血浆中6种初级胆汁酸HPLC–MS/MS测定

牟燕; 吴瑛瑕; 张易天; 曾颖炫; 周枝凤

doi:10.11847/zgggws1138567

血浆中6种初级胆汁酸HPLC–MS/MS测定

doi: 10.11847/zgggws1138567

南方医科大学公共卫生学院卫生检验检疫系，广东广州 510515

基金项目: 国家自然科学基金（21607070）

详细信息

作者简介:
牟燕（1995 – ），女，四川泸州人，硕士在读，研究方向：环境污染与健康

通讯作者:
周枝凤，E-mail：zfzhou@smu.edu.cn

中图分类号: Q 548⁺.5
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- 文章访问数: 30
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- 被引次数: 0
出版历程
- 收稿日期: 2022-03-20
- 网络出版日期: 2023-01-17
- 刊出日期: 2023-01-31

Simultaneous and rapid determination of 6 primary bile acids in plasma by high-performance liquid chromatography-tandem mass spectrometry

Department of Hygiene Inspection and Quarantine Science, School of Public Health, Southern Medical University, Guangzhou, Guangdong Province 510515, China

摘要

摘要: 目的建立高效液相色谱 – 串联质谱法快速检测血浆中6种初级胆汁酸的含量。方法血浆经甲醇沉淀蛋白后，用Thermo Accucore C₁₈色谱柱（100 mm × 4.6 mm，2.6 μm）进行色谱分离，流动相为甲醇 – 水（0.1 %甲酸 + 5 mmol/L乙酸铵），梯度洗脱。负离子模式下多反应监测模式检测。实测6名健康成年人和6只成年SD大鼠样本。结果 6种初级胆汁酸在6 min内实现基线分离，在5～2 000 μg/L范围内线性良好，相关系数＞0.995，检出限为0.25～0.45 μg/L，定量限为0.84～1.49 μg/L。日内、日间加标回收率分别为86.3 %～111.5 %和87.7 %～104.9 %；日内、日间相对标准偏差分别为5.2 %～11.9 %和6.6 %～14.3 %。健康成年人和成年SD大鼠血浆中初级胆汁酸的浓度显著不同。结论该方法简单、快速、灵敏、准确，可用于临床检验和动物试验中初级胆汁酸的快速检测。
- 高效液相色谱-串联质谱法 /
- 初级胆汁酸 /
- 血浆
Abstract: Objective To develop a rapid and accurate high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of 6 primary bile acids in plasma. Methods Plasma was separated on a Thermo Accucore C₁₈ column (100 mm × 4.6 mm; 2.6 μm) after being extracted by protein precipitation using methanol. The 6 primary bile acids were separated by using the mobile phase of methanol-water (0.1% formic acid + 5 mmol/L ammonium acetate) running in gradient elution. The mass spectrometer was operated on an electrospray ionization source in negative ion mode for the multiple reaction monitoring analysis. Plasma from 6 healthy adults and 6 adult Sprague-Dawley (SD) rats was detected. Results Baseline separation of 6 primary bile acids was achieved within 6 min. The linearity of the calibration curves of the 6 primary bile acids was excellent in the range of 5 – 2000 μg/L with a correlation coefficient higher than 0.995. The limits of detection and limits of quantitation were in the range of 0.25 – 0.45 μg/L and 0.84 – 1.49 μg/L, respectively. The recoveries of intra-day and inter-day were 86.3% – 111.5% and 87.7% – 104.9%, with relative standard deviations of 5.2% – 11.9% and 6.6% – 14.3%, respectively. The concentrations of primary bile acids in plasma of healthy adults and adult SD rats were significantly different. Conclusion This method is simple, rapid, sensitive, and accurate, and can meet the needs for rapid detection of primary bile acids in clinical tests and animal experiments.
- HPLC-MS/MS /
- primary bile acid /
- plasma

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图 1 水相中不同乙酸铵含量的响应强度

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图 2 6种初级胆汁酸及内标的色谱图（10 μg/L）

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表 1 6种初级胆汁酸及内标的母离子、子离子、去簇电压和碰撞电压

物质	母离子（m/z）	子离子（m/z）	去簇电压/（V）	碰撞电压/（V）
CA	407.9	407.9 ^a	– 141	– 17
		343.3		– 47
CDCA	391.3	391.3 ^a	– 151	– 21
		373.3		– 47
TCA	514.3	514.3 ^a	– 120	– 10
		80.0		– 120
TCDCA	498.2	498.2 ^a	– 90	– 13
		80.0		– 120
GCA	464.3	464.3 ^a	– 140	– 10
		402.4		– 50
GCDCA	448.3	448.3 ^a	– 110	– 10
		368.2		– 46
CA-d4	411.2	411.2 ^a	– 125	– 10
		347.4		– 48
TCA-d4	518.3	518.3 ^a	– 120	– 10
		80.0		– 120
GCA-d4	468.3	468.3 ^a	– 127	– 30
		404.6		– 48
注：a 为定量离子。

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表 2 6种初级胆汁酸的线性方程、相关系数、检出限和定量限

物质	内标	线性方程	相关系数	检出限/（μg/L）	定量限/（μg/L）
CA	CA-d₄	y = 1.13 × 10^{– 3}x + 1.22 × 10^{– 3}	0.998 3	0.45	1.49
CDCA	CA-d₄	y = 4.45 × 10^{– 3}x + 1.25 × 10^{– 2}	0.998 7	0.39	1.30
TCA	TCA-d₄	y = 6.19 × 10^{– 3}x + 9.46 × 10^{– 3}	0.995 1	0.35	1.17
TCDCA	TCA-d₄	y = 7.38 × 10^{– 3}x + 8.33 × 10^{– 3}	0.995 6	0.38	1.26
GCA	GCA-d₄	y = 8.09 × 10^{– 3}x + 3.02 × 10^{– 2}	0.997 0	0.38	1.28
GCDCA	GCA-d₄	y = 1.61 × 10^{– 2}x + 5.18 × 10^{– 2}	0.995 3	0.25	0.84

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表 3 血浆中6种初级胆汁酸的加标回收率及相对标准偏差

物质	加标量（μg/L）	日内		日间
物质	加标量（μg/L）	回收率（%）	相对标准偏差（%）	回收率（%）	相对标准偏差（%）
CA	6	94.1	8.7	98.6	14.1
	800	108.0	9.4	102.9	10.4
	1600	95.7	5.2	98.2	7.7
CDCA	6	101.8	11.9	101.0	6.6
	800	101.0	11.0	102.9	10.2
	1600	91.4	7.9	95.8	9.8
TCA	6	100.1	7.8	99.9	14.0
	800	101.8	6.5	96.5	10.9
	1600	95.4	11.2	91.5	8.2
TCDCA	6	99.8	10.1	104.9	12.1
	800	100.8	7.0	94.1	8.4
	1600	99.2	7.8	87.7	7.3
GCA	6	92.4	7.0	97.6	9.6
	800	102.0	11.1	101.6	11.1
	1600	97.0	9.7	99.2	14.3
GCDCA	6	86.3	8.5	94.1	11.7
	800	111.5	10.7	102.6	11.4
	1600	102.3	11.4	96.2	12.8

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表 4 6种初级胆汁酸在健康成年人和SD大鼠血浆中的含量（n = 6）

物质	检出量/（μg/L）		P 值
物质	健康成年人	SD大鼠	P 值
CA	9.7～270.0	248.0～467.0	0.031
CDCA	43.4～406.0	106.0～405.0	0.893
TCA	20.0～222.0	65.1～600.0	0.139
TCDCA	129.0～697.0	59.0～572.0	0.139
GCA	184.0～1020.0	5.9～128.0	0.005
GCDCA	1150.0～3144.0	ND～9.8	0.005
注： ND 为未检出。

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