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.

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.

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 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.

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.

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.

Long-term stability of gentamicin sulfate-ethylenediaminetetraacetic acid disodium salt (EDTA-Na_2) solution for catheter locks

A lock solution composed of gentamicin sulfate(5 mg/mL) and ethylenediaminetetraacetic acid disodium salt(EDTA-Na2, 30 mg/mL) could fully eradicate in vivo bacterial biofilms in totally implantable venous access ports(TIVAP). In this study, fabrication, conditioning and sterilization processes of antimicrobial lock solution(ALS) were detailed and completed by a stability study. Stability of ALS was conducted for12 months in vial(25 °C 7 2 °C, 60% 7 5% relative humidity(RH), and at 40 °C 7 2 °C, RH 75% 7 5%)and for 24 h and 72 h in TIVAP(40 °C 7 2 °C, RH 75% 7 5%). A stability indicating HPLC assay with UV detection for simultaneous quantification of gentamicin sulfate and EDTA-Na2 was developed. ALS was assayed by ion-pairing high performance liquid chromatography(HPLC) needing gentamicin derivatization, EDTA-Na2 metallocomplexation of samples and gradient mobile phase. HPLC methods to separate four gentamicin components and EDTA-Na2 were validated. Efficiency of sterility procedure and conditioning of ALS was confirmed by bacterial endotoxins and sterility tests. Physicochemical stability of ALS was determined by visual inspection, osmolality, pH, and sub-visible particle counting. Results confirmed that the stability of ALS in vials was maintained for 12 months and 24 h and 72 h in TIVAP.

A validated stability-indicating LC method for the separation of enantiomer and potential impurities of Linezolid using polar organic mode

Although a number of methods are available for evaluating Linezolid and its possible impurities, a common method for separation if its potential impurities, degradants and enantiomer in a single method with good efficiency remain unavailable. With the objective of developing an advanced method with shorter runtimes, a simple, precise, accurate stability-indicating LC method was developed for the determination of purity of Linezolid drug substance and drug products in bulk samples and pharmaceutical dosage forms in the presence of its impurities and degradation products. This method is capable of separating all the related substances of Linezolid along with the chiral impurity. This method can also be used for the estimation of assay of Linezolid in drug substance as well as in drug product. The method was developed using Chiralpak IA (250 mm 4.6 mm, 5 mm) column. A mixture of acetonitrile, ethanol, n-butyl amine and trifluoro acetic acid in 96:4:0.10:0.16 (v/v/v/v) ratio was used as a mobile phase. The eluted compounds were monitored at 254 nm. Linezolid was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, limit of detection, limit of quantification, precision, linearity, accuracy, robustness and system suitability.

Assay method for quality control and stability studies of a new antimalarial agent (CDRI 99/411)

CDRI compound no. 99/411 is a potent 1,2,4-trioxane antimalarial candidate drug under development at our Institute. An HPLC method for determination of CDRI 99/411 with its starting material and intermediates has been developed and validated for in process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection （LOD） and lower limit of quantification （LLOQ） were determined according to International Conference on Harmonization ICH Q2（R1） guidelines. HPLC separation was achieved on a RP-select B Lichrosphere~ column （250 mm x 4 ram, 5 lam, Merck） using water containing 0.1% glacial acetic acid and acetonitrile as the mobile phase in a gradient elution. The eluents were monitored by a photo diode array detector at 245 and 275 nm. Based on signal to noise ratio of 3 and 10 the LOD of CDRI 99/411 was 0.55 μg/mL, while the LLOQ was 1.05 μg/mL. The calibration curves were linear in the range of 1.05- 68 μg/mL. Precision of the method was determined by inter- and intra-assay variations within the acceptable range.

Determination of Fenofibrate and the Degradation Product Using Simultaneous UV-Derivative Spectrometric Method and HPLC

Two new selective, precise, and accurate methods were developed for the determination of fenofibrate in the presence of its basic degradation product. In the first method fenofibrate was determined using an algorithm bivariate calibration derivative method, in which an optimum pair of wavelengths was chosen for the determination of different binary mixtures. In the second method (HPLC), separation was achieved on RESTEK Pinnacle II phenyl column (5 μm, 250 × 4.6 mm) and Pinnacle II phenyl (5 μm, 10 × 4 mm) guard cartridge using a mobile phase consisting of methanol –0.1% phosphoric acid (60:40, v/v) at a flow rate 2 mL●min–1, and the column oven temperature was set at 50°C. The UV detector was time programmed at 302 nm and 289 nm for the internal standard (I.S.) and fenofibrate, respectively. The proposed methods were successfully applied for the determination of fenofibrate and its degradation product in the laboratory-prepared mixture and in pharmaceutical formulation. The assay results obtained using the bivariate method were statistically compared to those of the HPLC method and good agreement was observed.

A Validated Stability Indicating LC Method for Amlexanox in Bulk Drugs

A novel and sensitive stability indicating RP-HPLC method has been developed for the quantitative determination of amlexanox in bulk drugs. The separation was accomplished on C18 column using 10 mM ammonium dihydrogen orthophosphate (pH adjusted to 4.8 by using ortho phosphoric acid) and methanol (30:70 v/v) as mobile phase in an isocratic elution mode at a flow rate of 1.0 mL min-1. The eluents were monitored by PDA detector at 245 nm. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Significant degradation was found under basic, acidic stress and UV light. The resolution (Rs) between amlexanox and its degradation products was found to be greater than 2.5. Regression analysis shows correlation coefficient greater than 0.999 for amlexanox. The inter and intraday precision values for amlexanox were found to be within 1.0% RSD. The method has shown good and consistent recoveries for amlexanox in bulk drugs (98.86% - 101.05%). The developed method was validated with respect to linearity, accuracy, precision and robustness.

Effect of Excipients on Recombinant Interleukin-2 Stability in Aqueous Buffers

In order to retain structural and functional integrity, protein medicines are frequently stabilized with excipients in aqueous solutions. The goal of this investigation was to see how stable IL-2 is with excipients that are acceptable for cell therapy. We investigated the time-dependent stability of commercially available recombinant IL-2 in aqueous solutions (CTS, RPMI, PBS, and water) at different temperatures [2°C - 8°C, room temperature (20°C ± 2°C) and 37°C] in the presence of excipients (EDTA, methionine, histidine, and glycine) over a period of up to 30 days. To detect and quantify IL-2, reversed phase high performance liquid chromatography was employed. Electrophoresis on a sodium dodecyl sulfate polyacrylamide gel was used to assess conformational stability. We discovered that IL-2 stability was improved in aqueous solutions including excipients, and that it may have retained its biological activity and sterility in these conditions.

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。

RP-HPLC法测定胸腺五肽溶液的稳定性

目的考察胸腺五肽溶液的稳定性。方法采用RP-HPLC法，分别考察了温度（冷冻、冷藏、37℃、60℃）、缓冲液种类（磷酸盐、三羟甲基氨基甲烷-盐酸、枸橼酸盐、醋酸盐、碳酸盐、硼酸盐）、pH值（2．0～10．0）、光照及超声（200、400、600W）等因素对胸腺五肽溶液稳定性的影响。结果胸腺五肽含量的标准曲线方程为A=1．015×10^3 ρ 1．319×10^3，r=0．9998。胸腺五肽在质量浓度5～400mg·L^-1内与峰面积呈良好的线性关系，精密度与回收率符合要求。胸腺五肽溶液在冷冻条件下30d内基本稳定；在冷藏条件下7d内含量保持不变；放置于37℃时，48h内稳定性良好；60℃时，24h后药物开始降解。当溶液的pH值为5．0～9．2时，胸腺五肽溶液在4d内稳定性较好。缓冲液的种类对其稳定性的影响不大。在24h内光照对药物的稳定性基本无影响。以200W功率超声15min，胸腺五肽溶液基本稳定；以400W和600W功率超声时。7min和5min后药物开始降解。结论本实验中所建立的用于胸腺五肽溶液稳定性测定的RP-HPLC法，操作简便。精密度与回收率符合要求，且对降解产物具有很好的分离效果，适用于对胸腺五肽溶液稳定性的评价。

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。