Study on the determination of isoniazid and its metabolites concentration in plasma by high-performance liquid chromatography-mass spectrometry

doi:10.19982/j.issn.1000-6621.20240059

Abstract

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

CLC Number:

R521
R927

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

Figures/Tables 8

References 24

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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