药物中基因毒性杂质检测方法研究进展

基因毒性杂质由于对DNA的破坏作用而具有一定的致癌性,危害极大。因其结构众多,患者在服药过程中有摄入该类杂质的风险。部分国家对基因毒性杂质的限度控制已成为药品上市时必须考察的指标。本文概述了基因毒性杂质的基本概念、相关法规标准、部分杂质检测方法限度等内容,以期为控制药品中基因毒性杂质的含量提供参考和依据,保证患者用药安全。     

化学药品标准中杂质控制及其限度的建立

药品质量控制的关键之一是进行药物杂质控制。该文介绍了ICH(人用药品注册技术要求国际协调会)有关技术文件对于药物杂质控制的具体规定,并举例比较了各主要国际中药典收载化学药品标准中杂质控制限度的制定与ICH的差距,提出了进行药物杂质控制的标准要求、限度设置的依据,特别介绍了生化药品目前中国药典收载品种杂质控制的现状。。鉴于仪器分析的局限,有色杂质控制未被重视,是目前药物杂质控制中的薄弱环节,探讨了利用溶液颜色检查法进行有色杂质控制的必要性和需进一步改进检查方法的原因。     

LC-MS/MS法检测盐酸鲁拉西酮中磺酸酯基因毒杂质

目的：采用LC-MS/MS法检查盐酸鲁拉西酮中的痕量磺酸酯基因毒杂质。方法：采用Waters Atlantis T₃（2.1 mm×50 mm，3 μm）色谱柱，10 mmol·L^-1醋酸铵水溶液（55%）和甲醇（45%）作为流动相，等度洗脱，Agilent 1200色谱系统和Agilent 6460 MS/MS（ESI源）检测磺酸酯。结果：磺酸酯在0.05 ng·mL^-1至25.00 ng·mL^-1范围内线性良好，r＞0.999，加样回收率为85%～115%，样品溶液在恒温进样盘（4℃）中6 h稳定。结论：该方法灵敏度高、准确度良好，可用于盐酸鲁拉西酮中磺酸酯基因毒杂质的定量检测，并为质量控制提供参考。     

国外化合物结构-毒性数据库"TOPKAT"简介

在药品研发过程中,杂质的控制是保证药品安全性的一个重要方面.如何安全合理地确定杂质的限度,并将杂质控制在该限度内,是药品研发者面临的任务之一,也是药品评价者所关注的重点问题之一.由于杂质的毒性与其化学结构密切相关,所以,我国及ICH在杂质研究的指导原则中均引入了杂质的鉴定限度这一概念,要求对超过该限度的杂质均应进行定性研究,尽量确定其结构,然后再根据结构与毒性的关系,判断该杂质的毒性,从而为杂质限度的确定提供参考依据.     

ICH Q3D新药制剂元素杂质评估及控制的要点解读

目的：药品中可能有多个来源的元素杂质,由于元素杂质不能给病人提供任何治疗益处,因而药品生产中元素杂质含量应被控制在可接受的限度内。国际人用药物注册技术协调会议（ICH）Q3D元素杂质指南是新药制剂元素杂质控制的质量指南,旨在为新药制剂和其赋形剂中元素杂质的定性和定量控制提供全球性方针。本文介绍Q3D元素杂质指南,对相关要点进行解读。方法：详细描述元素杂质指南的主要内容,侧重于对元素风险评估及设定制剂中各组分元素杂质的限度进行具体分析。根据《美国药典》39和《欧洲药典》9.0中相关金属元素杂质的通则,汇总这些指导性文件与ICH Q3D的不同之处。结果与结论：Q3D主要包括潜在元素杂质的安全性评价、类别、元素杂质的风险评估和控制、日允许暴露量（Permitted Daily Exposure,简称PDE）与浓度限度之间的转换。元素杂质的风险评估应考虑潜在元素杂质的来源和药物服用方式,将特定元素杂质水平与PDE进行比较,评价该元素在药品中存在的可能性。经风险评估需要进行控制的元素杂质,可以根据药物服用剂量和PDE用3种方法设定元素浓度限度,这有利于帮助药品生产企业通过风险评估来决定对哪些元素进行额外控制,从而有效保障药品质量。     

我国药物研发中杂质研究面临的挑战与思考

目的：分析探讨化学药物杂质研究的基本思路与杂质限度确定的一般原则。方法：通过分析我国药物研发中杂质研究现状和国际上杂质研究发展趋势．阐述杂质研究的目的、内容及其一般过程,探讨其基本规律和相关要素。结果：杂质研究是药品研发中风险控制意识的重要体现．药品中的杂质是否能被全面准确地控制．直接关系到药品的质量可控与安全性。结论：通过选择合适的分析方法,准确分辨与定量杂质．综合药学、毒理学及临床研究的结果．确定杂质的合理限度．通过对起始原料．原料药辅料的源头控制、制备工艺的过程控制、包装材料的优选、贮藏条件及有效期的确立等终端控制措施,将杂质控制在安全合理的范围内．正是杂质研究的最终目的。     

浅谈杂质限度制定的一般原则

杂质研究及控制是药品安全保证的关键要素之一,是药品研发中风险控制意识的重要体现,药品临床使用中的不良反应除了与药品本身的药理活性有关外,有时还与药品中的杂质有关,须严格控制.文中结合SFDA(化学药物杂质研究技术指导原则>相关内容,探讨了杂质限度确定的一般原则.     

药品质量标准中杂质的限度确定方式探讨

杂质限度范围的确立是药品质量标准建立的关键环节之一,合理的杂质限度对于药品的安全性和可控性至关重要,关系着药品的质量。本文以几种典型杂质为实例,系统阐述和演示了药品中杂质的每日最大暴露量(permitted daily exposure, PDE)的确定方式、在具体药品中的换算方式以及在产品中的限度确定方式,可更好地理解和实施ICH相关规范,为药品研发者对产品质量标准的制定提供参考。     

亚硝胺类遗传毒性杂质的监管策略及思考

如何对药品中存在致癌风险的亚硝胺类杂质进行控制,已成为企业和监管部门关注的热点。本文对亚硝胺类杂质的常见类型、来源、致癌性作用特点进行梳理,并结合EMA、FDA、ICH及我国相关遗传毒性杂质控制指导原则,对制定符合我国国情的亚硝胺类杂质的监管限度和监管工作提出建议。尽管国外已陆续出台了一系列针对药品中亚硝胺类杂质的含量限定的指南性文件,但众多亚硝胺类杂质的毒性剂量、人体暴露量尚不明确,且药物合成工艺存在差异,我国无法完全照搬欧美等国的监管方法。当前应深入研究亚硝胺类杂质的遗传毒性和致癌性,从而制定符合我国国情的监管限度值和药品中亚硝胺杂质监管策略;此外,本文从解决实际问题的角度出发,讨论如何根据已有指导原则,确定在已知毒理学数据、毒理学数据不足和短期使用药物不同情况下亚硝胺类杂质的监管限度。本文将为药物生产和杂质评价与研究和监管领域相关人员提供借鉴。     

亚硝胺类遗传毒性杂质的监管策略及思考

如何对药品中存在致癌风险的亚硝胺类杂质进行控制,已成为企业和监管部门关注的热点。本文对亚硝胺类杂质的常见类型、来源、致癌性作用特点进行梳理,并结合EMA、FDA、ICH及我国相关遗传毒性杂质控制指导原则,对制定符合我国国情的亚硝胺类杂质的监管限度和监管工作提出建议。尽管国外已陆续出台了一系列针对药品中亚硝胺类杂质的含量限定的指南性文件,但众多亚硝胺类杂质的毒性剂量、人体暴露量尚不明确,且药物合成工艺存在差异,我国无法完全照搬欧美等国的监管方法。当前应深入研究亚硝胺类杂质的遗传毒性和致癌性,从而制定符合我国国情的监管限度值和药品中亚硝胺杂质监管策略;此外,本文从解决实际问题的角度出发,讨论如何根据已有指导原则,确定在已知毒理学数据、毒理学数据不足和短期使用药物不同情况下亚硝胺类杂质的监管限度。本文将为药物生产和杂质评价与研究和监管领域相关人员提供借鉴。     

Control and analysis of alkyl esters of alkyl and aryl sulfonic acids in novel active pharmaceutical ingredients (APIs)

This article reviews current regulatory guidelines and relevant scientific literature pertaining to the control and analysis of potential genotoxic impurities (PGIs) in new active pharmaceutical ingredients (APIs) with specific reference to a certain sub-class of PGIs, namely alkyl esters of alkyl and aryl sulfonic acids. Sulfonic acids are very important in pharmaceutical R&D employed both as counter-ions in the formation of acid-addition salts and also as reagents and catalysts in the synthesis of new drug substances. The article reviews the evolution of analytical methodology from early studies in the mid 1970s through development of direct injection GC and HPLC methods to liquid-liquid/solid phase extraction and headspace based techniques coupled to HPLC and GC methodologies employing UV and MS detection to new derivatisation-based techniques. The paper also reflects on the significant challenges in developing robust analytical methodology capable of the trace determination of sulfonate esters, the challenges in transferring methodology from R&D to QC labs and on the cost of inappropriate limits for genotox impurities. In so doing, the authors seek to inform the debate that the control of genotoxic impurities should be driven primarily by safety and risk/benefit considerations rather than by state-of-the-art analytical and process chemistry capabilities that drive controls to levels 'as low as practicable' regardless of the risk/safety requirements.     

Drug substances presented as sulfonic acid salts: overview of utility,safety and regulation

Objectives Controlling genotoxic impurities represents a significant challenge to both industry and regulators. The potential for formation of genotoxic short‐chain alkyl esters of sulfonic acids during synthesis of sulfonic acid salts is a long‐standing regulatory concern. This review provides a general overview of the utility of sulfonic acids as salt‐forming moieties and discusses strategies for effectively minimizing the potential for alkyl sulfonate formation during the synthesis and processing of sulfonate salt active pharmaceutical ingredients. The potential implications of the recent establishment of a substantial human threshold dose for ethyl methanesulfonate for the safety assessment of alkyl sulfonates in general are also discussed. Key findings The formation of alkyl sulfonates requires highly acidic conditions, possibly combined with long reaction times and/or elevated temperatures, to generate significant amounts, and these conditions are most unlikely to be present in the synthesis of active pharmaceutical ingredient sulfonate salts. It is possible to design salt formation conditions, using a short‐chain alcohol as solvent, to manufacture sulfonate salts that are essentially free of alkyl sulfonate impurities. Processes using non‐acidic conditions such as ethanol recrystallization or wet granulation should not raise any concerns of alkyl sulfonate formation. Summary An understanding of the mechanism of formation of alkyl sulfonates is critical in order to avoid restricting or over‐controlling sulfonic acid salts, which have many technical advantages as pharmaceutical counterions. Recent regulatory acceptance of a human threshold limit dose of 2 mg/kg per day for ethyl methanesulfonate, indicating that its toxicological risks have previously been considerably overestimated, could signal the beginning of the end over safety concerns on alkyl sulfonate residues, thus removing a major constraint from the exploitation of sulfonic acid counterions.     

Simultaneous determination of formic acid and formaldehyde in pharmaceutical excipients using headspace GC/MS

Formic acid and its esters, as well as formaldehyde, are trace impurities that are often present in pharmaceutical excipients. These trace impurities can potentially react with amino and/or hydroxyl groups in drugs to form significant levels of degradants. To select the appropriate excipients for a stable formulation, a gas chromatography/mass spectrometry (GC/MS) method was developed and validated for the rapid screening of trace amounts of residual formic acid, its esters and formaldehyde in pharmaceutical excipients. Samples were dissolved or dispersed in acidified ethanol to convert formic acid and formaldehyde to ethyl formate and diethoxymethane, respectively. Identification was conducted using a GC/MS system under scan mode and quantified using a selected ion monitoring (SIM) mode. Evaluation of the mass spectra of ethyl formate and diethoxymethane in the samples indicated that the method is specific. The limits of quantitation of the method were 0.5 ppm for formic acid and 0.2 ppm for formaldehyde. The precision of the method was demonstrated by the acceptable R.S.D. (相似文献   

Formation of reactive impurities in aqueous and neat polyethylene glycol 400 and effects of antioxidants and oxidation inducers

A 2,4-dinitrophenylhydrazine (DNPH) precolumn derivatization high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method was developed to quantify levels of formaldehyde and acetaldehyde in polyethylene glycol (PEG) solutions. Formic acid and acetic acid were quantified by HPLC-UV. Samples of neat and aqueous PEG 400 solutions were monitored at 40°C and 50°C to determine effects of excipient source, water content, pH, and trace levels of hydrogen peroxide or iron metal on the formation of reactive impurities. The effects of antioxidants were also evaluated. Formic acid was the major degradation product in nearly all cases. The presence of water increased the rate of formation of all impurities, especially formic acid as did the presence of hydrogen peroxide and trace metals. Acidic pH increased the formation of acetaldehyde and acetic acid. A distribution of unidentified degradation products formed in neat PEG 400 disappeared upon addition of HCl with corresponding increase of formic acid, indicating they were likely to be PEG-formyl esters. Other unidentified degradation products reacted with DNPH to form a distribution of derivatized products likely to be PEG aldehydes. Antioxidants butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate d-alpha tocopheryl polyethylene glycol-1000 succinate, and sodium metabisulfite were effective in limiting reactive impurity formation, whereas ascorbic acid and acetic acid were not.     

LCMS-QTOF鉴定卡络磺钠原料药的有关物质

目的 检测卡络磺钠原料药中有关物质，并进行结构鉴定，为工艺优化和质量控制提供参考。方法 采用Shim-pack Scepter C₁₈(4.6 mm×250 mm，5 µm)色谱柱，以50 mmol·L^–1乙酸铵水溶液为流动相A、乙腈为流动相B对卡络磺钠强制降解溶液中的有关物质进行分离。采用四极杆飞行时间高分辨质谱在m/z 100~800内进行正负离子扫描，数据依赖性采集获取二级质谱图。结合有关物质精确质量数、质谱裂解规律及卡络磺钠合成工艺推测其可能的结构。结果 卡络磺钠原料药经强制降解破坏得到8个主要的杂质，2种为已报道杂质，6种为本研究首次报道。强制降解试验表明卡络磺钠对高温和氧化相对较为稳定，光照、强酸和强碱条件对卡络磺钠影响较大。卡络磺钠原料药中检出杂质5，6和8，高温破坏的主要降解产物是杂质8。结论 本研究建立的液相色谱-四极杆飞行时间质谱联用法具有良好的分离能力，可有效地鉴定卡络磺钠原料药中有关物质，为其生产工艺和质量控制提供了参考。     

Control and analysis of alkyl and benzyl halides and other related reactive organohalides as potential genotoxic impurities in active pharmaceutical ingredients (APIs)

This paper continues the review of the relevant scientific literature associated with the control and analysis of potential genotoxic impurities (PGIs) in active pharmaceutical ingredients (APIs). The initial review [D.P. Elder, A. Teasdale, A.M. Lipczynski, J. Pharm. Biomed. Anal. 46 (2008) 1-8.] focused on the specific class of sulfonate esters but in this instance reference is made to the analysis of alkyl and benzyl halides and other related reactive organohalide alkylating agents. Such reactive materials are commonly employed in pharmaceutical research and development as raw materials, reagents and intermediates in the chemical synthesis of new drug substances. Consequently a great deal of attention and effort is extended by the innovative and ethical pharmaceutical industry to ensure that appropriate and practicable control strategies are established during drug development to ensure residues of such agents, as potential impurities in new drug substances, are either eliminated or minimized to such an extent so as to not present a significant safety risk to volunteers and patients in clinical trials and beyond. The reliable trace analysis of such reactive organohalides is central to such control strategies and invariably involves a state-of-the-art combination of high-resolution separation science techniques coupled to sensitive and selective modes of detection. This article reports on the most recent developments in the regulatory environment, overall strategies for the control of alkylating agents and the latest developments in analysis culminating in a literature review of analytical approaches. The literature is sub-categorized by separation technique (gas chromatography (GC), high-performance liquid chromatography (HPLC), thin layer chromatography (TLC) and capillary zone electrophoresis (CZE)) and further tabulated by API type and impurity with brief method details and references. As part of this exercise, a selection of relevant pharmacopoeial monographs was also reviewed. The continued reliance on relatively non-specific and insensitive TLC methodologies in several monographs was noteworthy.     

高效液相色谱法对新型大豆异黄酮磺酸酯的前药研究

目的:研究大豆异黄酮磺酸酯化学修饰物的理化性质,并预测其药代动力学性质。方法:建立高效液相色谱法进行药物的理化性质测试和体外水解动力学试验。结果:大豆苷元-7,4′-二苯磺酸酯(DBS1)和大豆苷元-7-苯磺酸酯(DBS2)的脂溶性与水溶性相对于原药大豆苷元(DZ)均有显著的提高,脂水分配系数分别为:3.57±0.75和1.97±0.45;水解反应半衰期分别为2.14 h和19.25 h。DBS1的水解反应中4′-的磺酸酯基更易断裂。结论:化合物DBS1和DBS2的脂溶性、水溶性、脂水分配系数、熔点、极性表面积等影响药物药代动力学的一些主要性质得以优化,预示这2个化合物可能有相对较好的过膜吸收性与转运分布特性,以及良好的口服生物利用度。     

胶束毛细管电泳在线富集技术测定那格列奈中痕量杂质时分峰现象的研究

目的:探究采用胶束毛细管电泳(MEKC)并结合在线推扫富集技术检测样品时出现分峰现象的原因。方法:以应用胶束毛细管电泳在线富集技术测定那格列奈原料中痕量杂质为例,讨论了背景缓冲液 pH、表面活性剂浓度(分别讨论了 SDS 及CTAB)、离子强度与进样时间对分峰现象的影响。结果:背景缓冲溶液 pH、表面活性剂浓度、离子强度不同时,出现分峰时的进样量不同,富集倍数不同;其他条件一定时,进样量对分峰的出现也有影响。应用此技术测定那格列奈原料中痕量杂质时,最佳富集倍数达到了200倍。结论:用 MEKC 并结合推扫法测定样品时,要对电泳条件进行逐一优化,以筛选出可获得最佳富集效果,满足检测灵敏度,并避免分峰的实验条件。     

Reversed-phase high-performance liquid chromatographic investigation of levodopa preparations I: Amino acid impurities

A reversed-phage high-performance liquid chromatographic procedure with UV detection at 280 mm is presented for the determination of 6-hydroxydopa, tyrosine, 3-O-methyldopa, and other trace amino acid impurities in levodopa preparations. The method is fast and sufficiently sensitive to determine trace impurities at 0.1% of the levodopa concentration with a relative standard deviation of 4-6%. The trace impurities can be estimated at less than or equal to 0.01%.     

Fenofibrate raw materials: HPLC methods for assay and purity and an NMR method for purity

HPLC methods for drug content and HPLC and NMR methods for related compounds in fenofibrate raw materials were developed. The HPLC methods resolved 11 known and six unknown impurities from the drug. The HPLC system was comprised of a Waters Symmetry ODS column (100 x 4.6 mm, 3.5 microm), a mobile phase consisting of acetonitrile water trifluoroacetic acid 700/300/l (v/v/v) at a flow rate of 1 ml min(-1). and a UV detector set at 280 nm. Minimum quantifiable amounts were about 0.1% for three of the compounds and less than 0.05% for the other eight. Individual impurities in 14 raw materials ranged from trace levels to 0.25%, and total impurities from 0.04 to 0.53% (w/w). Six unknown impurities were detected by HPLC, all at levels below 0.10%, assuming the same relative response as fenofibrate. An NMR method for related compounds was also developed and it was suitable for 12 known and several unknown impurities. It requires an NMR of 400 MHz, or greater, field strength. Individual impurities in the raw materials analyzed ranged from trace levels to 0.24%, and total impurities from trace levels to 0.59%. Several lots contained small amounts of unknown impurities at trace levels. Three lots, all from the same manufacturer, contained an unknown impurity, not detectable by HPLC, which was not present in the other raw materials. It was estimated to be present at a level greater than 0.2%. The results for related compounds by the two techniques were consistent. The main differences stem from the low sensitivity of the HPLC method for some of the related compounds at 280 nm, or from the higher limits of quantitation by the NMR method for several other impurities using the conditions specified. A fifteenth raw material was not homogeneous in its content of impurity VI, a synthetic intermediate and possible degradation product. The HPLC/MS results provided information on the peak purity (number of components) for minor HPLC peaks, as well as structural data such as the molecular ions and diagnostic fragment ions. The HPLC/MS results showed that there were five unknown drug related impurities, for which there were no standards available. Results for the assay of 15 raw materials by HPLC were within the range 98.5-101.5%.