Stress Degradation Studies on Aripiprazole and Development of a Validated Stability Indicating LC Method

The present paper describes the development of a stability indicating reversed phase column liquid chromatographic method for aripiprazole in the presence of its impurities and degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of aqueous hydrolysis, oxidative, photolytic and thermal stress degradation. The degradation of aripiprazole was observed under acid hydrolysis and peroxide. The drug was found to be stable to other stress conditions attempted. Successful separation of the drug from the synthetic impurities and degradation products formed under stress conditions was achieved on an Inertsil phenyl column using a mixture of 0.2% trifluoroacetic acid and acetonitrile (55:45, v/v). The developed LC method was validated with respect to linearity, accuracy, precision, specificity and robustness. The assay method was found linear in the range of 25–200 μg mL^?1 with a correlation coefficient of 0.9999 and the linearity of the impurities were established from LOQ to 0.3%. Recoveries of the assay and impurities were found between 97.2 and 104.6%. The developed LC method for the related substances and assay determination of aripiprazole can be used to evaluate the quality of regular production samples. It can also be used to test the stability samples of aripiprazole. To the best of our knowledge, the validated stability indicating LC method which separates all the impurities disclosed in this investigation was not published elsewhere.     

Automated statistical experimental design approach for rapid separation of coenzyme Q10 and identification of its biotechnological process related impurities using UHPLC and UHPLC–APCI‐MS

A novel ultra high performance liquid chromatography method development strategy was ameliorated by applying quality by design approach. The developed systematic approach was divided into five steps (i) Analytical Target Profile, (ii) Critical Quality Attributes, (iii) Risk Assessments of Critical parameters using design of experiments (screening and optimization phases), (iv) Generation of design space, and (v) Process Capability Analysis (Cp) for robustness study using Monte Carlo simulation. The complete quality‐by‐design‐based method development was made automated and expedited by employing sub‐2 μm particles column with an ultra high performance liquid chromatography system. Successful chromatographic separation of the Coenzyme Q10 from its biotechnological process related impurities was achieved on a Waters Acquity phenyl hexyl (100 mm × 2.1 mm, 1.7 μm) column with gradient elution of 10 mM ammonium acetate buffer (pH 4.0) and a mixture of acetonitrile/2‐propanol (1:1) as the mobile phase. Through this study, fast and organized method development workflow was developed and robustness of the method was also demonstrated. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance to the International Conference on Harmonization, Q2 (R1) guidelines. The impurities were identified by atmospheric pressure chemical ionization‐mass spectrometry technique. Further, the in silico toxicity of impurities was analyzed using TOPKAT and DEREK software.     

Determination of biogenic amines as dansyl derivatives in intestinal digesta and feces by reversed phase HPLC

Summary A new method was developed for the determination of fifteen biogenic amines in the intestinal digesta and feces of animal or human origin. The method involves the addition of an internal standard (heptylamine), extraction of the amines, and precipitation of the proteins with perchloric acid. The amines are derivatised with dansyl chloride, separated on a C18 column using gradient elution with 0.2M ammonium acetate at pH 5, water and acetonitrile, and detected with a fluorescence detector. The separation was achieved in a 40 min run. Recoveries ranged from 67 to 110%, the relative standard deviation for intra-assay precision being <5% and the limit of determination 1–5 mg kg⁻¹. The method is specific for biogenic amines in intestinal and fecal samples.     

Liquid chromatography–tandem mass spectrometry method for simultaneous quantification of urapidil and aripiprazole in human plasma and its application to human pharmacokinetic study

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of urapidil and aripiprazole in human plasma. A simple liquid–liquid extraction with ethyl acetate was used for the sample preparation. Chromatographic separation was achieved on a Phenomenex C₁₈ (4.6 × 50 mm, 5 µm) column with 0.1% formic acid–acetonitrile (10:90, v/v) as the mobile phase with flow rate of 0.6 mL/min. The quantitation of the target compounds was determined in a positive ion multiple reaction monitoring mode. Calibration plots were linear over the range of 2.0–2503.95 ng/mL for urapidil and 1.0–500.19 ng/mL for aripiprazole. The lower limit of quantitation for urapidil and aripiprazole was 2.0 and 1.0 ng/mL, respectively. Mean recovery was in the range of 69.94–75.62% for both analytes and internal standards. Intra‐day and inter‐day precisions of the assay at three concentrations were 2.56–5.89% with accuracy of 92.31–97.83% for urapidil, and 3.14–6.84% with accuracy of 91.38–94.42% for aripiprazole. The method was successfully applied to human pharmacokinetic study of urapidil and aripiprazole in healthy human male volunteers. Copyright © 2013 John Wiley & Sons, Ltd.     

蜂王浆中磷酸腺苷的提取及超高效液相色谱分析

比较了高氯酸提取、热水提取和热硫酸镁溶液提取3种提取方式对蜂王浆中磷酸腺苷三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和单磷酸腺苷(AMP)的提取效果,发现在低温(低于4 ℃)下以5%高氯酸的提取效果最佳。采用超高效液相色谱-紫外检测法分析蜂王浆中的ATP, ADP和AMP的含量。以BEH Shield RP18柱(100 mm×2.1 mm,1.7 μm)为分析柱,以50 mmol/L的磷酸二氢铵(pH 6.5)和乙腈为流动相进行梯度洗脱,3种磷酸腺苷在4 min内实现了较好的分离。以加标王浆样品作添加回收率测定,ATP, ADP和AMP的回收率分别为84.1%～94.3%,86.2%～93.7%和91.0%～104.3%,相对标准偏差均小于10%。方法已被用于一些实际样品的分析,以了解ATP, ADP和AMP在蜂王浆样品中的分布情况。     

Simultaneous determination of rocuronium and its eight impurities in pharmaceutical preparation using high-performance liquid chromatography with amperometric detection

A sensitive and selective HPLC method with amperometric detection (HPLC-ED) for the determination of rocuronium bromide and its eight impurities has been developed. The analysis was performed on Hypersil 100 Silica column 5 microm (250 mm x 4.6 mm; Thermo Electron). The mobile phase consisting of 4.53 g l(-1) solution of tetramethylammonium hydroxide adjusted to pH 7.4 with 85% phosphoric acid:acetonitrile (1:9), was found the best for the separation and determination of the studied compounds. The chromatograms were recorded over 10 min using the amperometric detection at a potential +0.9 V of the glassy carbon electrode versus the reference electrode Ag/AgCl. The limit of quantitation was 45 ng ml(-1) for rocuronium and from 25 to 750 ng ml(-1) for the examined impurities. The proposed HPLC-ED method was successfully applied to the analysis of rocuronium and its impurities in Esmeron solution for injection.     

An example of quality control of fine chemical intermediates: related impurity analysis of industrial phthalic anhydride by reversed-phase high-performance liquid chromatography

A reliable reversed-phase high-performance liquid chromatographic method has been developed for analysis of related impurities in industrial phthalic anhydride (PA). Maleic acid (hydrolysis product of maleic anhydride), phthalimide, and benzoic acid were separated from phthalic acid (Pa, hydrolysis product of PA) on a C₁₈ column by gradient elution with acetonitrile and 0.1% (v/v) aqueous perchloric acid solution. This method is simple, sensitive, and accurate, and has been successfully applied to quality control of PA for industrial use.     

Characterization of process‐related impurities including forced degradation products of alogliptin benzoate and the development of the corresponding reversed‐phase high‐performance liquid chromatography method

The characterization of process‐related impurities and forced degradants of alogliptin benzoate (Alb) in bulk drugs and a stability‐indicating HPLC method for the separation and quantification of all the impurities were investigated. Alb was found to be unstable under acid and alkali stress conditions and two major degradation products (Imp‐F and Imp‐G) were observed. The optimum separation was achieved on Kromasil C₁₈ (250 × 4.6 mm, 5 μm) using 0.1% perchloric acid (pH adjusted to 3.0 with triethylamine) and acetonitrile as a mobile phase in gradient mode. The proposed method was found to be stability indicating, precise, linear (0.10–75.0 μg/mL), accurate, sensitive, and robust for the quantitation of Alb and its process‐related substances and degradation products. The structures of 11 impurities were characterized and confirmed by NMR spectroscopy, MS, and IR spectroscopy, and the most probable formation mechanisms of all impurities were proposed according to the synthesis route.     

Reversed‐Phase High‐Performance Liquid Chromatographic Separation and Determination of 4‐Amino‐azobenzene‐4′,5‐disulfonic Acid and Related Products in Industrial Wastewater Effluents

A simple and rapid reversed‐phase high‐performance liquid chromatographic method for the separation and determination of 4‐amino‐azobenzene‐4′,5‐disulfonic acid (AABDS) and its process‐related impurities was developed. The separation was achieved on a μ‐Bondapak C₁₈ column using 0.15 M ammonium sulfate‐acetonitrile (55:45) (v/v) as eluent. A UV‐visible spectrophotometric detector fixed at 386 nm was used both for detection and quantitation. The method was used not only for quality assurance but also for process development and wastewater management of AABDS.     

Analysis of anthranilic acid by liquid chromatography

Analytical methods for the assay of anthranilic acid and for determination of the impurities methyl anthranilate, anthranoylanthranilic acid and 3- and 4-aminobenzoic acid are described. A Microbondapak C18 column is used for both the assay and the impurity determination. The assay is based on isocratic development with a mobile phase of 35:65 v v methanol/pH-3 phosphate buffer, with benzoic acid as internal standard. The impurities are separated by gradient elution. The standard deviation of the assay method is about 1% and the limit of detection for the impurities is about 0.01%.     

Identification of degradation impurities in aripiprazole oral solution using LC–MS and development of validated stability indicating method for assay and content of two preservatives by RP-HPLC

A simple and simultaneous reverse phase high-performance liquid chromatographic method was developed for the quantification of aripiprazole (ARI) and two preservatives, namely, methyl paraben and propyl paraben in ARI oral solution. The method was developed on ACE C18 (4.6?×?250?mm, 5?µm) column using gradient elution of 0.1% v/v trifluoroacetic acid in water and acetonitrile as mobile phase components. Flow rate of 1.0?mL/min and 30°C column temperature were used for the method at quantification wavelength of 254?nm. The developed method was validated in accordance with International Conference on Harmonization guideline for various parameters. Forced degradation study was conducted in acid, base, peroxide, heat, and light stress conditions. ARI was found to degrade in oxidation, acid hydrolysis, and heat while it was stable under the remaining conditions. Specificity of the method was verified using Photo Diode Array (PDA) detector by evaluating purity of peaks from degradation samples. Major degradation impurities formed during stress study were identified using liquid chromatography–mass spectrometry method. The present method was useful for determining the content of all the three main analytes present in the oral solution without interference from degradation impurities. The method was robust under the deliberately modified conditions.     

Detection of low-abundance impurities in synthetic thyroid hormones by stationary phase optimized liquid chromatography–mass spectrometry

The transfer of a gradient method to an isocratic or multistep gradient method employing stationary phase optimized liquid chromatography facilitated a reduction in analysis time by 50% and significantly improved the mass spectrometric detectability of impurities in synthetic thyroid hormones. Four column segments packed with different stationary phases were combined into a single chromatographic column, which allowed the separation and photometric as well as mass spectrometric detection of thyroid compounds in less than 30 min under isocratic- or step gradient elution conditions with 0.10% acetic acid/acetonitrile. Signal instability and baseline drift during detection by negative electrospray ionization time-of-flight mass spectrometry were minimized by optimizing the spray parameters for each individual elution step. This resulted in improved detectabilities and higher mass spectral quality, especially for low-abundance components in the sample mixture. The method was applied to the separation and detection of the low-abundance impurities formed upon the thermal stressing of a sample of synthetic levothyroxine.     

Improved method for direct high-performance liquid chromatography assay of angiotensin-converting enzyme-catalyzed reactions

A rapid and sensitive assay was developed for determination of the activity of angiotensin-converting enzyme (ACE) in the presence of inhibitory peptides present in soybean protein hydrolysates. The method utilizes reversed-phase high-performance liquid chromatography (HPLC) to separate and quantify hippuryl-histidyl-leucine (HHL) and hippuric acid (HA). HHL and HA were separated on a Symmetry C₁₈ column by gradient elution that used mixtures of trifluoroacetic acid (TFA)–acetonitrile and TFA–water as solvents. Analytical time and baseline separation of HA from HHL were improved over previous HPLC methods. In comparison to the standard spectrophotometric method, the new HPLC method obviates the need for ethyl acetate extraction of HA but requires direct injection of the ACE reaction mixture onto the HPLC column.     

高效液相色谱法测定硫酸伏拉帕沙原料药中杂质

建立了高效液相色谱法测定硫酸伏拉帕沙原料药中杂质的方法.采用ACE Excel C18(4.6 mmx 250 mm,5 pm)色谱柱;以pH 2.5的H3PO4溶液为流动相A,乙腈为流动相B,梯度洗脱;检测波长为260 nm;进样量为20 μL.结果表明,主成分与各杂质的分离度良好,各已知杂质在测定的范围内具有良好的...     

Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C₁₈ 250 × 4.6 mm column, 5 μm (1.0 ml min⁻¹, 280 nm, 50 μl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.     

A Validated and Stability-Indicating LC Assay Method for Valdecoxib

A gradient reversed-phase liquid chromatographic assay was developed for the quantitative determination of the non-steroidal anti-inflammatory drug valdecoxib. The developed method was also applicable to the determination of related substances in the bulk drug. Forced degradation studies were performed on bulk valdecoxib using acid (2.0 N hydrochloric acid), base (2.0 N sodium hydroxide), oxidation (6.0% v/v hydrogen peroxide), water hydrolysis, heat (60 °C) and photolysis. Mild degradation was observed using alkaline conditions and considerable degradation observed during oxidative stress. Chromatographic separation of process-related impurities and degradation products was achieved using a 5 micron Zorbax SB-CN LC column. The mobile phase consisted of aqueous potassium dihydrogen phosphate (pH 3.0) and acetonitrile. Stressed samples were assayed using the developed LC method and determination of the mass balance accounted for 99.5%, thus indicating the suitability of this stability-indicating method. Linearity, accuracy, precision and robustness have also been evaluated.     

Liquid-chromatographic separation and determination of process-related impurities, including a regio-specific isomer of celecoxib on reversed-phase C18 column dynamically coated with hexamethyldisilazane.

A simple and rapid reversed-phase high-performance liquid-chromatographic method for the separation and determination of process-related impurities of celecoxib (CXB) in bulk drugs and pharmaceuticals was developed. The separation of impurities viz., 4-methylacetophenone (I), 1-(4-methylphenyl)-4,4,4-trifluorobutane-1,3-dione (II), 4-hydrazinobenzene sulfonamide (III) and a regio-specific isomer [3-(4-methylphenyl)-5-trifluoromethyl-1H-pyrazole-1-yl]-benzenesulfonamide (IV), was accomplished on an Inertsil ODS-3 column dynamically coated with 0.1% hexamethyldisilazane (HMDS) in acetonitrile:water (55:45 v/v) as a mobile phase and detection at 242 nm using PDA at ambient temperature. The chromatographic conditions were optimized by studying the effects of HMDS, an organic modifier, time of silanization and column temperature. The method was validated and found to be suitable not only for monitoring the synthetic reactions, but also to evaluate the quality of CXB.     

液相色谱-串联质谱法测定鸡组织中沃尼妙林残留

建立了鸡组织中沃尼妙林残留的液相色谱-电喷雾串联质谱(LC-ESI-MS /MS) 检测方法.样品用乙腈提取后,过Strata-X-C固相萃取小柱净化.以乙腈-0.1%甲酸水溶液(35∶ 65,V/V)为流动相,经Luna C18色谱柱分离,采用电喷雾电离,多反应监测(MRM)正离子模式对沃尼妙林进行定性与定量分析.采用基质匹配法,对鸡皮肤、肌肉、肾脏和肝脏中沃尼妙林的含量进行标准校正,在5(7.5)～500 μg/kg范围内线性良好(r>0 998),在鸡肝脏中的检出限(LOD)及定量限(LOQ)分别为4.0和7.5 μg/kg;其它3种组织中的LOD及LOQ分别为2.5和5.0 μg/kg.在5(7.5), 50及500 μg/kg添加水平下,4中组织中沃尼妙林的平均回收率为78 5%～104.0%.日内相对标准偏差(RSD)为1.2%～9.8%,日间相对标准偏差为3.7%～13.2%.     

A Validated Stability Indicating RPLC Method for Tazarotene

A simple, isocratic, rapid and accurate reversed phase high performance liquid chromatography method was developed for the quantitative determination of tazarotene. The developed method is also applicable for the related substance determination in bulk drugs. The chromatographic separation was achieved on a Hypersil C18 (250 mm × 4.6 mm 5 μm) column using water pH 2.5 with orthophosphoric acid:acetonitrile (15:85, v/v) as a mobile phase. The chromatographic resolutions between tazarotene and its potential impurity A and B were found greater than three. The limit of detection and limit of quantification of impurities were found to be 25 and 75 ng mL⁻¹. The percentage recovery of impurities in bulk drug sample was ranged from 96.8 to 103.5.The percentage recovery of tazarotene in bulk drug sample was ranged from 98.4 to 100.9. The developed RPLC method was validated with respect to linearity, accuracy, precision and robustness.