三碘代-L-甲状腺原氨酸（T3）标准物质中结构相关杂质的分析

肖鹏; 宋丹; 李红梅

doi:10.12338/j.issn.2096-9015.2024.0134

三碘代-L-甲状腺原氨酸（T3）标准物质中结构相关杂质的分析

doi: 10.12338/j.issn.2096-9015.2024.0134

肖鹏^{1, 2,},
宋丹^{1, 2},
李红梅^{1, 2}

1.
中国计量科学研究院，北京 100029
2.
国家市场监管重点实验室（营养与健康化学计量及应用），北京 100029）

基金项目: 国家重点研发计划（2022YFF0710303）；国家科技基础条件平台（APT2302-1，APT2302-6）。

详细信息

作者简介:
肖鹏（1982-），中国计量科学研究院副研究员，研究方向：临床化学计量，邮箱：xiaop@nim.ac.cn

计量
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出版历程
- 收稿日期: 2024-04-19
- 录用日期: 2024-06-13
- 修回日期: 2024-06-13
- 网络出版日期: 2024-07-11

Analysis of the Structural-related Impurities Involved in 3,3',5-Triiodo-L-thyronine (T3) Purity Reference Material

XIAO Peng^{1, 2
,},
SONG Dan^{1, 2},
LI Hongmei^{1, 2}

1.
National Institute of Metrology, Beijing 100029, China
2.
Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation, Beijing 100029, China

摘要

摘要: 研制三碘代-L-甲状腺原氨酸（T3）纯度标准物质，可为临床测量结果提供溯源源头，为甲状腺疾病的精准诊断提供可靠依据。在质量平衡法定值过程中，对候选物结构相关杂质进行化学结构鉴定与定量，是降低主成分定值结果不确定度的有效方式。以核磁共振、高分辨质谱为化学分析工具，首先确认了T3标准物质主成分的结构；随后基于高效液相色谱、高分辨质谱、离子淌度等分析技术开发建立了结构相关杂质的分析方法，对T3纯度标准物质中的有机杂质进行分离、分析、鉴定。结果表明，NIM-RM3239标准物质中相对含量>0.1%的结构类似杂质共3种，分别为二碘代-L-甲状腺原氨酸、氯代三碘代-L-甲状腺原氨酸、L-甲状腺素，对映异构体拆分结果表明NIM-RM3239中不含主成分的对映异构体。研究结果可作为质量平衡法定值依据，用于研制纯度型T3国家一级有证标准物质。
- 计量学 /
- 三碘代-L-甲状腺原氨酸 /
- 标准物质 /
- 结构相关杂质 /
- 液相色谱 /
- 高分辨质谱
Abstract: Research and development of 3,3',5-Triiodo-L-thyronine (T3) purity reference material is a way to establish the primary material for clinical measurement value traceability, which can provide more accurate and comparable evidence in thyroid disease diagnosis. In the mass balance quantitative approach, determining and analyzing those structural-related impurities is essential. Such investigation could also be an effective way to reduce the uncertainty of the standard value. First, nuclear magnetic resonance and high-resolution mass spectrometry analytical tools have been employed to validate the chemical structure of the reference material. Then, the structural-related impurities are separated and qualitatively analyzed by using reliable analytical technologies, including high-performance liquid chromatography, high-resolution mass spectrometry, and ion mobility spectrometry. The results indicated that there are three kinds of structural-related impurities involved in the T3 purity reference material (NIM-RM3239), which are Diiodo-L-thyronine, Chloro-diiodo-L-thyronine, and L-Thyroxine. The relative content of each impurity is greater than 0.1% in UV 220nm. Furthermore, the chiral isomer separation result indicated that no dextroisomer (D-T3) existed in NIM-RM3239. These analytical results can be utilized as the foundations in the mass balance approach, applied in developing T3 primary certified reference material.
- metrology /
- 3,3',5-Triiodo-L-thyronine /
- reference material /
- structural-related impurity /
- liquid chromatography /
- high-resolution mass spectrometry

HTML全文

图 1 T3 标准物质在氘代 DMSO 中的核磁共振碳谱与氢谱

Figure 1. Carbon and proton nuclear magnetic resonance spectra of T3 reference material in deuterated DMSO

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图 2 高效液相色谱串联高分辨质谱分析 T3 标准物质（A）紫外吸收色谱图；（B）主成分质谱图

Figure 2. Impurities analysis of T3 reference material using UPLC coupled high-resolution MS. (A) UV spectrometry; (B)Mass spectra of main peak

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图 3 主成分及结构类似杂质二级质谱图（A）杂质 3；（B）主成分；（C）杂质 2；（D）杂质 1

Figure 3. The tandem product ions distribution for main compound and the structural-related impurities (A) Impurity 3; (B)Main compound; (C) Impurity 2; (D) Impurity 1

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图 4 杂质1鉴定结果验证:不同脱碘位置的二碘甲腺原氨酸色谱与质谱对比

Figure 4. Validation of impurity 1 identification: The LC chromatography and MS spectrum comparison between impurity 1 and three kinds of diiodothyroxines

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图 5 杂质3鉴定结果验证: 杂质3与L-甲状腺素色谱与质谱对比

Figure 5. Validation of impurity 3 identification: the LC chromatography and MS spectrum comparison between impurity 3 and L-Thyronine.

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图 6 L-/D-T3的离子淌度质谱图(A,B)波速300 m/s、波高40v下淌度谱图；(C,D)波速900 m/s、波高40v下淌度谱图；(E,F)波速300 m/s、波高40v下淌度谱图

Figure 6. Ion mobility spectrometry of L-/D-T3 solution under (A,B)wave velocity 300 m/s, wave height 40v; (C,D)wave velocity 900 m/s, wave height 40v, and (E,F)wave velocity 1200 m/s, wave height 40v, respectively

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图 7 基于环果聚糖色谱柱拆分L型、D型T3（A）L-T3基峰色谱图；（B）D-T3基峰色谱图；（C）L-/D-型T3混合样本

Figure 7. Separation of L-T3 and D-T3 using derivatized cyclofructan liquid chromatography. (A) Base peak ion chromatogram of L-T3;(B) Base peak ion chromatogram of D-T3; (C) Base peak ion chromatogram of L-/D-T3 mixture

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表 1 T3纯度标准物质中主要结构近似杂质分布

Table 1. The main impurities involved in T3 reference material

代号质荷比分子式

杂质1 525.89 C₁₅H₁₃I₂NO₄

杂质2 559.86 C₁₅H₁₁ClI₂NO₄

杂质3 777.69 C₁₅H₁₁I₄NO₄

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参考文献(32)

[1]	STERLING K, BRENNER M A, SAKURADA T. Rapid effect of triiodothyronine on the mitochondrial pathway in rat liver in vivo[J]. Science, 1980, 210(4467): 340-2. doi: 10.1126/science.7423197
[2]	KAHALY G J, DILLMANN W H. Thyroid hormone action in the heart[J]. Endocr Rev, 2005, 26(5): 704-28. doi: 10.1210/er.2003-0033
[3]	DAVIS P J, GOGLIA F, LEONARD J L. Nongenomic actions of thyroid hormone[J]. Nat Rev Endocrinol, 2016, 12(2): 111-21. doi: 10.1038/nrendo.2015.205
[4]	International Organization for Standardization. ISO 17511: 2020 In vitro diagnostic medical devices — Requirements for establishing metrological traceability of values assigned to calibrators, trueness control materials and human samples [S]. 2nd Edition. Switzerland: ISO, 2020.
[5]	GUZMAN D A, SOLARI G S. Traceability and harmonization in clinical laboratory[J]. Rev Med Chil, 2009, 137(5): 713-5.
[6]	JONES G R D, DELATOUR V, BADRICK T. Metrological traceability and clinical traceability of laboratory results - the role of commutability in External Quality Assurance[J]. Clin Chem Lab Med, 2022, 60(5): 669-74. doi: 10.1515/cclm-2022-0038
[7]	丁敏. 临床检验标准物质现状及研制方法 [J]. 上海计量测试. 2021, 48(6): 2-5.
[8]	杨振华. 建设我国检验医学参考体系任重道远[J]. 临床检验杂志. 2012, 30(10): 734-6.
[9]	焦慧, 李红梅, 阚莹, 等. 化学计量国际体系及发展趋势[J]. 计量科学与技术. 2021, 65(6): 65-70.
[10]	WESTWOOD S, CHOTEAU T, DAIREAUX A, et al. Mass balance method for the SI value assignment of the purity of organic compounds[J]. Anal Chem, 2013, 85(6): 3118-26. doi: 10.1021/ac303329k
[11]	涂梦灵, 杨冰鑫, 朱云霄, 等. 新烟碱类农药有证标准物质研究进展 [J]. 计量科学与技术. 2023, 67(3): 65-71.
[12]	XIAO P, CHEN J, WU P, et al. Development of an SI-traceable N-terminal pro-B-type natriuretic peptide certified reference material using structure-based impurity-corrected isotope dilution mass spectrometry approaches[J]. Analytical and Bioanalytical Chemistry, 2024, 416(14): 3447-58. doi: 10.1007/s00216-024-05295-9
[13]	JOSEPHS R D, LI M, SONG D, et al. Key comparison study on peptide purity—synthetic human C-peptide[J]. Metrologia, 2017, 54(1A): 08007. doi: 10.1088/0026-1394/54/1A/08007
[14]	JOSEPHS R D, LI M, DAIREAUX A, et al. Key comparison study on peptide purity - synthetic oxytocin[J]. Metrologia, 2020, 57(1A): 08014. doi: 10.1088/0026-1394/57/1A/08014
[15]	黄挺, 张伟, 全灿, 等. 定量核磁共振新方法在纯度定值的应用[J]. 计量技术. 2018(9): 8-9.
[16]	黄挺, 王静羽, 万康妮. 去除杂质干扰的定量核磁共振法进展综述[J]. 计量科学与技术. 2022, 66(6): 26-30.
[17]	JACQUEMIN C, MICHEL R, NUNEZ J, et al. Catalytic tritiation of iodized phenols. Preparation of tritiated estradiol, tyrosine and thyronine[J]. C R Hebd Seances Acad Sci, 1959, 249: 1904-6.
[18]	LISSITZKY S, ROQUES M. Oxidation of tyrosine, thyronine, and their iodo derivatives. I. Actions of the ascorbic acid-ferrous ion-oxygen system[J]. Bull Soc Chim Biol (Paris), 1957, 39(5-6): 521-37.
[19]	LISSITZKY S, BOUCHILLOUX S. Oxidation of tyrosine, thyronine & their iodinated derivatives. II. Effect of polyphenol oxidase on halogenated tyrosines & iodothyronines[J]. Bull Soc Chim Biol (Paris), 1957, 39(11): 1215-25.
[20]	阚莹, 卢晓华, 孟凡敏, 等. JJF1343-2012《标准物质定值的通用原则及统计学原理》的使用[J]. 中国计量. 2012(11): 122-4.
[21]	PIEHL S, HEBERER T, BALIZS G, et al. Development of a validated liquid chromatography/tandem mass spectrometry method for the distinction of thyronine and thyronamine constitutional isomers and for the identification of new deiodinase substrates[J]. Rapid Commun Mass Spectrom, 2008, 22(20): 3286-96. doi: 10.1002/rcm.3732
[22]	RUGGENTHALER M, GRASS J, SCHUH W, et al. Impurity profiling of liothyronine sodium by means of reversed phase HPLC, high resolution mass spectrometry, on-line H/D exchange and UV/Vis absorption[J]. J Pharm Biomed Anal, 2017, 143: 147-58. doi: 10.1016/j.jpba.2017.05.039
[23]	MAIER N M, FRANCO P, LINDNER W. Separation of enantiomers: needs, challenges, perspectives[J]. Journal of Chromatography A, 2001, 906(1): 3-33.
[24]	SHEN J, OKAMOTO Y. Efficient Separation of Enantiomers Using Stereoregular Chiral Polymers[J]. Chemical Reviews, 2016, 116(3): 1094-138. doi: 10.1021/acs.chemrev.5b00317
[25]	SANTOS L F, IGLESIAS A H, PILAU E J, et al. Traveling-wave ion mobility mass spectrometry analysis of isomeric modified peptides arising from chemical cross-linking[J]. J Am Soc Mass Spectrom, 2010, 21(12): 2062-9. doi: 10.1016/j.jasms.2010.08.017
[26]	EYERS C E, VONDERACH M, FERRIES S, et al. Understanding protein-drug interactions using ion mobility-mass spectrometry[J]. Curr Opin Chem Biol, 2018, 42: 167-76. doi: 10.1016/j.cbpa.2017.12.013
[27]	LALLI P M, IGLESIAS B A, TOMA H E, et al. Protomers: formation, separation and characterization via travelling wave ion mobility mass spectrometry[J]. J Mass Spectrom, 2012, 47(6): 712-9. doi: 10.1002/jms.2999
[28]	PHILLIPS S T, DODDS J N, ELLIS B M, et al. Chiral separation of diastereomers of the cyclic nonapeptides vasopressin and desmopressin by uniform field ion mobility mass spectrometry[J]. Chem Commun (Camb), 2018, 54(68): 9398-401. doi: 10.1039/C8CC03790F
[29]	XIAO P, LI H-M, LI M, et al. Structural characterization and thermally induced isomerization investigation of cis- and trans-vitamin K1 using ion mobility mass spectrometry[J]. Analytical Methods, 2015, 7(19): 8432-8. doi: 10.1039/C5AY01495F
[30]	CHANKVETADZE B. Recent developments on polysaccharide-based chiral stationary phases for liquid-phase separation of enantiomers[J]. Journal of Chromatography A, 2012, 1269: 26-51. doi: 10.1016/j.chroma.2012.10.033
[31]	李依璠, 刘龙婵, 李林楠, 等. 手性分离方法及在中药手性成分研究中的应用[J]. 药学学报. 2023, 58(6): 1566-76.
[32]	LEE J, ADHIKARI S, LEE W, et al. Development of Ultra High-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Enantiomer Resolution of Thyroxine on a Chiral Crown Ether Derived Chiral Stationary Phase[J]. Chromatographia, 2023, 86(1): 13-20. doi: 10.1007/s10337-022-04219-y