Neutral Hydrolysis Research Articles

Quantum Calculations of VX Ammonolysis and Hydrolysis Pathways via Hydrated Lithium Nitride.

Recently, lithium nitride (Li3N) has been proposed as a chemical warfare agent (CWA) neutralization reagent for its ability to produce nucleophilic ammonia molecules and hydroxide ions in aqueous solution. Quantum chemical calculations can provide insight into the Li3N neutralization process that has been studied experimentally. Here, we calculate reaction-free energies associated with the Li3N-based neutralization of the CWA VX using quantum chemical density functional theory and ab initio methods. We find that alkaline hydrolysis is more favorable to either ammonolysis or neutral hydrolysis for initial P-S and P-O bond cleavages. Reaction-free energies of subsequent reactions are calculated to determine the full reaction pathway. Notably, products predicted from favorable reactions have been identified in previous experiments.

Accelerated stability study of the ester prodrug remdesivir: Recently FDA-approved Covid-19 antiviral using reversed-phase-HPLC with fluorimetric and diode array detection.

Remdesivir (RDV) is the first antiviral drug, approved by the Food and Drug Administration, to treat severe acute respiratory syndrome coronavirus 2. RDV is a relatively new chemical entity, 'ester prodrug', with no reported stability profile. Due to the urgency of its use and thus fast production, it is important to develop a stability-indicating method for its assay. Chromatographic separation was carried out on a C18 column (250 × 4.6 mm, 5 µm) with dual detection: diode array at 240 nm and fluorescence at λex/em 245/390 nm. Isocratic elution of acetonitrile and distilled water (acidified with phosphoric acid, pH 4) in the ratio of 55:45 (v/v), respectively, was used. The linearity range using HPLC-diode array detection was 0.1-15 μg/mL, whereas that using fluorimetric detection was 0.05-15 μg/mL. As per the International Conference on Harmonization guidelines, RDV has been degraded by accelerated alkaline, acidic, neutral hydrolysis, oxidative, heat, and photolytic stress conditions. Possible degradation hypothesis of the parent molecule has been suggested and illustrated. The proposed methods have achieved selective determination of the intact drug with no peaks overlapping in all assumptions. Extensive degradation confirms threatened drug stability at thermal and basic hydrolytic stressing. The developed methods were fully validated and proved suitable for quality control routine analysis of RDV in raw material and pharmaceutical dosage forms.

Automated read-across workflow for predicting acute oral toxicity: I. The decision scheme in the QSAR toolbox

A decision-scheme outlining the steps for identifying the appropriate chemical category and subsequently appropriate tested source analog(s) for data gap filling of a target chemical by read-across is described. The primary features used in the grouping of the target chemical with source analogues within a database of 10,039 discrete organic substances include reactivity mechanisms associated with protein interactions and specific-acute-oral-toxicity-related mechanisms (e.g., mitochondrial uncoupling). Additionally, the grouping of chemicals making use of the in vivo rat metabolic simulator and neutral hydrolysis. Subsequently, a series of structure-based profilers are used to narrow the group to the most similar analogues. The scheme is implemented in the OECD QSAR Toolbox, so it automatically predicts acute oral toxicity as the rat oral LD50 value in log [1/mol/kg]. It was demonstrated that due to the inherent variability in experimental data, classification distribution should be employed as more adequate in comparison to the exact classification. It was proved that the predictions falling in the adjacent GSH categories to the experimentally-stated ones are acceptable given the variation in experimental data. The model performance estimated by adjacent accuracy was found to be 0.89 and 0.54 while based on R2. The mechanistic and predictive coverages were >0.85.

Development and validation of a stability‐indicating UPLC method for the determination of olmesartan medoxomil, amlodipine and hydrochlorothiazide degradation impurities in their triple‐combination dosage form using factorial design of experiments

The current work describes the development and validation of a stability-indicating UPLC method for the determination of olmesaratan medoxomil (OLM), amlodipine besylate (AMB), hydrochlorothiazide (HCT) and their degradation products in the triple-combination tablet dosage form. The separation was achieved using a Zorbax Eclipse plus C8 RRHD (100 mm × 3.0mm), 1.8μm column with gradient elution of mobile phase A containing 0.02m of sodium phosphate buffer (pH3.35) and mobile phase B as acetonitrile and water (90:10, v/v). The detector signal was monitored at UV 250 nm. Analytical performance of the optimized UPLC method was validated as per International Conference on Harmonization guidelines. The linearity ranges for OLM, AMB and HCT were 0.59-240, 0.30-60 and 0.37-150 μg/ml, respectively, with correlation coefficients >0.999. The dosage form was subjected to forced-degradation conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal and photodegradation. The method was proved to be stability indicating by demonstrating the specificity of the drugs from degradation products. The robustness of the method was evaluated through a two-level, three-factorial design with a multivariate approach. Statistical data analysis with best model fit P-value < 0.05 from an ANOVA test indicated that the influence of individual factors is relatively higher than the interaction effects. The method is useful for the analysis of drug products.

Development and greenness assessment of a stability-indicating HPLC-DAD method for simultaneous determination of allopurinol and benzbromarone

The growing interest in Green Analytical Chemistry (GAC) principles through the replacement of polluting analytical procedures with greener ones, has encouraged us to develop an eco-friendly stability-indicating HPLC with diode array detection method (HPLC-DAD) for simultaneous determination of allopurinol (ALP) and benzbromarone (BNZ). Effective separation was accomplished using Durashell C18 column (4.6 × 250 mm, 5 µm particle size) with gradient elution of the mobile phase composed of 0.02 M ammonium acetate (pH 5.0) and methanol. Quantification of ALP and BNZ was based on measuring their peak areas at 251 nm. ALP and BNZ peaks eluted at retention times 4.85 and 10.30 min respectively. The proposed HPLC procedure was carefully validated in terms of system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. The linearity range for both ALP and BNZ was 5–100 µg/mL with correlation coefficients >0.9999. Forced degradation conditions of neutral, acidic, and alkaline hydrolysis, oxidation, and thermal degradation were applied on both drugs. Good resolution of the drugs from their forced degradation products proved that the proposed method is stability-indicating. In addition, the resolution of both drugs from about 10 pharmacologically or chemically related pharmaceutical compounds of different medicinal categories showed the high specificity of the proposed method. The validated HPLC method was successfully applied to the simultaneous determination of both drugs in their tablet dosage forms. Furthermore, greenness assessment and comparison with previously published methods were carried out using two different GAC metrics, namely, the national environmental method index (NEMI) and the analytical Eco-Scale.

Development of a stability-indicating assay method by HPLC-DAD and MS characterization of forced degradation products of ravuconazole.

Ravuconazole (RAV) is a triazole antifungal with broad spectrum and a novel alternative in the treatment of systemic fungal infections. A stability-indicating method by high-performance liquid chromatography-diode array detection was developed and fully validated to assay ravuconazole in the presence of its degradation products. Separation was achieved with a Sunfire C18 column (250 mm × 4.6 mm id, 5 μm), mobile phase composed of acetonitrile and water (80:20), at 1 mL/min. The volume of injection was 5 μL and DAD detection was performed at 287 nm. RAV was well resolved from its degradation products and the method proved to be linear, selective, accurate, precise and robust. A forced degradation study was conducted on the pure drug under oxidative conditions in presence of H2O2 and metallic ions and under acid, alkaline and neutral hydrolysis. RAV was degraded mainly under alkaline hydrolysis, forming two main degradation products. The chemical structures were proposed according to the data obtained by liquid chromatography coupled to mass spectrometry (LC-MS) analysis. This study provided a new and selective stability-indicating method to evaluate the intrinsic stability of ravuconazole in active pharmaceutical ingredients. The developed method was found to be suitable for quality control routine analysis and to stability studies of ravuconazole.

Development of a Validated Stability Indicating Rp-Hplc Method for the Estimation of Pirfenidone in Bulk Drug and Tablet Dosage form

Objective: The present work focused on developing a validated stability indicating RP-HPLC method for the estimation of pirfenidone in bulk drug and tablet dosage form.&#x0D; Methods: The chromatographic separation was performed on symmetry C18 (150 mm x 4.6, 5 micron) with a 1 ml/min flow rate at 315nm. The mobile phase employed was orthophosphoric acid buffer: acetonitrile (65:35). Column temperature was maintained at 30ºC. Pirfenidone was subjected to different forced degradation conditions according to ICH guidelines, including acid, base and neutral hydrolysis, oxidation, photolysis and thermal degradation. &#x0D; Results: In alkali, acidic, oxidation and UV degradation conditions the drug shows considerable degradation. Pirfenidone was stable under neutral hydrolysis and thermal degradation. Pirfenidone was stable under extreme degradation conditions showing less than 8% of degradation in all degradation conditions. This result showed that pirfenidone was stable under stress degradation. Then the optimized method was validated for the parameters like linearity, accuracy, precision and robustness as per ICH guidelines.

Stability Indicating Assay Method (Siam) For Determination Of Semaglutide By Hplc Using Qbd Approach

An accurate, precise, robust and stability-indicating RP-HPLC method was developed for the estimation of Semaglutide using QbD approach. After conducting several trials using CCD method, one desirable method was optimized. Stationary phase selected was Kromasil C18 (250×4.6 mm, 5 µm) and potassium dihydrogen orthophosphate (pH 2) and Methanol used as mobile phase in the ratio of 61.2: 38.8. Detection was carried out at the wavelength 230nm. Flow rate selected for separation was 0.98ml/min and the temperature of 29.150C. The retention time was found to be 2.518 at the run time of 5min. The developed method was subjected to validation as per ICH guidelines. Semaglutide linear in the concentration range of 0-9µg/ml and the regression coefficient was found to be 0.999. The developed method was accurate, precise and robust. When a sample solution subjected to stress conditions like acid hydrolysis, neutral hydrolysis, photodegradation and thermal degradation, no degradation products were observed. In basic degradation studies two degradation products and oxidative conditions one degradation product was observed. From stress studies, we can observe that the degradation of Semaglutide was less than 10 in all conditions, and there is no impact of degradation products on system suitability parameters. The peak was homogenous I all conditions, thus proving the stability-indicating nature of the method. This method can be applied for the determination of Semaglutide in the drug substance.

DEVELOPMENT OF A VALIDATED STABILITY-INDICATING METHOD FOR ESTIMATION OF ETHIONAMIDE IN TABLETS BY RP-HPLC AND CHARACTERIZATION OF ITS ISOLATED ALKALI DEGRADATION PRODUCT

A simple, accurate, reproducible and specific stability-indicating RP-HPLC method was developed for estimation of ethionamide in tablets. Ethionamide was exposed to acid, alkali and neutral hydrolysis at elevated temperatures, to thermolytic degradation, peroxide-mediated oxidation at room temperature in dark and to photolytic degradation. The drug was found stable to thermolytic and photolytic conditions and to neutral hydrolysis. However, substantial degradation was obtained in acid and alkali hydrolysis and complete degradation in peroxide-medicated oxidation. Similar degradation behavior was observed when ethionamide tablets were exposed to the mentioned forced degradation conditions. The method showed adequate resolution of drug from its potential degradation products on C18 (250 × 4.6 mm, 5µ) column using mobile phase of methanol: water (50: 50 % V/V) at 1 mL/min. The drug and its potential degradation products were detected at 290 nm. The method was validated as per the ICH Q2(R1) guidelines. The enrichment of the alkali degradation product was performed and isolated by preparative TLC and further confirmed by NMR and IR spectroscopy.

Stability study of doripenem antibiotic applying LC-ESI-Q-TOF method and in silico prediction: An analytical investigation focused on degradation products

In the present study, the stability of the antibiotic doripenem was evaluated with a focus on identifying the degradation products generated. For this, a method by LC-ESI-Q-TOF was developed and validated. Also, as an auxiliary tool in the identification of degradation products, Zeneth 7 software was used in the prediction of drug degradation in silico. Under stress conditions six degradation products were detected and based on the literature and MS2 analyzes, their structures were proposed. Under hydrolytic conditions, in acid, neutral and alkaline media, the major degradation product was formed through the cleavage of the β -lactam ring. In neutral hydrolysis, the formation of two drug dimers was also observed. Oxidation of the drug using H2O2 (3%) led to the formation of three degradation products, not yet reported in the literature, generated by oxidation of the thioether group and by decarboxylation reaction. The most probable elemental compositions for degradations products were obtained with a high degree of confidence, where the error between the masses observed and calculated was less than 5 ppm. The stability of doripenem after reconstitution was studied in conditions that simulate its clinical use. At room temperature, the drug demonstrated a similar degradation profile when prepared using 0.9% NaCl and 5% glucose for infusion, keeping its content > 90% for at least 8 h, and the LC-ESI-Q-TOF analyzes detected the formation of three degradation products, highlighting two different dimers. Finally, all the data generated helps to understand the degradation behavior of doripenem and its important antibiotic class.

Natural depolymerization of waste poly(ethylene terephthalate) by neutral hydrolysis in marine water

Polyethylene terephthalate (PET) is one of the most widely used materials for food packaging and fishing nets. After use it become waste and, due to poor collection, most will be found floating in marine waters. This paper presents the results of a study of PET depolymerization by hydrolysis. We observed that marine water is a perfect reactant because it contains a multitude of metal ions that act as catalysts. A first-order kinetic model was developed and experimental data fitted to it. An activation energy of 73.5 kJ/mole and a pre-exponential factor of 5.33 × 107 h–1 were obtained. Considering that the global ocean is a huge batch reactor operating under isothermal conditions, the solution of the mathematical model shows that in tropical regions only 72 years is needed for total and only 4.5 years for 50% PET conversion.

Degradation of Organophosphates Promoted by 1,2,4-Triazole Anion: Exploring Scaffolds for Efficient Catalytic Systems.

Organophosphate (OP) pesticides are responsible for numerous human deaths every year. Nucleophilic substitution is an important method to mitigate the toxicity of obsolete stocks of OPs. Herein, the degradation of O,O-diethyl-2,4-dinitrophenyl phosphate (DEDNPP) and pesticide diethyl-4-nitrophenyl phosphate (Paraoxon) promoted by 1,2,4-triazole (TAZ) was investigated by means of kinetic studies, nuclear magnetic resonance (NMR) analyses, and theoretical calculations. Results showed fast degradation of OPs is promoted by the anionic form of the nucleophile (TAZ(-)) in pH > 8.5 (optimal at pH = 11). Rate enhancements of 106 and 105-fold in relation to neutral hydrolysis of DEDNPP and Paraoxon were observed, respectively, consistent with alpha-nucleophiles reactivity. TAZ(-) regioselectively promotes the degradation of DEDNPP via P-O bond break, forming a quickly hydrolyzable phosphorylated intermediate, regenerating the nucleophile. Calculations using M06-2X/6-311++G(d,p) level of theory revealed that the equivalent nitrogen atoms of TAZ(-) are the main nucleophilic center of the molecule. This study expands the knowledge on the reactivity of iminic compounds as detoxificant agents of OPs, indicating the efficiency and selectivity of TAZ(-) in aqueous medium, encouraging the design of novel TAZ-based catalysts.

Reaction Mechanisms Underlying Unfunctionalized Alkyl Nitrate Hydrolysis in Aqueous Aerosols

Alkyl nitrates (ANs) are both sinks and sources of nitrogen oxide radicals (NOx = NO + NO2) in the atmosphere. Their reactions affect both the nitrogen cycle and ozone formation and therefore air quality and climate. ANs can be emitted to the atmosphere or produced in the gas phase. In either case, they can partition into aqueous aerosols, where they might undergo hydrolysis, producing highly soluble nitrate products, and act as a permanent sink for NOx. The kinetics of AN hydrolysis partly determines the extent of AN contribution to the nitrogen cycle. However, kinetics of many ANs in various aerosols is unknown, and there are conflicting arguments about the effect of acidity and basicity on the hydrolysis process. Using computational methods, this study proposes a mechanism for the reactions of methyl, ethyl, propyl, and butyl nitrates with OH– (hydroxyl ion; basic hydrolysis), water (neutral hydrolysis), and H3O+ (hydronium ion; acidic hydrolysis). Using quantum chemical data and transition state theory, we follow the effect of pH on the contribution of the basic, neutral, and acidic hydrolysis channels, and the rate coefficients of AN hydrolysis over a wide range of pH. Our results show that basic hydrolysis (i.e., AN reaction with OH–) is the most kinetically and thermodynamically favorable reaction among our evaluated reaction schemes. Furthermore, comparison of our kinetics results with experimental data suggests that there is an as yet unknown acidic mechanism responsible for acidic catalysis of AN hydrolysis.

Unexpected Z/E isomerism of N-methyl-O-phosphothioyl benzohydroxamic acids, their oxyphilic reactivity and inertness to amines

Thiophosphinoylation of N-methyl p-substituted benzohydroxamic acids using disulfanes (method A) or diphenylphosphinothioyl chloride (method B) provides only one conformer of the respective O-phosphothioyl derivative (X-ray and NMR analysis). Undergoing the P-transamidoxylation reaction is an evidence of the reversibility of thiophosphinoylation. Only those products containing strong EWG substituents in the aroyl residue or bulky substituents at the phosphorus atom possess E conformation. DFT calculations confirmed the energetic domination of each isomer. The Z-isomers are distorted amides having both high degree of nitrogen pyramidalization (38–55°) and amide twist (12–30°). In solution they exist in a defined conformation that is evidenced by the presence of a sharp signal of N-methyl protons at low temperature. They do not isomerize in solutions. Some of them slowly undergo the N-O bond scission above 100 °C. Both isomers are not as sensitive to neutral hydrolysis as twisted amides can be and are inert toward amines. The rate of alkaline hydrolysis can be correlated with pKa of hydroxamic acid. Because of their outstanding oxyphilicity, these compounds can be defined as nerve agent surrogates and safer alternatives of phosphorus fluorides for serine-active enzyme inhibition studies.

Lipid Droplets as Regulators of Metabolism and Immunity

Abstract A hallmark of sterile and nonsterile inflammation is the increased accumulation of cytoplasmic lipid droplets (LDs) in non-adipose cells. LDs are ubiquitous organelles specialized in neutral lipid storage and hydrolysis. Originating in the ER, LDs are comprised of a core of neutral lipids (cholesterol esters, triglycerides) surrounded by a phospholipid monolayer and several LD-associated proteins. The perilipin (PLIN1-5) family are the most abundant structural proteins present on the surface of LDs. While PLIN1 is primarily expressed in adipocytes, PLIN2 and PLIN3 are ubiquitously expressed. LDs also acquire a host of enzymes and proteins that regulate LD metabolism. Amongst these are neutral lipases and selective lipophagy factors that promote hydrolysis of LD-associated neutral lipid. In addition, LDs physically associate with other organelles such as mitochondria through inter-organelle membrane contact sites that facilitate lipid transport. Beyond serving as a source of energy storage, LDs participate in inflammatory and infectious diseases, regulating both innate and adaptive host immune responses. Here, we review recent studies on the role of LDs in the regulation of immunometabolism.

Development of a UV-spectrophotometric method for study of degradation profile of tenofovir alafenamide

Tenofovir alafenamide is a nucleic acid analogue that is used for treatment of HIV infection and Hepatitis B. As compared with the other prodrug of tenofovir, tenofovir alafenamide is having less side effects, it is Steady in plasma and then quickly changed over into tenofovir once inside cells. The main aim and objective of present study was to perform the stability indicating studies as defined under ICH Guideline Q1A R2) by using UV spectrophotometer. According to ICH guidelines, of temperature, time, photo degradation, acid/base are the factors which are responsible for formation degradation product of a drug. UV-Vis spectroscopy method was intiated to study and compute the amount of drug in the presence of degradation products. The method was developed by using water as diluents and ? max was 260nm. Linearity was established for in the range of 2-10 ?g/mL with regression coefficient of 0.9912. The drug was subjected to acid, alkali and neutral hydrolysis, oxidation degradation conditions. The limits of detection (LOD) and quantification (LOQ) were calculated and found to be 1.43 ?g/mL and 4.33?g/mL, respectively. The method was found to be simple, economic and less time consuming as compared to other analytical techniques. Keywords: Stability indicating assay method, Tenofovir alafenamide fumarate.

Fatty Acids and Free Amino Acids Changes during Processing of a Mediterranean Native Pig Breed Dry-Cured Ham.

The aim of this work is to analyse the intramuscular fatty acids and the free amino (FAA) acids in Chato murciano dry-cured ham. There are several Mediterranean native pig breeds whose characteristics of derived products have been described, but the impact of lipolysis and proteolysis on Chato murciano dry-cured ham has not yet been studied. Fatty acids and free amino acids were determined in the fresh piece and at 14, 18, 22 and 24 months of manufacturing. Monounsaturated fatty acids are the majority in the neutral lipids and free fatty acid fractions. Lipolysis took place mainly until the 18th month, resulting in a decrease in the levels of fatty acids of neutral lipids (from 95.43% to 83.38%) and polar lipids (from 2.57% to 0.41%), accompanied by a corresponding increase in free fatty acids (from 2% to 16.21%). Neutral lipids hydrolysis provides the main free fatty acids as in other native breeds. Results for FAA showed an increase in concentration during the time preceding the 14th month. From this point onwards, until month 18, total FAA concentration remained stable, and the content decreased at the end of the processing (between months 22 and 24).

Development and validation of a stability indicating HPLC method for the determination of buprenorphine in transdermal patch

A rapid and sensitive stability indicating HPLC method has been developed and validated for the determination of buprenorphine (BPN) in transdermal patch. Chromatographic separation was achieved isocratically on an XBridge(superscript TM) Shield RP18 column with a mobile phase of acetonitrile/0.063 M ammonium bicarbonate buffer (pH 9.5) (58:42, v/v) at the flow rate of 1.5 mL/min with UV absorbance monitoring at 230 nm. The system performance was evaluated and the result showed that BPN and degradation products were separated. Buprenorphine was subjected to neutral, acidic and basic hydrolysis as well as chemical oxidation to evaluate the specificity. The calibration curve of buprenorphine was linear in the range of 30~70 μg/mL (r=0.9999, n=5). The values of RSD (%) for the intra-day and inter-day precision ranged from 0.04 to 0.22 and 0.65 to 0.88%, respectively. The average of the recovery percentage ranged from 98.86 to 99.36%. The detection limit (DL) and quantitation limit (QL) for buprenorphine were 0.008 and 0.024 μg/mL, separately. The robustness of this method was also evaluated on the small fluctuations of pH in the mobile phase, the mobile phase compositions, and the flow rate. The results of stability studies showed the hydrolysis reaction of buprenorphine followed zero-order kinetics model in acidic and basic environment and followed first-order kinetics model in the presence of hydrogen peroxide. This analytical method was successfully applied to the determination of buprenorphine in transdermal patch and can be used for routine quality control work.

Development and validation of high performance liquid chromatographic method for the determination of esomeprazole in tablets

A simple, selective and accurate high performance liquid chromatographic (HPLC) method was developed and validated for the analysis of esomeprazole magnesium trihydrate (ES) in tablets. Chromatographic separation was achieved isocratically on a C18 column utilizing a mobile phase of acetonitrile/phosphate buffer (60:40, v/v, pH 7) at a flow rate of 1.0 mL/min with UV detection at 205 nm. Lansoprazole was used as an internal standard (IS). The calibration curve of ES was linear in the range of 100-1000 ng/mL (r = 0.9992, n = 4). The RSD values for intra- and inter-day precision were 0.66-0.86% and 0.84-1.11%, respectively. The proposed method was successfully applied to the determination of ES in tablets. The mean recovery for ES from the tablets ranged between 97.82-98.22%. ES was subjected to neutral, acid and alkali hydrolysis as well as oxidation, dry heat treatment and photodegradation. Being simple, accurate and selective, the method can be used for routine quality control analysis.

Analytical investigation of ternary mixture of phenylephrine hydrochloride, dimetindene maleate and benzalkonium chloride using validated stability indicating HPLC-DAD method

A stability-indicating high performance liquid chromatography method with diode array detection (HPLC-DAD) was developed and validated for simultaneous determination of phenylephrine hydrochloride (PHR), dimetindene maleate (DMD) and benzalkonium chloride (BZM) in nasal drops and gel dosage forms. Effective liquid chromatographic separation was accomplished by employing Venusil XBP Cyano column (4.6 × 250 mm, 5 µm particle size) with gradient elution of the mobile phase consisting of buffer solution of potassium dihydrogen phosphate (0.025 M) and sodium 1-butane sulfonate (SBS) (0.025 M) (adjusted to pH 6.0) and acetonitrile. Peak areas of PHR, DMD and BZM at 271, 256 and 206 nm, respectively were measured and correlated to their concentrations. Peaks of PHR and DMD eluted at retention times 3.76 and 9.06 min, respectively, while BZM eluted as a couple of peaks at 11.88 and 12.51 min. The proposed HPLC procedure was carefully validated in terms of system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity range for both PHR and BZM was 10–400 μg/mL and DMD was 5–300 μg/mL with correlation coefficients >0.9999. The studied compounds were subjected to stress conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal degradation. Good resolution of the three compounds from their forced degradation products proves specificity and stability-indicating merits of the proposed method. In addition, resolution of the three drugs under investigation from some pharmaceutical compounds of different medicinal categories showed the high specificity of the described method.