	6.3	130.0
CORT	0.625~50	0.443	0.999 0	0.625 0	11.0	5.7	132.0
E1	0.05~2	0.052	0.998 0	0.031 3	10.0	13.5	137.0

表3 8种类固醇激素的精密度结果

分析物	批内			批间
分析物	$x ?$ (ng/mL)	s(ng/mL)	CV(%)	$x ?$ (ng/mL)	s(ng/mL)	CV(%)
DHEA	2.012	0.083	4.13	2.083	0.102	4.90
17α-OHP4	0.928	0.051	5.50	1.023	0.069	6.74
AD	0.675	0.013	1.93	0.631	0.048	7.61
P5	0.593	0.044	7.42	0.608	0.080	13.16
17-OHP5	2.702	0.138	5.11	2.793	0.304	10.88
CORT	6.777	0.488	7.20	6.673	0.696	10.43
DHT	0.387	0.036	9.30	0.316	0.039	12.34
E1	0.036 2	0.005	13.81	0.027 2	0.004 4	16.18

表3 8种类固醇激素的精密度结果

分析物	批内			批间
分析物	$x ?$ (ng/mL)	s(ng/mL)	CV(%)	$x ?$ (ng/mL)	s(ng/mL)	CV(%)
DHEA	2.012	0.083	4.13	2.083	0.102	4.90
17α-OHP4	0.928	0.051	5.50	1.023	0.069	6.74
AD	0.675	0.013	1.93	0.631	0.048	7.61
P5	0.593	0.044	7.42	0.608	0.080	13.16
17-OHP5	2.702	0.138	5.11	2.793	0.304	10.88
CORT	6.777	0.488	7.20	6.673	0.696	10.43
DHT	0.387	0.036	9.30	0.316	0.039	12.34
E1	0.036 2	0.005	13.81	0.027 2	0.004 4	16.18

表4 8种类固醇激素的加标回收率结果

分析物	加标量 (ng/mL)	测定值 (ng/mL)	RSD	加标回收率 (%)
DHEA	0	6.0±0.2	3.8	—
	1.0	7.2±0.3	4.9	120.0
	5.0	10.4±0.3	2.8	88.0
	50.0	55.5±1.1	2.0	99.0
17α-OHP4	0	1.7±0.2	10.1	—
	0.5	2.1±0.02	1.1	80.0
	1.0	2.9±0.2	6.7	120.0
	10.0	14.0±1.2	8.2	123.0
AD	0	1.8±0.1	4.7	—
	0.5	2.3±0.1	4.2	100.0
	1.0	3.0±0.06	1.8	120.0
	10.0	14.0±0.44	3.1	122.0
P5	0	0.86±0.06	6.6	—
	0.5	1.5±0.02	1.4	128.0
	1.0	2.1±0.04	1.7	124.0
	10.0	10.6±0.2	1.9	97.0
17-OHP5	0	3.8±0.16	4.2	—
	0.5	4.2±0.16	3.8	80.0
	1.0	5.0±0.1	2.6	120.0
	10.0	14.3±0.2	1.5	105.0
CORT	0	2.2±0.04	1.8	—
	0.5	2.6±0.04	0.6	80.0
	1.0	3.2±0.1	3.3	100.0
	10.0	12.6±0.2	1.7	104.0
DHT	0	0.35±0.02	9.1	—
	0.5	0.84±0.08	4.3	98.0
	1.0	1.43±0.1	7.0	108.0
	10.0	11.1±0.3	2.9	108.0
E1	0	0.04±0.005	12.7	—
	0.5	0.47±0.05	10.2	86.0
	1.0	1.23±0.06	4.8	119.0
	10.0	13.1±0.17	1.3	130.6

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