柱前衍生化高效液相色谱法测定氯氮平中肼含量

doi:10.19894/j.issn.1000-0518.210039

摘要/Abstract

摘要：

建立了柱前衍生高效液相色谱法准确测定氯氮平中游离肼的含量。该方法以对二甲氨基苯甲醛为衍生试剂，将游离肼衍生化为对二甲氨基苄连氮，然后进行高效液相色谱测定。采用Inertsil ODS-3色谱柱（250 mm×4.6 mm，5 μm）分离，以体积分数0.1%磷酸水溶液和乙腈为流动相进行梯度洗脱，检测波长为480 nm，流速为1.0 mL／min，进样量为30 μL，柱温为30℃。结果表明，游离肼在0.001～12.25 μg／mL范围内具有良好线性关系，线性相关系数r为0.9994，在低、中和高3个添加水平下目标物的加标回收率在97.4%～100.9%之间，RSD均低于1.2%（n＝9），游离肼的检出限为0.3125 μg／L，定量限为1.225 μg／L，该结果完全满足标准限度的要求。随机抽取的3个批次氯氮平样品均未检出游离肼，表明抽取的氯氮平药品中肼含量合格。与盐酸肼屈嗪中肼（USP39版）的检测方法相比较，本方法具有专属性强，灵敏度高，结果准确可靠等优点，为氯氮平样品中游离肼的常规定量检测提供了参考。

关键词: 高效液相色谱, 游离肼, 氯氮平, 对二甲氨基苯甲醛

Abstract:

A precolumn derivatization-high performance liquid chromatography (HPLC) method was developed for the accurate determination of hydrazine in clozapine. This method is based on precolumn derivatization with p-dimethylaminobenzaldehyde to form p-dimethylaminobenzene. The chromatographic separation is achieved on a Inertsil ODS-3 column (250 mm×4.6 mm, 5 μm) with gradient elution at 480 nm detection wavelength, the mobile phase consists of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, the injection volume is 30 μL, and the column temperature is 30℃. The results show that the linearity range for free hydrazide is 0.001~12.25 μg/mL with R²=0.9994. The recoveries at three spiking levels are over the range of 97.4%~100.9% with RSD≤1.2% (n=9). The limit of detection is estimated to be 0.3125 μg/L, and the limit of quantification is 1.225 μg/L. The results fully meet the requirements of the standard limit. The three batches of clozapine samples do not have free hydrazine, indicating that the content of hydrazine in clozapine is quantified. Compared with traditional detection method of hydrazine hydrochloride (USP39 version), the proposed method has prominent advantages, such as high specificity, sensitivity and reliability, and hence has great potential in routine quantitative analysis of hydrazine in clozapine samples.

Key words: High performance liquid chromatography, Hydrazine, Clozapine, p-dimethylaminobenzaldehyde

中图分类号:

O655

陈瑶, 唐英. 柱前衍生化高效液相色谱法测定氯氮平中肼含量[J]. 应用化学, 2022, 39(02): 322-331.

Yao CHEN, Ying TANG. Determination of Hydrazine in Clozapine by Precolumn Derivatization High Performance Liquid Chromatography Method[J]. Chinese Journal of Applied Chemistry, 2022, 39(02): 322-331.

图/表 14

图1 肼的衍生化反应

Fig.1 Derivatization reaction of hydrazine

表1 流动相梯度洗脱条件

Table 1 Gradient elution conditions

时间	流动相Ⅰ	流动相Ⅱ
Time／min	φ（mobile phaseⅠ）／%	φ（mobile phaseⅡ）／%
0.0	70	30
15.0	30	70
16.0	5	95
20.0	5	95
20.1	70	30
25.0	70	30

图2 衍生产物X（A）和Y（B）分别在0.1%乙酸水溶液（a）、0.1%甲酸水溶液（b）和0.1%磷酸水溶液（c）中的紫外吸收光谱图

Fig.2 UV absorption spectra of derivatives X（A）and Y（B）in aqueous 0.1% acetic acid（a），0.1% formic acid（b）and 0.1% phosphoric acid solutions（c），respectively

图3 对二甲氨基苄连氮（Y）结构随pH变化图

Fig.3 Structure of p-dimethylamine benzylidene（Y）with different pH

图4 衍生反应液（b）和氯氮平样品（a）在390 nm处流动相Ⅰ分别为0.1%乙酸水溶液（A）、0.1%甲酸水溶液（B）、0.1%磷酸水溶液（C）的色谱图

Fig.4 Chromatogram of aqueous 0.1% acetic acid（A），aqueous 0.1% formic acid（B），and aqueous 0.1% phosphoric acid（C）in the mobile phase A of the derivatization reaction solution（b）and clozapine sample（a）at 390 nm

图5 衍生反应液（b）和氯氮平样品（a）在480 nm处流动相Ⅰ分别为0.1%乙酸水溶液（A）、0.1%甲酸水溶液（B）、0.1%磷酸水溶液（C）的色谱图

Fig.5 Chromatogram of aqueous 0.1% acetic acid（A），aqueous 0.1% formic acid（B）and aqueous 0.1% phosphoric acid（C）in mobile phaseⅠof the derivatization reaction solution（b）and clozapine sample（a）at 480 nm

图6 进样量对主产物Y的峰形影响（A：THF溶剂，B：DMF溶剂）

Fig.6 Influence of injection volume on the peak shape of main product Y（A：THF solvent，B：DMF solvent）

表2 不同溶剂对衍生反应和临界进样体积的影响

Table 2 The influence of different solvents on the derivatization reaction and critical injection volume

溶剂 Solvent	面积百分比^／% Area percentage^／%		临界进样体积 Critical injection volume／μL
溶剂 Solvent	峰X Peak X	峰Y Peak Y	临界进样体积 Critical injection volume／μL
甲醇	13.25	18.75	—
Methanol	13.25	18.75	—
乙腈	15.7	84.3	—
Acetonitrile	15.7	84.3	—
THF	0.86	99.14	≤10
DMF	3.09	96.91	30≤

图7 主产物Y的峰面积（a）面积百分比（b）与醋酸用量的关系图

Fig.7 Relationship between the peak area（a）and the peak area percentage（b）of main product Y and the amount of acetic acid

图8 衍生试剂／肼的物质的量比与主产物Y峰面积的关系图

Fig.8 The plot of the molar ratio of derivatization reagent／hydrazine and the peak area of main product Y

图9 主产物Y峰面积（A）和峰面积百分比（B）与时间和温度的关系图

Fig.9 The diagrams of peak area（A）and peak area percentage（B）of main product Y with time and temperature

图10 游离肼的浓度与主产物Y的峰面积的关系图

Fig.10 The concentration of hydrazine versus the peak area of the main product Y

表3 肼在空白基质中的加标回收率（n＝9）

Table 3 Spiked recoveries of hydrazine in blank substrate（n＝9）

序号	空白基质中肼含量	加入量	测定量	回收率	平均回收率	RSD／%
No.	Hydrazine content in　blank substrate／μg	Addition／μg	Measured　quantity／μg	Recovery　rate／%	Average recovery　rate／%	RSD／%
1	N. D^*	1.018	1.018	100.0	99.2	1.2
2	N. D	1.018	1.013	99.5	?	?
3	N. D	1.018	1.004	98.6	?	?
4	N. D	1.272	1.263	99.3	?	?
5	N. D	1.272	1.277	100.4	?	?
6	N. D	1.272	1.284	100.9	?	?
7	N. D	1.527	1.509	98.8	?	?
8	N. D	1.527	1.496	98.0	?	?
9	N. D	1.527	1.488	97.4	?	?

表4 不同衍生方法测定游离肼浓度的结果

Table 4 Determination of the concentration of hydrazine by different derivatization methods

批号 Batch No	添加量 Additive amount／（mg·L^－1）	对比方法 Contrast method		本实验方法 Experimental method
批号 Batch No	添加量 Additive amount／（mg·L^－1）	含量 Content／（mg·L^－1）	信噪比 SNR（S／N）	含量 Content／（mg·L^－1）	信噪比 SNR（S／N）
190412	5.01	6.34	16.8	5.01	4 967.3
190412	5.01	6.59	8.8	5.06	4 394.1
190413	5.01	7.81	9.6	5.03	4 418.8
190413	5.01	7.91	10.1	5.09	3 677.9
190414	5.01	8.05	11.5	5.10	3 336.0
190414	5.01	7.35	9.7	5.01	3 878.7

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