Forced Degradation Study to Develop and Validate Stability-Indicating RP-LC for Quantification of Ropinirole Hydrochloride in Its Modified Release Tablets

A forced degradation study on ropinirole hydrochloride in bulk and in its modified release tablets was conducted under the conditions of hydrolysis, oxidation and photolysis in order to develop an isocratic stability-indicating LC-UV method for quantification of the drug in tablets. An impurity peak in standard solution was found to increase under acidic and neutral hydrolytic conditions while another degradation product was formed under alkaline condition. The drug and its degradation products were optimally resolved on a Hypersil C₁₈ column with mobile phase composed of diammonium hydrogen orthophosphate (0.05 M; pH 7.2), tetrahydrofuran and methanol (80:15:5% v/v) at a flow rate of 1.0 mL min^?1 at 30 °C using 250 nm as detection wavelength. The method was linear in the range of 0.05–50 μg mL^?1 drug concentrations. The %RSD of inter- and intra-day precision studies was <1. The system suitability parameters remained unaffected during quantification of the drug on three different LC systems. Excellent recoveries (101.59–102.28%) proved that the method was sufficiently accurate. The LOD and LOQ were found to be 0.012 and 0.040 μg mL^?1, respectively. Degradation behaviour of the drug in both bulk and tablets was similar. The drug was very unstable to hydrolytic conditions but stable to oxidative and photolytic conditions. The method can be used for rapid and accurate quantification of ropinirole hydrochloride in tablets during stability testing. Based on chemical reactivity of ropinirole in different media, the degradation products were suspected to be different from the known impurities of the drug.     

Development and validation of spectrophotometric, TLC and HPLC methods for the determination of lamotrigine in presence of its impurity

Three reliable, rapid and selective methods have been developed and validated for the determination of lamotrigine in the presence of its impurity, 2,3-dichlorobenzoic acid. The first method is spectrophotometric method using p-chloranilic acid forming a colored product with lambda(max) 519+/-2 nm. All variables affecting the reaction have been investigated and the conditions were optimized. Beer's law was obeyed over a concentration range of 10-200 microg ml(-1) with mean accuracy 100.13+/-0.44%. The molar ratio of the formed ion-association complex is found to be 1 : 1 as deduced by Job's method. The conditional stability constant (K(f)), standard free energy (DeltaG), molar absorptivity(epsilon), and sensitivity index were evaluated. The second method is based on TLC separation of the cited drug (Rf=0.75+/-0.01) from its impurity (Rf=0.23+/-0.01) followed by densitometric measurement of the intact drug spots at 275 nm. The separation was carried on silica gel plates using ethyl acetate : methanol : ammonia 35% (17 : 2 : 1 v/v/v) as a mobile phase. The linearity range was 0.5-10 microg/spot with mean accuracy 99.99+/-1.33%. The third method is accurate and sensitive stability-indicating HPLC method based on separation of lamotrigine from its impurity on a reversed phase C(18) column, using a mobile phase of acetonitrile : methanol : 0.01 M potassium orthophosphate (pH 6.7+/-0.1) (30 : 20 : 50 v/v/v) at ambient temperature 25+/-5 degrees C and UV detection at 275 nm in an overall analysis time of about 6 min., based on peak area. The injection repeatability, intraday and interday repeatability were calculated. The procedure provided a linear response over the concentration range 1-12 microg ml(-1) with mean accuracy of 99.50+/-1.30%. The proposed methods were successfully applied for the determination of lamotrigine in bulk powder, in dosage form and in presence of its impurity. The results obtained were analyzed by ANOVA to assess that no significant difference between each of the three methods and the reported one. The validation was performed according to USP guidelines.     

Development and validation of high-performance liquid chromatography method for analysis of β-CCT and its related substances

Compound 2β-carbomethoxy-3β-(4-chlorophenyl)tropane (β-CCT) is a key intermediate for the synthesis of some clinical dopamine transporter (DAT) imaging agents. Potential impurities from synthesis process of β-CCT and degradation during storage might have detrimental effect on the final imaging agents. Thus, it is necessary to guarantee the quality of β-CCT. In this study, a rapid, sensitive and accurate high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of β-CCT and its related substances. The chromatographic separation was achieved on a reverse-phase phenomenex? Gemini C18 column with an isocratic mobile phase consisted of methanol, water and TFA (30:70:0.1 v/v/v). The flow rate was 1.0 mL/min at 30 °C and samples were monitored at 220 nm. The method was validated concerning system suitability, linearity, accuracy, precision, specificity, robustness and stability. The limit of detection (LOD) and the limit of quantification (LOQ) of β-CCT were 0.5 and 1.5 μg/mL, respectively. The linearity range of β-CCT was 1.5–450 μg/mL with a good linear correlation coefficient (R² = 0.9999) between the peak response and concentration. Specificity investigation through forced degradation experiments displayed that β-CCT was stable in acidic, thermal and photolytic degradation conditions, but significantly unstable in alkaline and oxidative conditions. With the developed chromatographic method, possible impurity α-CCT from synthetic process and potential degradation products could be well separated from β-CCT. Good recovery and precision were manifested in the assay method. These results indicated that the present method would be suitable for not only the quality assurance of β-CCT in regular production sample assays but also the monitoring and determination of its related substances.     

Identification of a trace colored impurity in drug substance by preparative liquid chromatography and mass spectrometry

6-(Nitrooxy)hexyl-(2z)-4-(acetyloxy)-3-[4-(methylsulfonyl)phenyl]-2-phenylbut-2-enoate (enoate 1) was investigated as a novel therapy for pain relief. In a recent manufacturing run at the pilot plant scale, the enoate drug substance was found to have a yellowish color not observed previously. An unknown impurity at trace level was detected by high-performance liquid chromatographic (HPLC) analysis and found to be the primary cause for the color of the drug substance. The colored impurity was enriched by preparative HPLC and structurally elucidated by liquid chromatography/tandem mass spectrometry (LC/MS/MS). It was found that the colored impurity was derived from the product of oxidative dimerization of rofecoxib, an impurity present in the enoic acid intermediate. It was further revealed by the photodiode array and LC/MS/MS data that the colored impurity exists in the drug substance as a pair of double-bond isomers with one isomer at majority. These findings were also confirmed by synthesizing the colored impurity through the proposed pathway.     

Development and Validation for Quantification of 7-Nitroso Impurity in Sitagliptin by Ultraperformance Liquid Chromatography with Triple Quadrupole Mass Spectrometry

The purpose of this research study was to develop an analytical method for the quantification of 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4] triazolo [4,3-a] pyrazine (7-nitroso impurity), which is a potential genotoxic impurity. Since sitagliptin is an anti-diabetic medication used to treat type 2 diabetes and the duration of the treatment is long-term, the content of nitroso impurity must be controlled by using suitable techniques. To quantify this impurity, a highly sensitive and reproducible ultraperformance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-MS/MS) method was developed. The analysis was performed on a Kromasil-100, with a C18 column (100 mm × 4.6 mm with a particle size of 3.5 µm) at an oven temperature of approximately 40 °C. The mobile phase was composed of 0.12% formic acid in water, with methanol as mobile phases A and B, and the flow rate was set to 0.6 mL/min. The method was validated according to the current International Council for Harmonisation (ICH) guidelines with respect to acceptable limits, specificity, reproducibility, accuracy, linearity, precision, ruggedness and robustness. This method is useful for the detection of the impurity at the lowest limit of detection (LOD), which was 0.002 ppm, and the lowest limit of quantification (LOQ), which was 0.005 ppm. This method was linear in the range of 0.005 to 0.06 ppm and the square of the correlation coefficient (R²) was determined to be > 0.99. This method could help to determine the impurity in the regular analysis of sitagliptin drug substances and drug products.     

Nickel–poly(o-aminophenol)-modified carbon paste electrode; an electrocatalyst for methanol oxidation

In this work, a modified carbon paste electrode consisting of Nickel dispersed in poly(ortho-aminophenol) was used for electrocatalytic oxidation of methanol in alkaline solution. A carbon paste electrode bulk modified with o-aminophenol was used for polymer preparation by cyclic voltammetry method; then, Ni(II) ions were incorporated by immersion of the modified electrode in 1 M Ni(II) ion solution at open circuit. The electrochemical characterization of this modified electrode exhibits stable redox behavior of the Ni(III)–Ni(II) couple. Electrocatalytic oxidation of methanol on the surface of modified electrode was investigated with cyclic voltammetry and chronoamperometry methods, and the dependence of the oxidation current and shape of cyclic voltammograms on methanol concentration and scan rate were discussed. Also, long-term stability of modified electrode for electrocatalytic oxidation of methanol was investigated.     

Development and Validation of a Stability Indicating LC Method for the Determination of Faropenem in Pharmaceutical Formulations

A simple, selective and sensitive stability indicating LC method has been developed and validated for the determination of faropenem in bulk drug and pharmaceutical formulations in the presence of degradation products. The separation was achieved by using an isocratic mobile phase mixture of acetate buffer of pH 3.5 and methanol (65:35, v/v) and 250 mm × 4.6 mm I.D., 5 μm particle size SGE make Wakosil C-18 AR column at flow rate of 1.0 mL min^?1 with detection at 305 nm. The retention time of faropenem is 6.63 min and was linear in the range of 5–75 μg mL^?1 (r = 0.9999). The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation and was found to be unstable in all the stress conditions. The proposed method was successfully employed for quantification of faropenem in bulk drug and its pharmaceutical formulations.     

Photostability and solubility improvement of β-cyclodextrin-included tretinoin

In this work, we investigated the influence of β-cyclodextrin on the photostability of tretinoin and compared the photo-chemical stability of tretinoin, either in methanol or complexed with β-cyclodextrin, when exposed both to UV and fluorescent light. The physico-chemical characterization of tretinoin-β-cyclodextrin complexes, prepared by the freeze-drying process, using different tretinoin:β-cyclodextrin molar ratios (1:1 and 1:3), was carried out in solution by phase solubility studies, ¹H-NMR spectroscopy, and in solid state by infrared spectroscopy (FT-IR); these analyses confirmed the existence of an inclusion compound. Solubility study results showed that tretinoin solubility was enhanced by inclusion in β-cyclodextrin as a function of increasing concentrations of β-cyclodextrin in aqueous solution at different pH values (i.e., 3.0, 5.5, and 7.0). Moreover, the complexation of the tretinoin with β-cyclodextrin effectively protected the photolabile drug and reduced the degradation of tretinoin induced by UV and fluorescent light, improving its photo-chemical stability in comparison with free drug in methanol. Indeed, dissolved tretinoin in methanol degraded very quickly and completely, while β-cyclodextrin-included tretinoin decomposition was delayed and, after 30 days under UV exposure, the percentage of remaining drug was about 20–25% (depending on the tretinoin concentration). The photodegradation of tretinoin in methanol under fluorescent light was slower: after 5 days of irradiation it reached a photostationary state and intact tretinoin remained constant (6.6%). In conclusion, the β-cyclodextrin complexation always led to a reduction of degradation, depending on the tretinoin:β-cyclodextrin molar ratio and on the drug concentration (0.2 mg/ml or 0.4 mg/ml).     

Identification and characterization of the process‐related impurities in fasudil hydrochloride by hyphenated techniques using a quality by design approach

Following the underlying principles of quality by design mentioned in the ICH Q8 guidance, systematic approaches for the control of process‐related impurities have been taken in the manufacturing process of fasudil hydrochloride, a potent Rho‐kinase inhibitor and vasodilator. Three related impurities were found in fasudil hydrochloride lab samples by a newly developed RP‐HPLC with volatile mobile phase gradient elution and UV detection method. The elemental compositions of the impurities were determined by positive ESI high‐resolution TOF‐MS analysis of their [M + H]⁺ ions and their structures were identified through the elucidation of the product mass spectra obtained by a triple quadrupole mass spectrometer. The key impurity was further verified through synthesis and organic spectroscopy including NMR and IR spectroscopy. The origins of these impurities were located and the effective approaches to eliminate them were proposed based on the redesign of the synthetic conditions. The results obtained are important for quality control in the manufacture of fasudil hydrochloride bulk drug substance and injection.     

Development of chromatographic methods to monitor the synthesis of (tributylstannyl)methanol through unstable lithiated intermediates

Simple and robust derivatization methods for monitoring the formation of (tributylstannyl)methanol from tributyltin hydride via unstable lithiated intermediates have been developed and validated. These analytes present both chromatographic and detection difficulties in their native states due to low volatility, poor aqueous solubility/stability, and lack of a chromophore. Derivatization of these analytes to trimethylsilyl analogues for gas chromatographic analysis or to benzenesulfonyl urethane analogues for reversed-phase liquid chromatographic analysis was evaluated. The derivatization/gas chromatographic methods developed were demonstrated to be more specific and sensitive for impurity monitoring during (tributylstannyl)methanol preparation.     

Synthesis,characterization and control of eight process-related and two genotoxic fingolimod impurities by a validated RP-UPLC method

An HPLC method has been described in the European Pharmacopoeia and United States Pharmacopeia for the determination of nine organic impurities (imp A–I) in fingolimod hydrochloride, a synthetic sphingosine-1-phosphate receptor modulator. The manufacturing process of fingolimod hydrochloride consists of multistep chemical synthesis wherein controls of precursors, intermediates and process steps should be performed to assure the final quality of the drug substance. We synthesized and isolated eight process-related impurities (FINI imp A–H) of fingolimod, which were different from the pharmacopoeial impurities. One unknown process-related impurity was found as a key intermediate (FINI) and was identified by LC–MS. Characterization of all of the impurities were done using spectroscopic techniques (¹H and ¹³C NMR, FTIR, MS), and the mechanistic pathways to the formation of these impurities were also discussed. Two of these impurities were evaluated as potential genotoxic impurities owing to their alerting structures and alkylating properties (alkyl sulfonates and alkyl halides, class 3, ICH M7). We also developed and validated an RP-UPLC method in line with ICH Q2 guidelines for control these impurities (FINI imp A–H) and to assure the pharmacopoeial quality drug substance.     

A rapid and reversible colorimetric assay for the characterization of aminated solid surfaces

The covalent immobilization of synthetic or natural macromolecular compounds containing amino groups onto polystyrene (PS) solid surfaces is of great interest in diagnostic applications. A sensitive assay allowing the determination of reactive end groups is therefore a powerful tool for predicting the performance of the active surface. Recently, we reported the use of the Coomassie brilliant blue (CBB) colorimetric reagent to quantify protonated groups (N⁺) in linear and dendritic structures in solution (Coussot et al., Polym Int 58(5):511–518, 2009). In this work, a simple method using CBB dye for the characterization of PS aminated solid surfaces is developed. The proposed amino density estimation by colorimetric assay (ADECA) method is based on the reversible complexation of the dye with the N⁺ groups on solid surfaces. The assay measures the released dye thanks to the use of a unique sodium carbonate–methanol buffer. Thereby, for the first time, the same surface can be used for characterization and for further coupling applications. A surface density of four N⁺ groups per square nanometer can be measured in PS microwell format, the whole characterization being done within 30 min. Performances of this new colorimetric-based method are detailed. The ADECA method is further demonstrated to be useful for the characterization of aminated polypropylene and glass materials with various sizes and shapes.     

Quantitative Analysis of a Chloroquinolin-Ethenyl Phenyl Derivative Using Thin Layers Impregnated With Di-P-Toluyl Tartaricacid

Abstract

Thin Layer Chromatography (TLC) is an established method for the evaluation of final product drug and intermediate impurity profiles. Quantitative TLC has gained credibility within the Pharmaceutical industry as a result of the latest developments in an availability of scanning technology. In the present paper we wish to report a quantitative TLC method for the determination of some potential impurities which may exist in a final bulk drug MK0679. In order to improve the selectivity of the chromatographic method, di-p-toluyl tartaric acid was impregnated on the stationary phase. Utilizing the modified layer, complete separation of the known impurity was obtained. The calibration curves for all components studied were linear and the detection limits obtained were less than 5ng.     

Electrical double layer and adsorption of iodide ions at the Bi | acetonitrile interface

The adsorption of I^? ions on the Bi(111) single crystal plane from solutions in acetonitrile has been investigated by impedance measurement method. The ionic charge due to the specific adsorption has been obtained using the mixed electrolyte method applied for both electrode charge and electrode potential as the independent electrical variables. The Gibbs energy of ions adsorption has been calculated using the virial adsorption isotherm. It was found that under comparable conditions the results obtained at constant electrode potential and at constant electrode charge are coincident and the Gibbs energy of I^? anion adsorption increases in the sequence of solvents: methanol < ethanol < propylene carbonate < acetonitrile. The electrosorption valency has been calculated and it was found that this parameter increases in the sequence propylene carbonate < ethanol < methanol < acetonitrile.     

Validated HPTLC and HPLC methods for determination of fluorometholone and sodium cromoglycate in presence of their impurities and degradation products; application to kinetic study and on rabbit aqueous humor

Fluorometholone (FLM) and Sodium Cromoglycate (CMG) are co-formulated in ophthalmic preparation and showed marked instability under different conditions. Two specific, sensitive and precise stability-indicating chromatographic methods have been developed and validated for their determination in the presence of their degradation products and FLM impurity. Ten components were efficiently separated by them. The first method was HPTLC-spectrodensitometry, where the separation was achieved using silica gel 60?F₂₅₄ HPTLC plates and developing system of ethyl acetate: methanol (9:1, v/v). The second method was a reversed phase HPLC associated with kinetic study of the degradation process and was successfully applied for determination of the studied compounds in spiked rabbit aqueous humor. The mobile phase was acetonitrile: methanol: 0.05?M potassium dihydrogenphosphate (0.1% trimethylamine); pH 2.5, adjusted with orthophosphoric acid (20: 30: 50, by volume). In both methods, the separated components were detected at 240?nm and system suitability was checked. Good correlation was obtained in the range of 0.10–24.00 and 0.20–48.00?µg band^?1, for FLM and CMG by HPTLC. While for HPLC, the linearity ranges from 0.01–50.00 and 0.05–50.00?µg?mL^?1 for both drugs. The methods were applied in pharmaceutical formulation, where they were compared to the reported method with no significant difference.     

Photolysis of 6-ethoxy-2,2,4-trimethyl-8-nitro-1,2-dihydroquinoline. Unusual dependence on the irradiation wavelength

The spectral and kinetic parameters of transient species generated in the irradiation of 6-ethoxy-2,2,4-trimethyl-8-nitro-1,2-dihydroquinoline were examined by stationary and pulse photolysis in the solvents: heptane, acetonitrile, methanol, and ethanol. Upon excitation of the long-wavelength absorption band (λ_ex > 450 nm), a reversible photochemical reaction was revealed, and the spectral and kinetic parameters of three transient species observed in the photolysis were characterized (λ_max = 390, 400, and 420 nm (acetonitrile), k = 97, 500, and 2000 s⁻¹, respectively). The absorption spectra and the rate constants of the decay of transient species are almost independent of the medium polarity and the presence of oxygen in the system. The excited state generated during irradiation to the short-wavelength absorption band (290 < λ_ex < 350 nm) is inactive in the photochemical reaction and deactivates without the formation of transient species. The mechanism of the reversible photochemical reaction is suggested, which involves the opening of the heterocycle N-C bond upon photoexcitation of the long-wavelength absorption band and the thermal back reaction.     

Development of a Simple,Rapid, and Robust Isocratic Liquid Chromatographic Method for the Determination of Pyrimethamine and its Synthetic Impurities in Bulk Drugs and Pharmaceutical Formulations

Pyrimethamine is an important antiparasitic drug in the treatment of malaria and toxoplasmosis and is often used in combination with either sulfadoxine, sulfalene, or sulfadiazine. Determining the content of pyrimethamine and investigating the related substances is currently possible applying either a compendial monograph utilizing thin layer chromatography as well as liquid chromatographic methods used by the respective manufacturers. To provide a simple method which is capable of determining the content of pyrimethamine and of resolving four of its potential synthetic impurities a very simple, cheap, precise, and accurate isocratic RP-HPLC method was developed. All analytes can be separated within a total runtime of 30 min and the method was linear within the concentration ranges of 0.12–0.740, 0.104–0.621, 0.120–0.710, 2.0–11.8, and 1.01–5.80 µg mL^?1 for pyrimethamine, impurity A, impurity B, impurity C, and impurity D, respectively. These substances were separated by employing a Eurospher-II C₁₈H column (250 × 4.6 mm, 5 µm particle size), a mobile phase being a mixture of a 0.05 M KH₂PO₄ buffer solution (pH 2.6) and methanol in the ratio 40:60 (v/v). The analysis was carried out at 30 °C, applying a flow rate of 1.2 mL min^?1, and a detection wavelength of λ = 215 nm. The coefficients of determinations (R ²) for the five analytes were greater than 0.994 for pyrimethamine and all impurities. Results of recovery studies were within the range of 89.1–105.1% for all substances. In all tested genuine batches of pyrimethamine raw material impurities within the specified limits were present which is concurrent with results obtained from using the present manufacturer’s method.     

Use of alizarin red S as a chromogenic agent for the colorimetric determination of dothiepin hydrochloride in pharmaceutical formulations

The present study describes two simple, rapid, selective and cost-effective spectrophotometric methods for the determination of dothiepin hydrochloride (DOTH), an antidepressant drug, in bulk drug and pharmaceutical formulations. The first method (method A) is based on the formation of yellow colored ion-pair complex between DOTH and alizarin red S (ARS) in acid medium which was extracted into dichloromethane and the absorbance was measured at 445 nm. The second method (method B) is based on the breaking of the yellow DOTH–ARS ion-pair complex in alkaline medium followed by the measurement of the violet color free dye at 570 nm. Under the optimized conditions, Beer’s law is obeyed over the concentration ranges of 2.50–55.0 and 1.00–35.0 μg ml⁻¹ DOTH for method A and method B, respectively. The molar absorptivity, Sandell’s sensitivity, detection and quantification limits are also calculated. The methods were validated for intra-day and inter-day accuracy and precision; selectivity and robustness and ruggedness. The proposed methods were applied successfully to the determination of DOTH in pure drug and commercial formulations. The accuracy and reliability of the proposed methods were further established by parallel determination by the official method and also by recovery studies via standard addition technique.     

CF3‐Inspired Synthesis of Air‐Tolerant 9‐Phosphaanthracenes that Feature Fluorescence and Crystalline Polymorphs

9‐Phosphaanthracene (dibenzo[b,e]phosphorin, acridophosphine) has attracted interest as one of the heavier acenes. Herein, we demonstrate an efficient synthetic process that provides air‐tolerant 1,8‐bis(trifluoromethyl)‐9‐phosphaanthracenes. The sterically encumbered and electron‐withdrawing trifluoromethyl (CF₃) groups are quite advantageous not only to stabilize the intrinsically unstable heavier unsaturated phosphorus atom but also to facilitate construction of the phosphinine skeleton based on a putative increase in aromaticity. The isolated 9‐phosphaanthracenes allowed characterization of their fluorescence functionality and planar heteroanthracene frameworks. The crystal structures of 9‐phosphaanthracenes are remarkably dependent on the aryl substituents at the 10 position; anthryl‐substituted 9‐phosphaanthracene showed unique polymorphs that induced different‐colored crystals.     

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.