Determination of Hydrazine in Clozapine by Precolumn Derivatization High Performance Liquid Chromatography Method

doi:10.19894/j.issn.1000-0518.210039

Chinese Journal of Applied Chemistry ›› 2022, Vol. 39 ›› Issue (02): 322-331.DOI: 10.19894/j.issn.1000-0518.210039

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Determination of Hydrazine in Clozapine by Precolumn Derivatization High Performance Liquid Chromatography Method

Yao CHEN¹, Ying TANG¹^,²()

^1.Hunan Key Laboratory of Biomedical Nanomaterials and Devices, Hunan University of Technology, Zhuzhou 412007, China
^2.State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China

Received:2021-01-22 Accepted:2021-04-30 Published:2022-02-10 Online:2022-02-09
Contact: Ying TANG
Supported by:
National Natural Science Foundation of China(21705044);Natural Science Foundation of Hunan Province(2018JJ3114);Scientific Research Project of the Education Department of Hunan Province(19C0554);State Key Laboratory of Chemical and Biological Sensing and Metrology(2018015)

Abstract

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

CLC Number:

O655

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.

Figures/Tables 14

Fig.1 Derivatization reaction of hydrazine

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

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

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

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

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

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

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≤

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

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

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

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

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	?	?

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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Determination of Hydrazine in Clozapine by Precolumn Derivatization High Performance Liquid Chromatography Method

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