阅读说明：本技术 一种快速筛查药品及中间体中多种仲胺的lc-ms法 (LC-MS method for rapidly screening various secondary amines in medicines and intermediates ) 是由 顾凯 朱子丰 卢彦 嵇慧卿 于 2020-12-11 设计创作，主要内容包括：本发明提供一种快速筛查药品及中间体中多种仲胺的LC-MS法,具体涉及药物的样品前处理技术领域,S1、取十二种仲胺对照品适量,以乙腈-水(90：10)为稀释剂,配制适当浓度的仲胺混合溶液,作为混合对照品溶液；S2、配制系列仲胺对照品溶液,经LC-MS法检测,分别获得对照品溶液中十二种仲胺的峰面积,并与溶液浓度进行标准曲线拟合。本发明样品前处理过程简单,易操作,采用HILIC模式色谱柱,获得良好的保留及分离,分析时间短,灵敏度高,极大提高了分析效率,适用于大量样品中多种仲胺的筛查,通过控制样品中仲胺含量,评估产生亚硝胺与亚硝酰胺杂质的风险,为生产工艺提供指导。(The invention provides an LC-MS method for rapidly screening various secondary amines in medicines and intermediates, and particularly relates to the technical field of sample pretreatment of medicines, S1, taking a proper amount of twelve secondary amine reference substances, taking acetonitrile-water (90: 10) as a diluent, and preparing a secondary amine mixed solution with a proper concentration as a mixed reference substance solution; s2, preparing a series of secondary amine reference substance solutions, detecting by an LC-MS method, respectively obtaining peak areas of twelve secondary amines in the reference substance solutions, and performing standard curve fitting with the solution concentrations. The method has the advantages of simple sample pretreatment process, easy operation, good retention and separation by adopting the HILIC mode chromatographic column, short analysis time, high sensitivity, greatly improved analysis efficiency, suitability for screening of various secondary amines in a large number of samples, evaluation of the risk of generating nitrosamine and nitrosamide impurities by controlling the content of the secondary amines in the samples and guidance for the production process.)

1. An LC-MS method for rapidly screening various secondary amines in medicines and intermediates is characterized by comprising the following steps:

s1, taking a proper amount of twelve secondary amine reference substances, and taking acetonitrile-water (90: 10) as a diluent to prepare a secondary amine mixed solution with a proper concentration as a mixed reference substance solution;

s2, preparing a series of secondary amine reference substance solutions, detecting by an LC-MS method, respectively obtaining peak areas of twelve secondary amines in the reference substance solutions, and performing standard curve fitting with the solution concentrations.

2. The LC-MS method for rapidly screening various secondary amines in drugs and intermediates according to claim 1, wherein: the chromatographic conditions of the LC-MS method are as follows: adopting HILIC mode chromatographic column, with column temperature of 35 deg.C and sample amount of 10 μ l; the detector is a mass spectrum detector, the ion source is ESI, and the positive ion mode is adopted; taking 10mM ammonium acetate as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate is 0.3 ml/min; isocratic elution.

Technical Field

The invention belongs to the technical field of pretreatment of samples of medicines, and particularly relates to an LC-MS (liquid chromatography-mass spectrometry) method for rapidly screening various secondary amines in medicines and intermediates.

Background

N-nitrosamine compounds are strong carcinogens, and nitrosamine is listed as a 2A carcinogen by the International agency for research on cancer (IARC), namely, animal carcinogenesis is clear, but the effect on a human body is not clear. It is specifically proposed in the ICHM7 directive that nitrosamines are among the substances with higher carcinogenicity and that these compounds should be strictly controlled in medicine. The nitrosamine compound belongs to nitroso compounds, comprises two types of nitrosamine and nitrosamide, and is a large class of chemical substances synthesized by nitrite and amine, particularly secondary amine. The description of some of the root causes of nitrosamine formation and contamination as determined by examination of item 31 in valsartan may be a good reference for the evaluation of nitrosamines in pharmaceutical products. The core idea in the article is that in the presence of a secondary or tertiary amine, contact with nitrite is a potential cause of nitrosamine formation. Therefore, the logic of risk assessment is to check whether the two compounds are in the risk of existence or contact in the production and manufacturing process of the medicines such as the starting materials, the reagents, the intermediates, the bulk drugs, the auxiliary materials and the like. At present, mechanisms such as FDA, EMA and the like successively issue related test methods for nitrosamine compounds, but the measurement reports for one secondary amine compound from the nitrosamine compounds are few, and the secondary amine compounds are mostly measured by adopting pre-column derivatization-high performance liquid chromatography, gas chromatography, ion chromatography and the like.

For nitrosamine compounds, mechanisms such as FDA, EMA and the like issue related test methods in sequence, the limit requirement is strict, the method detection requirement is higher, two types of nitrosamine and nitrosamide are a large class of chemical substances synthesized by nitrite and amine, particularly secondary amine, and the defects and shortcomings of the prior art are as follows: 1. the prior method has the disadvantages of complex sample processing process and poor method reproducibility, and is not suitable for screening a large number of samples; 2. the ion chromatography has narrow application range, long balance time, fast consumption of consumables and high cost, and is not suitable for screening a large number of samples.

Disclosure of Invention

The invention provides an LC-MS method for rapidly screening twelve secondary amines in medicines and intermediates, which has the advantages of simple sample pretreatment process, easy operation, adoption of HILIC mode chromatographic columns, good retention and separation, short analysis time, high sensitivity, great improvement of analysis efficiency, suitability for screening of various secondary amines in a large number of samples, evaluation of risks of producing nitrosamines and nitrosamide impurities by controlling the content of the secondary amines in the samples, and guidance for production processes.

The invention provides the following technical scheme:

an LC-MS method for rapidly screening various secondary amines in medicines and intermediates comprises the following steps:

s1, taking a proper amount of twelve secondary amine reference substances, and taking acetonitrile-water (90: 10) as a diluent to prepare a secondary amine mixed solution with a proper concentration as a mixed reference substance solution;

s2, preparing a series of secondary amine reference substance solutions, detecting by an LC-MS method, respectively obtaining peak areas of twelve secondary amines in the reference substance solutions, and performing standard curve fitting with the solution concentrations;

preferably, the chromatographic conditions of the LC-MS method are as follows: adopting HILIC mode chromatographic column, with column temperature of 35 deg.C and sample amount of 10 μ l; the detector is a mass spectrum detector, the ion source is ESI, and the positive ion mode is adopted; taking 10mM ammonium acetate as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate is 0.3 ml/min; isocratic elution.

The invention has the beneficial effects that:

the method has the advantages of simple sample pretreatment process, easy operation, good retention and separation by adopting the HILIC mode chromatographic column, short analysis time, high sensitivity, greatly improved analysis efficiency, suitability for screening of various secondary amines in a large number of samples, evaluation of the risk of generating nitrosamine and nitrosamide impurities by controlling the content of the secondary amines in the samples and guidance for the production process.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 dimethylamine standard curve; FIG. 2A standard curve of methylethylamine; FIG. 3 pyrrolidine standard curve; FIG. 4 diethylamine standard curve; FIG. 5 piperidine standard curve; FIG. 6 Standard Curve for N-ethylisopropylamine; FIG. 7 morpholine standard curve; FIG. 8 diethanolamine standard curve; FIG. 9 dibutylamine standard curve; FIG. 10 Standard Curve for N-nitrodi-N-propylamine; FIG. 11a is a diagram of EIC of diisopropylamine and di-n-propylamine; FIG. 11bEIC diagram; FIG. 12 is a dibenzylamine EIC chart (molecular weight: 197.28); FIG. 13 dibutylamine EIC plot (molecular weight 129.24); FIG. 14 is a graph of EIC of diisopropylamine and di-n-propylamine (molecular weight 101.19); FIG. 15 is a diagram of N-ethyl isopropylamine and morpholine EIC; FIG. 16 diethylamine EIC chart (molecular weight 73.14); FIG. 17 piperidine EIC chart (molecular weight 85.15); FIG. 18 is a graph of the EIC extraction of methylethylamine (molecular weight 59.11); FIG. 19 pyrrolidine EIC extract diagram (molecular weight 71.12); FIG. 20 is a diethanolamine EIC extract graph (molecular weight 105.14); FIG. 21 is a dimethylamine EIC extraction profile (molecular weight 45.08).

Detailed Description

An LC-MS method for rapidly screening twelve secondary amines in medicines and intermediates comprises the following steps of taking a proper amount of twelve secondary amine reference substances, and taking acetonitrile-water (90: 10) as a diluent to prepare a secondary amine mixed solution with a proper concentration as a mixed reference substance solution; preparing a series of secondary amine reference substance solutions, detecting by an LC-MS method, respectively obtaining peak areas of twelve secondary amines in the reference substance solutions, and performing standard curve fitting with the solution concentration; the chromatographic conditions of the LC-MS method are as follows: adopting HILIC mode chromatographic column, with column temperature of 35 deg.C and sample amount of 10 μ l; the detector is a mass spectrum detector, the ion source is ESI, and the positive ion mode is adopted; taking 10mM ammonium acetate as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate is 0.3 ml/min; isocratic elution. The test results are shown in the attached drawings;

although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the invention as defined by the appended claims. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.