Alkaline Degradation Products Research Articles

Densitometric Determination of Ezetimibe in the Presence of its Alkaline Degradation Product

Objectives: A simple, accurate, selective and sensitive densitometric method was developed for the determination of ezetimibe in the presence of its alkaline degradation product. Methods: TLC-densitometric separation of ezetimibe from its degradation products was carried out on silica gel plates using ethyl acetate: n-hexane (2:1 v/v) as a developing system. This method depends on quantitative densitometric evaluation of ezetimibe at 230 nm over a concentration range of 1–8 µg/spot. Ezetimibe and its alkaline degradation product were resolved with Rf values of 0.49 and 0.68 respectively. Results: The proposed method has been successfully applied to the analysis of ezetimibe in pharmaceutical dosage form without interference from additives and the results were statistically compared with the reported method. Conclusion: TLC- densitometric technique has provided a simple, straightforward method for separating ezetimibe and its alkaline degradation product simultaneously.

Application of Computational Study in High Performance Liquid Chromatography for the Determination of Albendazole in the Presence of its Alkaline Degradation Product

A simple, sensitive, specific and accurate HPLC method was developed for the determination of albendazole (ALB) in presence of its alkaline degradation product. Computational and theoretical studies were done electronically and geometrically to investigate the affinity of ALB and its alkaline degradation product to the stationary phase. The analysis was carried out on an ODS SUPELCO C18 (25 cm x 4.6 mm, 5 μm particle size) using a mobile phase consisting of acetonitrile : methanol : water (40:30:30, by volume). The analysis was performed at ambient temperature with a flow rate of 1 ml/min and UV detection at 230 nm. The method showed good linearity over the concentration range of 1-7 μg/ml with a lower detection limit of 0.121 and quantification limit of 0.366 μg/ml. The proposed method can selectively analyse the drug in presence of up to 85.71 % of its alkaline degradation product with mean recovery ± RSD % of 99.78 ± 0.615. The proposed method was extensively validated according to the ICH guidelines and used for estimation of ALB in tablets and the obtained results were statistically compared with those of the reported method by applying t-test and F-test at 95 % confidence level and no significant difference was observed regarding accuracy and precision.

Stability Testing Followed by Manipulating Mean Centering Ratio and Derivative Ratio Spectrophotometric Methods for the Determination of Gliquidone in Presence of its Induced Degradation Products

Stability testing on gliquidone was conducted under different conditions followed by adoption of two smart and selective spectrophotometric methods namely; mean centering of ratio spectra and derivative ratio for the determination of gliquidone in presence of alkaline degradation products. Degradation products have been elucidated by LC/MS and IR spectroscopy. The spectrum of alkaline degradation products (10 μg/ml) was chosen as a divisor to resolve the overlapped spectra of gliquidone and its degradation products. Mean centering of ratio spectra method depends on using Matlab software and measuring the mean centered values at 260 nm. Derivative ratio method is based on the measurements of analytical signal at 253 nm. Instrumental parameters and divisor concentration were carefully studied. The adopted methods have been validated as per International Conference on Harmonization guidelines. Linearity was obeyed in the range 1-18 μg/ml and detection limit was found to be 0.180, 0.324 μg/ml for mean centering of ratio spectra and derivative ratio methods, respectively. The recommended procedures were successfully applied for the analysis of Glurenor® tablets with good respective recoveries of 103.40± 0.50 and 102.99± 0.62. The results were statistically analyzed using student’s t-test and F-ratio to confirm accuracy and precision.

New Triterpenoid Saponin C-20 Epimers from the Alkaline-Degradation Products of Ginsenoside Re and Their Cytotoxic Activities

Nine compounds, including two new triterpenoid saponin C-20 epimers (4 and 5) and seven known ginsenosides (1–3, 6–9), have been isolated and purified from the alkaline-degradation products of ginsenoside Re. Their structures were elucidated on the basis of spectroscopic analyses, including 1D and 2D NMR and HR-ESI-MS. Among them, (20S,22E)-6-O-β-D-glucopyranosyl-dammar-22(23),24-diene-3β,6α,12β-triol (4) and (20R,22E)-6-O-β-D-glucopyranosyl-dammar-22(23),24-diene-3β,6α,12β-triol (5) were identified as new triterpenoid saponins and they were assayed for cytotoxic activities against five human tumor cell lines.

In vitro toxicity assessment of rivaroxaban degradation products and kinetic evaluation to decay process

Degradation kinetics of oral anticoagulant rivaroxaban (RIV) was assessed in acid and alkaline media and while exposed to UVC radiation. Among all stress conditions tested, kinetic degradation process was better described by a zero-order model. A stability indicating method was validated for the analysis of the anticoagulant RIV in tablets by high-performance liquid chromatography. Robustness was evaluated with a two-level Plackett–Burman experimental design. The effect of acute exposition of the human hepatoblastoma HepG2 cell line to RIV stressed samples (100 and 500 µM) was assessed through in vitro toxicity tests. MTT reduction, neutral red uptake, mitochondrial membrane potential, and low molecular weight DNA diffusion assays were employed for cytotoxicity evaluation (5×104 cells/well). The genotoxic potential was assessed by comet assay (2×104 cells/well). Acute toxicity to HepG2 cells was assessed after 24 h incubation with sample solutions, for each test. A direct relationship between the increased amount of alkaline degradation products and higher cytotoxic potential was found. Results obtained by viability assay investigations support the concerns on risks associated with acute toxicity and genotoxicity of pharmaceutical samples containing degradation products as impurities.

Validated Stability-Indicating Methods for Determination of Mometasone Furoate in Presence of its Alkaline Degradation Product.

Two novel stability-indicating TLC densitometric and chemometric methods were developed for the determination of mometasone furoate (MF) in the presence of its alkaline degradation product (MF Deg). The developed TLC densitometric method (Method A) is based on the quantitative densitometric separation of MF from its alkaline degradation product on silica gel 60 F254 and measurement of the bands at 250 nm. The separation was carried out using hexane-chloroform-methanol-acetonitrile (6:6:1:0.3, by volume) as a developing system. A well-resolved and compact bands for (MF) and (MF Deg) at retention factors 0.36 and 0.66, respectively. Good resolution between (MF) and (MF Deg) assured the specificity of the proposed method. The method showed good linearity in the concentration range 0.5-5 μg/band with r2 = 0.9998. The method validation was performed according to ICH guidelines demonstrating to be accurate, precise, robust and sensitive. The LOD and LOQ were found to be 0.21 and 0.63 μg/band for MF, respectively. The developed TLC-densitometric method can be applied for identification and quantitative determination of MF in bulk drug and pharmaceutical dosage forms without any interference from excipients and degradates. Method B is a multivariate chemometric-assisted spectrophotometry, where classical least squares, principal component regression and partial least squares were applied. Statistical analysis of the results has been carried out revealing high accuracy and good precision.

Validated determination of diacerein and its active metabolite, rhein, by stability indicating constant pattern method as a novel manipulation of zero order spectra

A novel spectrophotometric method named constant absorbance pattern technique was proposed for simple determination of binary mixtures exemplified by diacerein, a treatment of osteoarthritis, and rhein, diacerein main alkaline degradation product and active metabolite. The proposed novel constant absorbance pattern technique was highly accurate, precise, sensitive, specific and stability indicating as well. The proposed technique allowed determination of diacerein and rhein in their binary mixtures manipulating zero order absorption spectra with no need for ratio spectra, derivatization steps, or even preliminary separation. Diacerein was determined at zero absorption spectra depending on the constant absorbance pattern between the two wavelengths 230 and 430 nm while rhein was directly determined at zero order at its λmax, 430 nm. The proposed methods showed obeying for Beer-Lambert’s law in the concentration range of 1–16 µg mL−1 for both diacerein and rhein. Validation of the proposed methods was carried out according to ICH guidelines with respect to accuracy and precision. Moreover, the proposed method was applied for determination of Diacerein in different pharmaceutical formulations and results were statistically compared with those of the reported HPLC one where no significant difference was found concerning Student’s t-test and F-value.

Application of quinone-based fluorophore and native fluorescence for the spectrofluorimetric determination of agomelatine in dosage form: Identification of acidic and alkaline- induced degradation products by LC-MS/TOF.

Three spectrofluorimetric methods were developed for agomelatine (AGM) determination in commercial tablets. Method A is based on measuring the native fluorescence of AGM aqueous solution at 230/360nm. Methods B and C are based on the formation of a charge transfer complex between AGM and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) with measurement of the formed fluorophore at 365/475nm and 250/304nm, respectively. The relative fluorescence intensity (RFI) of AGM-DDQ complex was greatly enhanced in the presence of methyl-β-cyclodextrin (CD). The methods were linear over the concentration ranges of 0.015-0.5, 0.5-8.0, 0.09-6.0 and 0.05-0.2 μg/ml for AGM-native fluorescence, AGM-DDQ, AGM-DDQ-CD and AGM-TCNQ complexes, respectively with excellent correlation coefficients (r=0.9999). The methods were validated as per the International Conference on Harmonization (ICH) guidelines and all validation requirements were satisfied. The developed methods were extended to the analysis of AGM in commercial tablets. Furthermore, the stability of AGM was studied under different stress conditions (alkaline, acidic, oxidative and photolytic). The potential alkaline and acidic degradation products were identified by LC-MS/TOF.

Stability Indicating RP-HPLC Method for Simultaneous Determination of Bambuterol HCl and Na Benzoate in the Presence of Bambuterol HCl Alkaline Degradation Product in Bulk and Syrups: Application to Degradation Kinetics

Bambuterol HCl (BMT) (bronchodilator) was subjected to stress alkaline hydrolysis. BMT alkaline degradation product (BMT-DP) was prepared, isolated and verified chemically using Fourier transform infrared spectroscopy (FTIR), Proton nuclear magnetic resonance (1H-NMR) as well as mass spectrometry. Na benzoate (BNZ) is commonly co-formulated in oral pharmaceutical preparations as a preservative. Subsequently, a simple, selective and valid stability indicating reversed phase high performance liquid chromatography (RP-HPLC) method was developed for simultaneous estimation of BMT and BNZ in the presence of BMT-DP. Chromatographic separation was achieved using an isocratic mobile phase consisting of a mixture of (20 mM)-KH2PO4 (containing 0.2% Triethylamine (TEA) v/v), pH adjusted to 3 with orthophosphoric acid]: acetonitrile (70:30, v/v) with a flow rate 1 mL/min at ambient temperature on a Eurospher II C18 (250 mm × 4.6 mm, 5 μm) column. Analysis was carried out with ultraviolet detection at 215 nm for BMT and BMT-DP, and 225 nm for BNZ. Linearity, accuracy and precision were found to be acceptable over a concentration range of (1-120 μg/mL) and (2-240 μg/mL) for BMT and BNZ, respectively. Limits of detection (LODs) for BMT and BNZ were 0.056 and 0.125 μg/mL, whereas limits of quantitation (LOQs) were 0.169 and 0.378 g/mL, respectively. Moreover, the kinetics of alkaline degradation of BMT was investigated. Arrhenius plot was constructed and the apparent pseudo first-order rate constant, half-life time, shelf-life time, and the activation energy of the degradation process could be calculated.

Application of normal fluorescence and stability-indicating derivative synchronous fluorescence spectroscopy for the determination of gliquidone in presence of its fluorescent alkaline degradation product

Simple, smart and sensitive normal fluorescence and stability-indicating derivative synchronous spectrofluorimetric methods have been developed and validated for the determination of gliquidone in the drug substance and drug product. Normal spectrofluorimetric method of gliquidone was established in methanol at λ excitation 225nm and λ emission 400nm in concentration range 0.2–3μg/ml with LOD equal 0.028. The fluorescence quantum yield of gliquidone was calculated using quinine sulfate as a reference and found to be 0.542. Stability-indicating first and third derivative synchronous fluorescence spectroscopy were successfully utilized to overcome the overlapped spectra in normal fluorescence of gliquidone and its alkaline degradation product. Derivative synchronous methods are based on using the synchronous fluorescence of gliquidone and its degradation product in methanol at Δ λ50nm. Peak amplitude in the first derivative of synchronous fluorescence spectra was measured at 309nm where degradation product showed zero-crossing without interference. The peak amplitudes in the third derivative of synchronous fluorescence spectra, peak to trough were measured at 316,329nm where degradation product showed zero-crossing. The different experimental parameters affecting the normal and synchronous fluorescence intensity of gliquidone were studied and optimized. Moreover, the cited methods have been validated as per ICH guidelines. The peak amplitude-concentration plots of the derivative synchronous fluorescence were linear over the concentration range 0.05–2μg/ml for gliquidone. Limits of detection were 0.020 and 0.022 in first and third derivative synchronous spectra, respectively. The adopted methods were successfully applied to commercial tablets and the results demonstrated that the derivative synchronous fluorescence spectroscopy is a powerful stability-indicating method, suitable for routine use with a short analysis time. Statistical comparison between the results obtained by normal fluorescence and derivative synchronous methods and the official one using student's t-test and F-ratio showed no significant difference regarding accuracy and precision.

Stability Indicating Spectrophotometric Methods for the Determination of Cefoxitin Sodium in Pure form and in Pharmaceutical Preparations

Four simple, accurate and precise stability-indicating spectrophotometric methods were developed and validated for the simultaneous determination of cefoxitin sodium in presence of its alkaliinduced degradation product without prior separation namely; Q-analysis, absorbance subtraction, absorbance ratio, and amplitude modulation. These methods were validated for accuracy and precision. The four proposed methods were successfully applied for the determination of the cited drug in pure form and in pharmaceutical preparation also in the presence of its alkaline degradation product. The obtained results were statistically compared with those of a reported method. The comparison showed that there are no significant differences between the proposed methods and the reported method.

Development and Validation of an RP-HPLC Method for the Determination of Vinpocetine and Folic Acid in the Presence of a Vinpocetine Alkaline Degradation Product in Bulk and in Capsule Form.

An alkaline-forced degradation hydrolytic product of vinpocetine was prepared and characterized by 1H-NMR, FTIR spectroscopy, and MS. Subsequently, a simple, selective, and validated reversed-phase HPLC method was developed for the simultaneous estimation of vinpocetine and folic acid in the presence of a vinpocetine alkaline degradation product. Chromatographic separation was achieved using an isocratic mobile phase consisting of acetonitrile-0.02 M KH2PO4 [containing 0.2% (v/v) triethylamine and adjusted to pH 6 with orthophosphoric acid; (80 + 20, v/v)] at a flow rate of 0.9 mL/min at ambient temperature on a Eurospher II C18 (250 × 4.6 mm, 5 μm) column, with UV detection at 280 nm for folic acid and 230 nm for vinpocetine and its alkaline hydrolytic product. Linearity, accuracy, and precision were found to be acceptable over a concentration range of 12.5-200 μg/mL for vinpocetine and 1-16 μg/mL for folic acid. The proposed method was successfully applied for the determination of both drugs and a vinpocetine hydrolysis product in a laboratory-prepared mixture and in a capsule containing both drugs.

Molecular and Quantum Mechanical Study for the Separation of Cefprozil in the Presence of Its Alkaline Degradation Product Using RP-HPLC with UV Detection.

A reversed-phase HPLC method (RP-HPLC) with UV detection was developed and validated for the quantitative determination of cefprozil, a second-generation cephalosporin. Due to β-lactam ring instability under alkaline conditions, this RP-HPLC method was applied for the determination of cefprozil in the presence of its possible degradation product. The interactions that govern the separation process with stationary phase were investigated at both molecular and quantum mechanical levels. Moreover, electrostatic potential maps were generated to determine the sites of interaction with mobile phase. The suggested method was validated in compliance with International Conference on Harmonization guidelines and successfully applied for the determination of cefprozil in its commercial pharmaceutical formulation.

Chromatographic Determination of Cyclopentolate Hydrochloride and Phenylephrine Hydrochloride in the Presence of Their Potential Degradation Products.

Two sensitive, selective, and precise stability-indicating methods have been developed for the simultaneous determination of the active pharmaceutical ingredients cyclopentolate hydrochloride (CLO) and phenylephrine hydrochloride (PHE) in their pure forms and in the presence of their degradation products. The methods were applied for the determination of CLO and PHE in a pharmaceutical formulation. Method A was based on isocratic elution HPLC determination. Separation was achieved using a Waters Spherisorb ODS2 C18 analytical column (5 μm particle size) and a mobile phase of 0.1% heptane-1-sulphonic acid sodium salt in methanol-water (80 + 20, v/v). The flow rate was 1.0 mL/min and detection was performed at 210 nm. Method B was an HPTLC- densitometric method using HPTLC silica gel 60 F254 plates and an optimized mobile phase of ethyl acetate-methanol-ammonia (8 + 2 + 0.1, v/v/v). The separated spots were densitometrically scanned at 210 nm. Polynomial equations were used for regression. The developed methods are suitable for the determination of CLO and PHE in their binary mixture and in the presence of their corresponding degradation products. The two methods were validated in compliance with International Conference on Harmonization guidelines and successfully applied for the determination of CLO and PHE as synthetically prepared in laboratory mixtures and in the presence of their possible degradation products. CLO alkaline degradation products were stated as potential impurities in British Pharmacopoeia. The degradation products were separated and identified by mass spectra. Postulation of a PHE oxidative degradation pathway was suggested. The obtained results were statistically analyzed and compared with those obtained by applying the official methods for both drugs.

Determination of Albendazole in the Presence of its Alkaline Degradation Product Using TLC-Densitometric and Chemometric Methods: A Comparative Study

Determination of Albendazole in the Presence of its Alkaline Degradation Product Using TLC-Densitometric and Chemometric Methods: A Comparative Study

Determination of Cefoxitin Sodium in Presence of its Alkaline Degradation Product Using Two Wavelengths Manipulating Methods

Four accurate and precise spectrophotometric methods were developedas stability-indicating methods for the determination of cefoxitin sodium in the presence of its alkaline degradation product. The proposed methods adopted for selective determination of intact cefoxitin sodium in the presence of up to 66.67% of its alkaline degradation product for dual wavelength, bivariate, and area under the curve methods and up to 83.33% for the simultaneous equation method. The methods obey Beers’s law in the range (4-36 μg ml-1) and were successfully applied for the determination of cefoxitin sodium in pure form and in pharmaceutical preparation. The results obtained were statistically analyzed and compared with those of a reported method, there are no significant differences between the proposed methods and the reported one.

Optimization of nano-hydroxyapatite/poly(lactic-co-glycolic acid) coatings on magnesium substrates using one-step electrophoretic deposition

This study focuses on optimizing nanocomposite coatings of nanophase hydroxyapatite (nHA) and poly(lactic-co-glycolic acid) (PLGA) on magnesium (Mg) substrates using one-step electrophoretic deposition (EPD). The nHA, PLGA and Mg are all biodegradable in the human body, which makes them favorable for temporary implant applications. Mg and its alloys possess the elastic modulus and strength comparable to cortical bone, and thus reduce the detrimental effects of stress-shielding while providing necessary strength for load-bearing applications. The nHA is an attractive coating material for bone healing due to its osteoconductivity. PLGA promotes coating adhesion, and may neutralize the alkaline degradation products of Mg substrates. The nHA_PLGA composite coatings were deposited on Mg substrates using EPD with acetone as the electrolyte solvent. The parameters for EPD process, such as deposition voltage, time and electrolyte concentration, were studied to determine the optimal condition for creating the nHA_PLGA coating on Mg. The nHA_PLGA coating showed a desirable thickness of 65.85±11.95 µm with a complete homogenous coverage on Mg substrates when the deposition voltage of 75 V was applied for 6min using an electrolyte containing 1mg/mL nHA and 0.2mg/mL PLGA in acetone. The characterization of the coating confirmed the presence of both nHA and PLGA.

Bivariate and Multivariate Spectrophotometric Methods for Determination of Ezetimibe with Kinetic Study of Its Alkaline Degradation

The first study concerned with the determination of ezetimibe EZE in presence of its alkaline degradation product were developed and validated using different chemometric techniques [bivariate, classical least square (CLS), and principle component regression (PCR), partial least square (PLS)], with kinetic study and calculation of half-life time and activation energy which essential for quality control of its product. Application of the suggested techniques were successfully applied in synthetic mixture and in pharmaceutical preparations, the results obtained by the proposed techniques were statistically comparable with those obtained by the reported method. All the proposed techniques were validated according to international conference on harmonization (ICH) guidelines.

Development and validation of stability indicating TLC densitometric and spectrophotometric methods for determination of Clobetasol propionate

Two simple analytical techniques that manipulate the inherent spectroscopic properties of the drug differently were developed for Clobetasol propionate (CP) determination in the presence of its alkaline hydrolytic degradation products. The first method depends on TLC-densitometric determination of the UV–visualized bands after TLC separation of CP in the presence of its alkaline degradation products in its bulk and pharmaceutical formulation. Separation was performed on preactivated silica gel 60 F254 TLC plates using ethyl acetate:hexane:ammonia (5:5:0.2, by volume) as a developing system followed by scanning at 240nm. Linear correlation was obtained in the range of 0.10–0.50μg/band. The second method was ratio difference spectrophotometry. It was applied by measuring the difference in peak amplitude of the ratio spectra at 243.40 and 256.40nm. The selectivity of both methods was checked by analyzing laboratory prepared mixtures containing different concentrations of CP and its alkaline degradation products. The methods were validated in compliance with ICH guidelines. The methods determined CP in its bulk powder with average percentage recoveries of 99.60%±1.09 and 99.44%±1.60 for densitometry and ratio difference, respectively. Both methods were successfully applied for quantification of CP in its commercial cream.

A validated inherent stability indicating HPTLC method for estimation of cyclobenzaprine hydrochloride in tablets and use of MS–QTOF in characterization of its alkaline stress degradation product

A simple, selective and sensitive stability indicating high-performance thin-layer chromatographic method was developed and validated for estimation of cyclobenzaprine hydrochloride (CBP) in bulk drug and commercial tablets according to ICH guidelines. A precoated silica gel 60F254 HPTLC plates were used for chromatographic separation using mobile phase toluene: ethyl acetate: methanol: glacial acetic acid in the ratio 4:2:3.5:0.5 v/v/v/v. A TLC scanner was set at detection wavelength 290nm for densitometric analysis of analyte on absorbance mode. The degradants were resolved satisfactorily in a mixture of stressed sample having Rf values 0.48±0.05, 0.52±0.05, 0.86±0.05, 0.66±0.05 and 0.27±0.05. The calibration curve of drug was found linear in the concentration range 200–1000ng/band. The (r2>0.999) correlation coefficient value indicated good correlation between the analyte concentrations and peak areas. The repeatability and intermediate precision study revealed the % RSD value less than 2.0 and was found to be satisfactory. The accuracy of developed method was ascertained by performing the recovery study using standard addition method and expressed by percent recovery (100.32%) which was found satisfactory. CBP was subjected to various stress conditions as per International Conference on Harmonization (ICH) guidelines. The drug showed significant degradation during hydrolytic and oxidative stress condition. CBP remained stable in thermal and photolytic stress testing. The validated chromatographic method was further utilized to isolate the alkaline degradation product using preparative HPTLC technique and extensive FT-IR, ESI-MS/TOF studies were performed to ascertain the structure of degradant.