A quality-by-design evaluated liquid chromatography method development and validation for the separation and quantification of nitroxoline and its impurities.

A novel, isocratic, sensitive, stability-indicating high-performance liquid chromatography method was developed for the separation and quantification of related substances in nitroxoline (NTL). The chromatographic separation has been achieved on Inertsil ODS-3 V, (250 × 4.6 mm, 5 µm) at 240 nm using ethylenediamine tetraacetic acid buffer and methanol in the ratio of 60:40 v/v as mobile phase. The performance of the method has been checked as per the International Conference on Harmonization guidelines for specificity, linearity, accuracy, precision, and robustness. Regression analysis showed a correlation coefficient value greater than 0.99 for NTL and its three impurities. The detection limit of impurities was in the range of 0.01% (0.05 µg/mL)-0.22% (1.1 µg/mL) indicating the sensitivity of the newly developed method. The accuracy of the method was established based on the recovery obtained between 94.7% and 104.1% for all the impurities. The percentage relative standard deviation obtained for the repeatability was less than 4.0% at the specification level for all impurities. Forced degradation was performed to establish the stability-indicating nature of the method and to know about the degradation products, the quality of a drug substance changes with time under the influence of stress conditions. Thus, the proposed method was validated and found to be specific, sensitive, linear, accurate, precise, reproducible, and beneficial for routine usage.

A new synthetic approach to cyclic ureas through carbonylation using di-tert-butyl dicarbonate (boc anhydride) via one pot strategy.

A new approach has been successfully employed to synthesize cyclic ureas via carbonylation, utilizing Boc anhydride and employing K2CO3 as a base along with N,N-dimethylformamide as the solvent. Remarkably high yields were achieved using K2CO3 in conjunction with (Boc)2O, enabling the streamlined preparation of benzimidazolones and 2-benzoxazolones within a single reaction vessel. Significantly, this approach obviates the necessity for using any dangerous reagents, rendering it environmentally friendly, and its key benefit lies in being a metal-free system. The method stands out for its efficiency, concise pathway, optimization from readily accessible starting materials, and ease of execution. The resulting benzimidazolones and 2-benzoxazolones were thoroughly characterized using techniques including LCMS, 1H NMR, and 13C NMR.

Valbenazine isomers and enantiomer determination by chiral normal phase liquid chromatography.

A novel, simple, specific, rapid, enantioselective normal phase chiral high-performance liquid chromatographic method with amylose-based Chiral Pak IG-3(250 × 4.6 mM) 3.0 µM column was developed and validated for separation and quantification of isomers and enantiomer of Valbenazine. The mobile phase composed of n-Heptane, isopropyl alcohol, dichloromethane, ethanol, and diethylamine in the ratio of 70:10:15:5:0.1 (V/V/V/VV) with a gradient flow rate was applied. The injection volume was 10 µl, and detection was carried out using a photodiode array detector at 282 nM. The column compartment was set at 35°C. The resolution between the enantiomer and isomers was found to be more than 2.0. The method was linear over the concentration range of limit of quantitation to 250% for isomers and enantiomers. The method was found to be robust with column temperature. The proposed chiral method is applicable for the determination of isomers and enantiomer of Valibenazine and was successfully used in the quality control of bulk drug manufacturing and pharmaceuticals.

Development, stability-indicating assessment, and evaluation of influential method conditions using a full factorial design for the determination of Nintedanib esylate-related impurities.

The design of an appropriate analytical method for assessing the quality of pharmaceuticals requires a deep understanding of science, and risk evaluation approaches are appreciated. The current study discusses how a related substance method was developed for Nintedanib esylate. The best possible separation between the critical peak pairs was achieved using an X-Select charged surface hybrid Phenyl Hexyl (150 × 4.6) mm, 3.5 µm column. A mixture of water, acetonitrile, and methanol in mobile phase-A (70:20:10) and mobile phase-B (20:70:10), with 0.1% trifluoroacetic acid and 0.05% formic acid in both eluents. The set flow rate, wavelength, and injection volumes were 1.0 ml/min, 285 nm, and 5 µl, respectively, with gradient elution. The method conditions were validated as per regulatory requirements and United States Pharmacopeia general chapter < 1225 >. The correlation coefficient for all impurities from the linearity experiment was found to be > 0.999. The % relative standard deviation from the precision experiments ranged from 0.4 to 3.6. The mean %recovery from the accuracy study ranged from 92.5 to 106.5. Demonstrated the power of the stability-indicating method through degradation studies; the active drug component is more vulnerable to oxidation than other conditions. Final method conditions were further evaluated using a full-factorial design. The robust method conditions were identified using the graphical optimization from the design space.

A combined qualitative and quantitative method development and validation of vancomycin hydrochloride injection formulation by HPLC and UV involving quality by design.

A selective, specific, precise, linear, accurate and robust analytical method was developed and validated for the assay of vancomycin HCl in vancomycin hydrochloride injection. Comparative UV spectrophotometric and reverse-phase HPLC were used to develop the quantitative determination. Acetonitrile and pH 2.2 phosphate buffer in the ratio 20:80 v/v were used as the mobile phase, and a flow rate of 1.0 ml/min with a 20 min run time. The detection was carried out at 235 nm with a Nucleosil C18 (250 × 4.6 mm) 10 µm column, and the ambient column temperature was maintained. The method uses a 20 µl injection volume and diluent as a blank solution in this connection. The method was validated as per the current regulatory guidelines. The linearity of this method was found to be linear in the range of 50-150% of the working concentration, and the correlation coefficient was >0.999. The method's accuracy was within the acceptable range, which was 98.1-101.5%. The method's precision was within an acceptable range of about 0.32% RSD. The analytical solution was stable for up to 48 h at room temperature. The method's robustness was proved by utilizing quality design tools. Stress studies demonstrated the method's stability-indicating nature.

Trace-level determination of potential genotoxic impurities in quetiapine fumarate using LC-MS.

A novel LC-MS method was developed and validated to determine three potential genotoxic impurities, namely 2-(2-aminophenylthio)benzoic acid hydrochloride, 2-aminothiophenol, and 2-(2-aminophenylthio)benzonitrile, at trace level (~1.6 ppm) in quetiapine fumarate drug substance, an antipsychotic drug. These impurities are potentially genotoxic and therefore should be controlled at or below specific acceptance limits. An InertSustain AQ-C18 column (250 × 4.6 mm, 5 µm) in reversed-phase mode with the column temperature at 45°C was used. The mobile phase was 0.1% trifluoroacetic acid in water and acetonitrile with gradient elution mode, and the run time was 45 min. The flow rate was 0.8 ml/min. A mass spectrometer was used to quantify the amount of impurities using electrospray ionization mode at specific m/z 245.9, 126.0, and 226.9 for 2-(2-aminophenylthio)benzoic acid hydrochloride, 2-aminothiophenol, and 2-(2-aminophenylthio) benzonitrile, respectively. The method was found to be sensitive and possessed excellent linearity in the concentration ranges from the limit of quantification to 150% of the permitted level (0.47-2.36 µg/ml) with correlation coefficients above 0.999. The results showed that the method was specific, precise, linear, and accurate for the estimation of these three impurities in quetiapine fumarate.

An effective and stability-indicating method development and optimization utilizing the Box-Behnken design for the simultaneous determination of acetaminophen, caffeine, and aspirin in tablet formulation.

Analytical techniques must be sensitive, specific, and accurate to assess the active pharmaceutical ingredients in pharmaceutical dosage forms. The quality-by-design (QbD) application has proven to be a practical method for magnifying HPLC operations. This article discusses the successfully developed QbD-based stability-indicative LC method for evaluating acetaminophen, caffeine, and aspirin (ASP) in tablet dosage form. To achieve the necessary chromatographic separation, Milli-Q water, methanol, and glacial acetic acid were employed in the following ratios: 63:35:2 (v/v/v) for mobile phase A and 18:80:2 (v/v/v) for mobile phase B. The flow rate, column temperature, and detecting wavelength were 1.0 ml/min, 40°C, and 275 nm, respectively, and an InertSustain C18 analytical column (150 × 4.6 mm, 3 µm) was used. Linearity was between 10.0 and 150.0 µg/ml for ASP and acetaminophen and between 2.6 and 39.0 µg/ml for caffeine. The accuracy findings were more than 97%, and the correlation coefficient for all three components was found to be greater than 0.999. The validated HPLC method yielded reliable and accurate results. ASP was shown to be vulnerable to both acid and alkaline hydrolysis in the forced degradation study. The described method is capable of separating the degradants produced during stress testing and is regarded as stability indicating. The proposed method can be used for a wider range of other formulations with an appropriate diluent selection and sample preparation procedure optimization.

Quality by design tool-evaluated stability-indicating ultra-performance liquid chromatography method for the determination of drugs (ritonavir and darunavir) used to treat the human immunodeficiency virus/acquired immunodeficiency syndrome.

Ritonavir and darunavir were examined using a ultra-performance liquid chromatography (UPLC) approach in pharmaceutical dosage forms. The small number of analytical studies that are currently available do not demonstrate the method's stability or nature. The study sought to assess both chemicals using a stability-indicating approach with a relatively short run time. The HSS C18 (100 × 2.1 mm), 2-mm column was used for the chromatographic separation, and isocratic elution was used to achieve this. In the mobile phase, methanol and 0.01 M phosphate buffer (pH 4.0) were included in a 60:40 (v/v) ratio. Throughout the analysis, the flow rate was kept at 0.2 mL min-1 , and a photodiode array detector set to 266 nm was used to find the major components. The proposed method showed a linear response (r2  > 0.999), and the accuracy was between 98.0% and 102.0%. The precision data showed relative standard deviation ≤1.0%. The UPLC method for quantification of ritonavir and darunavir in pharmaceutical dosage forms using a very short run time of under a minute is the subject of the proposed article. To meet current regulatory criteria, the quality by design idea was used in the method performance verification.

Estimation of purity of antimicrobial lead compound sulfonamide-anthranilate derivative methyl-ester-toluene-sulfonamide using LC to develop a new drug application.

The newly synthesized lead molecule methyl-ester-toluene-sulfonamide is the combined derivative of sulfonamide-anthranilate. It was estimated by gradient elution using 0.1% triethylamine in water with pH 2.0 as mobile phase A and the mixture of acetonitrile and tetrahydrofuran in the ratio of 975:25 (v/v) as mobile phase B at a flow rate of 0.8 ml/min and 210 nm wavelength on an Agilent 1260 infinity series HPLC system equipped with a diode array detector. The column used was ACE 3 C18-PFP (250 × 4.6 mm, 3 µm i.d.) operating at 40°C. The gradient program was time (min)/% B: 0.0/50, 3.0/50, 15.0/70, 25.0/90, 30.0/90, 31/50, and 38/50. The method is simple, accurate, rapid, and selective. The method was linear with a concentration range of 1.6-240 µg/ml. The accuracy data obtained were 98.5-100.5%. The method validation data and quality by design-based robustness study results indicate that the developed method is robust and fit for routine use in the quality control laboratory. Therefore, the ready availability of the method can be useful in pharmaceutical new drug development.

Unique green chromatography method for the determination of serotonin receptor antagonist (Ondansetron hydrochloride) related substances in a liquid formulation, robustness by quality by design-based design of experiments approach.

Serotonin receptor antagonist drug Ondansetron hydrochloride injectable formulation containing all related substances was identified and quantified by a single, simple, sensitive, eco-friendly, and green high-performance liquid chromatography method. The disseverment of all impurities was achieved with the Discovery Cyano (250 × 4.6) mm, 5 µm column. The gradient program was composed of pH 5.7 phosphate buffer as mobile phase A and acetonitrile as mobile phase B. The flow rate, column compartment temperature, and detection wavelengths were 0.9 mL/min, 30°C, and 216 nm, respectively. The method was validated as per current regulatory guidelines. The obtained %relative standard deviation for the precision results was between 0.55 and 2.72% for all impurities. The correlation coefficient values from the linearity experiment for impurities and analyte were more than 0.995. The accuracy results were obtained between 88.4 and 113.0% for all impurities. Both sample and standard solutions showed 24 h stability at benchtop and refrigerator conditions. All impurities and analytes met the specificity and mass balance for all forced degradation conditions. Quality-by-design-based design of experiments was utilized to establish the method's robustness. Method greenness was assessed by using the current advanced tool green analytical procedure index, National Environmental Methods Index, and analytical eco-scale.

Development and validation of rapid ultra high performance liquid chromatography with tandem mass spectroscopic method for the quantification of N-nitrosodimethyl amine and N-nitrosodiethyl amine in sitagliptin and metformin hydrochloride immediate and extended-release formulations.

A simple, effective LC-MS based method is developed and validated to determine N-nitrosodimethylamine and N-nitrosodiethylamine in pharmaceutical formulations of Sitagliptin and Metformin hydrochloride combination dosage forms. Atlantis T3 (100 × 3 mm, 3 µm) column, eluent-A (0.1% formic acid in water), and eluent-B (0.1% formic acid in methanol) were used to achieve chromatographic separation. A gradient program time (min)/%B: 0.01/3, 2/3, 4/55, 5/55, 5.5/90, 6.0/90, 6.5/3, and 7/3, and column flow rate: 0.75 mL/min was employed. The column oven and auto sample cooler temperatures were 40°C and 10°C, respectively. Atmospheric Pressure Ionisation positive mode with corona discharge potential as 4.0 V, drying gas (N2 ) flow as 110 mL/min, and nebulizer gas (N2 ) flow as 350 mL/min. Employing PerkinElmer triple quadrupole mass spectrometer, QSight 200 series, the source temperature was 450°C, and hot surface-induced desolvation temperature was 250°C. Under optimized conditions, diluent-1 and diluent-2 offered better recovery and improved peak shapes. The required method sensitivity of nitrosodimethylamine (LOQ 0.74 ng/mL) and nitrosodiethylamine (LOQ 0.37 ng/mL) for the nitrosamine impurities were achieved using an optimized test concentration of Metformin hydrochloride at 45.7 mg/mL.

Identification, isolation, and structural characterization of novel forced degradation products of apixaban using advanced analytical techniques.

The current research explains the stress degradation behavior of Apixaban, which is an anticoagulant or blood thinner. The degradation was conducted using hydrolytic, oxidative, thermal, and photolytic conditions. Apixaban is relatively stable in oxidative, thermal, and photolytic conditions; however, considerable degradation was observed in acid and base hydrolysis. Degradation products were identified using ultra-high performance liquid chromatography-mass spectrometry, isolated using semi-preparative high-performance liquid chromatography, and structural characterization by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. A total of five degradation products were identified and isolated in acid and base degradation. Degradation products 1, 2, and 3 were observed in acid conditions, whereas in base conditions, along with those three, two more degradation products 4 and 5 were identified. The representative thing was that among the five degradation products, two sets of positional isomers 1, 4, and 2, 5 were observed; out of which 2 and 5 are novel. The remaining degradation products 1, 3, and 4 are already reported tentatively using a single analytical technique of mass analysis without any evidence from nuclear magnetic resonance spectroscopy. Hence, the present study focused on using high-resolution mass, and nuclear magnetic resonance spectroscopy data for concrete confirmation of structures for degradation products.

Favipiravir (SARS-CoV-2) degradation impurities: Identification and route of degradation mechanism in the finished solid dosage form using LC/LC-MS method.

Favipiravir finished dosage was approved for emergency use in many countries to treat SARS-CoV-2 patients. A specific, accurate, linear, robust, simple, and stability-indicating HPLC method was developed and validated for the determination of degradation impurities present in favipiravir film-coated tablets. The separation of all impurities was achieved from the stationary phase (Inert sustain AQ-C18, 250 × 4.6 mm, 5-µm particle) and mobile phase. Mobile phase A contained KH2 PO4 buffer (pH 2.5 ± 0.05) and acetonitrile in the ratio of 98:2 (v/v), and mobile phase B contained water and acetonitrile in the ratio of 50:50 (v/v). The chromatographic conditions were optimized as follows: flow rate, 0.7 mL/min; UV detection, 210 nm; injection volume, 20 µL; and column temperature, 33°C. The proposed method was validated per the current International Conference on Harmonization Q2 (R1) guidelines. The recovery study and linearity ranges were established from the limit of quantification to 150% optimal concentrations. The method validation results were found to be between 98.6 and 106.2% for recovery and r2  = 0.9995-0.9999 for linearity of all identified impurities. The method precision results were achieved below 5% of relative standard deviation. Forced degradation studies were performed in chemical and physical stress conditions. The compound was sensitive to chemical stress conditions. During the study, the analyte degraded and converted to unknown degradation impurities, and its molecular mass was found using the LC-MS technique and established degradation pathways supported by reaction of mechanism. The developed method was found to be suitable for routine analysis of research and development and quality control.

Determination of progesterone (steroid drug) in the semi-solid dosage form (vaginal gel) using a stability-indicating method by RP-HPLC/PDA detector.

A simple stability-indicating method was developed and validated for the determination of progesterone (a steroid drug) in the semi-solid dosage form. All the impurities were separated from the main compound with a simple stationary phase (Eclipse XDB, C8, 150 × 4.6 mm, 5 µm). The mobile phase A contained phosphate buffer and acetonitrile in the ratio of 90:10, v/v, and mobile phase B contained purified water and acetonitrile in the ratio of 10:90, v/v. The optimized chromatographic conditions were as follows: flow rate, 1.0 mL min-1 ; UV detection, 241 nm; injection volume, 10 µL; and the column temperature, 30°C. The method was validated as per the current ICH Q2 guidelines. The recovery study and linearity ranges were established from 50 to 300% optimal concentrations. The method validation results were found between 98 and 102% for accuracy and r2  = 0.999 for linearity. Forced degradation in hydrolytic, oxidative, thermolytic, and photostability conditions was performed, and the stability indicating nature of the method was proved. Based on the validation and forced degradation results, the current method was found to be specific, precise, accurate, linear, robust, and stability-indicating method. The developed method was cost effective and easy to handle for quality control analysis.

A Quality by Design and green LC technique for the determination of mast cell stabilizer and histamine receptor antagonist (Olopatadine HCl) in multiple formulations.

Mast cell stabilizer and histamine receptor antagonist olopatadine hydrochloride (OPT) assay method predicated on LC have been established for the analysis in multiple formulations. The current method dealt with ophthalmic solution, nasal spray, and tablet formulation products. The isocratic chromatography method was optimized and validated with a Boston green C8 column (150 × 4.6 mm, 5 µm i.d.). Sodium dihydrogen phosphate buffer (pH 3.5) with acetonitrile in the ratio of 75:25 (v/v) was used as a mobile phase at a flow rate of 1.0 mL min-1 and at the column temperature of 30°C, and the detection was done at 299 nm. The method was validated as per International Council for Harmonisation (ICH) guidelines and United States Pharmacopoeia (USP). The accuracy results ranged from 99.9 to 100.7%, % relative standard deviation (RSD) from the precision was 0.5, and correlation coefficient from the linearity experiment was > 0.999. Solution stability was established for 24 h at room temperature and refrigerator conditions, and it was found that the solutions were stable. Using quality by design-based experiment designs, critical quality attributes were studied and it was found that the method was robust. In all the forced degradation studies peak purity was passed, and no interference was found at the retention time of the active component. The method validation data demonstrated that the developed method is linear, precise, accurate, specific, robust, and stable for the determination of OPT from multiple formulations. Analytical eco-scale tool, Green Analytical Procedure Index (GAPI) tool, and the National Environmental Method Index (NEMI) were used to evaluate the greenness of the method, and the analytical eco-score of 77 for the presented method was found to be excellent.

A simple isocratic LC method for quantification of trace-level inorganic degradation impurities (ferricyanide, ferrocyanide, nitrite, and nitrate) in sodium nitroprusside injection and robustness by quality using design approach.

This study developed and validated a trace-level quantification inorganic impurities method using reversed-phase HPLC and performed the robustness check using quality-by-design approach by varying the multiple factors simultaneously. This method is economical and simple and exhibits its stability-indicating nature [for the determination of ferrocyanide ([Fe(CN)6]4- ), ferricyanide ([Fe(CN)6 ]3- ), nitrate (NO3 - ), and nitrite (NO2 - )] in sodium nitroprusside (SNP) drug substance and liquid dosage form. Chromatographic separation was achieved using a USP L43 column (ACE PFP, 150 × 4.6 mm, 3 µm) with a simple isocratic elution. The buffer consists of potassium dihydrogen phosphate (50 mM), tetrabutylammonium hydrogen sulfate (9 mM), and tetrabutylammonium hydroxide (25 mM). The buffer pH was adjusted to 7.2 with tetrabutylammonium hydroxide. The mobile phase was mixed with the buffer and acetonitrile (68:32 v/v). The flow rate was 0.8 mL/min, column temperature was maintained at 30°C, and injection volume was 5.0 µL. The SNP impurities were monitored at 225 nm using a UV detector. Further, the method was validated per the International Council for Harmonisation (ICH) guidelines, and forced degradation studies were carried out under different stress conditions. The detector responses were plotted against concentrations, and correlation was linear (r > 0.999) over the range of 0.8-7.5 µg/mL for ferricyanide; 1.0-37.5 µg/mL for SNP; and 0.2-7.5 µg/mL for ferrocyanide, nitrite, and nitrate. The method repeatability was established for all the impurities with relative standard deviation (%), and the results were found to be less than 2.0.

A validated stability-indicating reversed-phase-HPLC method for dipyridamole in the presence of degradation products and its process-related impurities in pharmaceutical dosage forms.

In this study, we developed and validated a method to determine dipyridamole-related impurities in pharmaceutical dosage forms using the reversed-phase-HPLC technique. All impurities were separated on a YMC pack C8 (150 mm × 4.6 mm, 3.0 µm) analytical column using a suitable mobile phase. Mobile phase A was 10 mM concentration of phosphate buffer (pH adjusted to 4.7 by adding diluted orthophosphoric acid) and mobile phase B was buffer:acetonitrile:methanol (at the ratio of 30:40:30 v/v). The optimized chromatographic conditions used in the experiment were as follows: flow rate, 1.0 mL/min; injection volume, 10 µL and column temperature, 35°C. Chromatographic detection was performed at 295 nm. The stressed samples were analyzed for degradation under acidic, basic, peroxide, water hydrolysis, and physical degradation conditions. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines, and found to be specific, linear, accurate and have a robust stability-indicating nature. The method showed excellent linearity from limit of quantification (LOQ) to 150% level of concentrations for all impurities. The correlation coefficient (r2 ) for all impurities was between 0.995 and 0.999. The recovery study was performed from LOQ to 150% level concentrations, with mean recovery values between 92.9% and 103.2%, respectively. The developed method can be used to determine dipyridamole and its relative impurities. The degradation and validated study results indicate its stability-indicating nature. Therefore, the method can be used in pharmaceutical research and development and quality control departments.

Stability-indicating reversed-phase-HPLC method development and validation for sacubitril/valsartan complex in the presence of impurities and degradation products: Robustness by quality-by-design approach.

According to current regulatory guidelines, a stability-indicating method has been developed to determine the impurities in sacubitril (SCB) and valsartan (VLS) tablet dosage forms and perform robustness studies using the design of experiments approach. The present study was initiated to understand quality target product profile, analytical target profile, and risk assessment for method variables that affect the method response. A reversed-phase-HPLC system was equipped with a Phenomenex Gemini-NX C18 column (150 × 4.6 mm, 3 µm) and a photo diode array detector. A gradient mobile phase was used in this research work. The detection was performed at 254 nm; the flow rate was 1.5 mL/min, and the column temperature was maintained at 30°C. The proposed method was validated per the International Council for Harmonisation Q2 (R1) guidelines. The coefficient of correlation was >0.999 for all impurities. The limits of detection and quantification were evaluated for SCB, VLS, and all impurities. The precision and accuracy were obtained for SCB, VLS, and their related impurities. Intra- and inter-day relative standard deviation values were less than 10.0%, and the recoveries of impurities varied between 90.0 and 115.0%. Based on the validation results, the proposed DoE method can estimate SCB and VLS impurities in the finished dosage form.

Role of Natural Products in Developing Novel Anticancer Agents: A Perspective.

Cancer is a heterogeneous disease and is one of the significant health issues, especially in public health systems around the world. Natural products and their structural derivatives with outstanding chemical diversity have been investigated for potential anti-cancer agents. Many natural products revealing potential anti-cancer properties such as cytotoxicity, proliferation inhibition, induced apoptosis, retard metastasis, suppressing angiogenesis, and improved chemotherapy have been isolated from various plants and herbs. Several promising lead molecules have been identified recently; a few are in the clinical trial stage. This short communication summarises the role of natural products and their analogs in anti-cancer drug developments, especially plant, marine and microbial-based anti-cancer agents.

Molecular Hybrids of Pyazolo[3,4-b]pyridine and Triazole: Design, Synthesis and In Vitro Antibacterial Studies.

Antimicrobial resistance is on the rise, and there aren't enough new treatments to combat it. This might send the modern world back to the pre-antibiotic age. The molecular hybrids of pyrazolo[3,4-b]pyridine and triazole have been designed, synthesized, and analyzed for their drug-like molecule nature and in vitro analyses for their inhibition potentials against S. aureus and K. pneumoniae. The compounds 24 and 27 have been identified as the high potential molecules in this series based on in vitro experiments. Compound 24 has zone of inhibition values of 15 ± 0.82 mm and 14 ± 0.7 mm, whilst compound 27 has zone of inhibition values of 18 ± 0.95 mm and 16 ± 0.82 mm against S. aureus and K. pneumoniae, respectively. MIC and MIB values for compounds 24 and 27 against S. aureus and K. pneumoniae are 0.25 and 0.5, respectively.