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.

Stability-indicating liquid chromatographic method for the determination of Letrozole in pharmaceutical formulations

A stability-indicating high-performance liquid chromatographic method was developed and validated for the determination of Letrozole in tablet dosage forms. Reversed-phase chromatography was performed on Shimadzu Model LC-Class-Vp with Lichrocart/Lichrosphere 100 C-18 (250 mm 4.6 mm, 5 mm particle size) column with methanol: tetra butyl ammonium hydrogen sulfate (80:20V/V) as mobile phase at a flow rate of 1 mL/min with UV detection at 240 nm. Linearity was observed in the concentration range of 0.5-150 mg/mL (R 2 0.9998) with regression equation y 102582xt43185. The limit of quantitation (LOQ) and limit of detection (LOD) were found to be 0.043 and 0.012 mg/mL respectively. The forced degradation studies were performed by using HCl, NaOH, H 2 O 2 , thermal and UV radiation. Letrozole is more sensitive towards alkaline conditions and very much resistant towards acidic, oxidative and photolytic degradations. The method was validated as per ICH guidelines. The RSD for intra-day (0.78-0.97) and inter-day (0.86-0.96) precision were found to be lesser than 1%. The percentage recovery was in good agreement with the labeled amount in the pharmaceutical formulations and the method is simple, specific, precise and accurate for the determination of Letrozole in pharmaceutical formulations.

A Validated Stability-Indicating LC Method for Fluocinonide in the Presence of Degradation Products, Its Process-Related Impurities and Identification of Degradation Products

The objective of the current study was to develop a validated, specific and stability-indicating reverse phase liquid chromatographic method for the quantitative determination of fluocinonide 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 condi- tions of hydrolysis (acid and base), oxidation, photolysis and thermal degradation per International Confer- ence on Harmonization (ICH) prescribed stress conditions to show the stability- indicating power of the method. Significant degradation was observed during acid, base hydrolysis, and peroxide degradation. The major degradants were identified by LC-MS, FTIR and 1H/13C NMR spectral analysis. The chromatographic conditions were optimized using an impurity-spiked solution and the samples generated from forced degra- dation studies. In the developed HPLC method, the resolution between fluocinonide and its process-related impurities, (namely imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8) and its degradation products was found to be greater than 2.0.The chromatographic separation was achieved on a C18, 250 mm × 4.6 mm, 5 μm column. The LC method employed a linear gradient elution and the detection wavelength was set at 240 nm. The stress samples were assayed against a qualified reference standard and the mass balance was found to be close to 99.3%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

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.

Stability-indicating assay method for determination of actarit,its process related impurities and degradation products:Insight into stability profile and degradation pathways

The stability of the drug actarit was studied under different stress conditions like hydrolysis（acid,alkaline and neutral）,oxidation,photolysis and thermal degradation as recommended hy International Conference on Harmonization（ICH） guidelines.Drug was found to be unstable in acidic,basic and photolytic conditions and produced a common degradation product while oxidative stress condition produced three additional degradation products.Drug was impassive to neutral hydrolysis,dry thermal and accelerated stability conditions.Degradation products were identified,isolated and characterized by different spectroscopic analyses.Drug and the degradation products were synthesized by a new route using green chemistry.The chromatographic separation of the drug and its impurities was achieved in a phenomenex luna C18 column employing a step gradient elution by high performance liquid chromatography coupled to photodiode array and mass spectrometry detectors（HPLC-PDAMS）.A specilic and sensitive stability-indicating assay method for the simultaneous determination of the drug actarit.its process related impurities and degradation products was developed and validated.

A Validated Stability-Indicating UHPLC Method for Determination of Naproxen and Its Related Compounds in Bulk Drug Samples

A simple, rapid, precise, accurate, rugged and robust stability-indicating ultra-fast high performance liquid chromatographic (UHPLC) method has been developed for the estimation of related compounds (imp-A, imp-B, imp-C, imp-D and imp-E) in Naproxen and also the assay of Naproxen from bulk drug samples. The stability indicating capability of the method was proven by subjecting the samples to stress conditions such as acid, base, oxidation, photolysis and thermal degradation. The efficient chromatographic separation was achieved using mobile phase solution A prepared as buffer solution 10 mM monobasic potassium phosphate pH 4.0 ± 0.05 adjusted with diluted ortho phosphoric acid solution and solution B acetonitrile with linear gradient elution on poroshell 120 EC-C18 shot column (50 mm × 4.6 mm, 2.7 μm) and UV detection at 235 nm at a flow rate 1.0 mL/min, column oven temperature was set to 25?C. The above are all known impurities and degradation impurities are well resolved with Naproxen peak and these are eluted within a 10 min runtime of HPLC. The photo diode array detector was used for peak homogeneity testing during stress study experiments and the overall mass balance was found to be 99.2% to 100.2% in all stress conditions. The linear calibration range was found to be 0.05 μg/mL to 0.75 μg/mL for related compounds and 50 μg/mL to 150 μg/mL for Naproxen and the accuracy of the method was found to be 91.5% to 98.5% recovery for the related substance method and 95.4% to 97.4% recovery for the assay method. The Naproxen and related compounds were found to be stable up to 48 hours and the method validation data show excellent results for precision, linearity, specificity, limit of detection, limit of quantitation and robustness. The present method can be successfully used for routine QC and stability studies and it will help to reduce the analysis cost, time and effluent load compared to conventional HPLC methods.

Development and validation of a stability indicating RP-HPLC method for the determination of Rufinamide

A stability-indicating RP-HPLC method was developed and validated lbr the determination of Rufinamide in tablet dosage forms using C 18 column （250 mm x 4.6 mm, 5 μm） wilh mobile phase consisting of water acetonitrile （40：60, v/v） with a [low rate of 0.8 mL/min （UV detection 215 nm）. Linearity was observed over the concentration range 1.0 200 μg/mL （R2=0.9997） with regression equation y-113190 x＋63053. Rufinamide was subjected to stress conditions including acidic, alkaline, oxidation, photolysis and thermal degradation. Rufinaraide is more sensitive towards acidic degradation. The method was validated as per ICH guidelines.

Validated stability indicating methods for determination of nitazoxanide in presence of its degradation products

Three sensitive,selective and reproducible stability-indicating methods are presented for determination of nitazoxanide (NTZ),a new anti-protozoal drug,in presence of its degradation products.Method A utilizes the first derivative of ratio spectra spectrophotometry by measurement of the amplitude at 364.4 nm using one of the degradation products as a divisor.Method B is a chemometric-assisted spectrophotometry,where principal component regression (PCR) and partial least squares (PLS) were applied.These two approaches were successfully applied to quantify NTZ in presence of degradation products using the information included in the absorption spectra in the range 260-360 nm.Method C is based on the separation of NTZ from its degradation products followed by densitometric measurement of the bands at 254 nm.The separation was carried out on silica gel 60F 254,using chloroform-methanol-ammonia solution-glacial acetic acid (95:5:1:1 by volume,pH=5.80) as a developing system.These methods are suitable as stability-indicating methods for the determination of NTZ in presence of its degradation products either in bulk powder or in pharmaceutical formulations.Statistical analysis of the results has been carried out revealing high accuracy and good precision.

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.

Formulation, stability testing, and analytical characterization of melatonin-based preparation for clinical trial

A new institutional clinical trial assessed the improvement of sleep disorders in 40 children with autism treated by immediate-release melatonin formulation in different regimens(0.5 mg, 2 mg, and 6 mg daily) for one month. The objectives of present study were to(i) prepare low-dose melatonin hard capsules for pediatric use controlled by two complementary methods and(ii) carry out a stability study in order to determine a use-bydate. Validation of preparation process was claimed as ascertained by mass uniformity of hard capsules.Multicomponent analysis by attenuated total reflectance Fourier transformed infrared(ATR-FTIR) of melatonin/microcrystalline cellulose mixture allowed to identify and quantify relative content of active pharmaceutical ingredients and excipients. Absolute melatonin content analysis by high performance liquid chromatography in 0.5 mg and 6 mg melatonin capsules was 93.6% ± 4.1% and 98.7% ± 6.9% of theoretical value, respectively. Forced degradation study showed a good separation of melatonin and its degradation products. The capability of the method was 15, confirming a risk of false negative < 0.01%. Stability test and dissolution test were compliant over 18 months of storage with European Pharmacopoeia. Preparation of melatonin hard capsules was completed manually and melatonin in hard capsules was stable for 18 months, in spite of low doses of active ingredient. ATR-FTIR offers a real alternative to HPLC for quality control of highdose melatonin hard capsules before the release of clinical batches.

Development and validation of an RP-HPLC method for simultaneous determination of Ramipril and Amlodipine in tablets

AnRP-HPLC method for the simultaneous determination ofRamipril （RP） andAmlodipine （AL） in tablets was developed and validated by Chinese Pharmacopoeia 2010. The linearity of the proposed method was investigated in the range of 0.01-0.25 mg/mL （r^2=0.9998） for RP and 0.014-0.36 mg/mL （r^2=0.9997） for AL. The limits of detection （LOD） were 0.06 lag/mL and 0.02 μg/mL for RP and AL, and the limits of quantitation （LOQ） were 0.2 Dg/mL and 0.07 μg/mL, respectively. Some major impurities and degradation products did not disturb the detection of RP and AL and the assay can thus be considered stability-indicating.

Production and stability study of a hospital parenteral nutrition solution for neonates

Standard parenteral nutrition solutions are mixtures comprising interacting components that may degrade themselves over time. The objective of this study was to investigate the physicochemical and microbiological stability of a hospital preparation for parenteral nutrition in neonatology. The analyses were performed throughout the storage of the preparations at 2–8 °C(up to 4 months). The extent of stability was based on the determination of amino acids dosage, visual and physicochemical properties(glucose and electrolytes concentrations, pH and osmolality measurements, particle counting) and microbiological analysis(sterility test). A thermal degradation of ascorbic acid was conducted to evaluate the antioxidant properties of the parenteral mixture. Physicochemical and microbiological controls were found to comply with the specifications. Amino acids showed a good stability throughout the 4 months storage except for cysteine, which was progressively degraded to cystine, conferring a yellow coloration to parenteral solutions. Parenteral nutrition standards solutions remain stable for 4 months at 2–8 °C,ensuring safe administration in preterm infants.

指示稳定性的高效液相色谱方法和强制降解研究片剂型马西替坦中的杂质（英文）

Macitentan (MAC) is a pulmonary arterial hypertension (PAH) drug marketed as a tablet and often has stability issues in the final dosage form. Quantitative determination of MAC and its associated impurities in tablet dosage form has not been previously reported. This study quantified impurities present in Macitentan tablets using a binary solvent-based gradient elution method using reversed phase-high performance liquid chromatography.The developed method w as validated per International Conference on Harmonization (ICH) guidelines and the drug product w as subjected to forced degradation studies to evaluate stability. The developed method efficiently separated the drug and impurities (48 min) w ithout interference from solvents,excipients,or other impurities. The developed method met all guidelines in all characteristics w ith recoveries ranging from 85%-115%,linearity w ith r 2≥0. 996 6,and substantial robustness. The stability-indicating nature of the method w as evaluated using stressed conditions (hydrolysis:1 N HCl at 80℃/15 min; 1 N NaOH at 25℃/45 min; humidity stress (90%relative humidity) at 25℃for 24 h,oxidation:at 6%(v/v) H2O2,80℃/15 min,thermolysis:at105℃/16 h and photolysis:UV light at 200 Wh/m2; Fluorescent light at 1. 2 million luxh). Forced degradation experiments show ed that the developed method w as effective for impurity profiling. All stressed samples w ere assayed and mass balance w as> 96%. Forced degradation results indicated that MAC tablets w ere sensitive to hydrolysis (acid and alkali) and thermal conditions. The developed method is suitable for both assay and impurity determination,w hich is applicable to the pharmaceutical industry.

Selective separation, detection of zotepine and mass spectral characterization of degradants by LC–MS/MS/QTOF

A simple, precise, accurate stability-indicating gradient reversed-phase high-performance liquid chromatographic （RP-HPLC） method was developed for the quantitative determination of zotepine （ZTP） in bulk and pharmaceutical dosage forms in the presence of its degradation products （DPs）. The method was developed using Phenomenex C18 column （250 mm ~ 4.6 mm i.d., 5 mm） with a mobile phase containing a gradient mixture of solvents, A （0.05%trifluoroacetic acid （TFA）, pH ？ 3.0） and B （acetonitrile）. The eluted compounds were monitored at 254 nm;the run time was within 20.0 min, in which ZTP and its DPs were well separated, with a resolution of 41.5. The stress testing of ZTP was carried out under acidic, alkaline, neutral hydrolysis, oxidative, photolytic and thermal stress conditions. ZTP was found to degrade significantly in acidic, photolytic, thermal and oxidative stress conditions and remain stable in basic and neutral conditions. The developed method was validated with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness as per ICH guidelines. This method was also suitable for the assay determination of ZTP in pharmaceutical dosage forms. The DPs were characterized by LC-MS/MS and their fragmentation pathways were proposed.

Validated Gradient Stability Indicating UPLC Method for the Determination of Related Substances of Posaconazole in Bulk Drug

A stability-indicating UPLC method has been developed and validated for the determination of related substances of Posaconazole with its four related substances (Hydroxytriazole, Tosylated compound, Deshydroxy posaconazole and Benzylated posaconazole) in the drug substance. Forthwith simple UPLC chromatographic separations were achieved on a Waters Acquity BEH shield C18 (100 mm length, 2.1 mm internal diameter and 1.7 μm particle size) with a mobile phase containing 0.1% Orthophosphoric acid (i.e. 1 mL in 1000 mL water) in gradient combination with acetonitrile (ACN) at a flow rate of 0.5 mL/min and the eluent were monitored at 210 nm. As a result, the resolution of Posaconazole from any of impurities was found to be greater than 2.0. The test solution and spiked solutions were found to be stable in the diluent for 48 h. For the purpose method to be stability indicating, forced degradation studies were conducted and the method resolved the drug from its known impurities, stated above, and from additional impurities generated when POS subjected to forced degradation;the mass balance was found close to 100%. Regression analyses indicate correlation coefficient value greater than 0.999 for Posaconazole and its known impurities. The LOD for Posaconazole and the known impurities was at a level below 0.05%. The method has shown good, consistent recoveries for known impurities (89% - 106%). To summarise, the method was found to be accurate, precise, linear, specific, sensitive, rugged, robust, and stability-indicating.

Stability Indicating RP-HPLC Method for the Determination of Dronedarone Hydrochloride and Its Potential Process-Related Impurities in Bulk Drug and Pharmaceutical Dosage Form

Simple, sensitive and accurate stability indicating analytical method for dronedarone has been developed and validated using RP-HPLC technique. Developed method is used to evaluate the assay and related substances of dronedarone drug substance and tablets (Multaq?). The drug substance was subjected to the stress conditions such as hydrolysis (acid and base), oxidation, photolysis and thermal degradation as per International Conference on Harmonization (ICH) pre- scribed stress conditions to show the stability-indicating the nature of the method. Significant degradation was observed during acid and base hydrolysis, and peroxide degradation. The major degredants were identified by LC-MS, FTIR and 1H NMR spectral analysis. The chromatographic conditions were optimized using an impurity-spiked solution and the samples generated from forced degradation studies. In the developed HPLC method, the resolution between dronedar- one, process-related impurities, (namely Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7, Imp-8, Imp-9, Imp-10 and Imp-11) and degradation products were found to be greater than 1.5. The eleven potential process related impurities were separated on an Ascentis? Express C18 column (4.6 × 10 cm i.d., particle size 2.7 μm) at a flow rate of 1.2 mL.min-1. The LC method employed a linear gradient elution and the detection wavelength at 220 nm. The chroma- tographic behavior of all the impurities was examined under variable compositions of different solvents, temperatures and pH values.

A Novel Validated Stability Indicative UP-LC Method for Etravirine for the Determination of Process Related and Degradation Impurities

A novel stability indicating reverse phase ultra performance liquid chromatographic (UP-LC) method has been developed for Etravirine along with eight impurities (imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8) and validated as per ICH recommendations. Stress degradation conditions were established for Etravirine by subjecting it to stress conditions of acid, base, oxidation, humidity, thermal and photolysis. Significant degradation is observed in base stress condition and the major degradant (RRT at about 0.94) is identified by LC-MS and spectral analysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.0%. Efficient chromatographic separation was achieved on a Shimpack ODS-II stationary phase with a gradient mobile phase combination. Quantification was carried at 303 nm at a flow rate of 0.6 mL?min–1. The resolution between Etravirine and eight potential impurities is found to be greater than 2.0. Regression analysis shows as r value (correlation coefficient) of greater than 0.999 for Etravirine and eight potential impurities. This method is capable to detect the impurities of Etravirine at a level of 0.003% with respect to test concentration of 1.0 mg·mL–1.