Photolytic Conditions Research Articles

Bridging Pharmaceutical Analysis with Computational Chemistry to Ascertain the Pharmacokinetic and Toxicological Profiling of Febuxostat and its Diacid Impurity

Drug degradation may change the pharmacological action and results in altered therapeutic and toxicological profile of drug. Identification of degradation product is good opportunity in the drug discovery, as non-toxic impurities can be considered for the new pharmacological potentials. Additionally, development of computer based in-silico model and methods further assist in prediction of pharmacokinetic, dynamic as well as biological activities which make drug discovery process more unfailing. In present work, Febuxotat (FEB) was subjected to forced degradation experiments and the formed degradation products were separated and resolved using optimized HPLC method. Further the study was extended to enrich its alkali degradation product and was ascertained using 1H NMR spectroscopy and mass spectrometry. The ADME/Toxicological properties of alkali degradation product were determined by using Admet SAR 2.0. The method was validated as per ICH guidelines Q2 (R1). In forced degradation experiments, substantial degradation of FEB was observed under acid, alkali, wet heat, dry heat and oxidative conditions, however, FEB was found stable to photolytic conditions. The drug and its formed degradation products separated and resolved from each other on Waters SunFire C8 (250 × 4.6 mm, 5 µm) column using blend of methanol: 20 mM potassium phosphate buffer (pH 3.0) (80: 20 % v/v). From the 1H NMR spectroscopy and mass spectrometry the enriched alkali degradation product was confirmed as 2-[3-carboxy-4-(2-methylpropoxy) phenyl]-4-methyl-1,3-thiazole-5-carboxylic acid (diacid FEB). Febuxostat and diacid Febuxostat was found non-toxic for carcinogenicity (binary), eye corrosion, eye irritation and Ames mutagenesis during ADME/Tox studies. However, both were found hepatotoxic. A simple, accurate, precise, and robust stability indicating HPLC method was developed and validated for the estimation of FEB in bulk and tablet dosage form. Based on ADME/Tox both were found to hepatotoxic. Henceforth, based on our evidences, one should use FEB with precaution in patient suffering from liver disease.

A Stability Indicating Method Development of Lopinavir and Rotinavir in Combined Tablet Dosage Forms by RP-HPLC

A new stability indicating RP-HPLC method was developed for simultaneous estimation of Lopinavir and Rotinavir in combined tablet formulations. Chromatography was carried out on a Kromasil C18 HPLC Column (250 x 4.6mm; 5µm) eluting with a mobile phase consisting of a 30:70v/v mixture of Phosphate buffer and acetonitrile at a flow rate of 1.0mL/minute. The wavelength detection was fixed at 220nm. Using standard addition method accuracy was assessed. The developed HPLC method was validated with respect to precision, specificity, accuracy, linearity and robustness. Forced degradation studies on the formulation were conducted by adopting the proposed method to assess the stability of the analytes under acid, base, peroxide, thermal and photolytic conditions and suitability of the method to resolve the degradation products.

Force degradation studies of relugolix: identification, isolation and structure characterization of stress degradation products by using liquid chromatography-mass spectrometry, auto purification mass mediated preparative-high performance liquid chromatography, high resolution mass spectrometry, nuclear magnetic resonance spectroscopy

To assess the stability of Relugolix under stress conditions, identify and characterize the degradation products, it was subjected to hydrolytic and oxidative stress conditions, according to the International Council for Harmonization guideline Q1A (R2). The drug showed degradation in basic and oxidative conditions, while it was stable in acid conditions. Four degradation products were obtained, two from each basic and oxidative degradation, which was separated on a C18 column by using Auto Purification Mass Spectrometer. The four degradant products have not been reported earlier in any of the literature and are being reported here for the first time. HIGHLIGHTS Present work captures force degradation of Relugolix under acidic, basic, oxidative, photolytic, and thermal conditions as per International Council for Harmonization guidelines. Two degradation products were observed in basic condition marked as Degradation Product-1, Degradation Product-2 and two degradation products were formed in oxidative condition marked as Degradation Product-3, Degradation Product-4. All the four degradants are novel and are not published in any of the literature. Four degradation products were identified and characterized using various high-end analytical techniques such as Liquid Chromatography and Mass Spectrometer, Reversed Phase–High Performance Liquid Chromatography, High Resolution Mass Spectrometer and Nuclear Magnetic Resonance spectroscopy.

Identification, Isolation and Characterization of Degradation Products (DPs) in Osimertinib Mesylate (OSM) Tablets by UHPLC-IMS-QTOF-MS, and NMR: Evaluation of Genotoxicity for OSM and DPs

The aim of this study is identification, isolation, structural characterization and qualification of degradation products DP-1, DP-2, DP-3 and DP-4. These impurities are major acid hydrolysis degradation products identified i n Osimertinib M esylate ( OSM) Tablets. T he O SM i s o bserved s table u nder thermal, photolytic and neutral conditions. The degradation of drug is observed under acidic, basic and oxidative conditions. Basic and oxidative degradation products already been reported, however, four novel degradation products are formed in acid hydrolysis which are not reported in the literature. Therefore, Using HPLC/PDA gradient program with waters X-bridge (250 mm × 4.6 mm, 5 µm) C18 column, the four acid hydrolysis degradants were identified and further characterized using UHPLC-IMS-QTOF-MS (ion mobility quadrupole time-of-flight m ass spectrometer) a nd N MR. T he s tructures o f D P-1, D P-2, D P-3 a nd D P-4 w ere further elucidated as hydroxy OSM Impurity, Des-acryl OSM impurity, Chloro OSM impurity and OSM dimer impurity, respectively. The structural characterization was followed by qualification of Impurities DP-1, DP-2, DP-3 and DP-4 using several different in-silico methodologies. From the results obtained, it can be concluded that no new structural alerts have been generated for DP-1 and DP-4 relative to the parent compound OSM. However, the degradation products, DP-2 and DP-3 shows structural alert positive for genotoxicity, which were both potential genotoxic substances. The current study presents separation, isolation, characterization and in-silico toxicity study for major acid degradants of OSM. The OSM generates DP-1 around 3.1%, DP-2 around 14.1%, DP-3 around 18.3% and DP-4 around 2.8% under acid hydrolysis on HPLC. The stress study was carried out in order to properly define the quality specifications for OSM drug products.

Development and Validation of Novel Ultraviolet Spectrophotometric Method for Estimation of Antileishmanial Drug Buparvaquone

A novel, simple, accurate, precise, economical and reliable ultraviolet spectrophotometric method has been developed for the estimation of buparvaquone in bulk and in pharmaceutical dosage form. The drug shows maximum absorption at 251 nm by using acetonitrile as solvent. The method was validated as stated in International Council for Harmonisation Q2 (R1) guidelines. It obeys Beer’s law in the concentration range of 2-20 μg/ml with correlation coefficient of 0.998. The drug shows great accuracy close to 100 %. The method was found to be robust and precise as the relative standard deviation are less than 2 %. Limit of detection and limit of quantitation were found to be 0.60 μg/ml and 1.83 μg/ml respectively. From the results of specificity, the drug was found to be more degraded under alkaline, oxidative and photolytic conditions. The proposed method can be employed for the reliable quantification of buparvaquone in bulk and routine analysis of pharmaceutical formulations.

ISOLATION AND STRUCTURAL ELUCIDATION OF SACUBITRIL SODIUM DEGRADATION PRODUCTS BY MASS AND NMR SPECTROSCOPY

As per ICH Q1A (R2), stress testing (i.e., acidic, basic, oxidative, thermolytic, hydrolytic, and photolytic) was carried out at room temperature with a single batch of Sacubitril Sodium of the drug substance. Though the drug is resistant to oxidation, thermal, and photolysis conditions, two degradation products were formed during base stress. By using gradient preparative HPLC and a normal C-18 column, the degradants were separated, and the obtained SAC D-1 and SAC D-2 were precisely characterized by LCMS and extensive NMR (including 2D) spectroscopic methods. Study of NMR spectra (1D, 2D) and Mass spectrometry confirmed the suggested structures for the products 5-([1,1'-biphenyl]-4-yl)-4-(3-carboxy propanamide)-2-methyl pentatonic acid (SAC D-1) and (3S)-5-([1,1'- biphenyl]-4-ylmethyl)-3-methyl pyrrolidin-2-one (SAC D-2).

Method Development and Validation of a Specific Stability Indicating RP-HPLC Method for Molnupiravir API

A simple, accurate, isocratic stability indicating RP‐HPLC method was developed for the determination of Molnupiravir in bulk drug. This RP-HPLC was achieved on “Waters 2695 using an Agilent Zorbax Eclipse C18 (250 mm × 4.6 mm × 5 µm)” column with the mobile phase consisting of 30 mM, ammonium phosphate monobasic and Methanol in the ratio of 47:53 %v/v. The stress testing of Molnupiravir was carried out under acidic, alkaline, oxidative, thermal, and photolytic conditions and formed degradation products were well resolved from Molnupiravir API (active pharmaceutical ingredient). The suggested approach was validated according to ICH (International Council on Harmonisation) principles, and all of the validation parameters' findings were within acceptable limits. The method was proven to be appropriate for quality control of Molnupiravir in bulk and pharmaceutical dose forms, as well as stability testing.

Reactions of the unsaturated methyl-bridged complexes [Mo2Cp2(μ-CH3)(μ-PtBu2)(CO)x] (x = 1, 2) towards transition metal carbonyls: convenient dehydrogenative route to heterometallic methylidyne-bridged clusters

The unsaturated dicarbonyl complex [Mo2Cp2(μ-CH3)(μ-PtBu2)(CO)2] (1) reacted with [W(CO)6] under photolytic conditions to give three isomers of formula [Mo2WCp2(μ3-CH)(μ-PtBu2)(CO)7] (cis-3, cis-3´ and trans-3 in a ratio ca. 10:1:3), these differing in the relative disposition of the Cp, CO and methylidyne ligands, as inferred from the available spectroscopic data and the crystal structure of trans-3 (Mo−Mo = 2.8931(4) Å). The cis isomers are thermally unstable species which could not be isolated as pure solids as they either decomposed upon manipulation (cis-3´) or rearranged into the corresponding trans isomer (cis-3). In contrast, no reaction was observed between this compound and [W(THF)(CO)5] in the absence of photochemical activation, yet a rapid reaction occurred between this fragment and the monocarbonyl [Mo2Cp2(μ-CH3)(μ-PtBu2)(μ-CO)] (2) to give the cis-3 isomer exclusively. The reaction of 2 with [Fe2(CO)9] at room temperature led to a mixture of two trinuclear clusters: the 46-electron methylidyne-bridged [Mo2FeCp2(μ3-CH)(μ-PtBu2)(CO)5] (4a) and the acetyl-bridged [Mo2FeCp2{μ3-κ1:η2:κ1-C(O)CH3}(μ-PtBu2)(CO)5] (5) in a ca. 3:1 ratio. A symmetrical coordination of the methylidyne group is found in compound 4a, while the electronic unsaturation is mainly located at the Mo−Mo edge (2.667(1) Å). In contrast, an asymmetric 5-electron donor coordination of the acetyl group is observed in compound 5 and, accordingly, the intermetallic lengths are consistent with the formulation of single bonds in a 48-electron trinuclear cluster (Mo−Mo = 2.8235(4) Å and Mo−Fe ca. 2.76 Å). Finally, the reaction of compound 2 with [Ru3(CO)12] required photochemical activation and then led to the formation of the corresponding unsaturated methylidyne cluster [Mo2RuCp2(μ3-CH)(μ-PtBu2)(CO)5] (4b) in high yield.

Comprehensive degradation profiling and influence of different oxidizing reagents on tinoridine hydrochloride: Structural characterization of its degradation products using HPLC and HRMS.

Stress testing on tinoridine hydrochloride was carried out using a multidimensional approach. This included different conditions: hydrolytic (acidic, alkaline, and neutral conditions), different oxidative reagents, thermal, photolytic conditions, HPLC method development, and structural elucidation using high-resolution mass spectrometry (HRMS). It provides the basis for quality control of tinoridine hydrochloride and its derivatives during storage conditions. The tinoridine hydrochloride was subjected to a variety of stress conditions. A gradient reversed-phase HPLC method was developed on a X-Bridge C18 column (250 × 4.6mm, 5μm) to separate all the degradation products (DPs). HRMS studies have been performed to elucidate the structure of DPs. HPLC-PDA study revealed that significant degradation products were formed in hydrolytic, AIBN (radical initiator at 40°C), thermal, and solid-state photolight stress conditions, but the drug was stable under oxidative conditions (H2 O2, Fenton's reagent at room temperature and ferric chloride at 40°C). The structure of degradation products was elucidated, and mechanism of their formation was explained. Stress study was successfully carried out as per ICH Q1A (R2) guideline on tinoridine hydrochloride. A total of six new degradation products were characterized, DP 2 and DP 6 formed by the effect of co-solvent. This study provides the scientifically sound basis for quality monitoring and storage conditions of tinoridine hydrochloride.

Stability-Indicating Reverse-Phase High-Performance Liquid Chromatographic Method Development and Validation of Lamotrigine in Bulk and Pharmaceutical Dosage Form

In the current study, analytical method has been validated by system suitability parameters, linearity, accuracy and percent recovery, precision, ruggedness, robustness, limit of detection, and limit of quantification. The present study of lamotrigine was achieved using Cosmosil C18 (250 nm×4.6 ID, particle size: 5 Micron) Column with mobile phase methanol: water (60:40) pH: 3 at a flow rate 0.8 ml/min with UV detection at 308 nm. The retention time for lamotrigine was found to be 4.979 min. In linearity, the correlation coefficient (R2) for lamotrigine was found to be 0.999, slope is 39,801, and intercept was found to be 51,862 which are well within the acceptance criteria. The mean percent recovery for lamotrigine at three different levels for 50%, 100%, and 150% was found to be 99.28%, 99.30%, and 100.40%. The % RSD should not more than 2%. In precision study, interday (RSD is 0.41%) and intraday (RSD is 0.26%) are found. Forced degradation experiments were carried out by exposing standard form of lamotrigine for acid-base, oxidative, photolytic, and thermal stress conditions. Hence, the developed method is accurate, precise, repeatable, and reproducible and can be used for routine analysis of lamotrigine.

New stability indicating RP-HPLC-PDA method for determination of mifepristone in bulk and tablet formulation

BackgroundMifepristone is progestational and glucocorticoid hormone antagonist. The objective of this study is to develop simple and economical stability indicating RP-HPLC method for the determination of mifepristone in bulk and tablet formulation.ResultThe chromatographic separation was achieved on Qualisil BDS C8 column with mobile phase containing of mixture of Buffer (Potassium dihydrogen ortho phosphate, pH to 3.0 with ortho phosphoric acid) and Organic Solvent (Acetonitrile) 60:40 v/v pumped at flow rate 0.6 mL min−1. The detection of elute was performed using PDA detector at 305 nm. Mifepristone was eluted at 8.67 min. According to international conference on harmonization Q2(R1) guideline, method was validated and shows satisfactory results for accuracy, precision, linearity, ruggedness, robustness, detection limit, quantitation limit. The method indicated to be linear in the series of concentration 3–18 µg mL−1, and correlation coefficient was 0.9997. In acidic, basic, oxidative, thermal, photolytic forced degradation conditions, the peak of degradation product was clearly and well separated from drug peak without any interference in quantitative analysis. This represents stability indicating nature of established method.ConclusionThe established RP-HPLC method is simple, accurate, specific, precise, robust, rugged, sensitive, and economical in nature which can be utilized for routine analysis of mifepristone in bulk and pharmaceutical formulation.

Implementation of design of experiments for optimization of forced degradation conditions and development of a stability-indicating high-performance liquid chromatography method for sepiwhite.

Sepiwhite is a novel anti-pigmenting agent that is derived from fatty acid and phenylalanine and used for hyperpigmentation induced by light exposure or inflammation. In this study, a simple and validated high-performance liquid chromatography method for the quantitation of sepiwhite was developed. Optimized forced degradation of sepiwhite at thermal, acid/base, photolysis, oxidative, and heavy metal ions conditions were evaluated and the effect of each of them on production of specific 10%-30% degradants was studied by the approach of design of experiments. Sepiwhite accelerated study was conducted and toxicity of sepiwhite at each condition was tested. An optimized high-performance liquid chromatography method was validated by a face-centered central composition design. Ten different degradants were identified from sepiwhite and degradation behavior under different conditions was studied. Sepiwhite and its degradant products show no cytotoxicity. This optimized high-performance liquid chromatography method can be applied for quality control assay and sepiwhite degradation behavior may be considered in the manufacturing of sepiwhite products.

STABILITY INDICATING HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF EMTRICITABINE, DOLUTEGRAVIR AND TENOFOVIR ALAFENAMIDE IN THEIR COMBINED PHARMACEUTICAL DOSAGE FORM

A simple, rapid, precise and accurate reversed phase HPLC stability indicating method was developed for simultaneous determination of emtricitabine (ECT), dolutegravir (DOL) and tenofovir alafenamide (TEN) in their combined dosage marketed formulation. The chromatographic separation was achieved on Hypersil BDS C18 (250  4.6 mm, 5.0 µ) column using 0.5 M potassium dihydrogen phosphate buffer: acetonitrile (80: 20, v/v), pH 3.5 ± 0.1 maintained by 0.1 % o-phosphoric acid as mobile phase at a flow rate of 1.0 mL/min and monitored at 260 nm. All three analytes were resolved at retention times of 5.777, 6.783, 8.393 min for ECT, DOL, and TEN respectively. The assay performance of the present developed method was validated for various parameters like system suitability, linearity, accuracy, precision, robustness, limit of detection and quantification. The calibration curves were linear over concentration ranges of 12.5-75.0, 50.0-300, and 6.25-37.5 μg/ml for DOL, ECT, and TEN, respectively with correlation coefficients  0.9995. All three analytes were subjected to various stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found in alkaline condition. The present method demonstrated to be effective stability-indicating by resolution of the analytes from their forced-degradation products. The validated method was successfully applied for the simultaneous estimation three drugs in their combined pharmaceutical dosage form Spegra.

Synthesis of [(CO)5MS=CFcCH3] and exploration of the nature of M-S vs. M-O bonds in [(CO)5ME=CFcCH3]; (M = Cr, Mo, W and E = O, S) complexes

Photolysis of a methanol solution containing substituted alkyne, group VI metal hexacarbonyl and P4S10 at low temperature under an inert atmosphere leads to metal-sulfur coordinated complexes of chromium, molybdenum, and tungsten. The structure of pentacarbonylthioacetylferrocene-S-chromium (1) is confirmed by single crystal x-ray diffraction study and both the complexes (1 and 2) are confirmed using IR and NMR spectral studies. The sulfur-coordinated metal carbonyl complexes are thought to be formed by the initial activation of metal carbonyl with acetylenic ligand which then rearranges into metal coordinated thioacetylferrocene by dithiophosphoryl radical (formed in situ by reaction of P4S10 and methyl alcohol under photolytic condition). The stability of pentacarbonylthioacetylferrocene of group VI metal complexes are further compared with their acetylferrocene derivatives of Group VI metal complexes using DFT studies. Density functional theory was performed on six complexes [(CO)5ME=CFcCH3] (where M = Cr, Mo and W; while E = O and S) to assess their geometry and electronic structure aspects. Natural bonding orbital (NBO) and energy-decomposition analysis (EDA) were carried out to probe the nature and strength of the M-E interactions. NBO and EDA analysis predict M-E bond strength in the following order: Cr-E < Mo-E < W-E. The M-E bond covalency increases as we move down the group from Cr, Mo to W, which rationalizes the observed trends in the M-E bonds. While comparing the nature of the E (O and S) linkage in bonding, we found that -S analogue shows exceptionally robust σ and π interactions compared to the -O analogue. The reasonably strong M-S interactions resulted in the isolation of these -thio analogues.

A Stability Indicating Novel UPLC-PDA Method for the Estimation in Bulk and Capsule Dosage form of Isavuconazole Effective for Mucormycosis Caused by COVID-19 Pandemic

The present research provides a new proven ultra-high-performance liquid chromatographic technique for isavuconazole in both bulk and capsule dosage forms. DIKMA Waters BEH C18(50 x 2.6mm, 1.7m) column was used for chromatographic separation. With the isocratic elution mode, a mixture phosphate buffer: methanol 40:60 v/v, was used as the mobile phase, and the eluent was measured at 253 nm using a UV detector. The strategy has been maintained and validated in accordance with the International Conference on Harmonization Guidelines. Stressed deterioration has also been investigated in acidic, alkaline, peroxide, thermal, and photolytic conditions. Isavuconazole was eluted with the retention time 0.861 minute in this procedure. The calibration curve plots for isavuconazole were found to be linear within the concentration ranges of 1-25µg/mL. With a percentage recovery of 100.18 percent, the limit of detection was 0.025g/ml and the value for limit of quantification was 0.50µg/mL. In a force degradation analysis, the current approach was also determined to be stable. Based on the experiential evidence, the present method was found appropriate of isavuconazole estimation in the form of bulk and capsule.

VALIDATED AND QUANTIFIED STABILITY INDICATING STRESS DEGRADATION STUDY OF ORAL ANTI-DIABETIC AGENT CANAGLIFLOZIN BY RP-HPLC METHOD

Objective: The present investigation is aimed to develop and validate, a simple, consistent and sensitive stability-indicating reverse phase-high performance liquid chromatography (RP-HPLC) method for the determination of oral anti-diabetic drug Canagliflozin in bulk and pharmaceutical dosage form as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH-Q2 (R1)). Methods: The chromatographic separation was achieved by using Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) ZORBAX C18 (250 mm x 4.6 mm, 5μm particle size) with a mobile phase consisting of Acetonitrile: Water in a ratio of 53:47% v/v at a flow rate of 1 ml/min with an injection volume of 20 μl. Results: The Retention time of the drug Canagliflozin was found to be 2.36±0.05 min and detected at 214 nm UV wavelength. The linear regression equation was found to be y = 60702x–2156.2 with a correlation coefficient 0.9999. Stress degradation studies were performed by exposing the Canagliflozin into acidic, alkaline, oxidative, thermal and photolytic stress conditions with active samples withdrawn at different time intervals as per ICH guidelines. Conclusion: The proposed Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was found to be robust, precise and specific for estimation of Canagliflozin in pharmaceutical dosage forms.

A Stability-Indicating Ultra Performance Liquid Chromato-Graphic (UPLC) Method for the Determination of a Mycophenolic Acid-Curcumin Conjugate and Its Applications to Chemical Kinetic Studies.

A simple, precise, and accurate reversed-phase ultra-performance liquid chromatographic (UPLC) method was developed and validated for the determination of a mycophenolic acid-curcumin (MPA-CUR) conjugate in buffer solutions. Chromatographic separation was performed on a C18 column (2.1 × 50 mm id, 1.7 µm) with a gradient elution system of water and acetonitrile, each containing 0.1% formic acid, at a flow rate of 0.6 mL/min. The column temperature was controlled at 33 °C. The compounds were detected simultaneously at the maximum wavelengths of mycophenolic acid (MPA), 254 nm, and curcumin (CUR), or MPA-CUR, at 420 nm. The developed method was validated according to the ICH Q2(R1) guidelines. The linear calibration curves of the assay ranged from 0.10 to 25 μg/mL (r2 ≥ 0.995, 1/x2 weighting factor), with a limit of detection and a limit of quantitation of 0.04 and 0.10 μg/mL, respectively. The accuracy and precision of the developed method were 98.4–101.6%, with %CV < 2.53%. The main impurities from the specificity test were found to be MPA and CUR. Other validation parameters, including robustness and solution stability, were acceptable under the validation criteria. Forced degradation studies were conducted under hydrolytic (acidic and alkaline), oxidative, thermal, and photolytic stress conditions. MPA-CUR was well separated from MPA, CUR, and other unknown degradation products. The validated method was successfully applied in chemical kinetic studies of MPA-CUR in different buffer solutions.

A New Stability-indicating Chiral RP-HPLC Method for the Determination of Degradation Products in Meclizine Hydrochloride

Background:An enantiomeric separation of the stability-indicating high-performance liquid chromatographic method was developed and validated for the analysis of Meclizine enantiomers. The degradation behavior of Meclizine Hydrochloride was investigated under different stress conditions recommended by the International Conference on Harmonization (ICH). Methods:Enantiomeric resolution of the drug and complete separation from its degradation products were successfully achieved on a Phenomenex® lux cellulose 1 C18 (250 mm × 4.6 mm i.d, 5 μm particle size) column, using UV detector at a wavelength of 230 nm, with a mobile phase consisting of acetonitrile, 20mM ammonium bicarbonate at the ratio of 75:25 (v/v), and a flow rate of 1 mL/min. The drug was subjected to alkaline, acidic, neutral, oxidative and photolytic conditions in order to mimic stress conditions. Result:The degradation products were well resolved from main peak and proving the stability-indicating power of the method. The developed method provided linear responses within the concentration range 1-5 µg/mL, and regression analysis showed a correlation coefficient value (r2) of 0.999. The HPLC method was validated as per ICH guidelines with respect to specificity, precision, linearity and robustness. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.25 µg/mL and 1.00 µg/mL respectively. Conclusion: The method provides good sensitivity and excellent precision and reproducibility. The method was highly selective, where degradation products and co formulated compounds did not interfere. The proposed method was successfully applied in pharmaceutical preparations.

Evaluation of Perampanel in the Presence of its Degradation Products and Process-Related Impurities by Validated Stability-Indicating Reverse Phase High Performance Liquid Chromatography Method.

A novel stability-indicating isocratic reverse phase high-performance liquid chromatographic method was developed for the quantitative determination of perampanel in the presence of degradation products and its process-related impurities. Good resolution was achieved between the peaks corresponding to process-related impurities and degradation products from the analyte using Shim-pack GIST C18 column with mobile phase, potassium dihydrogen phosphate (pH 2.5; 30 mM)-acetonitrile (45: 55, v/v) at a wavelength of 294nm. Perampanel and its impurities were well-separated within a run time of 7min. To show the stability-indicating power of the method, forced degradation studies were performed on bulk samples of perampanel as per ICH prescribed stress condition using acid, base, hydrolytic, oxidative and photolytic degradation conditions. The degradation products were resolved from main peak and its impurities. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also applied for assay and related substances determination of perampanel in bulk and pharmaceutical dosage form.

Azole fungicides: (Bio)degradation, transformation products and toxicity elucidation

The increasing consumption of azole antifungal agents leads to their uncontrolled release into the environment. Therefore, it is crucial to remove their residues from natural ecosystems. This study aimed to examine the biological and chemical degradation of four typical azole fungicides: fluconazole (Fc), clotrimazole (Cl), climbazole (Cb), and epoxiconazole (Ep). The biodegradation was investigated using activated sludge and two novel Gram-negative bacterial strains. The chemical degradation experiments aimed to assess the efficiency of fungicides removal through UV treatment, the Fenton reaction, and a combination of these methods. Transformation products of Cb, Ep, and Cl photocatalytic removal were identified by mass spectrometry. In addition, the AlamarBlue® Assay and the MTT Assay allowed careful evaluation of the toxicity of azole derivatives and their transformation products towards newly isolated strains, Stenotrophomonas maltophilia AsPCl2.3 and Pseudomonas monteilii LB2. Among all azole fungicides, Cb was the most susceptible to biological removal while Fc, Ep, and Cl were basically resistant to biodegradation. Cl and Ep showed a significant biosorption on the activated sludge. Under optimized photolysis conditions, the removal efficiency of Cl, Cb, and Ep was significantly higher than that of biodegradation. The Fenton reaction supported by the UV-irradiation offered the best results of fungicides elimination. After 1 min of the experiment, Cl was almost completely removed while Cb and Ep removal rates reached an average of 60%. The proposed main degradation route of azole fungicides during UV-irradiation includes halogen atoms substitution by hydroxyl moieties. The final degradation product was imidazole or triazole. Azole fungicides and their transformation products differently affected the metabolic activity of Gram-negative bacteria. Cl and Cb intermediates showed lower toxicity than parent compounds. The findings help better understand the environmental impact of azole fungicides, their degradation, and toxicity. They also stress the need for reducing their uncontrolled release to the environment.