Abstract In this study, a novel hydrophilic interaction liquid chromatography (HILIC) technique was established for the concurrent analysis of four active ingredients; chlorpheniramine maleate (CM), dextromethorphan hydrobromide (DHB), ephedrine hydrochloride (EHC) and guaifenesin (GUA). The developed method was characterized by its simplicity, accuracy and reliability. It has been successfully applied to quantify these compounds both in their pure forms and within combined pharmaceutical formulations containing all four active pharmaceutical ingredients APIs. A high-purity silica L3 column provided sharp and well-resolved peaks. Separation was achieved using an isocratic mixture of methanol and a 0.05 M acetate buffer at pH 4.0 in a ratio of 95.5:4.5 (v/v) with detection at ƛ max 258 nm. This method demonstrated better separation than conventional reversed-phase HPLC techniques. The method validation followed the requirements outlined in ICH Q2 (R2) and USP. The results showed high linearity, with correlation coefficients of 0.998 for GUA, 0.996 for EHC, 0.996 for DHB and 0.998 for CM in the concentration range of 50–750, 7.5–112.5, 6.25–93.75 and 4.0–60.0 μg mL −1 for GUA, EHC, DHB and CM respectively. LODs of the method were 2.70, 1.00, 0.76 and 0. 33 μg mL −1 and LOQs were 8.10, 3.00, 2.28 and 0.99 μg mL −1 for GUA, EHC, DHB and CM respectively. The method showed robust performance and minimal matrix interference and it was suitable for routine quality control of multi-ingredient pharmaceutical formulations containing the quaternary mixture of the cited antitussive APIs specifically as syrups.

Abstract Cigarettes are a product that contains thousands of dangerous chemicals that can disrupt the function of body organs and even threaten life. Nicotine is the most dangerous compound of cigarettes due to its addictive effect, and one way to reduce nicotine dependence is to use citronella oil. This study began with a quality test of citronella oil from Jatijejer Village using Thin Layer Chromatography (TLC), Stahl Distillation, and Gas Chromatography-Mass Spectrometry (GC-MS) instruments. Next, an in silico study was conducted to assess drug-likeness, bond energy, and bond interactions. The results of the quality test showed that citronella oil was proven to have good quality, and there were 5 spots indicating the presence of essential oils through TLC testing with a total essential oil content of 0.82% w/v, which was determined by the Stahl distillation method. The chemical content of citronella oil from Jatijejer Village has a content profile and chromatogram pattern that is quite similar to PT.N, as proven by GC-MS testing. The results of in silico testing show that one of the chemical contents, namely Geraniol (−6.3 kcal/mol), has a lower binding energy value (ΔG) than nicotine and can bind to important amino acids, so that it has the potential to replace nicotine. Based on the results of the drug-likeness analysis, the compound has also met Lipinski's rule of five and Veber's rule but does not meet the Ghose Filter Law. Meanwhile, based on the interaction, the chemical content of (1R)-cis-Verbenol and Geranial dimethyl acetal has the most appropriate %similarity to nicotine, and the chemical content of Chavibetol has the most appropriate %similarity to varenicline.

Abstract A selective UPLC-MS/MS assay was established and fully validated to quantify segetalin A, segetalin B, and hypaphorine in mouse plasma, supporting subsequent pharmacokinetic evaluation. Plasma samples were prepared by protein precipitation using methanol, with only 10 μL of plasma required. A UPLC HSS T3 column was used for separation with an 0.1% formic acid-acetonitrile as mobile phase, with elution completed within 3.5 min. Multiple reaction monitoring coupled with electrospray ionization was used for quantitative analysis. Mice received segetalin A, segetalin B, and hypaphorine intravenously (5 mg kg −1 ) or orally (10 mg kg −1 ), and the pharmacokinetics of all three compounds were evaluated. The UPLC-MS/MS method showed excellent linearity in mouse plasma: 2.0–2,100 ng mL −1 for segetalin A, 2.0–2,080 ng mL −1 for segetalin B, and 2.2–2,280 ng mL −1 for hypaphorine (all R 2 > 0.995). It was used to evaluate the pharmacokinetics of all three compounds after intravenous and oral dosing in mice. The oral bioavailability of segetalin A, segetalin B, and hypaphorine was determined to be 3.18, 1.17, and 63.44% in mice, respectively.

Abstract A simple, rapid, and environmentally friendly reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous determination of ketoprofen and famotidine in combined dosage forms. Chromatographic separation was achieved on a Roc C18 column (250 × 4.6 mm, 5 μm) using an isocratic mobile phase of methanol:water:acetonitrile (35:35:30, v/v/v) adjusted to pH 3.1 with formic acid, at a flow rate of 2 mL/min and detection at 264 nm. Retention times were 2.86 min for ketoprofen and 8.56 min for famotidine. The method was validated following ICH Q2(R2) guidelines, demonstrating excellent linearity (R 2 = 0.9996 for ketoprofen, R 2 = 0.9993 for famotidine), accuracy (98.35–101.26%), precision (%RSD < 1%), and sensitivity (LOD: 18.3 and 25 ng/mL; LOQ: 61.8 and 84 ng/mL, respectively). Robustness testing confirmed reliability under varied conditions. Importantly, the method minimizes solvent usage and employs short run times, aligning with green analytical chemistry principles. The proposed RP-HPLC method is precise, accurate, and suitable for routine quality control of ketoprofen–famotidine formulations, offering a sustainable alternative for pharmaceutical analysis.

Abstract 2-diethylamino-N-2,5-dimethylphenyl acetamide (JM25-1), a novel anti-inflammatory agent with epithelial barrier protective function, requires comprehensive pharmacokinetic evaluation to support clinical translation. A quantitative analysis method was developed to characterize the pharmacokinetics of JM25-1 in Wistar rats after oral and intraperitoneal ( i.p ) administration. Plasma, obtained via centrifugation and methanol protein precipitation, was analyzed using HPLC-MS/MS. Chromatographic separation was achieved on a Kinetex column embedded with C 18 stationary phase, using a binary-gradient mobile phases consisting of water containing 0.1% formic acid (phase A) and methanol containing 0.1% formic acid (phase B). The flow rate was set at 0.6 mL min −1 with a total run time of 4 min. Mass spectrometric detection was performed by monitoring the m/z transitions of 235.1/86.2 and 235.1/58.1 for JM25-1, 241.2/86.2 and 241.2/58.1 for Lidocaine-d6 (used as internal standard) in the electrospray ionization positive mode. The method exhibited excellent linearity over concentration ranges of 1–200 ng mL −1 ( r 2 = 0.9998). All validation parameters, including specificity, extraction recovery (89.65–98.44%), matrix effect (absolute: 97.22–99.82%; relative: <15%), accuracy (87.93–104.18%), and precision (intra- and inter-day RSD ≤7.26%), met bioanalytical acceptance criteria. Stability assessments confirmed that JM25-1 remained stable under all tested conditions. Pharmacokinetically, oral administration showed rapid absorption ( t max = 10 min) but significantly lower systemic exposure compared to i.p , resulting in low bioavailability. This study developed a rapid, sensitive, and reproducible HPLC-MS/MS method for JM25-1 quantification in rat plasma, and the validated method was successfully applied to the pharmacokinetic study of JM25-1 following oral and i.p administration.

Abstract High-performance liquid chromatography (HPLC) is a critical analytical technique in the pharmaceutical sector, valued for its accuracy, speed and efficiency in drug quantification. This study aimed to develop and validate a novel reversed-phase HPLC (RP-HPLC) method for quantifying triamcinolone base (TRb) in solid pharmaceutical forms, using consistent chromatographic conditions for both dosage determination and dissolution studies. The method was validated in accordance with regulatory guidelines and demonstrated excellent precision, accuracy, and robustness. The mobile phase consisted of methanol and water (50:50), with a flow rate of 1.5 mL min −1 , detection at 254 nm and a column temperature of 30 °C. Key performance parameters included selectivity, linearity ( R = 0.9998 across 2–12 μg mL −1 ), precision (RSD < 2%), accuracy (98–102% recovery in the presence of two placebo matrices), and robustness (seven variables evaluated without significant impact). The retention time was 4.61 min. The method proved effective for both dosage quantification and formulation dissolution studies. Applying the same methodology across various stages of pharmaceutical development enhances standardization, traceability, and data reliability. As such, this RP-HPLC method is highly recommended for quality control laboratories seeking practical, sustainable, and scientifically robust analytical solutions.

Abstract A sensitive and selective LC–MS/MS method was developed and validated for the simultaneous quantification of pretomanid and linezolid in BALB/c mouse plasma and lung tissue in accordance with ICH M10 bioanalytical guidelines. Chromatographic separation was achieved on a C18 column with gradient elution using methanol and 0.1% formic acid in water. The method demonstrated linearity ( r 2 > 0.995), accuracy, precision, and recovery within the validated range of 10–20,000 ng mL −1 , without significant matrix effect. Stability under various storage and handling conditions was also confirmed. The validated assay was successfully applied to a pharmacokinetic study using a sparse sampling design, demonstrating reliable quantification of both analytes in plasma and lung tissue and confirming the applicability of the method to preclinical bioanalysis. The developed method provides a robust and validated analytical platform for the simultaneous determination of co-administered anti-tuberculosis agents and is suitable for future pharmacokinetic and tissue distribution studies in preclinical models.

Abstract Ezetimibe and atorvastatin calcium are widely used antihyperlipidemic agents to reduce blood cholesterol levels and prevent cardiovascular disorders. Reliable analytical methods are essential for estimating these drugs simultaneously in combined pharmaceutical formulations. The current research work aimed to develop and validate a new, simple RP-HPLC method for estimating ezetimibe and atorvastatin calcium simultaneously. Chromatographic analysis was carried out using an Agilent 1,260 Infinity system fitted with a C-18 column. The mobile phase was 0.1 M ammonium acetate buffer (pH 6.5) and acetonitrile in a 28:72 v/v ratio. The rate of flow of mobile phase was set at 1.0 mL min −1 , detected at a wavelength of 242 nm. The validation of the method was carried out according to ICH guidelines for parameters including precision, linearity, accuracy, robustness, repeatability, reproducibility, specificity, and system suitability. Ezetimibe and atorvastatin calcium were well separated, with retention times of approximately 3.2 and 5.8 min, respectively. Excellent linearity was shown by the method, high recovery rates confirming accuracy, and precision with a %RSD below 2%. The limits of detection (LOD) and quantification (LOQ) were also established. The method remained robust against deliberate variations in temperature, wavelength, and flow rate. Based upon the outcomes, it can be concluded that the validated RP-HPLC method is precise, simple, and reproducible, making it appropriate for routine quality control and simultaneous estimation of ezetimibe and atorvastatin calcium in bulk materials and pharmaceutical products.

Abstract Despite extensive development and widespread use, existing methods for analysis of caffeine and paracetamol overlook their environmental impact. Furthermore, these methods were not developed using the Analytical Quality by Design approach, leading to a significant gap in understanding critical separation factors. Moreover, the absence of a hydrophilic interaction liquid chromatography (HILIC) method for the simultaneous analysis of caffeine and paracetamol, integrating Analytical Quality by Design and a comprehensive green approach, presents an exciting opportunity for innovation and advancement in the field. In this paper, we report an efficient and sustainable HILIC method for caffeine and paracetamol analysis, which is based on Analytical Quality by Design, green and white chemistry principles. Optimal chromatographic conditions were adjusted using an InertSil Diol column with the mobile phase of acetonitrile and ammonium acetate buffer. The method was validated according to the International Council for Harmonisation guidelines, and its applicability in the analysis of Panadol Extra® tablets was confirmed. Relative standard deviation values were 0.07–0.28% for accuracy and 0.45–0.54% for precision. The environmental footprint and effectiveness of the method were evaluated through assessments of triple-color analysis which showed the dominance of green, blue, and white with scores of 0.66, 85.0, and 83.3, respectively. These parameters confirm that the method is eco-friendly, reliable, accurate, and precise for future use in the analysis of dosage forms containing caffeine and paracetamol. This is the first time that the Analytical Quality by Design approach has been combined with green and white chemistry principles, creating a strong synergy for the development of environmentally friendly methods.

Abstract The aim of the proposed study was to build and verify a rapid, sensitive, greener, and stability indicating Ultra-performance liquid chromatography photodiode array detector (UPLC-PDA) method for the estimation of paracetamol (PRL) in marketed formulations. Before developing and validating the UPLC method, a comprehensive evaluation of the quality parameters of the tablet formulations was conducted to ensure their compliance with pharmacopeial standards. This preliminary assessment included general appearance, weight variation, hardness, friability, and disintegration tests. These tests were essential in confirming the overall quality and integrity of the tablets before proceeding with the analytical method development using UPLC and they were found to be within the limits of pharmacopeial standards. The developed UPLC method was validated for several validation parameters and greenness. The greener mobile phase was composed of water and isopropyl alcohol (IPA) (98:2, v/v). The maximum absorbance of PRL was detected at a wavelength of 244.3 nm. The developed UPLC method was found to be linear 5.5–110 ng mL −1 range. And the LOD and LOQ of the PRL were found to be 0.63 ± 0.00 ng mL −1 and 1.91 ± 0.00 ng mL −1 , respectively. In addition, the developed UPLC method for PRL estimation was fast, accurate, precise, robust and highly sensitive. The excellent greenness profile was revealed by the computed values of the analytical Eco-Scale, ChlorTox and AGREE scales for the current technique, which are 93, 0.006 g and 0.82, respectively. We further employed the concept of ‘blueness’ assessment using the recently introduced Blue Applicability Grade Index (BAGI) algorithms. The obtained high BAGI score (77.5) demonstrated excellent applicability of the method. This novel UPLC-PDA approach provides a rapid, environmentally friendly, and highly applicable alternative to conventional techniques for analyzing active constituents in marketed formulations. The degradation of products appears, the current UPLC method was still able to identify PRL, exhibiting its selectivity and stability qualities. Five different marketed formulations named Tbt-1, Tbt-2, Tbt-3, Tbt-4 and Tbt-5 contained 97.35, 96.50, 105.27, 97.65 and 98.46% w/w of PRL, respectively. Moreover, out of five marketed formulations, four were scored, including Tbt-1, Tbt-3, Tbt-4 and Tbt-5 were found to contain 97.93, 98.47, 96.68, and 96.85% w/w of PRL, respectively. These findings exhibited that PRL in marketed formulations can be consistently standardized and analyzed their content by using the proposed UPLC-PDA method.