Study of the Degradation Kinetics of Carvedilol by Use of a Validated Stability-Indicating LC Method

The objectives of this investigation were to establish a validated stability-indicating LC method for assay of carvedilol and to study the degradation behaviour of the drug under different ICH-recommended stress conditions. Chromatographic separation was achieved on a C₁₈ column with 55:45 (%, v/v) acetonitrile–0.02 m phosphate buffer, pH 3.5, as mobile phase at a flow rate of 1.0 mL min^?1; detection was by UV absorbance at 242 nm. The method was validated for linearity, precision, accuracy, robustness, specificity, and sensitivity, with the bulk drug. The drug was subjected to forced degradation and peaks of all the degradation products were well resolved from that of the pure drug, with significantly different retention times, which indicates the specificity and stability-indicating properties of the method. First-order degradation kinetics of carvedilol were observed under acidic and alkaline conditions. When the utility of the method was verified by analysis of the drug in marketed tablets and a nano-emulsion formulation, the assay was found to be 98.60–99.61 and 99.52–99.87, respectively. These results indicate the method can be successfully used for routine analysis of carvedilol in the bulk drug and in pharmaceutical dosage forms.     

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.     

Study of Forced Degradation Behaviour of Brinzolamide Using LC–ESI–Q-TOF and In Silico Toxicity Prediction

Brinzolamide (BZA) is a topical ophthalmic drug which is generally used to lower the intraocular pressure during glaucoma. It was subjected to forced degradation studies under hydrolytic (acidic, basic), oxidative, photolytic and thermal stress conditions; the drug degraded significantly in hydrolytic and oxidative conditions, leading to the formation of seven degradation products in total. It was stable on exposure to light and dry heat in the solid state. An ultra-performance liquid chromatography (UPLC) method was developed on a Waters CSH phenyl hexyl column (100 × 2.1 mm, 1.7 µm), using gradient elution of 0.1 % formic acid and methanol as mobile phase. The method was extended to quadrupole time-of-flight mass spectrometry (Q-TOF–MS) for the structural characterisation. All degradation products were comprehensively characterised by UHPLC–ESI/MS/MS experiments. The most probable mechanisms for the formation of degradation products were also proposed. In silico toxicity of the drug and its degradation products was determined using TOPKAT toxicity prediction software.

     

Validated LC Method for Simultaneous Analysis of Tramadol Hydrochloride and Aceclofenac in a Commercial Tablet

This paper describes development and validation of a high-performance liquid chromatographic method for simultaneous analysis of tramadol hydrochloride (TR) and aceclofenac (AC) in a tablet formulation. When the combination formulation was subjected to ICH-recommended stress conditions, adequate separation of TR, AC, and the degradation products formed was achieved on a C₁₈ column with 65:35 (v/v) 0.01 M ammonium acetate buffer, pH 6.5—acetonitrile as mobile phase at a flow rate of 1 mL min^?1. UV detection was performed at 270 nm. The method was validated for specificity, linearity, LOD and LOQ, precision, accuracy, and robustness. The method was specific against placebo interference and also during forced degradation. The linearity of the method was investigated in the concentration ranges 15–60 μg mL^?1 (r = 0.9999) for TR and 40–160 μg mL^?1 (r = 0.9999) for AC. Accuracy was between 98.87 and 99.32% for TR and between 98.81 and 99.49% for AC. Because degradation products were well separated from the parent compounds, the method was stability-indicating.     

Stability-Indicating HPTLC Method for Analysis of Moclobemide, and Use of the Method to Study Degradation Kinetics

A sensitive, selective, precise, and stability-indicating HPTLC method for analysis of moclobemide in the bulk drug and in formulations has been established and validated. Aluminium TLC plates precoated with silica gel 60 F₂₅₄ were used with benzene–methanol–40% ammonia 7:3:0.1 (v/v) as mobile phase. Densitometric analysis was performed in absorbance mode at 238 nm. Compact bands were obtained for moclobemide (R _F 0.67 ± 0.02; n = 6). The drug was subjected to acidic and alkaline hydrolysis, oxidation, dry heat treatment, and photodegradation. The drug undergoes degradation under acidic, basic, and oxidising conditions. The degradation products were well resolved from the pure drug with significantly different R _F values, so the method can be regarded as stability-indicating. Response to moclobemide was a linear function of amount in the range 50–600 ng per band, with a correlation coefficient, r ², of 0.9967 ± 0.51. LOD and LOQ, determined experimentally, were 10 and 30 ng per band, respectively. Statistical analysis proves the method is repeatable and specific for analysis of moclobemide. The method was used to investigate the kinetics of alkaline degradation. The Arrhenius plot was constructed and the activation energy calculated.     

Stability-Indicating LC-PDA Method for Determination of Idebenone in Nanoparticles Based on Chitosan and N-Carboxymethylchitosan

A forced degradation study of idebenone was conducted under conditions of UV irradiation, acid, basic and oxidative hydrolysis and in order to develop an isocratic stability-indicating LC-UV method for drug quantification in chitosan and N-carboxymethylchitosan nanoparticles obtained by spray drying. The drug was more labile to alkaline treatment than under the other forced degradation conditions. Idebenone and its degradation products were optimally resolved (resolution >4) on a Luna Phenomenex C₁₈ column with mobile phase composed by methanol:water: (80:20% v/v) at a flow rate of 1.0 mL min^?1, at 30 °C, using wavelength of 279 nm for drug detection. The method was linear, over a drug concentration range of 2 to 10 μg mL^?1. The RSD% value of intra- and inter-day precision studies was <1.5. The method showed excellent recoveries (99.4 to 101.1%). The LOD and LOQ values were found to be 0.18 and 0.59 μg mL^?1, respectively. In conclusion this method can be used as a rapid and accurate assay of idebenone in the nanoparticles during stability tests.     

Validated LC Method for Simultaneous Analysis of Tramadol Hydrochloride and Aceclofenac in a Commercial Tablet

This paper describes development and validation of a high-performance liquid chromatographic method for simultaneous analysis of tramadol hydrochloride (TR) and aceclofenac (AC) in a tablet formulation. When the combination formulation was subjected to ICH-recommended stress conditions, adequate separation of TR, AC, and the degradation products formed was achieved on a C₁₈ column with 65:35 (v/v) 0.01 M ammonium acetate buffer, pH 6.5—acetonitrile as mobile phase at a flow rate of 1 mL min⁻¹. UV detection was performed at 270 nm. The method was validated for specificity, linearity, LOD and LOQ, precision, accuracy, and robustness. The method was specific against placebo interference and also during forced degradation. The linearity of the method was investigated in the concentration ranges 15–60 μg mL⁻¹ (r = 0.9999) for TR and 40–160 μg mL⁻¹ (r = 0.9999) for AC. Accuracy was between 98.87 and 99.32% for TR and between 98.81 and 99.49% for AC. Because degradation products were well separated from the parent compounds, the method was stability-indicating.

     

Study of degradation behavior of besifloxacin,characterization of its degradation products by LC–ESI–QTOF–MS and their in silico toxicity prediction

Knowledge and understanding of the stability profile of a drug is important as it affects its safety and efficacy. In the present work, besifloxacin, a new, fourth‐generation fluoroquinolone antibiotic, was subjected to different forced‐degradation conditions as per International Conference on Harmonization (ICH) guidelines such as hydrolysis (acid, base and neutral), oxidation, thermal and photolysis. The drug degraded under acidic, basic, oxidative and photolytic conditions while it was found to be stable under dry heat and neutral hydrolytic conditions. In total, five degradation products (DPs) were formed under different conditions—DP1 and DP2 (photolysis), DP3 (oxidation), DP4 (acidic), DP3 and DP5 (basic). The chromatographic separation of besifloxacin and its degradation products was achieved on a Sunfire C₁₈ (250 mm × 4.6 mm, 5 μm) column with 0.1% aqueous formic acid–acetonitrile as a mobile phase. The gradient RP‐HPLC method was developed and validated as per ICH guidelines. The degradation products were characterized with the help of LC–ESI–QTOF mass spectrometric studies and the most likely degradation pathway of the drug was proposed. In silico toxicity assessment of the drug and its degradation products was carried out, which indicated that DP3 and DP4 carry a mutagenicity alert.     

UV-assisted degradation of propiconazole in a TiO2 aqueous suspension: identification of transformation products and the reaction pathway using GC/MS

Photocatalytic degradation of propiconazole, a triazole pesticide, in the presence of titanium dioxide (TiO₂) under ultraviolet (UV) illumination was performed in a batch type photocatalytic reactor. A full factorial experimental design technique was used to study the main effects and the interaction effects between operational parameters in the photocatalytic degradation of propiconazole in a batch photo-reactor using the TiO₂ aqueous suspension. The effects of catalyst concentration (0.15–0.4 gL^?1), initial pH (3–9), initial concentration (5–35 mg L^?1) and light conditions were optimised at a reaction time duration of 90 min by keeping area/volume ratio constant at 0.919 cm² mL^?1. Photocatalytic oxidation of propiconazole showed 85% degradation and 76.57% mineralisation under UV light (365 nm/30 Wm^?2) at pH 6.5, initial concentration 25 mg L^?1 and constant temperature (25 ± 1 °C). The Langmuir–Hinshelwood kinetic model has successfully elucidated the effects of the initial concentration on the degradation of propiconazole and the data obtained are consistent with the available kinetic parameters. The photocatalytic transformation products of propiconazole were identified by using gas chromatography–mass spectrometry (GC/MS). The pathway of degradation obtained from mass spectral analysis shows the breakdown of transformation products into smaller hydrocarbons (m/z 28 and 39).     

Comparison of Two Stability-Indicating Chromatographic Methods for the Determination of Mirabegron in Presence of Its Degradation Product

Mirabegron is a novel β₃-adrenoceptor agonist containing an amide group. It was subjected to stress conditions of acidic and alkaline hydrolyses. The hydrolytic degradation product was isolated and its structure was confirmed using mass and IR spectrometry. Two stability-indicating chromatographic methods have been proposed for the determination of mirabegron. TLC method was applied using silica gel as stationary phase and chloroform–methanol–ammonia (9.0:1.0:0.1 by volume) as the mobile phase, and chromatograms were scanned at 250 nm. Accurate determination of the drug was achieved over the concentration range of 2–12 μg per band. In addition, an isocratic HPLC method was developed on Agilent C18 column (150 mm × 4.5 mm I.D., particle size 5 µm) using ethanol-phosphate buffer pH 2.5 (30:70, by volume) as a mobile phase with flow rate of 1 mL min^?1.The intact drug was detected at 250 nm with running time less than 5 min. Mirabegron was determined accurately in a concentration range of 1–25 µg mL^?1. The proposed chromatographic methods were applied successfully for the assay of mirabegron in pharmaceutical dosage form and both methods were validated as per the International Conference on Harmonization guidelines and statistically compared with a reported gradient HPLC method.     

Fast Determination of Sudan I-IV in Chili Products Using Automated On-Line Solid Phase Extraction Coupled with Liquid Chromatography-Mass Spectrometry

A rapid, accurate and precise method for the determination of sudan I-IV in chili products using on-line solid phase extraction and LC-MS has been developed. Chili products were extracted with acetone and the analytes were cleaned up and enriched on an SPE column (C₁₈, 15–40 µm) through on-line SPE. Chromatographic separation was performed on a C₁₈ analytical column (2.1 × 150 mm, 3 µm) with gradient elution programming of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. All four sudan dyes were separated in less than 8 min. Using in-house validation data, linearity coefficients of determination (R²) of more than 0.9997 were obtained. The limits of detection (LOD) and limits of quantitation (LOQ) for sudan I, II and IV were 0.03 and 0.05 mg kg^?1, respectively, and 0.04 and 0.1 mg kg^?1 for sudan III. The intra- and inter-day recoveries of the four sudan dyes in chili powder were between 90.1–101.6% and 90.2–102.0%, respectively, with relative standard deviation (RSD) between 0.014–0.164% and 0.011–0.202%, respectively. Therefore, this proposed method could be an alternative assay for the determination of sudan I-IV in chili products due to its rapidness, sensitivity, less sample and solvent consumption.     

SPE and LC–ESI–MS for Quantitative Analysis of Mitiglinide in Human Plasma in a Bioequivalence Study

Liquid chromatography–electrospray ionization mass spectrometry has been used for rapid, selective, and sensitive quantitative analysis of mitiglinide in human plasma. Sample pretreatment involved solid-phase extraction from plasma with gliclazide as internal standard. Separation was performed on a C₁₈ column (150 × 2.0 mm) with 71:29 (v/v) acetonitrile–water (containing 0.1% formic acid and 0.2 mmol L^?1 ammonium acetate) as mobile phase at a flow rate of 0.2 mL min^?1. The method was validated then successfully applied to a clinical bioequivalence study of mitiglinide in 20 healthy volunteers after oral administration.     

LC Method for Studies on the Stability of Sibutramine in Soft Gelatin Capsules

A sensitive and rapid liquid chromatographic method was successfully developed and validated for the determination of sibutramine hydrochloride in bulk and capsules. Sibutramine in the presence of its degradation products was analyzed using UV detection at 225 nm. Chromatography was performed on a reversed-phase C₈ (150 × 4.0 mm I.D., 5 μm) analytical column under isocratic conditions. The mobile phase was composed of acetonitrile:water (aqueous phase containing 0.3% triethylamine and pH adjusted to 7.0) (75:25, v/v) at a flow-rate of 1.1 mL min⁻¹. No chromatographic interference was found during the analysis. Light was the stress condition which most contributed to sibutramine degradation. The method showed a linear response (r > 0.999) from 30 to 90 μg mL⁻¹. The mean recovery for capsules was 101.2%. Inter-day assays showed relative standard deviations of 0.42 and 1.62% for bulk and capsules, respectively. The developed method is able to separate sibutramine from its major degradation products and it may be used in the quality control of this active pharmaceutical ingredient in both bulk and capsules.

     

Development of a Stability-Indicating LC Assay Method for Determination of Chloroquine

A simple and rapid development of a stability-indicating LC method for determination of chloroquine diphosphate in the presence of its hydrolysis, oxidative and photolysis degradation products is described. Stress testing showed that chloroquine diphosphate was degraded under basic conditions and by photolytic treatment but was stable under the other stress conditions investigated. Separation of the drug from its degradation products was achieved with a Nova Pack C18 column, 0.01 M PIC B7 and acetonitrile (40:60 v/v) pH 3.6, as mobile phase. Response was linear over the range 0.08–5.70 μg mL⁻¹ (r = 0.996), with limits of detection and quantification (LOD and LOQ) of 0.17 and 0.35 μg mL⁻¹, respectively.

     

Preconcentration of radioiodine as AgI and purification from radiotellurium waste

A radiochemical method was investigated for separation and preconcentration of radioiodine from alkaline radiotellurium waste solution as Ag¹²⁵I followed by recovery of ¹²⁵I into aqueous NH₃ solution and final purification by wet distillation. ¹²⁵I–^123mTe radiotracer solution (5 M NaOH) was equilibrated with prepared silver granules for different times and it was found that ¹²⁵I was quantitatively removed from the aqueous phase after 7.0 h. The ¹²⁵I-loaded silver was then equilibrated with ammonia solution in the presence of one of different reducing agents (namely, sodium borohydride, dextrose and zinc dust). Different concentrations of NH₃ solution and reducing agents were studied. Quantitative recovery of ¹²⁵I in the aqueous phase was achieved after 1.0 h of equilibration of ¹²⁵I-loaded silver granules with 2.7M NH₃ solution in the presence of Zn dust with a Zn:Ag molar ratio of 0.5. Purification of the recovered ¹²⁵I was carried out by wet distillation from 20 % H₂SO₄ in the presence of H₂O₂. The distilled off ¹²⁵I was received in a mixture solution of 0.1M NaOH and 0.01M Na₂S₂O₃ with a radionuclidic purity of ≥99.99 %, radiochemical purity of ~98.8 % (as I^? anions) and pH-value of ~13.     

Destruction of 2,4 Dichlorophenol in an Atmospheric Pressure Dielectric Barrier Discharge in Oxygen

The processes of degradation of 2,4-dichlorophenol (2,4-DCP) under the action of atmospheric pressure of dielectric barrier discharge (DBD) in oxygen were studied. It was shown that the degradation of 2,4-DCP proceeds efficiently. Degree of decomposition reaches 90%. The degradation kinetics of 2,4-DCP obeys the formal first-order kinetic law on concentration of 2,4-DCP. The effective rate constants depend weakly on the experimental conditions and are equal to ~0.2 s^?1. Based on experimental data, the energy efficiency of decomposition of 2,4-DCP was determined. Depending on the conditions, the energy efficiency was in the range of (8–90) × 10^?3 molecules per 100 eV. The composition of the products was studied by gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection-fourier transform infrared (ATR-FTIR) spectroscopy, electron spin resonance (ESR) spectroscopy and UV/Visible spectroscopy. It was shown that about ~20% of 2,4-DCP is converted to CO₂, while the other part forms an organic film on the reactor wall. The substance formed is close to the carboxylic acids in chemical composition and exhibits electrical conductivity and paramagnetic properties. Almost all of the chlorine contained in the 2,4-DCP is released into the gas phase. The active species of the afterglow react with liquid hexane, forming the products of its oxidation. Some assumptions regarding the pathway of the process are discussed.     

Kinetic studies on H+- and OH−-catalysed aquation of bis-aspartatochromium(III)

A potential synthetic biochromium source, bis-aspartatochromium(III) ion (where Asp is a tridentate N,O,O′-ligand, bonded via amine nitrogen and carboxylate oxygen atoms) has been obtained and characterized in aqueous solution. Kinetics of partial dechelation of the complex catalysed by H⁺ and OH^? ions has been studied spectrophotometrically within 0.1–1.0 M HClO₄ and 0.1–1.0 M NaOH ranges under first-order conditions. A linear dependence of the k _obs,H on [H⁺] and independence of the k _obs,OH on [OH^?] were established. The derived rate expression and identification of components of the reaction mixture provide evidence for a reaction mechanism, where the key role in the overall process is the formation of an intermediate species with bidentate N,O-bonded Asp via both spontaneous and H⁺(OH^?)-catalysed reaction paths. The intermediate is meta-stable and at pH 5–7 restores the substrate.     

Simultaneous Quantification of Benazepril, Gliclazide and Valsartan in Human Plasma by LC–MS–MS and Application for Rapidly Measuring Protein Binding Interaction between Rhein and These Three Drugs

For new drug candidates with high protein binding in the treatment of diabetic nephropathy, their influence on the protein bindings of angiotensin-converting enzyme inhibitors, sulfonylurea drugs, and angiotensin receptor blockers should be predicted to prevent side effects. To provide an efficient tool for this study, a sensitive and rapid LC–MS–MS method was developed for the simultaneous quantification of representative drugs, benazepril, gliclazide and valsartan in human plasma. Chromatographic separation was performed on a Shim-pack VP-ODS C18 column (250 × 2.0 mm i.d., 5 μm) using methanol–0.05% formic acid (90:10, v/v) as mobile phase. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in SRM mode. Lower limits of quantification were 2, 2 and 20 ng^?1 mL for benazepril, gliclazide and valsartan with 0.1 mL plasma sample. The method fulfills the precision, accuracy, linearity, sensitivity, selectivity requirements to quantify the three drugs and has been successfully used to studying protein binding of benazepril, gliclazide and valsartan in the presence of rhein.     

The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile–0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min⁻¹. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid–base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method’s linearity was investigated in the ranges 0.40–1.40 µg mL⁻¹ for isomer I and 0.40–2.40 µg mL⁻¹ for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL⁻¹ for isomer I and 0.0356 and 0.1078 µg mL⁻¹ for isomer II, respectively.

     

RP-LC Analysis and Hydrolytic Degradation Profile of Racecadotril

A sensitive, stability-indicating liquid-chromatographic method for analysis of racecadotril in the presence of its degradation products has been developed and validated. Efficient chromatographic separation was achieved on a C₁₈ column with a simple isocratic mobile phase—60:40 methanol–water. Quantification was by photo-diode array (PDA) detection at 220 nm. The linearity of the method was excellent over the range 1–32 μg mL^?1. The method was sensitive, with low limits of detection (20 ng mL^?1) and quantification (100 ng mL^?1). The recovery of the method was consistently good (98.7–100.9%), with low (<1%) intra-day and inter-day relative standard deviation. Robustness studies confirmed that peak area was unaffected by small changes in temperature, and mobile phase composition and flow rate. Both alkaline and acidic hydrolytic degradation were performed in methanolic solution. In alkaline medium the drug was degraded immediately; it was degraded within 90 min in acidic medium. The validated, stability-indicating, method was used for analysis of racecadotril in pharmaceutical dosage form and also to reveal the hydrolytic degradation profile of the racecadotril.