浅谈强制降解试验

er 强制降解测定方法验证报告一、引言强制降解测定方法是指在一定条件下，将样品经过一系列处理后，测定样品在降解过程中产生的物质或物质质量的变化。

本报告旨在验证强制降解测定方法的可靠性和有效性，并验证其在实际应用中的适用性。

二、实验方法1. 材料准备在本实验中，使用了实验室中提供的样品A作为研究对象。

样品A是一种有机化合物。

还需要准备一系列溶剂、试剂和仪器设备，包括pH计、离心机、温控水浴槽等。

2. 实验步骤a. 前处理：将样品A按照固定比例配制成所需浓度。

b. 强制降解：将样品A置于特定条件下进行降解处理。

条件包括温度、湿度、光照等。

c. 样品采集：在一定时间间隔内，采集降解过程中的样品。

d. 分析测定：采用相关仪器设备对采集到的样品进行检测和测定。

3. 数据处理利用所得数据，进行统计学分析和结果的计算。

包括平均值、标准差等。

三、实验结果通过实验，我们获得了样品A在不同温度下的降解情况，在一定时间范围内样品质量的变化情况。

我们还进行了若干次重复实验，以验证实验结果的可靠性。

1. 样品降解率样品A在不同条件下经过一定时间的降解后，测得其降解率。

根据实验数据统计，我们得到了降解率与时间的变化关系曲线。

2. 降解产物分析从样品A降解后的样品中，我们采集到了降解产物，并利用质谱仪、红外光谱仪等设备进行了分析。

通过对降解产物的鉴定和分析，确定了样品A在降解过程中产生的物质。

四、讨论与分析根据实验结果，我们可以得出以下结论：1. 样品A在特定条件下确实发生了降解反应，且降解率可达到一定程度。

2. 样品A降解产物的种类和含量与降解条件有关，不同条件下可能产生不同的降解产物。

3. 强制降解测定方法对样品A的降解过程进行了有效监测和测定，结果具有可靠性和重复性。

在实际应用中，我们可以利用强制降解测定方法对样品的稳定性和降解性进行研究，在药物开发、环境监测等领域中具有一定的应用前景。

五、结论通过本次实验，我们验证了强制降解测定方法的可靠性和有效性，并且确定了该方法在样品A的测定中的适用性。

Scientific Considerations of Forced degradation Studies in aNda SubmissionsaBStraCta well-designed stress study can provide insight in choos-ing the appropriate formulation for a proposed product prior to intensive formulation development studies. it can prevent re-development or re-validation of a stabil-ity indicating analytical method. this paper outlines the scientific aspects of forced degradation studies that should be considered in relation to aNda submissions. iNtroduCtioNForced degradation is synonymous with stress test-ing and purposeful degradation. Purposeful degra-dation can be a useful tool to predict the stability of a drug substance or a drug product with effects on purity, potency, and safety. it is imperative to know the impurity profile and behavior of a drug substance under various stress conditions. Forced degradation also plays an important role in the development of analytical methods, setting specifications, and design of formulations under the quality-by-design (Qbd) paradigm. the nature of the stress testing depends on the individual drug substance and the type of drug product (e.g., solid oral dosage, lyophilized powders, and liquid formulations) involved (1).the international Conference on Harmonisation (iCH) Q1B guideline provides guidance for perform-ing photostability stress testing; however, there are no additional stress study recommendations in the iCH sta-bility or validation guidelines (2). there is also limited information on the details about the study of oxidation and hydrolysis. the drug substance monographs of analytical Profiles of drug Substances and excipients provide some information with respect to different stress conditions of various drug substances (3).the forced degradation information provided in the abbreviated new drug application (aNda) submissions is often incomplete and in those cases deficiencies are cited. an overview of common deficiencies cited through-out the chemistry, manufacturing, and controls (CMC) section of the aNdas has been published (4-6). Some examples of commonly cited deficiencies related to forced degradation studies include the following:• y our d rug s ubstance d oes n ot s how a ny d egrada-tion under any of the stress conditions. Pleaserepeat stress studies to obtain adequate degra-dation. if degradation is not achievable, pleaseprovide your rationale.• P lease note that the conditions employed forstress study are too harsh and that most of yourdrug s ubstance h as d egraded. P lease r epeat y ourstress s tudies u sing m ilder c onditions o r s horterexposure t ime t o g enerate r elevant d egradationproducts.• i t is noted that you have analyzed your stressedsamples as per the assay method conditions.For the related substances method to be sta-bility indicating, the stressed samples shouldbe analyzed using related substances methodconditions.• P lease state the attempts you have made toensure that all the impurities including thedegradation p roducts o f t he u nstressed a nd t hestressed samples are captured by your analyti-cal method.ragine MaheswaranaBout tHe autHorragine Maheswaran, Ph.d., is a CMC reviewer at the office of generic drugs within the office of Pharmaceutical Science, under the uS Food and drug administration’s Center for drug evaluation and research and may be reached by e-mail at ragine.Maheswaran@.[For more author information, go to /bios• P lease provide a list summarizing the amountof d egradation p roducts (known a nd u nknown)in your stressed samples.• P lease verify the peak height requirement ofyour s oftware f or t he p eak p urity d etermination.• P lease e xplain t he m ass i mbalance o f t he s tressedsamples.• P lease identify the degradation products thatare formed due to drug-excipient interactions.• y our photostability study shows that the drugproduct is very sensitive to light. Please explainhow this is reflected in the analytical method,manufacturing process, product handling, etc.in an attempt to minimize deficiencies in the aNda submissions, some general recommendations to conduct forced degradation studies, to report relevant information in the submission, and to utilize the knowledge of forced degradation in developing stability indicating analytical methods, manufacturing process, product handling, and storage are provided in this article.StreSS CoNditioNStypical stress tests include four main degradation mecha-nisms: heat, hydrolytic, oxidative, and photolytic degrada-tion. Selecting suitable reagents such as the concentration of acid, base, or oxidizing agent and varying the conditions (e.g., temperature) and length of exposure can achieve the preferred level of degradation. over-stressing a sample may lead to the formation of secondary degradants that would not be seen in formal shelf-life stability studies and under-stressing may not serve the purpose of stress test-ing. therefore, it is necessary to control the degradation to a desired level. a generic approach for stress testing has been proposed to achieve purposeful degradation that is predictive of long-term and accelerated storage condi-tions (7). the generally recommended degradation varies between 5-20% degradation (7-10). this range covers the generally permissible 10% degradation for small molecule pharmaceutical drug products, for which the stability limit is 90%-110% of the label claim. although there are refer-ences in the literature that mention a wider recommended range (e.g., 10-30%), the more extreme stress conditions often provide data that are confounded with secondary degradation products.PhotostabilityPhotostability testing should be an integral part of stress testing, especially for photo-labile compounds. Some recommended conditions for photostability testing are described in iCH Q1B Photostability testing of New drug Substances and Products (2). Samples of drug substance, and solid/liquid drug product, should be exposed to a minimum of 1.2 million lux hours and 200 watt hours per square meter light. the same samples should be exposed to both white and uv light. to minimize the effect of temperature changes during exposure, tempera-ture control may be necessary. the light-exposed samples should be analyzed for any changes in physical proper-ties such as appearance, clarity, color of solution, and for assay and degradants. the decision tree outlined in the iCH Q1B can be used to determine the photo stability testing conditions for drug products. the product label-ing should reflect the appropriate storage conditions. it is also important to note that the labeling for generic drug products should be concordant with that of the reference listed drug (rld) and with united States Pharmacopeia (uSP) monograph recommendations, as applicable. Heatthermal stress testing (e.g., dry heat and wet heat) should be more strenuous than recommended iCH Q1a accel-erated testing conditions. Samples of solid-state drug substances and drug products should be exposed to dry and wet heat, whereas liquid drug products can be exposed to dry heat. it is recommended that the effect of temperature be studied in 10ºC increments above that for routine accelerated testing, and humidity at 75% rela-tive humidity or greater (1). Studies may be conducted at higher temperatures for a shorter period (10). testing at multiple time points could provide information on the rate of degradation and primary and secondary degrada-tion products. in the event that the stress conditions pro-duce little or no degradation due to the stability of a drug molecule, one should ensure that the stress applied is in excess of the energy applied by accelerated conditions (40º for 6 months) before terminating the stress study. acid and Base Hydrolysisacid and base hydrolytic stress testing can be carried out for drug substances and drug products in solution at ambient temperature or at elevated t emperatures. the selection of the type and concentrations of an acid or a base depends on the stability of the drug substance.a strategy for generating relevant stressed samples for hydrolysis is stated as subjecting the drug substance solution to various pHs (e.g., 2, 7, 10-12) at room tem-perature for two weeks or up to a maximum of 15% degradation (7). Hydrochloric acid or sulfuric acid (0.1 M to 1 M) for acid hydrolysis and sodium hydroxide or potassium hydroxide (0.1 M to 1 M) for base hydrolysis are suggested as suitable reagents for hydrolysis (10). For lipophilic drugs, inert co-solvents may be used tosolubilize the drug substance. attention should be given to the functional groups present in the drug molecule when selecting a co-solvent. Prior knowledge of a com-pound can be useful in selecting the stress conditions. For instance, if a compound contains ester functionality and is very labile to base hydrolysis, low concentrations of a base can be used. analysis of samples at various intervals can provide information on the progress of degradation and help to distinguish primary degradants from secondary degradants.oxidationoxidative degradation can be complex. although hydro-gen peroxide is used predominantly because it mimics possible presence of peroxides in excipients, other oxi-dizing agents such as metal ions, oxygen, and radical initiators (e.g., azobisisobutyronitrile, aiBN) can also be used. Selection of an oxidizing agent, its concentration, and conditions depends on the drug substance. Solutions of drug substances and solid/liquid drug products can be subjected to oxidative degradation. it is reported that subjecting the solutions to 0.1%-3% hydrogen peroxide at neutral pH and room temperature for seven days or up to a maximum 20% degradation could potentially generate relevant degradation products (10). Samples can be analyzed at different time intervals to determine the desired level of degradation.different stress conditions may generate the same or different degradants. the type and extent of degradation depend on the functional groups of the drug molecule and the stress conditions.aNalySiS MetHodthe preferred method of analysis for a stability indicating assay is reverse-phase high-performance liquid chroma-tography (HPlC). reverse-phase HPlC is preferred for several reasons, such as its compatibility with aqueous and organic solutions, high precision, sensitivity, and ability to detect polar compounds. Separation of peaks can be carried out by selecting appropriate column type, column temperature, and making adjustment to mobile phase pH. Poorly-retained, highly polar impurities should be resolved from the solvent front. as part of method development, a gradient elution method with varying mobile phase composition (very low organic composi-tion to high organic composition) may be carried out to capture early eluting highly polar compounds and highly retained nonpolar compounds. Stressed samples can also be screened with the gradient method to assess poten-tial elution pattern. Sample solvent and mobile phase should be selected to afford compatibility with the drug substance, potential impurities, and degradants. Stress sample preparation should mimic the sample preparation outlined in the analytical procedure as closely as possible. Neutralization or dilution of samples may be necessary for acid and base hydrolyzed samples. Chromatographic profiles of stressed samples should be compared to those of relevant blanks (containing no active) and unstressed samples to determine the origin of peaks. the blank peaks should be excluded from calculations. the amount of impurities (known and unknown) obtained under each stress condition should be provided along with the chromatograms (full scale and expanded scale show-ing all the peaks) of blanks, unstressed, and stressed samples. additionally, chiral drugs should be analyzed with chiral methods to establish stereochemical purity and stability (11, 12).the analytical method of choice should be sensitive enough to detect impurities at low levels (i.e., 0.05% of the analyte of interest or lower), and the peak responses should fall within the range of detector’s linearity. the analytical method should be capable of capturing all the impurities formed during a formal stability study at or below iCH threshold limits (13, 14). degradation product identifica-tion and characterization are to be performed based on for-mal stability results in accordance with iCH requirements. Conventional methods (e.g., column chromatography) or hyphenated techniques (e.g., lC-MS, lC-NMr) can be used in the identification and characterization of the degradation products. use of these techniques can provide better insight into the structure of the impurities that could add to the knowledge space of potential structural alerts for genotoxicity and the control of such impurities with tighter limits (12-17). it should be noted that structural characterization of degradation products is necessary for those impurities that are formed during formal shelf-life stability studies and are above the qualification threshold limit (13).various detection types can be used to analyze stressed samples such as uv and mass spectroscopy. the detec-tor should contain 3d data capabilities such as diode array detectors or mass spectrometers to be able to detect spectral non-homogeneity. diode array detection also offers the possibility of checking peak profile for multiple wavelengths. the limitation of diode array arises when the uv profiles are similar for analyte peak and impurity or degradant peak and the noise level of the system is high to mask the co-eluting impurities or degradants. Compounds of similar molecular weights and functional groups such as diastereoisomers may exhibit similar uv profiles. in such cases, attempts must be made to modify the chromatographic parameters to achieve necessaryseparation. an optimal wavelength should be selected to detect and quantitate all the potential impurities and degradants. use of more than one wavelength may be necessary, if there is no overlap in the uv profile of an analyte and impurity or degradant peaks. a valuable tool in method development is the overlay of separation signals at different wavelengths to discover dissimilarities in peak profiles.Peak Purity analysisPeak purity is used as an aid in stability indicating meth-od development. the spectral uniqueness of a compound is used to establish peak purity when co-eluting com-pounds are present.Peak purity or peak homogeneity of the peaks of interest of unstressed and stressed samples should be established using spectral information from a diode array detector. when instrument software is used for the determination of spectral purity of a peak, relevant parameters should be set up in accordance with the man-ufacturer’s guidance. attention should be given to the peak height requirement for establishing spectral purity. uv detection becomes non linear at higher absorbance values. thresholds should be set such that co-eluting peaks can be detected. optimum location of reference spectra should also be selected. the ability of the soft-ware to automatically correct spectra for continuously changing solvent background in gradient separations should be ascertained.establishing peak purity is not an absolute proof that the peak is pure and that there is no co-elution with the peak of interest. limitations to peak purity arise when co-eluting peaks are spectrally similar, or below the detec-tion limit, or a peak has no chromophore, or when they are not resolved at all.Mass BalanceMass balance establishes adequacy of a stability indicat-ing method though it is not achievable in all circum-stances. it is performed by adding the assay value and the amounts of impurities and degradants to evaluate the closeness to 100% of the initial value (unstressed assay value) with due consideration of the margin of analytical error (1).Some attempt should be made to establish a mass balance for all stressed samples. Mass imbalance should be explored and an explanation should be provided. varying responses of analyte and impurity peaks due to differences in uv absorption should also be examined by the use of external standards. Potential loss of volatile impurities, formation of non-uv absorbing compounds, formation of early eluants, and potential retention of compounds in the column should be explored. alternate detection techniques such as ri lC/MS may be employed to account for non-uv absorbing degradants. terMiNatioN oF StudyStress testing could be terminated after ensuring adequate exposure to stress conditions. typical a ctivation energy of drug substance molecules varies from 12-24 kcal/mol (18). a compound may not necessarily degrade under every single stress condition, and general guideline on exposure limit is cited in a review article (10). in cir-cumstances where some stable drugs do not show any degradation under any of the stress conditions, specificity of an analytical method can be established by spiking the drug substance or placebo with known impurities and establishing adequate separation.otHer CoNSideratioNSStress testing may not be necessary for drug substances and drug products that have pharmacopeial methods and are used within the limitations outlined in uSP <621>. in the case where a generic drug product uses a different polymorphic form from the rld, the drug substance should be subjected to stress testing to evaluate the physiochemical changes of the polymorphic form because different polymorphic forms may exhibit dif-ferent stability characteristics.ForCed degradatioNiN QBd ParadigMa systematic process of manufacturing quality drug prod-ucts that meet the predefined targets for the critical quality attributes (CQa) necessitates the use of knowledge obtained in forced degradation studies.a well-designed, forced degradation study is indis-pensable for analytical method development in a Qbd paradigm. it helps to establish the specificity of a stability indicating method and to predict potential degradation products that could form during formal stability studies. incorporating all potential impurities in the analytical method and establishing the peak purity of the peaks of interest helps to avoid unnecessary method re-development and revalidation.Knowledge of chemical behavior of drug substances under various stress conditions can also provide useful information regarding the selection of excipients for formu-lation development. excipient compatibility is an integral part of understanding potential formulation interactions during product development and is a key part of product understanding. degradation products due to drug-excipi-ent interaction or drug-drug interaction in combina-tion products can be examined by stressing samples of drug substance, drug product, and placebo separately and comparing the impurity profiles. information obtained regarding drug-related peaks and non-drug-related peaks can be used in the selection and devel-opment of more stable formulations. For instance, if a drug substance is labile to oxidation, addition of an antioxidant may be considered for the formulation. For drug substances that are labile to acid or undergo stereochemical conversion in acidic medium, delayed-release formulations may be necessary. acid/base hydrolysis testing can also provide useful insight in the formulation of drug products that are liquids or suspensions.Knowledge gained in forced degradation studies can facilitate improvements in the manufacturing process. if a photostability study shows a drug substance to be photolabile, caution should be taken during the manufacturing process of the drug product. useful information regarding process development (e.g., wet versus dry processing, temperature selection) can be obtained from thermal stress testing of drug substance and drug product.additionally, increased scientific understanding of degradation products and mechanisms may help to determine the factors that could contribute to stability failures such as ambient temperature, humidity, and light. appropriate selection of packaging materials can be made to protect against such factors. CoNCluSioNan appropriately-designed stress study meshes well with the Qbd approaches currently being promoted in the pharmaceutical industry. a well-designed stress study can provide insight in choosing the appropriate formulation for a proposed product prior to inten-sive formulation development studies. a thorough knowledge of degradation, including mechanistic understanding of potential degradation pathways, is the basis of a Qbd approach for analytical method development and is crucial in setting acceptance criteria for shelf-life monitoring. Stress testing can provide useful insight into the selection of physical form, stereochemical stability of a drug substance, packaging, and storage conditions. it is important to perform stress testing for generic drugs due to allowable qualitative and quantitative differences in formulation with respect to the rld, selection of manufacturing process, processing parameters, and packaging materials.reFereNCeS1. iCH, Q1a(r2) Stability testing of New drug Substances andProducts, geneva, February 2003.2. iCH, Q1B Stability testing: Photostability testing of New drugSubstances and Products, geneva, November 1996.3. H. Brittain, analytical Profiles of drug Substances and excipients,academic Press, london.4. a. Srinivasan and r. iser, Pharm. technol. 34(1), 50-59, 2010.5. a. Srinivasan, r. iser, and d. gill, Pharm. technol. 34(8), 45-51, 2010.6. a. Srinivasan, r. iser, and d. gill, Pharm. technol. 35(2), 58-67, 2011.7. S. Klick, et al., Pharm.technol. 29(2) 48-66, 2005.8. K. M. alsante, l. Martin and S. w. Baertschi, Pharm.technol.27(2) 60-72, 2003.9. d. w. reynolds, K. l. Facchine, J. F. Mullaney, K. M. alsante,t. d. Hatajik, and M. g. Motto, Pharm.technol. 26(2), 48-56, 2002.10. K. M. alsante, a. ando, r. Brown, J. ensing, t. d. Hatajik, w.Kong, and y. tsuda, advanced drug delivery reviews 59, 29-37 (2007).11. Fda, guidance for industry on analytical Procedures and methodsvalidation Chemistry, Manufacturing, and C ontrols documenta-tion (draft), rockville, Md, august 2000.12. iCH, Q6a: Specifications: test Procedures and acceptance Crite-ria for New drug Substances and New drug Products: Chemical Substances, geneva, october 1999.13. iCH, Q3a(r2) impurities in New drug Substances, geneva,october 2006.14. iCH, Q3B(r2) impurities in New drug Products, geneva, June2006.15. Fda, guidance for industry aNdas: impurities in drug Sub-stances (draft), rockville, Md, august 2005.16. Fda, guidance for industry aNdas: impurities in drug Products(draft), rockville, Md, November 2010.17. eMea, guideline on the limits of genotoxic impurities, Com-mittee for Medical Products for Human use (CHMP) (doc. ref eMea/CHMP/QwP/251344/2006), Jan. 1, 2007.18. K. a. Conners et al., Chemical Stability of Pharmaceuticals,wiley and Sons, New york, New york, 2nd ed., 1986) p.19.JvtaCKNowledgMeNtS aNd diSClaiMerthe author would like to thank Bob iser, Naiqi ya, dave Skanchy, Bing wu, and ashley Jung for their scientific input and support.disclaimer: the views and opinions in this articleare only those of the author and do not necessarily reflect the views or policies of the uS Food and drug administration.。

如何分析强降解试验质量不守恒强降解试验的目的并非是为了实现分析结果的质量平衡，而是对降解化学有一个比较全面的了解：1）了解药物的降解路径及分子内稳定性；2）建立稳定性指示分析方法，使其适用于样品检测；3）为药品的处方、工艺、包装、贮藏条件的确定提供有益支持，以便于稳定性试验的顺利进行。

在一些情况下，降解产物（杂质）质量（或摩尔数）的增加小于母体化合物（主成分）质量（或摩尔数）相应的减少。

问题的潜在来源和解决方法如下所述：（一）降解产物在色谱柱上未被洗脱假定母体化合物和所有降解产物都是完全可溶的，并且可以通过HPLC-UV 检测。

有以下方法可以诊断此问题：（a）可以修改HPLC方法以洗脱其他杂质可以通过增加流动相的强度（即增大有机相比例）或增加分析时间来修改HPLC方法，以洗脱保留较强的非极性化合物。

（b）可以使用紫外分光光度法将部分降解的样品与未降解进行分析，并将结果与通过HPLC分析获得的结果进行比较。

此方法对于使用UV检测器的HPLC方法很有用。

由于紫外分光光度法不涉及分离，因此不会由于化合物保留在色谱柱上而被遗漏。

使用这种方法，可以将部分降解的样品在流动相中溶解或稀释。

获得部分降解样品完整的UV（或VIS）光谱，并将其与未降解样品的光谱进行比较。

在HPLC方法中使用的波长下获得部分降解样品与未降解样品的吸光度之比。

然后将该吸光度比与通过HPLC方法获得的部分降解样品与未降解样品的总峰面积之比进行比较。

如果使用HPLC方法检测到所有杂质，则部分降解样品的总HPLC峰面积除以未降解样品的HPLC 峰面积应等于吸光度比。

如果HPLC方法利用光电二极管阵列（PDA）检测器，则可以根据需要在多个波长下确定比较。

如果HPLC面积比明显小于分光光度法吸光度比，则存在一种或多种降解产物没有从色谱柱上洗脱。

（c）可以使用不接色谱柱的HPLC系统分析样品（即流动注射分析）。

在该实验中，用双通代替HPLC系统中的色谱柱，将获得的总峰面积与使用色谱柱时获得的总峰面积进行比较。

破坏试验的具体做法与要求破坏试验的具体做法与要求破坏试验，也称为强制降解试验(stressing test),在人为设定的特殊条件下，如酸、碱、氧化、高温、光照等，引起药物的降解，通过对降解产物的测定，验证检测方法的可行性，同时分析药物可能的降解途径和降解机制。

破坏性试验的设计常结合具体药物的特点，选择合适的条件，使药物在每种环境下尽可能都有一定量的降解，并根据剂型的特点充分分析药物的降解途径和降解机制，保证试验的意义。

对于相对稳定的药物，可增强破坏的强度，至少在1种条件下的降解达到10%,很少有药物对所有条件都稳定。

1、酸破坏试验一般选择0、1〜lmol/L的盐酸，在室温或加热条件下进行考察。

一般配制2倍浓度溶液，测定时以方便用等浓度的碱溶液调节pH值至中性。

2、碱破坏试验一般选择0、1〜1 mol/L的氢氧化钠溶液，在室温或加热条件下进行考察。

一般配制2倍浓度溶液，测定时以方便用等浓度的酸溶液调节pH值至中性。

3、高温破坏试验(热破坏)分别在固体和溶液状态下进行考察。

固体一般6(TC 或者8(TC下15〜30天，溶液可水浴或者1309烘箱下放置数小时。

4、光破坏试验分别在固体和溶液状态下进行考察(考察15〜30天)。

5、氧化破坏试验主要在溶液状态下进行考察，氧化剂可采用饱和的氧气或不同浓度的过氧化氢（双氧水），分别在室温或加热条件下进行考察。

需要同法进行空白试验，如为原料破坏可仅进行氧化破坏空白。

试验结束后需要报告得出明确的结论：药品在各种条件下的稳定特性、降解途径与降解产物，有关物质分析方法是否可用于检查降解产物等。

破坏试验常规要求：1、对于采用IIPLC法测定降解产物时，以主成分计算一般降解10%即可。

并需要采用有效的方法对降解产物进行检测，需要报告测定的回收量，通常应达到90%左右，以证明检测方法的有效性。

2、对于破坏性试验时降解量较大的降解产物，建议结合稳定性研究中加速试验和长期试验的具体杂质数据，参考ICII对新原料药中杂质的规定（每日服用最大剂量不超过2克时，鉴定阈值为0、10%；每日服用最大剂量超过2克时，鉴定阈值为0、05%）,必要时进行定性分析，并作为已知杂质，根据安全性数据，采用已知杂质对照，确定合理的限度，订入质量标准。

酸破坏、碱破坏、高温破坏、高湿破坏、光照破坏、氧化破坏酸破坏样品中酸的浓度一般为1mol/L，量一般2ml，具体多少量以样品能分解就行，产生降解产物本来酸破坏试验就是检验该色谱条件能否使杂质峰与主峰分开，是否能达到控制产品质量的目的。

破坏后加入适量的碱使pH值基本为中性，加入适量的磷酸二氢钾使溶液的pH为4~5，这样可以延长色谱柱的使用寿命。

破坏试验的具体做法与要求破坏试验，也称为强制降解试验（stressing test），在人为设定的特殊条件下，如酸、碱、氧化、高温、光照等，引起药物的降解，通过对降解产物的测定，验证检测方法的可行性，同时分析药物可能的降解途径和降解机制。

破坏性试验的设计常结合具体药物的特点，选择合适的条件，使药物在每种环境下尽可能都有一定量的降解，并根据剂型的特点充分分析药物的降解途径和降解机制，保证试验的意义。

对于相对稳定的药物，可增强破坏的强度，至少在1种条件下的降解达到10%，很少有药物对所有条件都稳定。

1.酸破坏试验一般选择0.1～1mol/L的盐酸，在室温或加热条件下进行考察。

一般配制2倍浓度溶液，测定时以方便用等浓度的碱溶液调节pH值至中性。

2.碱破坏试验一般选择0.1～1 mol/L的氢氧化钠溶液，在室温或加热条件下进行考察。

一般配制2倍浓度溶液，测定时以方便用等浓度的酸溶液调节pH值至中性。

3.高温破坏试验（热破坏）分别在固体和溶液状态下进行考察。

固体一般60℃或者80℃下15～30天，溶液可水浴或者130℃烘箱下放置数小时。

4.光破坏试验分别在固体和溶液状态下进行考察（考察15～30天）。

5.氧化破坏试验主要在溶液状态下进行考察，氧化剂可采用饱和的氧气或不同浓度的过氧化氢（双氧水），分别在室温或加热条件下进行考察。

需要同法进行空白试验，如为原料破坏可仅进行氧化破坏空白。

试验结束后需要报告得出明确的结论：药品在各种条件下的稳定特性、降解途径与降解产物，有关物质分析方法是否可用于检查降解产物等。

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破坏性试验，也称为强制降解试验（stressing test），它是在人为设定的特殊条件下，如酸、碱、氧化、高温、光照等，引起药物的降解，通过对降解产物的测定，验证检测方法的可行性，分析药物可能的降解途径和降解机制。

每项破坏性试验通常包括以下内容：酸降解一般采用L－1mol/L盐酸或硫酸；碱降解采用L－1mol/L的氢氧化钠溶液；氧化降解采用合适的过氧化氢溶液。

以上三种试验，为了加快反应或者提高降解强度，必要时可以加热或提高浓度；高温试验通常温度高于加速试验温度的10℃，如50℃、60℃等，对于原料药有时需考虑水溶液或混悬液的降解，或者考虑在不同的pH值条件下的降解；光照试验条件可采用4500LX。

破坏性试验的具体条件，与具体药物密切相关，需结合具体药物的特点，选择合适的条件，使药物有一定量的降解，并对可能的降解途径和降解机制进行分析，保证实验的意义。

药物经强力破坏产生的降解产物通常采用色谱法测定，需结合药物和可能降解产物的理化性质，选择不同的色谱方法（HPLC、GC、TLC）或检测器，有时可采用不同分离机理的色谱系统。

下面以HPLC法分析降解产物为例，说明在进行破坏性试验时的关注点和存在的问题：1、在选定的破坏条件下，药物应有一定量的降解。

虽然不是每一种破坏性条件都使药物产生降解产物，但一般情况下，很少有一种化合物对每一种破坏性试验条件都稳定，因此，可以通过试验，选择合适的条件，如提高酸、碱、氧化的浓度或者通过加热等，使药物降解。

对于采用HPLC法测定降解产物时，以主成分计算，一般降解10%左右。

应采用有效的方法对降解产物进行检测，关注测定的回收量，通常应达到90%左右，证明检测方法的有效性。

对于破坏性试验时降解量较大的降解产物，建议结合稳定性研究中加速试验和长期试验的具体杂质数据，参考ICH对新原料药中杂质的规定（每日服用最大剂量不超过2克时，鉴定阈值为%；每日服用最大剂量超过2克时，鉴定阈值为%。

FDA关于ANDA强制降解试验的观点：强制降解试验为方法学验证中的重要内容，为了解国外对强制降解试验的要求，根据Pharmaceutical Technology第36卷5期中“FDAPerspectives: Scientific Considerations of Forced DegradationStudies in ANDA Submissions”一文（发布时间为2012年5月2日，作者为Ragine Maheswaran），对FDA关于强制降解试验的相关要求进行了翻译整理，具体内容如下：一、强制降解试验简介强制降解试验也称破坏性试验，其试验目的明确。

强制降解试验可预测原料药的稳定性或影响制剂的纯度、有效性和安全性的因素。

了解不同破坏条件下药物的降解产物和降解途径是非常必要的。

强制降解试验可以为分析方法的建立、说明书的制定和处方设计的确定等提供有益的参考。

样品破坏的程度取决于药物本身的性质和产品的剂型。

ICHQ1B为光稳定性试验提出了一些建议，在ICH稳定性指导原则和验证指南中，没有可以参考的关于其他降解条件的建议，对于氧化和水解降解研究也仅有有限的信息。

原料药与辅料分析方面的药物专著可以为不同原料药的各降解条件提供参考。

二、仿制药强制降解试验研究存在的问题仿制药申请时提供的强制降解试验研究数据不完整是申报的一大缺陷。

美国仿制药申报常见缺陷解读（CMC部分）已经出版，常见的一些例子说明，强制降解试验的缺陷包括以下几个方面：原料药在各破坏条件下均不产生降解。

请重复破坏试验以获得足够的降解产物,若没有产生降解,请提供依据。

破坏条件过于剧烈，导致大部分药物均被降解。

请用温和的破坏条件或减少样品暴露时间以产生相关的降解产物。

请注意即使你已经用含量测定的方法对破坏的样品进行了检测，为了验证有关物质的检测方法具有稳定性指示功能，破坏的样品也应用有关物质的方法进行测定。

请提供所做的验证试验数据，以证明用以检测未破坏样品和破坏样品的方法能够检测出所有的降解杂质。

不合理的强制降解实验近年来，科学界对于环境保护和可持续发展的重要性有了更深刻的认识。

然而，令人担忧的是，一些不合理的强制降解实验正在进行，给生态环境带来了严重的破坏。

本文将探讨这些实验的问题，并呼吁采取措施来阻止这种不负责任的行为。

不合理的强制降解实验往往忽视了生态系统的复杂性和脆弱性。

生态系统是一个相互依存、相互作用的系统，其中的每个组成部分都发挥着重要的作用。

然而，一些实验将生态系统简化为几个因素，忽略了其他重要的环境因素，导致实验结果的不准确性和不可靠性。

这些实验往往缺乏科学的依据和合理的设计。

科学研究应该建立在充分的理论基础上，并遵循科学方法的原则。

然而，一些实验仅仅是凭主观判断和个人意愿进行，缺乏科学的严谨性和可重复性。

这种不合理的实验设计不仅浪费了宝贵的资源，还可能导致错误的结论和误导他人。

不合理的强制降解实验往往忽视了伦理和道德的考量。

生态环境是我们赖以生存的基础，我们有责任保护和维护它的完整性。

然而，一些实验将生态环境当作实验场地，对其进行强制性的破坏，这是对生态环境的不尊重和不负责任的表现。

我们应该意识到，保护环境是我们每个人的责任，不能为了一己私利而损害整个生态系统的健康。

为了解决这些问题，我们需要采取一系列的措施。

首先，科学界应加强对不合理实验的监管和评估，确保实验的合理性和科学性。

其次，政府和相关部门应加强对环境保护的法律法规的制定和执行，对违规行为进行严厉的处罚。

此外，公众应加强环境保护意识，积极参与到环境保护行动中来，共同守护我们的家园。

不合理的强制降解实验给生态环境带来了严重的破坏，需要引起我们的高度关注和重视。

我们应该认识到生态环境的重要性，采取行动来保护和维护它的完整性。

只有通过科学的研究和合理的实践，我们才能实现可持续发展的目标，为子孙后代留下一个美好的家园。

让我们共同努力，为环境保护贡献自己的一份力量。

二羟丙茶碱强制降解实验方案一、实验目的:验证二羟丙茶碱中的相应杂质峰出现的依据。

二、实验方案：1、仪器方法：照高效液相色谱法实验，用十八烷基硅烷键合硅胶为填充剂；以磷酸二氢钾溶液（取磷酸二氢钾1.0g，加水溶解并稀释至1000ml）--甲醇（72：28）为流动相；检测波长为254nm，流速为0.8，保留时间为90min。

2、实验方法及步骤：2.1、酸性溶液中高温实验：酸性溶液高温下样品处理，分别精密称取样品1.0g，置25ml 量瓶中，加10ml的1mol/L的盐酸溶液溶解。

置100℃水浴中加热约2小时，4小时，分别取出放冷至室温，取1ml加1mol/L的氢氧化钠溶液，用水稀释至100ml。

2.2、碱性溶液中高温实验：碱性溶液高温下样品处理，分别精密称取样品 1.0g，置25ml 量瓶中，加10ml的1mol/L的氢氧化钠溶液溶解。

置100℃水浴中加热约2小时，4小时，分别取出放冷至室温，取1ml加1mol/L的盐酸溶液，用水稀释至100ml。

2.3氧化降解实验：精密称取样品0.1g两份，分别置于2个100ml容量瓶中，分别加入5ml 水使溶解，加入10%过氧化氢溶液2ml，摇匀，一份放置2小时，另一份放置4小时，加水溶解稀释至刻度，摇匀，即得。

三、实验结果：通过对二羟丙茶碱样品20190605、20190306以及茶碱样品进行上述实验，样品20190605中正常样品以及经过处理的样品均未检出相应的杂质峰；样品20190306中正常样品、氧化降解2h、4h以及酸性高温2h、4h均检出相应的杂质峰（65min左右）；在碱性高温2h、4h均未检出相应的杂质峰；茶碱样品中正常样品以及经过处理的样品均未检出相应的杂质峰。

四、实验结论：经过实验未得出影响相应杂质峰出现的原因；在碱性高温条件下可导致相应的杂质峰消失。