Development and Validation of Stability Indicating HPLC Method for Simultaneous Estimation of Amoxicillin and Clavulanic Acid in Injection

A simple, fast, precise, accurate and rugged stability indicating high performance liquid chromatography (HPLC) method has been developed for simultaneous estimation of Amoxicillin and Clavulanic acid from injectable dosage form. The stability indicating capability of the method was proven by subjecting the drugs to stress conditions as per ICH recommended test conditions such as alkaline and acid hydrolysis, oxidation, photolysis, thermal degradation and resolution of the degradation products formed therein. The separation was obtained using a mobile phase composition at a ratio of 95:5 (v/v) of pH 5.0 buffer and methanol on Inertsil C18 column (250 × 4.0 mm, 4 μm) with UV detection at 220 nm at a flow rate of 1 ml/minute. The photodiode array detector was used for stress studies. The order of elution of peaks was Clavulanic acid followed by Amoxicillin. The linear calibration range was found to be 79.51 to 315.32 μg/ml for Amoxicillin and 17.82 to 67.90 μg/ml for Clavulanic acid. The Amoxicillin and Clavulanic acid were found to be stable in solution up to 24 hours. The method validation data showed excellent results for precision, linearity, specificity, limit of detection, limit of quantification and robustness. The present method can be successfully used for routine quality control and stability studies.

Development and Validation of Stability Indicating RP-HPLC Method on Core Shell Column for Determination of Degradation and Process Related Impurities of Apixaban—An Anticoagulant Drug

A rapid, specific, sensitive, and precise reverse-phase HPLC method for the quantitative determination of process related and degradation impurities of Apixaban, an anticoagulant drug is described. The developed RP-HPLC method was successfully applied to the analysis of both Apixaban drug substance and drug product. The chromatographic separation was achieved on a Sigma-Aldrich’s Ascentis Express®C18 (4.6 mm × 100 mm, 2.7 μ) HPLC column with a runtime of 40 min. Mobile phase-A and mobile phase-B were phosphate buffer and acetonitrile respectively. The column oven temperature was set at 35°C and photodiode array detector was set at 225 nm. Nine process related impurities (Imp-1 to Imp-9) have been detected in test sample of Apixaban by using newly developed RP-HPLC method. Forced degradation study was carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability-indicating nature of the developed RP-HPLC method. The developed method was validated as per ICH guideline and found to be specific, precise, sensitive and robust.

Validated gradient stability indicating HPLC method for determining Diltiazem Hydrochloride and related substances in bulk drug and novel tablet formulation

A stability-indicating liquid chromatographic method has been developed and validated for the determination of Diltiazem Hydrochloride(DTZ) together with its six related substances(Diltiazem sulphoxide,Imp-A,Imp-B,Imp-D,Imp-E,and Imp-F) in a laboratory mixture as well as in a novel tablet formulation developed in-house.Efficient chromatographic separation was achieved on a Hypersil BDS C18(150 mm*4.6 mm,5.0 μm) with mobile phase containing 0.2% Triethylamine(TEA) in gradient combination with acetonitrile(ACN) at a flow rate of 1.0 mL/min and the eluent was monitored at 240 nm.In the developed method,the resolution of DTZ from any pair of impurities was found to be greater than 2.0.The test solution and related substances were found to be stable in the diluent for 24 h.The developed method resolved the drug from its known impurities,stated above,and also from additional impurities generated when the formulation was subjected to forced degradation;the mass balance was found close to 99.9%.Regression analyses indicate correlation coefficient value greater than 0.997 for DTZ and its six known impurities.The LOD for DTZ and the known impurities was at a level below 0.02%.The method has shown good,consistent recoveries for DTZ(99.8-101.2%) and also for its six known impurities(97.2-101.3%).The method was found to be accurate,precise,linear,specific,sensitive,rugged,robust,and stability-indicating.

Stability study on an anti-cancer drug 4-(3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid (CLEFMA) using a stabilityindicating HPLC method

CLEFMA, 4-(3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid, is a new chemical entity with anti-cancer and anti-inflammatory activities. Here, we report its stability in solution against stress conditions of exposure to acid/base, light, oxidant, high temperature, and plasma. The identity of the degradation products was ascertained by mass and proton nuclear magnetic resonance spectroscopy. To facilitate this study, we developed and validated a reverse phase high performance liquid chromatography method for detection of CLEFMA and its degradation. The method was linear over a range of 1–100 μg/m L; the accuracy and precision were within acceptable limits; it was stability-indicating as it successfully separated cis-/trans-isomers of CLEFMA as well as its degradation product. The major degradation product was produced from amide hydrolysis at maleic acid functionality caused by an acidic buffer, oxidant(3% hydrogen peroxide),or temperature stress(40–60 °C). The log k-p H profile showed that CLEFMA was most stable at neutral p H. In accelerated stability study we found that the shelf-life(T_(90%)) of CLEFMA at 25 °C and 4 °C was 45 days and220 days, respectively. Upon exposure to UV-light(365 nm), the normally prevalent trans-CLEFMA attained cis-configuration. This isomerization also involved the maleic acid moiety. CLEFMA was stable in plasma from which it could be efficiently extracted by an acetonitrile precipitation method. These results indicate that CLEFMA is sensitive to hydrolytic cleavage at its maleic acid moiety, and it is recommended that its samples should be stored under refrigerated and light-free conditions, and under inert environment.

Development and Validation of a Stability-Indicating RP-HPLC Method for Determination of Darifenacin Hydrobromide in Bulk Drugs

An isocratic stability-indicating reversed phase high performance liquid chromatographic method (RP-HPLC) was developed for determination of process related impurities and assay of darifenacin hydrobromide (DRF) in bulk drugs. DRF was subjected to various stress conditions such as hydrolysis (acid, base, and neutral), oxidation, photolysis and thermal degradation as per International Conference on Harmonization (ICH Q1A(R2) and Q1B) prescribed conditions to investigate the stability-indicating ability of the method. Significant degradation was observed during acidic hydrolysis and oxidative stress conditions. The chromatographic separation was accomplished on a Prodigy C8 column (250 × 4.6 mm, 5 μm) with mobile phase consisting of 0.05 M ammonium acetate (pH adjusted to 7.2 by using ammonia solution) and methanol (36% acetonitrile) in 35:65 v/v ratio in an isocratic elution mode at a flow rate of 1.0 mL/min at 25°C. Detection of analytes was carried out using photo diode array detector at a wavelength of 215 nm. The developed LC method was validated with respect to accuracy, linearity, precision, limits of detection and quantitation and robustness as per ICH guidelines.

Evaluation of stability and simultaneous determination of fimasartan and amlodipine by a HPLC method in combination tablets

A simple,rapid,accurate,precise and robust HPLC method was developed for the simultaneous determination of fimasartan and amlodipine in tablet dosage form.Furthermore,stability of active ingredients was evaluated under normal and stress conditions.The isocratic elution was accomplished by Nucleosil C18 column(250 mm×4.6 mm,5 mm)at 40℃.The mobile phase consisted of acetonitrile and 0.02 M monopotassium phosphate buffer(pH 2.2)in the ratio of 50:50(v/v)was eluted at 1.0 ml/min.The eluent was monitored by the UV detector for fimasartan and amlodipine at 237 nm for 8 min,detection time.The validation of HPLC method was carried out in accordance with the ICH guidelines.

A Stability Indicating HPLC Method for Dronedarone in Bulk Drugs and Pharmaceutical Dosage Forms

The objective of the current study was to develop a validated, specific and stability-indicating reverse phase HPLC method for the quantitative determination of Dronedarone and its related substances. The determination was done for active pharmaceutical ingredient and its pharmaceutical dosage forms in the presence of degradation products, and its process-related impurities. The drug was subjected to stress conditions of hydrolysis (acid and base), oxidation, photolysis and thermal degradation per International Conference on Harmonization (ICH) prescribed stress conditions to show the stability-indicating power of the method. Significant degradation was observed during acid, oxidative and photo stress studies. In the developed HPLC method, the resolution between Dronedarone and its process-related impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.999 for Dronedarone and it’s all the five impurities. The chromatographic separation was achieved on a C8 stationary phase. The method employed a linear gradient elution and the detection wavelength was set at 288 nm. The stress samples were assayed against a qualified reference standard and the mass balance was found to be close to 99.6%. The developed HPLC method was validated with respect to linearity, accuracy, precision and robustness.

Development and validation of a stability-indicating RP–HPLC method for estimation of atazanavir sulfate in bulk

A stability-indicating reverse phase–high performance liquid chromatography（RP–HPLC） method was developed and validated for the determination of atazanavir sulfate in tablet dosage forms using C_（18） column Phenomenix（250 mm×4.6 mm, 5 μm） with a mobile phase consisting of 900 mL of HPLC grade methanol and100 mL of water of HPLC grade. The pH was adjusted to 3.55 with acetic acid. The mobile phase was sonicated for 10 min and filtered through a 0.45 μm membrane filter at a flow rate of 0.5 mL/min. The detection was carried out at 249 nm and retention time of atazanavir sulfate was found to be 8.323 min. Linearity was observed from 10 to 90 μg/mL（coefficient of determination R^2 was 0.999） with equation, y=23.427x＋37.732.Atazanavir sulfate was subjected to stress conditions including acidic, alkaline, oxidation, photolysis and thermal degradation, and the results showed that it was more sensitive towards acidic degradation. The method was validated as per ICH guidelines.

Development and Validation of Stability Indicating RP-HPLC-PDA Method for Tenatoprazole and Its Application for Formulation Analysis and Dissolution Study

In the present study, comprehensive stress testing of tenatoprazole was carried out according to ICH guide-line Q1A (R2). Tenatoprazole was subjected to stress conditions of hydrolysis, oxidation, photolysis and neutral decomposition. Extensive degradation was found to occur in acidic, neutral and oxidative conditions. Mild degradation was observed in basic conditions. The drug is relatively stable in the solid-state. Successful separation of drug from degradation products formed under stress conditions was achieved on a Kromasil C18 column (250 mm × 4.6 mm, 5.0 μ particle size) using methanol: THF: acetate buffer (68:12:20 v/v) pH adjusted to 6.0 with acetic acid as mobile phase, flow rate was 1.0 mL●min–1 and column was maintained at 45°C. Quantification and linearity was achieved at 307 nm over the concentration range of 0.5 - 160 μg●mL–1 for tenatoprazole. The method was validated for specificity, linearity, accuracy, precision, LOD, LOQ and robustness.

炎琥宁的HPLC测定

A HPLC method for determination of potassium sodium 14-deoxy-11,12-didehydroandrographolidesuccinate was established. A C18 column was used with the mobile phase of 0.04mol/L KH2PO4-methanol(3∶7) at thedetection wavelength of 250nm. The calibration curve was linear in the range of 0.005 - 2mg/ml and the detection limit was4.4ng. The average recovery was 99.8%, with RSD of 0.64%,

HPLC法测定栀子药材中栀子苷含量的能力验证研究

目的开展栀子药材中栀子苷含量测定的能力验证研究,评价药品相关领域检验检测实验室开展中药材中指标成分含量测定的能力,提高相关实验室含量测定的质控能力。方法依据CNAS-RL02《能力验证规则》和国际标准ISO/IEC 17043《合格评定能力验证的通用要求》进行实验室能力验证活动。根据CNAS-GL003《能力验证样品均匀性和稳定性评价指南》对自制样品均匀性和稳定性试验结果进行分析,试验结果合格后作为能力验证样品,随机分发给参加者,回收结果,并对测定结果进行稳健统计分析,以Z比分数对各实验室结果进行判定,评价结果分为满意、可疑、不满意。结果共有403家实验室提交试验结果,其中能力验证结果为满意的有367家,满意率为91.07%;可疑的有17家,占4.22%;不满意的有19家,不满意率为4.71%。结论403家实验室中,大多数具备HPLC法测定栀子中栀子苷含量的能力,其中药品监管系统实验室检测能力和质量管理水平较高。本次能力验证为了解我国药品检验检测实验室的技术储备能力、管理水平提供依据,为今后的政府监管提供技术支撑。

HPLC测定静脉给药后大鼠血清中5-氟尿嘧啶的含量

目的 建立HPLC用于测定微量 (5 0 μL)血清样品中 5 氟尿嘧啶 (5 Fu)的含量。方法 采用 2 5 0mm× 4.6mm (5 μm)C18色谱柱 ,流动相 5 %甲醇 ,流速 0 .7mL·min-1,紫外检测波长 2 70nm ,内标为对氨基苯甲酸。结果 本方法最低检测浓度为 2 0ng·mL-1,线性范围是 0 .1～ 5 0 μg·mL-1,日内和日间RSD分别是 2 .2 2 %～ 3.81% (n =4)和 2 .42 %～ 3.19% (n =4) ,萃取率为 90 .2 1%～ 95 .6 9%。用本方法成功地测定了大鼠在静脉给药后 5 Fu的时量曲线 ,并计算了药动学参数。结论 本方法简便、快速、灵敏度高、重现性好 ,可适用于 5 氟尿嘧啶的血药浓度测定和药动学的研究。

生物样品中氟尿嘧啶的HPLC测定方法建立及鼠体内药物动力学研究

采用离子抑制技术,以5-溴尿嘧啶(5-Bru)为内标,建立了血 浆及组织中5-氟尿嘧啶(5-Fu)的高效液相色谱测定方法。色谱柱: NOVA PAK C18( 3.9 mm×15 cm, 5 μm); 流动相: 0.013 mol/L K2HPO4; 流速: 0.7 ml/min; 检 测波长 : 265 nm。样品经乙酸乙酯提取后, 血浆、心、肝、肺、肾、脾、脑的平均回收率分别为9 5.3％，89.6％，97.4％，92.5％，91.7％，86.1％，79.6％;5-Fu最低检测浓度分别为 2 0，36，28，16，25，30，20 ng/ml。 运用本文建 立的方法进行了5-Fu鼠体内药物动力学研究。

HPLC法测定河豚毒素的含量及稳定性

目的:建立测定河豚毒素含量的反相高效液相色谱法,并用该方法对河豚毒素及注射剂的稳定性进行测定。方法:以ZORBAX C8(250 mm×4.6 mm,5μm)为色谱分析柱;0.02 mol·L-1磷酸氢二钠-0.02 mol·L-1磷酸二氢钾(1:1)为流动相;流速0.4 mL·min-1;UV检测波长210 nm,柱温为25℃。结果:河豚毒素在0.5～80μg·mL-1范围内线性关系良好,通过该方法的测定,河豚毒素及注射剂在光、热等因素的影响下,含量明显下降,杂质明显增加。结论:该方法可以为河豚毒素原料及注射剂的稳定性研究提供灵敏、准确的检测方法。河豚毒素及注射剂对光和热的稳定性较差,需避光,低温(0～4℃)保存。但河豚毒素原料对光的稳定性优于注射剂。

红花药材黄色素A含量的RP-HPLC测定及其资源的质量评价

目的：为红花药材质量评价体系的建立、红花优良品种的筛选提供依据。方法：选择有代表性的红花品种22个，在不同生态地区和不同年份进行栽培试验，并采用RP-HPLE测定各品种中主要活性成分黄色素A含量，建立药材质量评价标准，比较品种间差异，考察其遗传稳定性。结果：红花不同品种黄色素A的含量在0．70％-1．85％，品种间存在非常显著差异（P〈0．01），其中，鱼台红花、合肥红花、芮城红花含量居前3位。结论：红花药材中的主要活性成分黄色素A为质量评价的重要指标，鱼台红花、合肥红花、芮城红花为红花优良种质资源。

白芍HPLC特征指纹图谱的稳定性考察

目的考察白芍特征指纹成分的稳定性,拟定适用性强的白芍特征指纹成分群。方法采用HPLC法,色谱柱为ZorbaxSB-Aq;流动相为乙腈-0.05%磷酸溶液(梯度洗脱);检测波长为230nm;流速为1mL·min-1;柱温为35℃。主要考察不同溶媒与提取方法、不同热处理时间对指纹图谱的影响。结果共标示出具有代表性的11个共有峰,鉴别了8个成分。不同溶媒与提取方法对没食子酸、苯甲酸、五没食子酰基葡萄糖影响较大,回流法较超声处理法易促进鞣质类水解生成没食子酸或五没食子酰基葡萄糖、苷类成分水解生成苯甲酸,分析白芍指纹图谱以50%乙醇超声处理法制备供试液较稳定;传统煎煮法易促进鞣质类成分转化为没食子酸,苷类成分水解生成苯甲酸,但鞣质水解程度差异则造成五没食子酰基葡萄糖的稳定性差;随加热时间延长,白芍煎液中儿茶素逐步下降,苯甲酸逐步上升,五没食子酰基葡萄糖先明显上升,然后明显下降。结论该研究为白芍及含白芍复方煎剂或制剂指纹图谱提供了一定参考。

HPLC-电化学检测法用于黄芩苷和黄芩素大鼠血浆稳定性研究

目的:考察黄芩苷(BG)和黄芩素(B)在缓冲液及大鼠血浆中的稳定性,建立测定二者在大鼠血浆中含量的样品处理方法,并初步探讨二者在血浆中稳定性的影响因素。方法:采用HPLC-电化学检测法,分别考察BG,B在不同条件下的稳定性及使二者稳定的条件。结果:BG,B在大鼠血浆中较在pH7.4的缓冲液中更不稳定。在pH7.4缓冲液中加入抗氧化剂可使BG和B的稳定性明显提高,剩余百分含量达到102%和100%;在大鼠血浆中加入抗氧化剂及1 mol.L-1HCl可使BG和B的稳定性明显增加,剩余百分含量可分别达到98%和103%。结论:BG和B在pH7.4缓冲液中的稳定性主要受氧化影响,BG和B在血浆中的稳定性不仅与氧化有关,还受血浆中的内源物质影响,最终确定每1 mL大鼠血浆中加入抗氧剂及100μL1 mol.L-1HCl为血浆样品的处理条件,此条件可使样品稳定。

HPLC法测定姜黄素及同系物

目的:建立反相高效液相色谱法测定姜黄素及其同系物并对其稳定性进行考察。方法:采用 Kromasil 色谱柱,以1%柠檬酸-四氢呋喃(55:45,pH=3.0)为流动相,检测波长264 nm 测定姜黄素含量。考察姜黄素甲醇液及姜黄素固体粉末的光(4500 lx)稳定性;原料固体粉末分别对热(40℃和60℃)和强湿(75%RH)状态下的稳定性;考察强酸、强碱及强氧化剂对姜黄素原料甲醇液的影响。结果:姜黄素 t_R=12.2 min;姜黄素、去甲氧基姜黄素和双去甲氧基姜黄素能够基线分离。姜黄素在0.5～40μg·mL^(-1)范围内峰面积与浓度呈线性关系,r=0.9999。姜黄素甲醇液光照下稳定8 h,之后按照一级动力学降解;姜黄素原料溶液光稳定72 h;原料固体对光、热(40℃)、湿在10 d 内稳定;强酸、强碱及氧化剂对姜黄素原料甲醇液有破坏作用。结论:该方法测定姜黄素及同系物含量,具有快速、简便、准确的特点;姜黄素在有机溶液中具有光、热(60℃)不稳定性的特性,易被氧化。

HPLC法考察制霉素甘油的稳定性

目的 :考察制霉素甘油的稳定性。方法 :制霉素甘油在不同光线及温度条件下放置 ,定时取样 ,采用 HPL C法测定制霉素中三个主组分的含量。结果 :放置三个月后 ,制霉素甘油于避光条件下稳定 ,自然光条件下降解近 2 0 % ,强光条件下降解近30 % ;5℃条件下稳定 ,2 5℃基本稳定 ,4 5℃下降解近 80 %。结论 :制霉素甘油易被光线破坏 。

不同品种荷叶HPLC指纹图谱研究

研究用高效液相色谱法建立荷叶指纹图谱的分析方法。采用依利特Sino Chrom色谱柱(4.6 mm×200mm,5μm),以乙腈-缓冲溶液(H3PO4-NaH2PO4,0.1 mol/L,pH 2.5)为流动相,梯度洗脱,流速1.5 mL/min,柱温30℃,检测波长254 nm。用上述方法确定了10批不同品种荷叶的12个共有峰,各色谱峰分离效果良好,精密度、稳定性、重复性试验中各共有峰相对保留时间和相对峰面积的RSD均小于3%,符合指纹图谱要求。该方法精密度、稳定性和重复性均较好,为荷叶的质量控制标准提供了参考。