ABSTRACT Citrus flavonoids are well known for their health‐promoting effects and contribution to daily nutritional dietary supplements. Hesperetin (HST), a citrus flavonoid, has gained special attention due to its multiple pharmacological actions, including anticancer, antioxidant, and anti‐inflammatory effects. However, identification of degradation products (DPs) is a key concern for preclinical development of HST. To achieve this, stability studies are essential to establish appropriate storage conditions and optimize the formulations accordingly. Despite this, substantial chemical stability data for HST applying RP‐HPLC, LC‐ESI‐MS/MS, and NMR, along with in vitro cytotoxicity, have not yet been investigated. To address these gaps, the impact of different stress conditions, namely hydrolytic (basic, acidic, and neutral), oxidative, thermal, and light exposure, was tested on HST. HST and its DPs were separated using a developed RP‐HPLC‐based stability‐indicating method. The developed method revealed that HST was highly stable under all stress conditions except basic conditions, where three DPs were formed. Out of the three DPs, two major DPs were isolated and enriched using mass‐triggered LC and subsequently characterized thoroughly by LC‐ESI‐MS/MS, and 1D/2D NMR spectroscopy. The DPs were identified as DP1 [(E)‐3‐(3‐hydroxy‐4‐methoxyphenyl) acrylic acid] and DP2 [(E)‐5‐(3‐hydroxy‐4‐methoxystyryl) benzene‐1,3‐diol], which are novel and have not been reported in the literature to date. The isolated DPs were evaluated for cytotoxicity using an invitro MTT assay on Dalton's lymphoma (DL) cancer cell line and peripheral blood mononuclear cells, along with in silico absorption, distribution, metabolism, excretion, and toxicity profiling. Both DPs induce minimal cytotoxicity in healthy cells, with cell death remaining below 10% throughout the treatment period. Additionally, Greenness assessment using the Analytical Greenness and Analytical Green Star Area tools indicated an acceptable environmental impact. The developed method effectively assessed HST stability under stressed conditions, supporting its use in routine quality control and formulation development. The study also offers valuable insights for the preclinical development of HST as a novel anticancer agent.
ABSTRACT Diazoxide is a benzothiadiazine‐derivative potassium channel opener that carries FDA orphan‐drug designation for the management of persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a life‐threatening neonatal condition with few pharmacological alternatives. Despite renewed clinical interest in this molecule, the analytical literature still lacks a validated, stability‐indicating reversed‐phase HPLC method capable of resolving diazoxide alongside all four of its related impurities, namely deschloro diazoxide, sulfonamide urea, benzene sulfonamide, and urea impurity, in a complex capsule matrix. A gradient RP‐HPLC method was developed on a Fortis Endurus C18‐Tri Alpha column (250 mm × 4.6 mm, 3 µm) using a binary mobile phase system: 0.1% aqueous formic acid as Phase A, and a ternary blend of acetonitrile, methanol, and water (40:40:20, v/v/v) as Phase B, with UV detection at 254 nm. Full validation followed ICH Q2(R2) guidelines, covering specificity, linearity, precision, accuracy, LOD/LOQ, and robustness. Relative response factors (RRF) were calculated individually for each impurity. All five analytes resolved cleanly, with PDA purity angles well below purity thresholds, confirming spectral homogeneity. Linearity was established over 0.050 to 0.786 µg/mL ( r 2 ≥ 0.9971). Precision yielded %RSD ≤ 0.50% across six replicate injections. Accuracy ranged from 91.9% to 105.1% recovery at LOQ, 100%, and 150% spike levels. Both LOD and LOQ were uniformly achieved at 0.017 µg/mL and 0.050 to 0.052 µg/mL, respectively. This is the first RP‐HPLC method to report experimentally determined relative response factors for all four related impurities together with a full ICH Q1A(R2) forced‐degradation evaluation, validated specifically for the diazoxide capsule dosage form.
ABSTRACT Herein, a rapid, sensitive, and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) method was developed and validated for the quantification of everolimus. The method utilizes a reverse‐phase Zorbex SB C18 column with a mobile phase consisting of acetonitrile and methanol, that requires a total run time of ≤ 30 min. Everolimus was detected at a wavelength of 276 nm that ensures clear separation from its related impurities, which included everolimus A, everolimus B, everolimus C, sirolimus B, and sirolimus C. Method validation was performed in accordance with the International Council for Harmonisation, ICH Q2(R1), validation of analytical procedures guidelines; which assesses critical parameters such as linearity, accuracy, precision, and robustness. The method demonstrated excellent linearity ( R 2 ≥ 0.999) over a concentration range of 1–400 ppm, with recovery rates between 99%–100%. The limit of detection and limit of quantification were established at 0.01 ppm (10 ppb) and 0.1 ppm (100 ppb), respectively. The method has been optimized with the following parameters: UV wavelength at 276 nm, column temperature maintained at 55°C, run time of 30 min, ternary solvent system of methanol–water–acetonitrile maintained at a solvent ratio of 61.5%:26%:12.5%. This validated HPLC method provides a rapid, accurate, and reliable analytical tool for the routine quality control of everolimus in pharmaceutical and medical device applications.
ABSTRACT While developing a topical gel 1% w/w dosage form, significant unknown impurities were observed above their threshold during shelf‐life determination, with relative retention time (RRT) of 0.66, 0.67, and 0.71 in accelerated stability conditions of 40°C temperature and 75% relative humidity sample. Due to the importance of pharmaceutical quality and purity, impurities during manufacture or storage might cause serious side effects. If an unknown impurity exceeds its threshold limits during product stability testing, its structure must be determined to assess the potential risks. Impurities were separated by gradient HPLC on a Kinetex C18 column with 0.1% formic acid mobile phases, detected at 245 nm. Structural characterization was done using liquid chromatography with mass spectrometry (LC–MS) and 1 H NMR (400 MHz). This study investigated and characterized degradation impurities observed during stability testing of a testosterone topical gel formulation. Three degradation impurities, identified as 6β‐hydroxy‐testosterone, 6β‐hydroperoxy‐testosterone, and 6‐keto‐testosterone impurities with molecular masses of 304.42, 320.20, and 302.41, were isolated and characterized using preparative chromatographic, high‐resolution mass spectrometric, and nuclear magnetic resonance techniques, followed by in silico (Q)SAR assessment of their potential genotoxicity using Deductive Estimate of Risk from Existing Knowledge (Derek) Nexus and Toxicity Prediction using Komputer Assisted Technology (TOPKAT) silico tools‐based knowledge and statistical methods. In silico toxicity evaluation with TOPKAT and Derek Nexus indicated that the testosterone impurities at RRT of 0.66, 0.67, and 0.71 are non‐mutagenic, corroborating their designation as ICH M7 Class 5 impurities.
ABSTRACT Azadirachta indica A. Juss. (neem) is widely recognized for its bioactive phytochemicals, including flavonoids and proanthocyanidins, which contribute to antioxidant and enzyme modulatory activities. This study investigated leaf extracts prepared with water and ethanol‐based solvents (30%–100%) to systematically correlate chemical composition with biological activities. Liquid chromatography coupled to ion‐trap mass spectrometry using electrospray ionization in negative ion mode was used to identify the individual bioactive compounds in the extracts; 25 major compounds were separated and characterized, predominantly proanthocyanidin dimers and trimers of (epi)catechin and (epi)gallocatechin. The most abundant compounds, mainly proanthocyanidins, were quantified, observing that the 50% ethanol extract showed the highest cumulative concentration of 63 mg/g. Enzyme inhibition was solvent‐dependent: The 100% and 80% ethanol extracts exhibited the strongest acetylcholinesterase, butyrylcholinesterase, and tyrosinase inhibition, while the water extract showed the highest α ‐amylase inhibition. These results highlight the critical influence of solvent polarity on phytochemical recovery and bioactivity, providing mechanistic insights to guide the optimized application of neem leaf extracts in functional foods, nutraceuticals, complementary therapies, and therapeutic formulations.
