高效液相色谱-质谱联用法测定血浆中异烟肼及其代谢物浓度的研究

doi:10.19982/j.issn.1000-6621.20240059

摘要/Abstract

摘要：

目的: 建立高效液相色谱-质谱联用法(HPLC-MS/MS)同时测定肺结核患者血浆中异烟肼及其代谢物乙酰肼和肼的浓度的方法。方法: 收集首都医科大学附属北京胸科医院确诊的104例肺结核患者(均接受规范抗结核治疗,服用异烟肼的剂量为1次/d,每次300~500mg,于服药后2h采集静脉血)的血浆样本作为研究样本。血浆样品经甲醇沉淀蛋白后,以异烟肼-D4为内标,以对甲基苯甲醛作为衍生化试剂,对待测物进行衍生化处理,采用InfinityLab Poroshell 120 HILIC-Z色谱柱(100mm×2.1mm,2.7μm)分离,二元洗脱系统(A相为含0.1%甲酸和5mmol甲酸铵的水溶液;B相为乙腈)进行梯度洗脱,柱温30℃,流速0.4ml/min;采用电喷雾电离正离子模式检测,多反应监测模式扫描,运行时间为8min。结果: 本测定方法不受血浆中内源性物质干扰,异烟肼、乙酰肼、肼分别在50~6000、25~3000、1~120ng/ml范围内线性良好[决定系数(R²)均>0.99];日内和日间精密度<15%;准确度绝对值<15%;提取回收率均>85%;基质效应在85%~115%;稳定性良好。应用该方法检测104例肺结核患者的异烟肼、乙酰肼及肼的浓度,分别为(2402.33±1248.57)ng/ml、(1902.51±596.82)ng/ml、(26.50±17.13)ng/ml。结论: 本研究建立的HPLC-MS/MS法操作简便、分析快速、专属性强、敏感度高,可应用于异烟肼及其代谢物乙酰肼和肼在肺结核患者中的治疗药物浓度监测。

关键词: 结核,肺, 异烟肼, 液相色谱-质谱法, 药物监测

Abstract:

Objective: To establish a method for simultaneous determination of isoniazid and its metabolites— acetylhydrazine and hydrazine in the plasma of pulmonary tuberculosis patients using high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Methods: Plasma samples were collected from 104 confirmed pulmonary tuberculosis patients (all receiving standardized anti-tuberculosis treatment, taking isoniazid at a dose of 300-500 mg once a day, and venous blood was collected 2 hours after medication) from Beijing Chest Hospital, Capital Medical University. After protein precipitation in methanol, plasma samples were subjected to derivatization using isoniazid-D4 as the internal standard and p-methylbenzaldehyde as the derivatization reagent. The analyte was separated using an InfinityLab Poroshell 120 HILIC-Z column (100 mm×2.1 mm, 2.7 μm). The binary elution system (A phase was an aqueous solution containing 0.1% formic acid and 5 mmol ammonium formate; B phase was acetonitrile) was used for gradient elution, with a column temperature of 30 ℃ and a flow rate of 0.4 ml/min); electro spray ionization positive ion mode detection and multi-reaction monitoring mode scanning was adopted, and the operation time was 8 min. Results: This determination method was not affected by endogenous substances in plasma. Isoniazid, acetylhydrazine, and hydrazine had good linearity in the range of 50-6000, 25-3000, and 1-120 ng/ml, respectively (determination coefficients (R²) >0.99); intra-day and inter-day precision <15%; absolute accuracy value <15%; the extraction recovery rates were all above 85%; the matrix effect ranges from 85% to 115%; the stability was good. The method was applied to detect the concentrations of isoniazid, acetylhydrazine, and hydrazine in 104 patients with pulmonary tuberculosis, which were (2402.33±1248.57) ng/ml, (1902.51±596.82) ng/ml, and (26.50±17.13) ng/ml, respectively. Conclusion: The HPLC-MS/MS method established in this study was easy to operate, rapid, specific, and sensitive. It could be applied to monitor the therapeutic drug concentration of isoniazid and its metabolites (acetylhydrazine and hydrazine) in patients with pulmonary tuberculosis.

Key words: Tuberculosis, pulmonary, Isoniazid, Liquid chromatography-mass spectrometry, Drug monitoring

中图分类号:

R521
R927

葛菲, 朱慧, 程凯, 陆宇, 徐建. 高效液相色谱-质谱联用法测定血浆中异烟肼及其代谢物浓度的研究[J]. 中国防痨杂志, 2024, 46(5): 549-556. doi: 10.19982/j.issn.1000-6621.20240059

Ge Fei, Zhu Hui, Cheng Kai, Lu Yu, Xu Jian. Study on the determination of isoniazid and its metabolites concentration in plasma by high-performance liquid chromatography-mass spectrometry[J]. Chinese Journal of Antituberculosis, 2024, 46(5): 549-556. doi: 10.19982/j.issn.1000-6621.20240059

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参考文献 24

[1]	Wang P, Pradhan K, Zhong XB, et al. Isoniazid metabolism and hepatotoxicity. Acta Pharm Sin B, 2016, 6(5): 384-392. doi:10.1016/j.apsb.2016.07.014. pmid: 27709007
[2]	Elder DP, Snodin D, Teasdale A. Control and analysis of hydrazine, hydrazides and hydrazones--genotoxic impurities in active pharmaceutical ingredients (APIs) and drug products. J Pharm Biomed Anal, 2011, 54(5): 900-910. doi:10.1016/j.jpba.2010.11.007. pmid: 21145684
[3]	Smolenkov AD, Shpigun OA. Direct liquid chromatographic determination of hydrazines: a review. Talanta, 2012, 102: 93-100. doi:10.1016/j.talanta.2012.07.005. pmid: 23182580
[4]	Sun M, Bai L, Liu DQ. A generic approach for the determination of trace hydrazine in drug substances using in situ derivati-zation-headspace GC-MS. J Pharm Biomed Anal, 2009, 49(2): 529-533. doi:10.1016/j.jpba.2008.11.009.
[5]	刘兵, 刘岩, 李斯琪, 等. 基于超高效液相色谱—质谱技术血浆神经酰胺含量分析. 临床军医杂志, 2022, 50(11): 1121-1124, 1128. doi:10.16680/j.1671-3826.2022.11.05.
[6]	Isenberg SL, Carter MD, Crow BS, et al. Quantification of Hydrazine in Human Urine by HPLC-MS-MS. J Anal Toxicol, 2016, 40(4): 248-254. doi:10.1093/jat/bkw015. pmid: 26977107
[7]	Song L, Gao D, Li S, et al. Simultaneous quantitation of hydrazine and acetylhydrazine in human plasma by high performance liquid chromatography-tandem mass spectrometry after derivatization with p-tolualdehyde. J Chromatogr B Analyt Technol Biomed Life Sci, 2017, 1063: 189-195. doi:10.1016/j.jchromb.2017.08.036. pmid: 28881295
[8]	Ky Anh N, My Tung P, Kim MJ, et al. Quantitative Analysis of Isoniazid and Its Four Primary Metabolites in Plasma of Tuberculosis Patients Using LC-MS/MS. Molecules, 2022, 27(23): 8607. doi:10.3390/molecules27238607.
[9]	张亮, 冯枭, 林霏申, 等. 异烟肼血药浓度的影响因素分析. 药学与临床研究, 2018, 26(6): 413-416. doi:10.13664/j.cnki.pcr.2018.06.004.
[10]	Staden DV, Haynes RK, Van der Kooy F, et al. Development of a HPLC Method for Analysis of a Combination of Clofazimine, Isoniazid, Pyrazinamide, and Rifampicin Incorporated into a Dermal Self-Double-Emulsifying Drug Delivery System. Methods Protoc, 2023, 6(6): 104. doi:10.3390/mps6060104.
[11]	Maekawa M, Mano N. Cutting-edge LC-MS/MS applications in clinical mass spectrometry: Focusing on analysis of drugs and metabolites. Biomed Chromatogr, 2022, 36(5): e5347. doi:10.1002/bmc.5347.
[12]	Zheng YZ, Wang S. Advances in antifungal drug measurement by liquid chromatography-mass spectrometry. Clin Chim Acta, 2019, 491: 132-145. doi:10.1016/j.cca.2019.01.023. pmid: 30685359
[13]	Zhang YV, Wei B, Zhu Y. et al. Liquid Chromatography-Tandem Mass Spectrometry: An Emerging Technology in the Toxicology Laboratory. Clin Lab Med, 2016, 36(4): 635-661. doi:10.1016/j.cll.2016.07.001. pmid: 27842783
[14]	Adaway JE, Keevil BG. Therapeutic drug monitoring and LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci, 2012, 883-884: 33-49. doi:10.1016/j.jchromb.2011.09.041.
[15]	Pourmohamadi N, Pour Abdollah Toutkaboni M, Hayati Roodbari N, et al. Association of Cytochrome P450 2E1 and N-Acetyltransferase 2 Genotypes with Serum Isoniazid Level and Anti-Tuberculosis Drug-Induced Hepatotoxicity: A Cross-Sectional Study. Iran J Med Sci, 2023, 48(5): 474-483. doi:10.30476/ijms.2023.96145.2765.
[16]	Boelsterli UA, Lee KK. Mechanisms of isoniazid-induced idiosyncratic liver injury: emerging role of mitochondrial stress. J Gastroenterol Hepatol, 2014, 29(4): 678-687. doi:10.1111/jgh.12516.
[17]	Brewer CT, Yang L, Edwards A, et al. The Isoniazid Metabo-lites Hydrazine and Pyridoxal Isonicotinoyl Hydrazone Modulate Heme Biosynthesis. Toxicol Sci, 2019, 168(1): 209-224. doi:10.1093/toxsci/kfy294.
[18]	Song SH, Jun SH, Park KU, et al. Simultaneous determination of first-line anti-tuberculosis drugs and their major metabolic ratios by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom, 2007, 21(7): 1331-1338. doi:10.1002/rcm.2961.
[19]	Mercier T, Desfontaine V, Cruchon S, et al. A battery of tandem mass spectrometry assays with stable isotope-dilution for the quantification of 15 anti-tuberculosis drugs and two metabolites in patients with susceptible-, multidrug-resistant- and extensively drug-resistant tuberculosis. J Chromatogr B Analyt Technol Biomed Life Sci, 2022, 1211: 123456. doi:10.1016/j.jchromb.2022.123456.
[20]	Gao S, Wang Z, Xie X, et al. Rapid and sensitive method for simultaneous determination of first-line anti-tuberculosis drugs in human plasma by HPLC-MS/MS: Application to therapeutic drug monitoring. Tuberculosis (Edinb), 2018, 109: 28-34. doi:10.1016/j.tube.2017.11.012.
[21]	Yilmaz E, Soylak M. Ultrasound assisted-deep eutectic solvent based on emulsification liquid phase microextraction combined with microsample injection flame atomic absorption spectrometry for valence speciation of chromium (Ⅲ/Ⅵ) in environmental samples. Talanta, 2016, 160: 680-685. doi:10.1016/j.talanta.2016.08.001. pmid: 27591663
[22]	Smith KA, Merrigan SD, Johnson-Davis KL. Selecting a Structural Analog as an Internal Standard for the Quantification of 6-Methylmercaptopurine by LC-MS/MS. J Appl Lab Med, 2018, 3(3): 384-396. doi:10.1373/jalm.2018.026187. pmid: 33636909
[23]	Anderson G, Vinnard C. Diagnostic Accuracy of Therapeutic Drug Monitoring During Tuberculosis Treatment. J Clin Pharmacol, 2022, 62(10): 1206-1214. doi:10.1002/jcph.2068.
[24]	Verbeeck RK, Günther G, Kibuule D, et al. Optimizing treatment outcome of first-line anti-tuberculosis drugs: the role of therapeutic drug monitoring. Eur J Clin Pharmacol, 2016, 72(8): 905-916. doi:10.1007/s00228-016-2083-4. pmid: 27305904

化合物	母离子(m/z)	子离子(m/z)	驻留时间(ms)	碎裂电压(eV)	去簇电压(eV)	保留时间(min)
异烟肼	240.0	118.0	100	23.0	65.0	3.65
乙酰肼	177.0	118.0	100	18.9	49.0	3.69
肼	237.3	119.0	100	28.8	55.0	4.24
异烟肼-D4	244.0	127.0	100	27.0	85.0	3.66

化合物	回归方程	决定系数 (R²)	线性范围 (ng/ml)
异烟肼	y=0.00548x-0.0004516	0.99974	50~6000
乙酰肼	y=0.02353x-0.07664	0.99914	25~3000
肼	y=0.31689x+0.23540	0.99949	1~120

化合物及浓度 (ng/ml)	日内(6份)			日间(6份)
化合物及浓度 (ng/ml)	日内实测值 ($\bar{x}±s$,ng/ml)	相对误差 (%)	相对标准差(%)	日间实测值 ($\bar{x}±s$,ng/ml)	相对误差 (%)	相对标准差(%)
异烟肼
50	50.97±1.28	1.93	2.49	50.35±4.40	0.70	8.74
150	143.03±2.46	-4.65	1.91	144.84±8.11	-3.44	5.60
2500	2339.24±38.19	-6.43	1.44	2351.34±184.65	-5.95	7.85
4500	4343.54±66.61	-3.48	1.55	4276.56±273.83	-4.97	6.40
乙酰肼
25	24.43±0.62	-2.29	1.57	25.18±1.87	0.73	7.44
75	73.45±3.47	-2.07	4.74	74.04±4.31	-1.28	5.83
1250	1246.71±26.09	-0.26	2.34	1203.44±81.71	-3.72	6.79
2250	2272.32±27.16	0.99	1.34	2173.46±153.35	-3.40	7.06
肼
1	0.99±0.08	-1.00	7.05	1.02±0.09	1.43	11.76
3	3.08±0.12	2.53	3.90	3.03±0.24	0.96	7.94
50	51.67±2.76	3.33	5.81	48.96±2.91	-2.08	5.95
90	93.48±1.94	3.86	2.32	88.87±5.23	-1.25	5.89

化合物理论浓度 (ng/ml)	提取回收率 ($\bar{x}±s$,%)	相对标准差 (%)
异烟肼
150	97.07±4.88	5.03
2500	100.59±3.58	3.56
4500	98.40±4.70	4.78
乙酰肼
75	94.66±3.13	3.31
1250	104.72±4.34	4.15
2250	100.77±5.38	5.33
肼
3	91.21±5.49	6.02
50	100.94±5.05	5.00
90	101.71±4.80	4.71

化合物理论浓度 (ng/ml)	内标归一化基质效应($\bar{x}±s$,%)	相对标准差 (%)
异烟肼
150	99.11±6.43	6.49
2500	105.95±6.01	5.68
4500	103.06±5.77	5.60
乙酰肼
75	101.09±5.41	5.35
1250	94.25±8.20	8.70
2250	103.80±4.87	4.69
肼
3	96.74±8.88	9.18
50	95.24±5.43	5.70
90	93.86±5.72	6.10