Research on the determination of serum steroid hormones by isotope dilution HPLC-MS/MS

doi:10.3969/j.issn.1673-8640.2015.05.005

Abstract

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

CLC Number:

R446.1

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.

Figures/Tables 5

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

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

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

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

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

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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