头发中甲状腺激素和类固醇激素的高效液相色谱串联质谱检测

doi:10.19894/j.issn.1000-0518.220025

摘要/Abstract

摘要：

基于高效液相色谱-串联质谱（HPLC-MS/MS），开发了一种灵敏、可靠的方法，用于同时测定人类头发中的甲状腺和类固醇激素，包括甲状腺素（T4）、3，3'，5-三碘甲状腺原氨酸（T3）、3，3'，5'-三碘甲状腺原氨酸（rT3）、3，3'-二碘甲状腺原氨酸（3，3'-T2）、L-甲状腺原氨酸（T0）、皮质醇（F）和可的松（E）。头发用量20 mg，使用甲醇/氨水（体积比95∶5）在40 ℃下孵育24 h。采用电喷雾离子源，在多反应监测模式和正离子模式下进行定量分析。在甲醇/纯水（体积比80∶20，含0.2 mmol/L乙酸铵）的等度洗脱条件下，7种待测物分离良好。该方法检测上述7种激素的检测限在0.3~1.9 pg/mg，定量限在1.1~6.5 pg/mg；日内、日间变异系数均小于10%，回收率在89.4%~109.1%之间；稳定性、残留效应均符合检测标准要求。运用该方法检测140名被试的头发样本，所有被试的T0、F和E均可定量检测，而T4、T3、rT3和3，3'-T2的含量非常低，均在定量限附近，仅在少量被试中可被检出。

关键词: 甲状腺激素, 类固醇激素, 液相色谱串联质谱法, 头发含量

Abstract:

A sensitive and reliable method based on high performance liquid chromatography-tandem mass spectrometry （LC-MS/MS） is developed for the simultaneous determination of thyroids and steroids in human hair， including thyroxine （T4）， 3，3′，5-triiodothyronine （T3）， 3，3'，5'-triiodothyronine （rT3）， 3，3'-diiodothyronine （3，3'-T2） L-thyronine （T0）， cortisol （F） and cortisone （E）. The seven endogenous compounds are extracted from 20 mg hair sample incubated in the mixture solution of methanol and ammonia （volume ratio is 95∶5） for 24 hours at 40 ℃， and then analyzed with an electrospray ionization source in multiple reaction monitoring mode and positive ion mode. Under the isocratic elution conditions with methanol/pure water （volume ratio is 80∶20， containing 0.2 mmol/L ammonium acetate） as mobile phase， seven analytes are separated and well resolved. The limits of detection for the seven hormones range from 0.3 to 1.9 pg/mg and the limits of quantification from 1.1 to 6.5 pg/mg. The intra- and inter-day coefficients of variation are less than 10% and the recoveries range from 89.4% to 109.1% for all seven compounds. The stability and carryover also meet the detection criteria. The population analysis on hair samples from 140 participants reveals that T0， F and E could be quantified in all subjects， while T4， T3， rT3 and 3，3'-T2 could be detected in a small number of subjects， showing the contents around their limits of quantification.

Key words: Thyroids, Steroids, Liquid chromatography tandem mass spectrometry, Hair concentrations

中图分类号:

O657.6

袁琳, 吴彦, 楚刘喜, 王为, 朱敏惠, 张贺, 杨瑾, 邓慧华. 头发中甲状腺激素和类固醇激素的高效液相色谱串联质谱检测[J]. 应用化学, 2022, 39(11): 1703-1715.

Lin YUAN, Yan WU, Liu-Xi CHU, Wei WANG, Min-Hui ZHU, He ZHANG, Jin YANG, Hui-Hua DENG. Determination of Thyroids and Steroids in Hair by High Performance Liquid Chromatography Tandem Mass Spectrometry[J]. Chinese Journal of Applied Chemistry, 2022, 39(11): 1703-1715.

图/表 10

参考文献 22

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化合物 Compound	母离子 Precursor ion/Da	子离子 Product ion/Da	去簇电压 Declustering potential DP/V	入口电压 Entrance potential EP/eV	碰撞室入口电压 Collision cell entrance potential CEP/V	碰撞能 Collision energy CE/eV	碰撞室出口电压 Collision cell exit potential CXP/V
T4	777.2	731.2	68.0	7.6	57.5	32.0	7.0
T4^*	777.2	633.4	68.0	7.6	57.5	36.0	7.5
T4?¹³C₆	783.2	737.2	40.0	8.4	43.5	36.2	9.5
T3	651.5	605.3	41.0	8.4	41.8	30.7	6.2
T3^*	651.5	507.6	41.0	8.4	41.8	30.8	6.5
T3?¹³C₆	657.2	611.2	39.5	8.7	41.0	32.8	9.1
rT3	651.2	605.2	39.0	7.9	32.1	31.4	8.4
rT3^*	651.2	507.6	39.0	7.9	32.1	32.6	7.0
rT3?¹³C₆	657.1	611.3	34.0	8.4	54.5	31.0	8.5
3，3′?T2	525.6	479.6	30.7	6.9	56.1	28.3	7.5
3，3′?T2^*	525.6	381.9	30.7	6.9	56.1	29.5	5.6
3，3′?T2?¹³C₆	531.6	485.6	30.2	7.5	40.7	28.4	18.3
T0	274.0	215.0	51.0	5.8	37.5	24.5	1.2
T0^*	274.0	212.1	51.0	5.8	37.5	26.4	1.3
F	362.9	121.0	38.6	7.1	28.3	32.8	2.2
F^*	362.9	327.1	38.6	7.1	28.3	22.4	4.3
F?D2	365.0	122.0	41.6	6.4	30.4	38.1	1.4
E	360.9	163.0	48.9	7.8	26.4	31.6	0.9
E^*	360.9	121.1	48.9	7.8	26.4	43.2	0.8
E?D8	368.2	169.0	48.0	5.8	24.4	32.0	3.5

化合物 Compound	母离子 Precursor ion/Da	子离子 Product ion/Da	去簇电压 Declustering potential DP/V	入口电压 Entrance potential EP/eV	碰撞室入口电压 Collision cell entrance potential CEP/V	碰撞能 Collision energy CE/eV	碰撞室出口电压 Collision cell exit potential CXP/V
T4	777.2	731.2	68.0	7.6	57.5	32.0	7.0
T4^*	777.2	633.4	68.0	7.6	57.5	36.0	7.5
T4?¹³C₆	783.2	737.2	40.0	8.4	43.5	36.2	9.5
T3	651.5	605.3	41.0	8.4	41.8	30.7	6.2
T3^*	651.5	507.6	41.0	8.4	41.8	30.8	6.5
T3?¹³C₆	657.2	611.2	39.5	8.7	41.0	32.8	9.1
rT3	651.2	605.2	39.0	7.9	32.1	31.4	8.4
rT3^*	651.2	507.6	39.0	7.9	32.1	32.6	7.0
rT3?¹³C₆	657.1	611.3	34.0	8.4	54.5	31.0	8.5
3，3′?T2	525.6	479.6	30.7	6.9	56.1	28.3	7.5
3，3′?T2^*	525.6	381.9	30.7	6.9	56.1	29.5	5.6
3，3′?T2?¹³C₆	531.6	485.6	30.2	7.5	40.7	28.4	18.3
T0	274.0	215.0	51.0	5.8	37.5	24.5	1.2
T0^*	274.0	212.1	51.0	5.8	37.5	26.4	1.3
F	362.9	121.0	38.6	7.1	28.3	32.8	2.2
F^*	362.9	327.1	38.6	7.1	28.3	22.4	4.3
F?D2	365.0	122.0	41.6	6.4	30.4	38.1	1.4
E	360.9	163.0	48.9	7.8	26.4	31.6	0.9
E^*	360.9	121.1	48.9	7.8	26.4	43.2	0.8
E?D8	368.2	169.0	48.0	5.8	24.4	32.0	3.5

	平均值 Mean/%	标准差 SD/%
外标选择性 Selectivity for ESs
T4	4.6	0.4
rT3	7.5	1.2
T3	4.2	0.1
3，3'?T2	5.3	0.7
T0	7.2	1.2
F	8.8	1.3
E	5.7	1.1
内标选择性 Selectivity for ISs
T4?¹³C₆	0.5	0.3
rT3?¹³C₆	1.2	0.3
T3?¹³C₆	0.3	0.1
T2?¹³C₆	0.5	0.2
F?D2	1.6	0.5
E?D8	3.1	0.6

	平均值 Mean/%	标准差 SD/%
外标选择性 Selectivity for ESs
T4	4.6	0.4
rT3	7.5	1.2
T3	4.2	0.1
3，3'?T2	5.3	0.7
T0	7.2	1.2
F	8.8	1.3
E	5.7	1.1
内标选择性 Selectivity for ISs
T4?¹³C₆	0.5	0.3
rT3?¹³C₆	1.2	0.3
T3?¹³C₆	0.3	0.1
T2?¹³C₆	0.5	0.2
F?D2	1.6	0.5
E?D8	3.1	0.6

化合物 Compound	标准曲线 Standard curve	相关系数的平方（R²）	线性范围 Linear range/（pg·mg^-1）	检测限 LOD/（pg·mg^-1）	定量限 LOQ/（pg·mg^-1）
T4	y = 0.0016x-0.0035	0.996 7	6.5 ~ 1 000	1.9	6.5
rT3	y = 0.0063x-0.0111	0.999 7	3.9 ~ 1 000	1.2	3.9
T3	y = 0.0047x-0.0078	0.999 8	4.3 ~ 1 000	1.3	4.3
3，3'?T2	y = 0.0020x-0.0036	0.998 5	5.4 ~ 1 000	1.6	5.4
T0	y = 0.0021x-0.0037	0.997 2	3.9 ~ 1 000	1.2	3.9
F	y = 0.0167x-0.0098	0.996 2	1.8 ~ 1 000	0.6	1.8
E	y = 0.0623x-0.0373	0.999 2	1.1 ~ 1 000	0.3	1.1