应用化学 ›› 2022, Vol. 39 ›› Issue (02): 322-331.DOI: 10.19894/j.issn.1000-0518.210039
收稿日期:
2021-01-22
接受日期:
2021-04-30
出版日期:
2022-02-10
发布日期:
2022-02-09
通讯作者:
唐英
作者简介:
*E-mail:yingtang@hnu.edu.cn基金资助:
Received:
2021-01-22
Accepted:
2021-04-30
Published:
2022-02-10
Online:
2022-02-09
Contact:
Ying TANG
Supported by:
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。
中图分类号:
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.
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 |
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
溶剂 Solvent | 面积百分比*/% Area percentage*/% | 临界进样体积 Critical injection volume/μL | |
---|---|---|---|
峰X Peak X | 峰Y Peak Y | ||
甲醇 | 13.25 | 18.75 | — |
Methanol | |||
乙腈 | 15.7 | 84.3 | — |
Acetonitrile | |||
THF | 0.86 | 99.14 | ≤10 |
DMF | 3.09 | 96.91 | 30≤ |
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 | |
---|---|---|---|
峰X Peak X | 峰Y Peak Y | ||
甲醇 | 13.25 | 18.75 | — |
Methanol | |||
乙腈 | 15.7 | 84.3 | — |
Acetonitrile | |||
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
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/% | |
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/% | |
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 | ? | ? |
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 | ||
---|---|---|---|---|---|
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 |
5.01 | 6.59 | 8.8 | 5.06 | 4 394.1 | |
190413 | 5.01 | 7.81 | 9.6 | 5.03 | 4 418.8 |
5.01 | 7.91 | 10.1 | 5.09 | 3 677.9 | |
190414 | 5.01 | 8.05 | 11.5 | 5.10 | 3 336.0 |
5.01 | 7.35 | 9.7 | 5.01 | 3 878.7 |
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 | ||
---|---|---|---|---|---|
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 |
5.01 | 6.59 | 8.8 | 5.06 | 4 394.1 | |
190413 | 5.01 | 7.81 | 9.6 | 5.03 | 4 418.8 |
5.01 | 7.91 | 10.1 | 5.09 | 3 677.9 | |
190414 | 5.01 | 8.05 | 11.5 | 5.10 | 3 336.0 |
5.01 | 7.35 | 9.7 | 5.01 | 3 878.7 |
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