液质联用技术在药物降解产物及杂质分析中的应用

发布时间：2018-08-25 12:38

【摘要】：药物的质量与人类健康密切相关，药物中降解产物及微量杂质的鉴定分析也受到越来越多的重视。药物中杂质的存在会降低药物的活性，影响药物的稳定性，甚至产生副作用和不良反应，对人体健康产生危害，因此，对药物中杂质的控制是保证药物质量的重要环节。而对药物降解产物进行研究，不仅可以预测药物的稳定性，指导药物的生产过程并选择合适的包装和贮藏条件，而且有助于建立药物的降解途径并对所建分析方法的可行性进行验证。拉科酰胺是一种新型的抗癫痫药，常作为辅助药物用于治疗成年患者的癫痫部分性发作，但有关拉科酰胺中杂质及降解产物的研究仅有1篇报道，仍有降解产物未被鉴定。氨苄西林钠属于半合成的β-内酰胺类抗生素，主要用于抗感染的治疗，前期研究发现氨苄西林钠一些降解产物并不能根据已有杂质对照品或相关文献数据进行鉴定。因此，为更好地控制这些药物的质量，须建立有效的分析方法对其降解产物及未知杂质进行快速定性分析。高效液相色谱-质谱联用（HPLC-MS）技术集液相色谱的高分离能力与质谱的强结构解析能力于一体，已成为药物杂质分析的有力工具。本研究分别以拉科酰胺和氨苄西林钠为研究对象，以HPLC-MS技术为分析手段，对拉科酰胺和氨苄西林钠多种降解条件下的降解产物以及杂质进行了结构解析和推断，并分析了其降解特征，期望为其质量控制提供新的信息，也为药物杂质的研究提供新的手段。第一部分拉科酰胺降解产物的HPLC-MS/MS分析目的：采用高效液相色谱-三重四极杆串联离子阱质谱（HPLC-QTrap-MS）和高效液相色谱串联离子阱飞行时间质谱（HPLC-IT-TOF-MS）技术对拉科酰胺样品在酸、碱和氧化降解条件下的降解特征进行研究，并对其降解产物进行结构解析和推断。方法：将拉科酰胺样品分别在酸（1mol/L盐酸）、碱（1mol/L氢氧化钠）和氧化（30%H2O2）条件下进行强降解，采用HPLC-QTrap-MS技术对其降解产物进行分析，根据一级和二级质谱信息对未知降解产物的分子量和可能分子结构进行推断；然后采用HPLC-IT-TOF-MS技术对各降解样品进行分析，获得降解产物及其碎片离子的准确分子质量数和可能分子组成，进一步验证推断结果。实验采用Agilent Zorbax SB-C18色谱柱（150mm×4.6mm，5μm）进行分离，梯度洗脱，流动相为乙腈-2mmol/L乙酸铵（含0.1%甲酸），流速为0.8mL/min。结果：本实验对拉科酰胺所有降解样品中的7个降解产物进行了分析，推断出其中4个降解产物的结构，并对其余降解产物进行了部分定性分析；4个降解产物分别鉴定为：（R）-2-氨基-N-苯基-3-甲氧基丙酰胺（m/z209），2-乙酰基-N-苄基丙烯酰胺（m/z219），2-乙酰胺基-N-苯甲醇基-3-甲氧基丙酰胺（m/z267）和2-乙酰氨基-N-苯基-3-羟基丙酰胺（m/z237）。结论：1本实验所用方法灵敏、有效，能够将拉科酰胺与其降解产物完全分离；在所用质谱条件下可获得丰富、准确的质谱信息，从而快速对降解产物进行定性分析。2拉科酰胺在酸、碱和氧化条件下均可发生不同程度的降解，其中化合物（R）-2-氨基-N-苯基-3-甲氧基丙酰胺（m/z209）为其主要降解产物。3化合物2-乙酰基-N-苄基丙烯酰胺（m/z219），2-乙酰胺基-N-苯甲醇基-3-甲氧基丙酰胺（m/z267）和2-乙酰氨基-N-苯基-3-羟基丙酰胺（m/z237）为首次报道。第二部分氨苄西林钠降解产物的HPLC-MS/MS分析目的：采用HPLC-QTrap-MS技术和HPLC-Q-TOF-MS（液相色谱串联四极杆飞行时间质谱）技术对氨苄西林钠样品在各种强降解条件下的降解特征进行研究，并对其降解产物进行结构解析和推断。方法：将氨苄西林钠样品分别在酸、碱、加热、光照和高湿条件下进行降解，联合应用HPLC-QTrap-MS技术的EMS-ER-IDA-EPI（增强全扫描-增强分辨率扫描-信息依赖-增强型子离子扫描）、PREC-ER-IDA-EP（I母离子扫描-增强分辨率扫描-信息依赖-增强型子离子扫描）和EPI（增强型子离子扫描）三种扫描模式对降解样品中的未知降解产物进行分析，并根据获得的质谱信息对其结构进行推断；通过HPLC-Q-TOF-MS技术获得各降解产物及其碎片离子的准确分子质量数和可能分子组成，进一步验证推断结果。实验采用Agilent Zorbax SB-C18（150mm×4.6mm，5μm）色谱柱进行分离，梯度洗脱，流动相为乙腈-1mmol/L乙酸铵溶液（加0.1%乙酸），，流速为0.8mL/min。结果：本实验对氨苄西林钠样品在各种降解条件下的19个杂质和降解产物进行了鉴定和定性分析，其中有4个降解产物未曾报道，为新鉴定化合物，分别为：(Z)-2-氧-2-(((2-氧-3-苯基-2,3,6,7-四氢-1H-1,4-二氮杂卓-5-基)亚甲基)氨基-1-苯乙铵（m/z349），(E)-2-(((2-(2-氨基-2-苯乙酰胺基)-2-苯乙酰基)亚氨基)羧基)甲基)-5,5-二甲基四氢噻唑-4-羧酸（m/z499），(E)-1-羰基-N-((3,6-二氧-5-苯基-1,6-二氢二氮杂苯-2(3H)-取代基)甲基)-2-巯基-2-甲基丙烷-1-胺（m/z348）和5-苯甲氨基-7-甲酰基-2,2-二甲基-2,3-二氢咪唑并[5,1-b]噻唑-3-羧酸（m/z332）。结论：1HPLC-Q-TOF-MS方法灵敏度高、专属性好、准确度高，在未知杂质定性方面可获得更为丰富的结构信息；本实验所建方法能够将氨苄西林钠与其降解产物很好的分离，并可快速、有效地对其降解产物进行鉴定和定性分析。2HPLC-QTrap-MS中EMS-ER-IDA-EPI、PREC-ER-IDA-EPI和EPI模式的联合应用可有效地对药物中未知杂质和降解产物进行定性分析。第三部分氨苄西林钠中杂质的HPLC-MS/MS分析目的：建立灵敏、有效的HPLC-QTrap-MS方法，对氨苄西林钠样品中的杂质进行检测和分析，并与国外市售氨苄西林钠样品进行比较。方法：应用HPLC-QTrap-MS技术的增强全扫描（EMS）模式分别对氨苄西林钠及其国外市售样品进行分析：1通过与系统适用性对照品（包含氨苄西林，氨苄西林噻唑酸，二酮哌嗪氨苄西林，氨苄西林开环二聚体，氨苄西林闭环二聚体和氨苄西林开环三聚体）中的已知杂质进行色谱和质谱信息的比较，从而对样品中存在的这些杂质进行鉴定和确认；2对于样品中的未知杂质，可依据一级质谱图中的[M+H]+，[M+Na]+和[M+K]+离子峰来判断其分子量，再结合质谱图中的碎片离子信息对其进行结构解析和推断，并对碎片离子进行归属。实验采用Kromasil C18（150mm×4.6mm，5μm）色谱柱进行分离，梯度洗脱，流动相为甲醇-乙酸水溶液（pH3.4），流速为0.8mL/min。结果：1系统适用性对照品中的杂质氨苄西林噻唑酸、二酮哌嗪氨苄西林、氨苄西林开环二聚体和氨苄西林闭环二聚体在氨苄西林钠及其国外市售样品中均可被检测到；杂质氨苄西林开环三聚体仅在国外市售样品中被检测到。2根据色谱峰的质谱信息推断出系统适用性对照品中未包含的3个杂质分别为：L-氨苄西林、D-苯甘氨酸氨苄西林和氨苄西林闭环三聚体。结论：1通过比较，在氨苄西林钠样品中检测到了比其国外市售样品更少的杂质，该结果可为氨苄西林钠的质量控制提供相关依据。2本实验所建立的方法灵敏、有效，可将氨苄西林钠与其杂质很好地分离，并可根据质谱信息对样品中的未知杂质进行快速定性分析。
[Abstract]:The quality of drugs is closely related to human health. More and more attention has been paid to the identification and analysis of degradation products and trace impurities in drugs. The study of degradation products can not only predict the stability of drugs, guide the production process of drugs and select appropriate packaging and storage conditions, but also help to establish the degradation pathway of drugs and verify the feasibility of the analytical methods.
Lacosamide is a new type of antiepileptic drug, often used as an adjuvant in the treatment of epileptic partial seizures in adult patients. However, only one report on the impurities and degradation products of Lacosamide has been reported, and the degradation products have not been identified. Ampicillin sodium belongs to semi-synthetic beta-lactam antibiotics, mainly used in the treatment of infections. Previous studies have found that some of the degradation products of ampicillin sodium can not be identified by reference substances or related literature data. Therefore, in order to better control the quality of these drugs, it is necessary to establish effective analytical methods for rapid qualitative analysis of the degradation products and unknown impurities.
High performance liquid chromatography-mass spectrometry (HPLC-MS) has become a powerful tool for the analysis of drug impurities. Lacosamide and ampicillin sodium were used as the research objects and para-lactam and ampicillin sodium were used as the analytical means by HPLC-MS. The structure of degradation products and impurities were analyzed and deduced under different degradation conditions, and the degradation characteristics were analyzed. It is expected to provide new information for quality control and new means for the research of drug impurities.
Part one HPLC-MS/MS analysis of degradation products of lacacamide
OBJECTIVE: To study the degradation characteristics of Lacosamide under acid, alkali and oxidative degradation conditions by high performance liquid chromatography-triple quadrupole tandem ion trap mass spectrometry (HPLC-QTrap-MS) and high performance liquid chromatography-time of flight mass spectrometry (HPLC-IT-TOF-MS).
METHODS: Lacosamide samples were strongly degraded by acid (1mol/L hydrochloric acid), alkali (1mol/L sodium hydroxide) and oxidation (30% H2O2), respectively. The degradation products were analyzed by HPLC-QTrap-MS, and the molecular weight and possible molecular structure of the unknown degradation products were deduced according to the first and second-order mass spectrometry information. T-TOF-MS technique was used to analyze the degraded samples to obtain the exact molecular weight and possible molecular composition of the degraded products and their fragments, and further validate the inference results. Agilent Zorbax SB-C18 column (150mm *4.6mm, 5um) was used for separation and gradient elution. The mobile phase was acetonitrile-2mmol/L ammonium acetate (containing 0.1% formic acid) with flow rate. For 0.8mL/min.
RESULTS: Seven degradation products of Lacosamide in all degradation samples were analyzed, and the structures of four degradation products were deduced, and the remaining degradation products were analyzed qualitatively. Four degradation products were identified as (R) - 2-amino-N-phenyl-3-methoxypropionamide (m/z209), 2-acetyl-N-benzylacryloyl. Amine (m/z219), 2-acetamide-N-benzyl-3-methoxypropylamide (m/z267) and 2-acetamide-N-phenyl-3-hydroxypropylamide (m/z237).
CONCLUSION: 1. The method is sensitive and effective, and can completely separate Lacosamide from its degradation products. Rich and accurate mass spectrometric information can be obtained under the conditions of mass spectrometry. 2. Lacosamide can be degraded in varying degrees under the conditions of acid, alkali and oxidation. 2-amino-N-phenyl-3-methoxypropionamide (m/z209) was the main degradation product. 3 compounds 2-acetyl-N-benzyl acrylamide (m/z219), 2-acetamide-N-benzyl-3-methoxypropionamide (m/z267) and 2-acetamido-N-phenyl-3-hydroxypropionamide (m/z237) were first reported.
Second part HPLC-MS/MS analysis of degradation products of ampicillin sodium
OBJECTIVE: To study the degradation characteristics of ampicillin sodium under various strong degradation conditions by HPLC-QTrap-MS and HPLC-Q-TOF-MS, and to analyze and deduce the degradation products.
METHODS: Ampicillin sodium samples were degraded under acid, alkali, heating, light and high humidity conditions. The EMS-ER-IDA-EPI (Enhanced Full Scan-Enhanced Resolution Scanning-Information Dependent-Enhanced Subion Scanning) and PREC-ER-IDA-EP (I Master Ion Scanning-Enhanced Resolution Scanning-Information Dependent-Enhanced Subion Scanning) techniques were used in combination with HPLC-QTrap-MS. The unknown degradation products in the degraded samples were analyzed by three scanning modes of ion scanning and EPI (Enhanced Subion Scanning), and their structures were deduced according to the mass spectrum information obtained. The accurate molecular weight and possible molecular composition of the degraded products and their fragments were obtained by HPLC-Q-TOF-MS, and further verified. Agilent Zorbax SB-C18 (150mm *4.6mm, 5um) column was used for separation and gradient elution. The mobile phase was acetonitrile-1mmol/L ammonium acetate solution (with 0.1% acetic acid) and the flow rate was 0.8mL/min.
RESULTS: Nineteen impurities and degradation products of ampicillin sodium under various degradation conditions were identified and qualitatively analyzed. Among them, four degradation products were not reported. They were new identified compounds: (Z) - 2 - O - 2 - (((2 - O - 3 - phenyl - 2, 3, 6, 7 - tetrahydro - 1H - 1, 4 - diazepine - 5 - methylene) amino - 1 - phenylethylammonium (m) (m/z349), (E) - 2 - ((2- (2 amino 2 phenylacetamino amino) - 2 phenylacetamino amino amino amino amino amino amino amino amino amino amino amino amino) carboxymethyl) - 5,5 dimethyltetrahydrothiazole 4 carboxylic acid (m/z499), (E) - 1 carbony1 carbonyl N - ((3,6 diodiodiodiodiodioxy5 phenyl 1,6 dihydrodiadiadiadiadiadiadiazobenzene 1 2 (3H) - 2 (3H) - substituent methyl) - 2 mermermermercapcapcapto 2 2 methmethyl2 Methylpropane 1 methyl 1 methyl 1 methyl 1 methyl methyl 1 methyl methyl 1 methyl methyl 1 methyl 1 methyl methyl methyl 1 methmethylamino-7-formyl-2,2-dimethyl-2,3- Two hydrogen imidazo [5,1-b] thiazole -3- carboxylic acid (m/z332).
CONCLUSION: 1HPLC-Q-TOF-MS has high sensitivity, specificity and accuracy, and can obtain more abundant structural information in the qualitative analysis of unknown impurities. The method can separate ampicillin sodium from its degradation products, and identify and analyze the degradation products quickly and effectively. 2HPLC-QTrap-MS The combination of MS-ER-IDA-EPI, PREC-ER-IDA-EPI and EPI can effectively analyze the unknown impurities and degradation products in drugs.
HPLC-MS/MS analysis of impurities in ampicillin sodium in the third part
OBJECTIVE: To establish a sensitive and effective HPLC-QTrap-MS method for the determination and analysis of impurities in ampicillin sodium samples and compare it with those of ampicillin sodium sold abroad.
METHODS: Enhanced full scan (EMS) mode of HPLC-QTrap-MS was used to analyze ampicillin sodium and its foreign commercial samples respectively: 1. The reference substances (including ampicillin, ampicillin thiazolic acid, diacetylpiperazine ampicillin, ampicillin ring-opening dimer, ampicillin closed-loop dimer and ampicillin) were compared with the system suitability. The known impurities in the ring-opening trimer are identified and identified by comparing their chromatographic and mass spectrometric information. 2 For unknown impurities in the sample, their molecular weight can be determined by the peaks of [M+H]+, [M+Na]+ and [M+K]+ ions in the first-order mass spectrometry, and then combined with the fragment ion information in the mass spectrometry. Kromasil C18 (150mm *4.6mm, 5um) column was used for separation and gradient elution. The mobile phase was methanol-acetic acid aqueous solution (pH 3.4), and the flow rate was 0.8mL/min.
Results: 1 The impurities of ampicillin thiazolic acid, diacetylpiperazine ampicillin, ampicillin ring-opening dimer and ampicillin closed-loop dimer were detected in ampicillin sodium and its foreign market samples; the impurities of ampicillin ring-opening trimer were detected only in foreign market samples.2 According to color. Mass spectra of the peaks revealed that the three impurities not included in the reference substances were L-ampicillin, D-phenylglycine ampicillin and ampicillin closed-loop trimer.
CONCLUSION: 1. By comparison, fewer impurities were detected in ampicillin sodium samples than in the samples sold abroad. The results can provide a basis for the quality control of ampicillin sodium. 2 The method is sensitive and effective, and can be used to separate ampicillin sodium from its impurities, and can be used to detect the impurities in the samples according to the mass spectrometry information. Fast and qualitative analysis of impurities.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R917;O657.63

【引证文献】