【摘要】：藥物的質(zhì)量與人類健康密切相關(guān)，藥物中降解產(chǎn)物及微量雜質(zhì)的鑒定分析也受到越來越多的重視。藥物中雜質(zhì)的存在會降低藥物的活性，影響藥物的穩(wěn)定性，甚至產(chǎn)生副作用和不良反應(yīng)，對人體健康產(chǎn)生危害，因此，對藥物中雜質(zhì)的控制是保證藥物質(zhì)量的重要環(huán)節(jié)。而對藥物降解產(chǎn)物進(jìn)行研究，不僅可以預(yù)測藥物的穩(wěn)定性，指導(dǎo)藥物的生產(chǎn)過程并選擇合適的包裝和貯藏條件，而且有助于建立藥物的降解途徑并對所建分析方法的可行性進(jìn)行驗(yàn)證。 拉科酰胺是一種新型的抗癲癇藥，常作為輔助藥物用于治療成年患者的癲癇部分性發(fā)作，但有關(guān)拉科酰胺中雜質(zhì)及降解產(chǎn)物的研究僅有1篇報(bào)道，仍有降解產(chǎn)物未被鑒定。氨芐西林鈉屬于半合成的β-內(nèi)酰胺類抗生素，主要用于抗感染的治療，前期研究發(fā)現(xiàn)氨芐西林鈉一些降解產(chǎn)物并不能根據(jù)已有雜質(zhì)對照品或相關(guān)文獻(xiàn)數(shù)據(jù)進(jìn)行鑒定。因此，為更好地控制這些藥物的質(zhì)量，須建立有效的分析方法對其降解產(chǎn)物及未知雜質(zhì)進(jìn)行快速定性分析。 高效液相色譜-質(zhì)譜聯(lián)用（HPLC-MS）技術(shù)集液相色譜的高分離能力與質(zhì)譜的強(qiáng)結(jié)構(gòu)解析能力于一體，已成為藥物雜質(zhì)分析的有力工具。本研究分別以拉科酰胺和氨芐西林鈉為研究對象，以HPLC-MS技術(shù)為分析手段，對拉科酰胺和氨芐西林鈉多種降解條件下的降解產(chǎn)物以及雜質(zhì)進(jìn)行了結(jié)構(gòu)解析和推斷，并分析了其降解特征，期望為其質(zhì)量控制提供新的信息，也為藥物雜質(zhì)的研究提供新的手段。 第一部分拉科酰胺降解產(chǎn)物的HPLC-MS/MS分析 目的：采用高效液相色譜-三重四極桿串聯(lián)離子阱質(zhì)譜（HPLC-QTrap-MS）和高效液相色譜串聯(lián)離子阱飛行時間質(zhì)譜（HPLC-IT-TOF-MS）技術(shù)對拉科酰胺樣品在酸、堿和氧化降解條件下的降解特征進(jìn)行研究，并對其降解產(chǎn)物進(jìn)行結(jié)構(gòu)解析和推斷。 方法：將拉科酰胺樣品分別在酸（1mol/L鹽酸）、堿（1mol/L氫氧化鈉）和氧化（30%H2O2）條件下進(jìn)行強(qiáng)降解，采用HPLC-QTrap-MS技術(shù)對其降解產(chǎn)物進(jìn)行分析，根據(jù)一級和二級質(zhì)譜信息對未知降解產(chǎn)物的分子量和可能分子結(jié)構(gòu)進(jìn)行推斷；然后采用HPLC-IT-TOF-MS技術(shù)對各降解樣品進(jìn)行分析，獲得降解產(chǎn)物及其碎片離子的準(zhǔn)確分子質(zhì)量數(shù)和可能分子組成，進(jìn)一步驗(yàn)證推斷結(jié)果。實(shí)驗(yàn)采用Agilent Zorbax SB-C18色譜柱（150mm×4.6mm，5μm）進(jìn)行分離，梯度洗脫，流動相為乙腈-2mmol/L乙酸銨（含0.1%甲酸），流速為0.8mL/min。 結(jié)果：本實(shí)驗(yàn)對拉科酰胺所有降解樣品中的7個降解產(chǎn)物進(jìn)行了分析，推斷出其中4個降解產(chǎn)物的結(jié)構(gòu)，并對其余降解產(chǎn)物進(jìn)行了部分定性分析；4個降解產(chǎn)物分別鑒定為：（R）-2-氨基-N-苯基-3-甲氧基丙酰胺（m/z209），2-乙�；�-N-芐基丙烯酰胺（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-羥基丙酰胺（m/z237）。 結(jié)論：1本實(shí)驗(yàn)所用方法靈敏、有效，能夠?qū)⒗契０放c其降解產(chǎn)物完全分離；在所用質(zhì)譜條件下可獲得豐富、準(zhǔn)確的質(zhì)譜信息，從而快速對降解產(chǎn)物進(jìn)行定性分析。2拉科酰胺在酸、堿和氧化條件下均可發(fā)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）為其主要降解產(chǎn)物。3化合物2-乙�；�-N-芐基丙烯酰胺（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-羥基丙酰胺（m/z237）為首次報(bào)道。 第二部分氨芐西林鈉降解產(chǎn)物的HPLC-MS/MS分析 目的：采用HPLC-QTrap-MS技術(shù)和HPLC-Q-TOF-MS（液相色譜串聯(lián)四極桿飛行時間質(zhì)譜）技術(shù)對氨芐西林鈉樣品在各種強(qiáng)降解條件下的降解特征進(jìn)行研究，并對其降解產(chǎn)物進(jìn)行結(jié)構(gòu)解析和推斷。 方法：將氨芐西林鈉樣品分別在酸、堿、加熱、光照和高濕條件下進(jìn)行降解，聯(lián)合應(yīng)用HPLC-QTrap-MS技術(shù)的EMS-ER-IDA-EPI（增強(qiáng)全掃描-增強(qiáng)分辨率掃描-信息依賴-增強(qiáng)型子離子掃描）、PREC-ER-IDA-EP（I母離子掃描-增強(qiáng)分辨率掃描-信息依賴-增強(qiáng)型子離子掃描）和EPI（增強(qiáng)型子離子掃描）三種掃描模式對降解樣品中的未知降解產(chǎn)物進(jìn)行分析，并根據(jù)獲得的質(zhì)譜信息對其結(jié)構(gòu)進(jìn)行推斷；通過HPLC-Q-TOF-MS技術(shù)獲得各降解產(chǎn)物及其碎片離子的準(zhǔn)確分子質(zhì)量數(shù)和可能分子組成，進(jìn)一步驗(yàn)證推斷結(jié)果。實(shí)驗(yàn)采用Agilent Zorbax SB-C18（150mm×4.6mm，5μm）色譜柱進(jìn)行分離，梯度洗脫，流動相為乙腈-1mmol/L乙酸銨溶液（加0.1%乙酸），，流速為0.8mL/min。 結(jié)果：本實(shí)驗(yàn)對氨芐西林鈉樣品在各種降解條件下的19個雜質(zhì)和降解產(chǎn)物進(jìn)行了鑒定和定性分析，其中有4個降解產(chǎn)物未曾報(bào)道，為新鑒定化合物，分別為：(Z)-2-氧-2-(((2-氧-3-苯基-2,3,6,7-四氫-1H-1,4-二氮雜卓-5-基)亞甲基)氨基-1-苯乙銨（m/z349），(E)-2-(((2-(2-氨基-2-苯乙酰胺基)-2-苯乙酰基)亞氨基)羧基)甲基)-5,5-二甲基四氫噻唑-4-羧酸（m/z499），(E)-1-羰基-N-((3,6-二氧-5-苯基-1,6-二氫二氮雜苯-2(3H)-取代基)甲基)-2-巰基-2-甲基丙烷-1-胺（m/z348）和5-苯甲氨基-7-甲酰基-2,2-二甲基-2,3-二氫咪唑并[5,1-b]噻唑-3-羧酸（m/z332）。 結(jié)論：1HPLC-Q-TOF-MS方法靈敏度高、專屬性好、準(zhǔn)確度高，在未知雜質(zhì)定性方面可獲得更為豐富的結(jié)構(gòu)信息；本實(shí)驗(yàn)所建方法能夠?qū)逼S西林鈉與其降解產(chǎn)物很好的分離，并可快速、有效地對其降解產(chǎn)物進(jìn)行鑒定和定性分析。2HPLC-QTrap-MS中EMS-ER-IDA-EPI、PREC-ER-IDA-EPI和EPI模式的聯(lián)合應(yīng)用可有效地對藥物中未知雜質(zhì)和降解產(chǎn)物進(jìn)行定性分析。 第三部分氨芐西林鈉中雜質(zhì)的HPLC-MS/MS分析 目的：建立靈敏、有效的HPLC-QTrap-MS方法，對氨芐西林鈉樣品中的雜質(zhì)進(jìn)行檢測和分析，并與國外市售氨芐西林鈉樣品進(jìn)行比較。 方法：應(yīng)用HPLC-QTrap-MS技術(shù)的增強(qiáng)全掃描（EMS）模式分別對氨芐西林鈉及其國外市售樣品進(jìn)行分析：1通過與系統(tǒng)適用性對照品（包含氨芐西林，氨芐西林噻唑酸，二酮哌嗪氨芐西林，氨芐西林開環(huán)二聚體，氨芐西林閉環(huán)二聚體和氨芐西林開環(huán)三聚體）中的已知雜質(zhì)進(jìn)行色譜和質(zhì)譜信息的比較，從而對樣品中存在的這些雜質(zhì)進(jìn)行鑒定和確認(rèn)；2對于樣品中的未知雜質(zhì)，可依據(jù)一級質(zhì)譜圖中的[M+H]+，[M+Na]+和[M+K]+離子峰來判斷其分子量，再結(jié)合質(zhì)譜圖中的碎片離子信息對其進(jìn)行結(jié)構(gòu)解析和推斷，并對碎片離子進(jìn)行歸屬。實(shí)驗(yàn)采用Kromasil C18（150mm×4.6mm，5μm）色譜柱進(jìn)行分離，梯度洗脫，流動相為甲醇-乙酸水溶液（pH3.4），流速為0.8mL/min。 結(jié)果：1系統(tǒng)適用性對照品中的雜質(zhì)氨芐西林噻唑酸、二酮哌嗪氨芐西林、氨芐西林開環(huán)二聚體和氨芐西林閉環(huán)二聚體在氨芐西林鈉及其國外市售樣品中均可被檢測到；雜質(zhì)氨芐西林開環(huán)三聚體僅在國外市售樣品中被檢測到。2根據(jù)色譜峰的質(zhì)譜信息推斷出系統(tǒng)適用性對照品中未包含的3個雜質(zhì)分別為：L-氨芐西林、D-苯甘氨酸氨芐西林和氨芐西林閉環(huán)三聚體。 結(jié)論：1通過比較，在氨芐西林鈉樣品中檢測到了比其國外市售樣品更少的雜質(zhì)，該結(jié)果可為氨芐西林鈉的質(zhì)量控制提供相關(guān)依據(jù)。2本實(shí)驗(yàn)所建立的方法靈敏、有效，可將氨芐西林鈉與其雜質(zhì)很好地分離，并可根據(jù)質(zhì)譜信息對樣品中的未知雜質(zhì)進(jìn)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R917;O657.63

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 董曉亞;原料藥SR的質(zhì)量標(biāo)準(zhǔn)研究[D];河南大學(xué);2016年

2 音袁;2,4-滴丁酸的殘留分析研究及其在水稻和土壤中的降解規(guī)律[D];安徽農(nóng)業(yè)大學(xué);2016年

本文編號：2202911