Enrichment of Relevant Oxidative Degradation Products in Pharmaceuticals With Targeted Chemoselective Oxidation

The ability to produce and isolate relatively pure amounts of relevant degradation products is key to several aspects of drug product development: (a) aid in the unambiguous structural identification of such degradation products, fulfilling regulatory requirements to develop safe formulations (International Conference on Harmonization Q3B and M7); (b) pursue as appropriate safety evaluations with such material, such as chronic toxicology or Ames testing; (c) for a specified degradation product in a late-stage regulatory filing, use pure and well-characterized material as the analytical standard. Producing such materials is often a resource- and time-intensive activity, either relying on the isolation of slowly formed degradation products from stressed drug product or by re-purposing the drug substance synthetic route. This problem is exacerbated if the material of interest is an oxidative degradation product, because typical oxidative stressing (H₂O₂ and radical initiators) tends to produce a myriad of irrelevant species beyond a certain stress threshold, greatly complicating attempts for isolating the relevant degradation product. In this article, we present reagents and methods that may allow the rapid and selective enrichment of active pharmaceutical ingredient with the desired oxidative degradation product, which can then be isolated and used for purposes described above.     

Oxidative Degradation of a Sulfonamide-Containing 5,6-Dihydro-4-Hydroxy-2-Pyrone in Aqueous/Organic Cosolvent Mixtures

Purpose. To predict the oxidative stability of a sulfonamide-containing 5,6-dihydro-4-hydroxy-2-pyrone in lipid-based delivery systems, N-(3-{1[(3,6R)-4-hydroxy-2-oxo-6-phenyl-6-propyltetrahydro-2H-pyran-3-yl]propyl}phenyl)-5-(trifluoromethyl)-2-pyridinylsulfonamide (DHP) was oxidized by peroxides and peroxyl radicals in binary mixtures of water and organic cosolvents. Methods. DHP was oxidized by hydrogen peroxide, t-butylhydroperoxide, or peroxyl radicals derived from the thermal decomposition of 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) in 40% (v/v) organic cosolvent and 5 mM buffer at or near 40°C. Interactions between DHP and ]propane sulfonic acid and imidazole) and DH^– were assessed by ¹H-NMR spectroscopy. The formation of CO likely involves a free radical mechanism. Results. The reaction of DHP with peroxides in 40% (v/v) acetonitrile yields epimeric monohydroxylation products, R-OH and S-OH, at C-3 of the pyrone ring, and a keto-derivative (CO). Hydroxylation rates depend on the protonation state of DHP, and the nature of buffer and the organic cosolvent. Organonitriles accelerate the oxidation through formation of peroxycarboximidic acid. Peroxyl radicals do not yield significant amounts of R/S-OH or CO. Conclusions. The hydrogen peroxide-induced degradation of DHP in the presence of acetonitrile involves two reactions, hydroxylation and carbonyl formatin. Hydroxylation proceeds via nucleophilic attack by the monodeprotonated form of DHP (DH^–) on peroxycarboximidic acid. The oxidation rate is slowed by ion pairing between nitrogen-containing buffers ([3-N-morpholino]propane sulfonic acid and imidazole) and DH^–. The formation of CO likely involves a free radical mechanism.     

Forced Oxidative Degradation Pathways of the Imidazole Moiety of Daclatasvir

Daclatasvir hydrochloride (DCV) is the active pharmaceutical ingredient of Daklinza, a marketed product for the treatment of hepatitis C viral infection. The intrinsic stability of daclatasvir was evaluated via a forced degradation study. DCV was found to be stable in the solid state. In solution, its carbamate moiety is susceptible to basic hydrolysis, whereas its imidazole is liable to base-mediated autoxidation to form degradants 1 and 3, 7-8, respectively. The imidazole moiety can also be oxidized to form degradants 6-7 in the presence of hydrogen peroxide or azobisisobutyronitrile. The chloro-adduct degradant 9 was also observed in hydrogen peroxide solution. Furthermore, the imidazole moiety is sensitive to photodegradation in solution. Degradants 2-8 were observed in a solution of DCV exposed to high intensity light/UV light; the formation of degradants 2 and 5-8 was postulated through 4 degradation pathways. The degradants 3 and 4 were deemed to be secondary degradants of 7 and 5, respectively.     

迷迭香酸在不同溶剂中的热稳定性及其降解产物研究

目的 探讨迷迭香酸对照品在不同溶剂中加热温度和加热时间对其稳定性的影响,并对其在最不稳定的溶剂中的主要降解产物进行定性分析以阐释其降解途径。方法 将迷迭香酸对照品分别溶于水、70%乙醇、甲醇中,分别在不同水浴温度（20~100 ℃）下加热4 h,每隔1 h取样。采用HPLC测定迷迭香酸加热前后色谱峰的峰面积,计算其剩余率,同时采用HPLC-MS对其主要降解产物进行鉴定。结果 当加热温度≤60 ℃时,水溶液、70%乙醇溶液、甲醇溶液中的迷迭香酸剩余率均>95%,且甲醇溶液中的迷迭香酸剩余率高于水溶液和70%乙醇溶液;当迷迭香酸加热时间达到4 h时,水溶液和70%乙醇溶液中的迷迭香酸剩余率显著减小,甲醇溶液中的迷迭香酸剩余率几乎不变。迷迭香酸在水溶液中最不稳定,在水溶液中的降解途径主要有酯键水解、双键加成和氧化反应,降解后产生丹参素、咖啡酸、原儿茶醛、丹参酸C及其异构体等产物。结论 迷迭香酸的稳定性在甲醇中较好,70%乙醇中次之,水中最差;加热温度越高或加热时间越长,迷迭香酸的稳定性越差。因此,在迷迭香酸的研究过程中推荐首选甲醇为溶剂;迷迭香酸及含有该成分的中药在提取分离、加工制备和贮存过程中,温度不宜超过60 ℃,加热时间不宜超过4 h。     

草乌甲素的降解动力学

目的研究草乌甲素的降解动力学,考察影响草乌甲素降解的因素。方法采用经典恒温法进行实验,以高效液相色谱法测定草乌甲素的含量。结果草乌甲素的降解呈伪一级动力学,主要受OH-催化。温度升高,草乌甲素降解速率增加。草乌甲素的最稳pH值范围为3~5。草乌甲素在pH 7~9时的降解反应活化能分别为148.04、137.65和102.36 kJ.mol-1,室温(25℃)时的t0.9分别为1 054.00、95.82、7.42 h。碱性条件下,离子强度增加,表观降解速率常数减小。结论草乌甲素的降解反应为伪一级反应。温度、pH值和离子强度显著影响草乌甲素的降解反应。     

Degradation of Synthetic Salmon Calcitonin in Aqueous Solution

Pharmaceutical Research -     

左旋多巴的强降解实验研究

目的为了顺利进行左旋多巴原料药物的工业化大生产，了解左旋多巴的降解因素，为包装材料的选择和药品储存提供科学依据，确保其使用期内的化学稳定性。方法通过碱降解、氧化降解、酸降解、加热降解、光照降解等强制降解试验，测定其含量，考察左旋多巴的化学稳定性。结果左旋多巴在酸溶解静置7h和110℃加热15h条件下稳定；在光照条件下放置5d和10d基本稳定，粉末颜色略有变黄；在氧化条件下不稳定；在碱性奈件下极不稳定。结论左旋多巴原料药物应该密闭，避光保存。     

达比加群酯盐的降解产物研究

目的研究6种达比加群酯盐在不同条件下的降解产物,寻找不同酸根与达比加群酯盐稳定性之间的相互关系。方法将6种盐在高温、高湿和光照条件下分别放置10 d后,利用HPLC和LC-MS法对不同盐的降解产物进行深入分析。结果通过LC-MS法解析得到达比加群酯盐的3种关键降解产物,初步推断出不同杂质的产生与所选用的酸根结构密切相关。结论为了寻找更加稳定的达比加群酯盐,需要选用氧化还原性弱,结构中含氧基团少及p Ka值较低的酸根。     

人参皂苷水溶液热稳定性研究

目的 研究加热时间对人参皂苷水溶液的影响及受热后人参皂苷含量的变化情况。 方法 红参须浓缩液加热不同时间,采用高效液相色谱法测定其人参皂苷Rg₁、Re、Rb₁、Rc和Rd的含量。 结果 5种人参皂苷在加热6 h内,发生不同程度的降解反应,二醇类人参皂苷Rb₁、Rc和Rd在加热2~3 h时,含量呈明显下降趋势,人参皂苷Rd降解速率最慢。三醇类人参皂苷Rg₁和Re在加热3 h内含量快速下降,3 h后趋于平缓。 结论 在常压受热条件下,人参皂苷水溶液主要成分人参皂苷Rg₁、Re、Rb₁、Rc和Rd的含量,随加热时间延长而不断下降,3 h后下降速率减缓。三醇类人参皂苷较二醇类对热更为敏感。     

齐多夫定棕榈酸酯及其脂质体在小鼠各组织中的降解动力学

目的考察齐多夫定棕榈酸酯及其脂质体在小鼠各组织中的降解动力学。方法将齐多夫定棕榈酸酯溶液、齐多夫定棕榈酸酯脂质体分别与小鼠各组织匀浆混合,37℃恒温孵育后,采用HPLC法测定齐多夫定棕榈酸酯浓度。结果齐多夫定棕榈酸酯在小鼠心、肝、脾、肺、肾、脑中的表观降解速率常数(kobs,min-1)依次为:7.5×10-3、5.43×10-2、9.8×10-3、9.9×10-3、2.48×10-2、6.4×10-3。经脂质体包裹的齐多夫定棕榈酸酯在小鼠心、肝、脾、肺、肾、脑组织匀浆中的表观降解速率常数(kobs,min-1)依次为:1.18×10-3、6.70×10-3、1.20×10-3、1.70×10-3、3.00×10-3和1.10×10-3。结论齐多夫定棕榈酸酯及其脂质体在小鼠各组织匀浆中的降解速率不同。经脂质体包裹后,齐多夫定棕榈酸酯在小鼠各组织匀浆中的降解速率降低,表明脂质体对其具有较好的保护作用。     

Early Prediction of Pharmaceutical Oxidation Pathways by Computational Chemistry and Forced Degradation

PURPOSE: To show, using a model study, how electronic structure theory can be applied in combination with LC/UV/MS/MS for the prediction and identification of oxidative degradants. METHODS: The benzyloxazole 1, was used to represent an active pharmaceutical ingredient for oxidative forced degradation studies. Bond dissociation energies (BDEs) calculated at the B3LYP/6-311+G(d,p)//B3LYP/6-31G(d) level with isodesmic corrections were used to predict sites of autoxidation. In addition, frontier molecular orbital (FMO) theory at the Hartree-Fock level was used to predict sites of peroxide oxidation and electron transfer. Compound 1 was then subjected to autoxidation and H2O2 forced degradation as well as formal stability conditions. Samples were analyzed by LC/UV/MS/MS and degradation products proposed. RESULTS: The computational BDEs and FMO analysis of 1 was consistent with the LC/UV/MS/MS data and allowed for structural proposals, which were confirmed by LC/MS/NMR. The autoxidation conditions yielded a number of degradants not observed under peroxide degradation while formal stability conditions gave both peroxide and autoxidation degradants. CONCLUSIONS: Electronic structure methods were successfully applied in combination with LC/UV/MS/MS to predict degradation pathways and assist in spectral identification. The degradation and excipient stability studies highlight the importance of including both peroxide and autoxidation conditions in forced degradation studies.     

Factors Influencing Polysorbate's Sensitivity Against Enzymatic Hydrolysis and Oxidative Degradation

The aim was to compare the sensitivity of different grades of polysorbate 20 (PS20) and polysorbate 80 (PS80) against enzymatic hydrolysis and oxidative degradation in pharmaceutically relevant buffer systems. For this purpose, a fast liquid chromatography charged aerosol detection method was developed which allows to (1) differentiate between hydrolytic and oxidative PS degradation and (2) to monitor the PS decay over time. Systematic enzymatic and oxidative forced degradation studies were conducted with multicompendial PS20 and PS80, as well as all-laurate PS20 and all-oleate PS80 (with >98% oleic acid, as required by the Chinese Pharmacopoiea since 2015). No differences in the sensitivity toward enzymatic degradation were observed between multicompendial PS and high purity grade PS. However, all-laurate PS20 and all-oleate PS80 have a higher predisposition for oxidative degradation as compared to multicompendial PS20 and PS80. The buffer system used within the study played thereby a key role: histidine showed a protective effect against hydrogen peroxide–induced oxidation, whereas hydrogen peroxide oxidation of PS in acetate buffer was severe under the experimental conditions. Furthermore, ethylenediaminetetraacetic acid protected PS20 and PS80 against oxidative degradation in histidine buffer.     

头孢美唑钠的体外降解稳定性

目的:研究头孢美唑钠的体外降解稳定性,为保证临床使用的安全性提供理论依据。方法:通过经典恒温试验,应用HPLC法测定头孢美唑钠在不同温度、不同光照、不同相对湿度、不同pH值、不同离子强度条件下以及不同配伍溶液中的杂质总量来确定其稳定性。结果:头孢美唑钠在不同温度和光照的条件下呈现一级动力学特征,而在不同湿度条件下呈非线性动力学特征。随着离子强度和温度的增加,头孢美唑钠的降解加快;其在pH值5～9之间相对稳定;在注射用水、0.9%氯化钠注射液以及5%葡萄糖注射液此3种不同配伍溶液中8 h内均不稳定。结论:头孢美唑钠对光、热、湿均不稳定,应于凉暗干燥处密封保存。配制成溶液后应尽快使用。     

Degradation Study on Sulfasalazine and a Validated HPLC-UV Method for its Stability Testing

Sulfasalazine (SSZ) was subjected to degradation under the conditions of hydrolysis (acid, alkali, and water), oxidation (30% H₂O₂), dry heat, and photolysis (UV-VIS light) in accordance with the ICH guidelines. An RP-HPLC method was developed to study the degradation behavior. No degradation was noted under any condition except alkaline hydrolysis where SSZ was degraded to a single minor product. SSZ was optimally resolved from this product on an XTerra^® RP18 column with a mobile phase composed of methanol and an ammonium acetate buffer (10 mM, pH 7.0) (48:52, v/v) delivered at a rate of 0.8 mL/min in an isocratic mode. The method was validated and found to be linear (r²=0.99945), precise (%RSD <2), robust, and accurate (94–102%) in the concentration range of 0.5–50 μg/mL of SSZ. The PDA analysis of the degraded sample revealed the SSZ peak purity to be 998.99 and the drug peak eluted with a resolution factor of >2 from the nearest resolving peak, indicating the method to be selectively stability-indicating for the drug analysis. The method was applied successfully for the stability testing of the commercially available SSZ tablets that were under varied ICH-prescribed conditions. An explanation for the unusual stability of the drug when exposed to acidic hydrolysis, despite the presence of the sulfonamide linkage, is also discussed.     

HPLC法测定阿奇霉素在水溶液中的降解动力学

目的研究阿奇霉素在水溶液中的降解动力学,为阿奇霉素液体制剂的开发提供参考。方法通过经典恒温试验,应用HPLC法测定阿奇霉素在不同pH值、不同温度、不同离子强度、不同缓冲液条件下的降解动力学参数。结果阿奇霉素在水溶液中的降解呈现一级动力学特征,其最稳定pH值(pHm)为6.41;随着离子强度和温度的增加,阿奇霉素的降解加快;阿奇霉素在磷酸盐缓冲液中比在醋酸盐、枸橼酸盐缓冲液中相对稳定。结论阿奇霉素降解速率与溶液pH值、缓冲液种类、离子强度以及温度有关;溶液pH值与温度对阿奇霉素降解作用的影响较为明显。     

十二烷基键合固定相的制备及在青霉素降解研究中的应用

采用2，4-戊二酮与正十二烷基二甲基氯硅烷在咪唑的催化下制成活漏的中间体-硅醚烷基中间体，再与硅胶进行键合，用元素分析法测得该固定相中C的质量分数为9.68%。利用该固定相，在流动相为乙腈，0.02mol/L磷酸盐缓冲液（pH6.0)(30:70,含0.006mol/L氯化四丁基铵）的条件下成功地分离了青霉素G及其三种主要降解产物。     

Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS

Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itopride degraded in acid, alkali and oxidative stress conditions. The stability indicating method was developed and validated. The degradation pathway of the drug to products II-VIII is proposed.     

Mechanoradical-Induced Degradation in a Pharmaceutical Blend during High-Shear Processing

Mechanically generated radicals were shown to affect short-term stability of a model pharmaceutical formulation during high-shear processing. A formulation containing an oxidatively sensitive drug, either amorphous or crystalline, and a polymeric excipient was high-shear mixed and the resulting short-term degradation was determined with HPLC. High-shear mixing of the excipients was also carried out before drug addition to isolate effects on excipients versus those directly on the drug. Short-term drug stability was found to be strongly dependent on the amount of shear added to excipients prior to drug addition, regardless of morphology. A mechanism for the observed degradation based on mechanically generated radicals from microcrystalline cellulose is proposed. These results indicate that excipient high-shear exposure needs to be considered in regards to drug stability.     

N1-羧酰-5-氟尿嘧啶系列前体药物的体外降解动力学研究

采用HPLC法测定N1－羧酰－5－氟尿嘧啶系列前体药物在不同pH磷酸盐缓冲溶液中、不同的组织匀浆中的含量以考察降解情况，并数学拟合求取降解动力学方程。同时对光敏感的N1－维甲酰－5－氟尿嘧啶进行了光解动力学研究，还测定了该系列前体药物在正辛醇－水两相中的分配系数。结果表明各前体药物在缓冲溶液和组织匀浆中的降解均为表观一级反应，光解动力学方程为对数曲线，前体药物的分配系数均高于5－氟尿嘧啶。其中N1－维甲酰－5－氟尿嘧啶和N1－山梨酰－5－氟尿嘧啶的药物稳定性较好。     

The Novel Endomorphin Degradation Blockers Tyr-Pro-DClPhe-Phe-NH2 (EMDB-1) and Tyr-Pro-Ala-NH2 (EMDB-2) Prolong Endomorphin-2 Action in Rat Ileum In Vitro

The endogenous opioid system is involved in the control of gastrointestinal (GI) motility. The potential use of endogenous MOR ligands, endomorphins (EMs), as therapeutics is limited because of their rapid enzymatic degradation and short duration of action. Targeting enzymatic degradation is an approach to prolong EM activity. In the present study, we characterized the effects of novel blockers of EM degradation in GI tissue preparation in vitro. The effects of actinonin, diprotin A (DIP) and the novel peptide EM degradation blockers Tyr-Pro-D ClPhe-Phe-NH₂ (EMDB-1), Tyr-Pro-Ala-NH₂ (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3) on EM-2-mediated inhibition of electrically induced cholinergic twitch contractions were compared in rat ileum in vitro using an organ bath. EMDB-1 and EMDB-2 significantly prolonged the inhibitory effect of EM-2 on smooth muscle contractility in rat ileum. EMDB-2 extended the EM-2 action for up to 60 min compared to 10 min in controls and was more potent than the conventional peptidase inhibitor DIP. EMDB-1 and EMDB-2 are potent EM degradation blockers, which prolong the inhibitory effects of EM-2 on smooth muscle contractility in rat ileum. These novel compounds may be of future use when targeting the endogenous opioid system in the treatment of GI motility disorders such as diarrhea.