HPLC Method For Analysis Research Articles

Inhibitory effects of cuminaldehyde on human liver cytochrome P450 enzymes

This work explored the influence of cuminaldehyde on the metabolic activity of four primary human cytochrome P450 (CYP) enzymes. Human liver microsomes (HLM) with and without cuminaldehyde were incubated with specific substrates of different CYP enzymes. The influence of cuminaldehyde on “CYP1A2, CYP2C9, CYP2D6 and CYP3A4” activities was examined. The formation of specific metabolites was analyzed by HPLC analytical methods. It was observed that cuminaldehyde appears to have a negligible influence on CYP1A2 activity. However, CYP2C9 activity was significantly and potently inhibited by cuminaldehyde at all investigated concentrations. It was found that cuminaldehyde (1 µM) inhibited the most CYP2C9 enzyme activity with 64.87 % inhibition followed by CYP3A4 (38.90 %), CYP2D6 (25.76 %) and CYP1A2 (4.99 %). Inhibition of both CYP2D6 and CYP3A4 enzyme activity was observed in response to cuminaldehyde concentrations. Findings of the current investigation indicate that herb-drug interactions are very possible when cuminaldehyde is concurrently taken with medicines predominantly metabolized by CYP2C9 and CYP3A4. Cuminaldehyde should be tested further to examine how it affects CYP2C9 and CYP3A4-metabolized drugs.

Analysis of chemical composition, antimicrobial, antioxidant activities of pomegranate fruit thick and green tea leaf extracts

Infection diseases is a worldwide important problem for medicine and pharmacy. The purpose of this work was to study and compare phytochemical composition, antimicrobial, antioxidant potential of pomegranate fruit thick and green tea leaf liquid extracts. The quantification of biologically active substances (BAS) was performed using spectrophotometric, titrimetric, and HPLC analysis methods. Antioxidant activity was measured through a potentiometric method, while antimicrobial and antifungal effects were assessed using the well method and determining the minimum inhibition concentration. The total content of phenolic compounds was 0.40 and 10.10%, organic acids – 5.80 and 1.60% for pomegranate fruit thick and green tea leaf extract. The total content of catechins in the green tea leaf extract was 10500.0 mg/100 g, where epicatechin-3-O-gallate was dominated (3730.0±2.00 mg/100 g). The total content of anthocyanins in the pomegranate fruit thick extract was 107.10 mg/100 g, where cyanidin-3-O-glucoside was dominated (41.74 ± 0.10 mg/100 g), whereas delphinidin-3-O-glucoside had a lowest content (4.99 ± 0.10 mg/100 g). Both extracts possessed a high antioxidant potential, and effective antimicrobial effects. The antioxidant, antimicrobial activity of pomegranate fruit extract was higher than green tea leaf extract. In addition, we assumed that anthocyanins had higher antioxidant, antimicrobial properties than catechins. These findings would promote application of pomegranate fruits extract as pharmaceuticals and nutraceuticals.

Analytical HPLC Method for Determination of Miconazole Nitrate and Eugenol in Formulated Emulgel

A diabetic foot ulcer (DFU) is the prime cause of poor quality of life among diabetic patients. DFU leads to physical disability and an enormous economic burden on diabetic patients. The development and prognosis of DFU are preventable in most patients by modifying the risk factors for DFU. Programs intervened by clinical pharmacists can enhance rational drugs use and improve the early identification and prevention of DFU. This study intended to prove the beneficial outcome in identifying the risk factors and the provision of patient-tailored referral advice regarding DFU among diabetic patients. This cross-sectional study assessed the neuropathy through Michigan Neuropathy Screening Instrument (MNSI), Monofilament and Vibration test using Tuning Fork and Handheld Doppler for assessing the lower limb vascular disease as per NICE guideline among 137 diabetic patients. 64% of patients were found to have symptoms of neuropathy through the neuropathy assessment test whereas 36% did not have the symptoms. Foot sensory using monofilament test was found to be normal in 49.64% of DFU patients, reduced in 43.80%, and absent in 6.57% of DFU patients. The Ankle Brachial Index (ABI) was found to be normal in 48% of patients, mild in 34% of patients, moderate in 12% of patients, and incompressible in 6% of patients. Clinical Pharmacist intervened risk assessment through Hand-held Doppler, MNSI instrument, and Vibration test helped in screening the DFU patients for Neuropathy, and implementation of effective per patient-tailored education was found to be beneficial in the early identification of DFU and referral to specialty hospitals

Analytical HPLC Method for Determination of Miconazole Nitrate and Eugenol in Formulated Emulgel

Analytical HPLC Method for Determination of Miconazole Nitrate and Eugenol in Formulated Emulgel

Identification and quantification of the antioxidants in Ginkgo biloba leaf.

The antioxidant activity of Ginkgo biloba leaf (GBL) extract is closely related to its efficacy against various diseases; however, the antioxidant activities of the specific constituents of GBL remain unclear. In this study, 194 GBL constituents were identified using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, including 97 flavonoids, 37 terpenoids, 29 lignans, 19 carboxylic acids, 5 alkylphenolic acids, 5 alkylphenols, and 2 other compounds. The cleavage rules of the main constituents of GBL were dissected in detail. The 36 GBL constituents with high antioxidant activity were subsequently discovered using the oxygen radical absorbance capacity assay, including 30 flavonoids and six carboxylic acids. Finally, an HPLC analysis method was established to determine the content of the nine major antioxidants in the three batches of GBL. Among them, kaempferol 3-O-β-D-(6″-p-coumaroyl) glucopyranosyl-(1-2)-α-L-rhamnopyranoside, kaempferol-3-O-rutinoside, and rutin exhibited high antioxidant activity and were found in significant amounts in GBL, with concentrations greater than 0.7mg/g. These results provide an important reference for the development of pharmaceuticals and health products containing GBL.

Development of Uv-visible and Hplc Analytical Methods for Quantitative Analysis of Antiulcer Drugs in Dosage Forms

Precise measurement of active pharmaceutical ingredients (APIs) in dosage forms is crucial in the pharmaceutical business to guarantee the quality, effectiveness, and safety of the product. This research is focused on creating and confirming analytical techniques for accurately measuring the amount of antiulcer medicines in different types of medication. The measurement of antiulcer medicines in their respective formulations was carried out using two analytical techniques: UV-Visible spectroscopy and High-Performance Liquid Chromatography (HPLC). UV-Visible spectroscopy is a straightforward and cost-effective method that allows for quick examination. It is particularly useful for regular quality control applications. HPLC, in contrast, offers exceptional sensitivity, specificity, and precision, facilitating the isolation and measurement of many constituents in complex matrices. The technique development procedure included optimizing experimental parameters, including solvent systems, detection wavelengths, column types, mobile phase composition, and flow rates. The reliability and robustness of the suggested procedures were ensured by validation studies done in accordance with ICH recommendations. The adequacy of the procedures for their intended purpose was assessed by evaluating parameters such as linearity, precision, accuracy, specificity, and robustness. The UV-Visible and HPLC techniques that were developed showed exceptional linearity over the examined concentration range, with correlation values (R²) above 0.99. The precision experiments showed low relative standard deviations (RSDs), suggesting excellent repeatability and moderate accuracy. The accuracy checks demonstrated that the recovery values were within acceptable ranges, therefore verifying the dependability of the methodologies used for quantitative analysis.

An isocratic HPLC-UV analytical procedure for assessment of glutathione and its related substances

Reduced glutathione (GSH) is an endogenous tripeptide antioxidant which plays a crucial role in a variety of physiological and pathological activities. Although GSH is not present in any FDA-approved drug product, GSH dietary supplement products and compounded GSH drugs are available to patients in the US. Several incidents of toxicity have occurred in recent years due to endotoxin or otherwise contaminated GSH in compounded drugs. Efficient and sensitive analytical methods are needed for assessing and ensuring the quality of GSH substance and associated drug or dietary supplement products. Impurities A (L-cysteinylglycine), B (cysteine), C (oxidized L-glutathione) and D (γ-L-glutamyl-L-cysteine) are the main related impurities for GSH drug substance which have been detected and quantified by capillary electrophoresis and qNMR analytical procedures. However, there are no reported HPLC methods for detecting or quantifying the three main related impurities A, B and D even though numerous HPLC analytical methods have been reported for analyzing GSH and impurity C. In this report, an isocratic HPLC-UV analytical procedure was developed and validated for separating and identifying GSH and related impurities A-D as well as a newly identified degradant, L-pyroglutamic acid (pGlu), within 10 minutes with resolution (RS) more than 3. The LOD and LOQ were determined to be 0.02 % w/w and 0.05 % w/w, respectively, for impurities A-D and pGlu. Importantly, the optimized HPLC analytical procedure for GSH assay does not have interference from impurities A, B and D, providing highly specific results compared to the commonly used iodine titration method. The newly validated analytical procedure was applied to assess different commercial GSH bulk substance samples. The results suggest that the analytical procedure described in this work is suitable for quality assessment of GSH samples.

In Vitro Biological Evaluation of an Alginate-Based Hydrogel Loaded with Rifampicin for Wound Care.

We report a biocompatible hydrogel dressing based on sodium alginate-grafted poly(N-vinylcaprolactam) prepared by encapsulation of Rifampicin as an antimicrobial drug and stabilizing the matrix through the repeated freeze-thawing method. The hydrogel structure and polymer-drug compatibility were confirmed by FTIR, and a series of hydrogen-bond-based interactions between alginate and Rifampicin were identified. A concentration of 0.69% Rifampicin was found in the polymeric matrix using HPLC analysis and spectrophotometric UV-Vis methods. The hydrogel's morphology was evaluated by scanning electron microscopy, and various sizes and shapes of pores, ranging from almost spherical geometries to irregular ones, with a smooth surface of the pore walls and high interconnectivity in the presence of the drug, were identified. The hydrogels are bioadhesive, and the adhesion strength increased after Rifampicin was encapsulated into the polymeric matrix, which suggests that these compositions are suitable for wound dressings. Antimicrobial activity against S. aureus and MRSA, with an increased effect in the presence of the drug, was also found in the newly prepared hydrogels. In vitro biological evaluation demonstrated the cytocompatibility of the hydrogels and their ability to stimulate cell multiplication and mutual cell communication. The in vitro scratch assay demonstrated the drug-loaded alginate-grafted poly(N-vinylcaprolactam) hydrogel's ability to stimulate cell migration and wound closure. All of these results suggest that the prepared hydrogels can be used as antimicrobial materials for wound healing and care applications.

Chemical composition, antioxidant and antimicrobial activities of Vaccinium macrocarpon (Ericaceae) and Camellia sinensis (Theaceae) extracts

In the modern scientific community, considerable attention is given to studying the pharmacological activity of catechin and anthocyanin derivatives. However, a comparative analysis of the antioxidant and antimicrobial activities of extracts rich in anthocyanins and catechins has not been previously conducted. So, the purpose of this work was to study and compare phytochemical composition, antimicrobial and antioxidant potential of Vaccinium macrocarpon L., Ericaceae fruit thick and Camellia sinensis L., Theaceae leaf liquid extracts. The quantification of biologically active substances (BAS) was accomplished with spectrophotometric, titrimetric and HPLC methods of analysis; antioxidant activity was determined by the potentiometric method; antimicrobial and anti-fungi effects was evaluated by the well method and minimum inhibition concentration. The total content of phenolic compounds was 0.47% and 10.10%, organic acids – 4.27% and 1.60% for V. macrocarpon fruit thick and C. sinensis leaf extract. The total content of catechins in the C. sinensis leaf extract was 105,000 mg/kg, where epicatechin-3-O-gallate dominated (37300 mg/kg). The total content of anthocyanins in the V. macrocarpon fruit thick extract was 1280 mg/kg, where peonidin-3-O-galactoside dominated (408 mg/kg). Both extracts possessed a high antioxidant potential, and effective antimicrobial and anti-fungi effects. The antioxidant, antimicrobial and anti-fungi activity of V. macrocarpon fruit extract was higher than C. sinensis leaf extract. In addition, we assumed that anthocyanins had higher antioxidant, antimicrobial and anti-fungi properties than catechins. These findings would promote application of V. macrocarpon fruits extract as pharmaceuticals and nutraceuticals.

A comprehensive study of Tianma Toutong Tablets from the dual dimensions of quality and efficacy using fingerprint techniques and network pharmacology

BackgroundThe quality of traditional Chinese medicine (TCM) is the prerequisite for ensuring its safe and effective clinical application. With the increasing popularity of TCM worldwide, the quality control of TCM products has become increasingly crucial. Tianma toutong tablet (TMTTT) is mainly used for migraine caused by external wind and cold, blood stasis, or deficiency of blood and nourishment. However, the mechanism of action of TMTTT is still unclear, and there has been a lack of in vitro antioxidant activity research and migraine treatment mechanism research. Therefore, it is urgent to establish a set of comprehensive and effective evaluation methods. Resultsthree fingerprint profiles were established using HPLC, UV, and DSC analysis methods, and established three digital parameters simple complexity index (SX), simple clarity index (SY), simple complexity clarity ratio (Sω), 22 batches of samples were evaluated using a comprehensive linear quantitative fingerprint method (CLQFM). In addition, the antioxidant activity of the samples was determined using the DPPH method, and the relationship between fingerprint peaks in different fingerprints and antioxidant capacity was explored using Pearson correlation coefficients. Finally, network pharmacological research was conducted to investigate the potential targets, compounds, and pathways involved in the treatment of migraines with TMTTT. The results showed that the 22 batches of samples were classified into different quality grades. TMTTT exhibited good antioxidant activity, the fingerprint-efficacy relationship showed that gastrodin, chlorogenic acid, ferulic acid, imperatorin and isoimperatorin had strong antioxidant capacity, providing directions for the identification of active compounds. A total of 36 core targets were identified and screened by network pharmacology, which AKT serine/threonine kinase 1 (AKT1), albumin (ALB), insulin (INS), tumor necrosis factor (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2) and interleukin-6 (IL-6), and compounds such as β-sitosterol, chrysophanol, vanillin are the key to the treatment of migraine, providing references for subsequent clinical research and new drug development. Significance: This study examined the consistency of the quality of TMTTT and the mechanism of action in treating migraines from both quality and efficacy perspectives, providing a favorable direction for further research on TMTTT and offering new ideas for the quality control of TCM compound formulations.

Research on iron removal on carbon nanotube materials synthesized by the CVD method using 2,4-D adsorption in aqueous solution

In this study, the technique of oxidation in air and treatment with HCl was chosen to remove iron in carbon nanotube materials synthesized by the CVD method to apply 2,4-D adsorption in aqueous solution at initial concentration 52.2 mg/L. The oxidation process was repeated twice at 440 ˚C for 4 hours and 40 minutes, respectively. XRD, TEM, BET, AAS, HPLC analysis methods have shown that the MWCNTs tube tips are open and not shortened, can handle 94.42 % Fe, removing Fe has increased the surface area. BET alone increased from 170 to 262 m2/g and pore volume increased from 0.897 to 1.402 cm3/g, increasing the adsorption capacity of the material from 64.39 to 80.16 %.

Development and validation of a HPLC method simultaneously assisted with diode array and evaporative light scattering detection for the quantification of clinical mAb infusion bags in hospital pharmacy

To secure the supply chain of the monoclonal antibody (mAb) standardized infusion bags, a reversed phase (RP) HPLC analytical method assisted with a diode array detector (DAD) and an evaporative light scattering detector (ELSD) was implemented in the pharmacy department of the university hospital center of Besançon (UHCB). For a well-defined batch, the concordance between the two detections evaluated using the Lin’s concordance correlation coefficient (CCC) (Lin’s CCC = 0.9858) and a Bland–Altman plot was excellent. The analytical quantification of the different dose-banded (DB) of the infusion bags prepared in advance were achieved using this new reversed HPLC-DAD/ELSD analytical method in less than 4 min. For a same batch of mAbs, DAD generated nearly uniform response and showed a slightly superior accuracy than did ELSD. Their agreement in terms of calculated dose is excellent. In the event of a breakdown of one of the two detectors, this allowed the mAb infusion bags to continue to be monitored and thus improved the drug supply chain safety. As well our optimized method was able to monitor degradation mAbs created by accidental stress during the time period of stockage.

High-Performance Liquid Chromatography-Fluorescence Detection Method for Ochratoxin A Quantification in Small Mice Sample Volumes: Versatile Application across Diverse Matrices Relevant for Neurodegeneration Research.

Ochratoxin A (OTA) is a mycotoxin commonly found in various food products, which poses potential health risks to humans and animals. Recently, more attention has been directed towards its potential neurodegenerative effects. However, there are currently no fully validated HPLC analytical methods established for its quantification in mice, the primary animal model in this field, that include pivotal tissues in this area of research, such as the intestine and brain. To address this gap, we developed and validated a highly sensitive, rapid, and simple method using HPLC-FLD for OTA determination in mice tissues (kidney, liver, brain, and intestine) as well as plasma samples. The method was rigorously validated for selectivity, linearity, accuracy, precision, recovery, dilution integrity, carry-over effect, stability, and robustness, meeting the validation criteria outlined by FDA and EMA guidelines. Furthermore, the described method enables the quantification of OTA in each individual sample using minimal tissue mass while maintaining excellent recovery values. The applicability of the method was demonstrated in a repeated low-dose OTA study in Balb/c mice, which, together with the inclusion of relevant and less common tissues in the validation process, underscore its suitability for neurodegeneration-related research.

Chemical Relationship among Genetically Authenticated Medicinal Species of Genus Angelica.

The genus Angelica comprises various species utilized for diverse medicinal purposes, with differences attributed to the varying levels or types of inherent chemical components in each species. This study employed DNA barcode analysis and HPLC analysis to genetically authenticate and chemically classify eight medicinal Angelica species (n = 106) as well as two non-medicinal species (n = 14) that have been misused. Nucleotide sequence analysis of the nuclear internal transcribed spacer (ITS) region revealed differences ranging from 11 to 117 bp, while psbA-trnH showed variances of 3 to 95 bp, respectively. Phylogenetic analysis grouped all samples except Angelica sinensis into the same cluster, with some counterfeits forming separate clusters. Verification using the NCBI database confirmed the feasibility of species identification. For chemical identification, a robust quantitative HPLC analysis method was developed for 46 marker compounds. Subsequently, two A. reflexa-specific and seven A. biserrata-specific marker compounds were identified, alongside non-specific markers. Moreover, chemometric clustering analysis reflecting differences in chemical content between species revealed that most samples formed distinct clusters according to the plant species. However, some samples formed mixed clusters containing different species. These findings offer crucial insights for the standardization and quality control of medicinal Angelica species.

Development of a method for the determination of alkylpyrazine in traditional vinegar and its formation kinetics

Alkylpyrazine (APZ), as one of the crucial components in food flavor, are extremely important for the sensory evaluation of foods. For the purpose of simultaneously identifying multiple APZ in food items, a HPLC analytical method was created. The chromatography was carried out on an XBridge C18 column at a flow rate of 1 mL/min, with a detection wavelength of 275 nm. The ratio of mobile phase A (acetonitrile) to mobile phase B (0.1% formic acid and 0.1% aqueous trifluoroacetic acid) was 1:99. Compared to the conventional assay, RSD of precision less than 3%, average recoveries ranged from 96% to 110%, quantification limits were between 0.052 and 0.278 mg/L, detection limits were between 0.016 and 0.084 mg/L, and total analysis time is 35 min in the HPLC method. Meanwhile, we found that prolonging the aging time could increase the flavor characteristics of vinegar by analyzing the composition of APZ in vinegar and its formation pattern during the brewing process. The above results demonstrated that HPLC is an effective method for the rapid determination of pyrazines in vinegar. This study provides a technical way for further focusing on the characteristic flavor components of vinegars or other fermentation products.

Simultaneous quantitation of 15 bioactive components in Yupingfeng granules by LC-MS/MS.

Yupingfeng granules (YPFG) is commonly used in the treatment of immunological diseases, inflammations, and pulmonary diseases. Several studies have found that chromones, flavones, and saponins were the major bioactive compounds of YPFG. However, few studies have reported accurate quantification methods of these compounds. This study aimed to establish a simple and rapid method by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine 15 bioactive compounds in YPFG. The experimental parameters including extraction methods, extraction solvents, extraction time, solid-liquid ratio, and LC-MS/MS condition were optimized. The linearity, precision, repeatability, stability, and recovery of the established method were evaluated. The contents of 15 bioactive compounds in seven batches of YPFG samples were analyzed by the established method and the results were compared with the values determined by HPLC. The optimal extraction condition was to extract 0.1 g of YPFG by ultrasound with 50 mL 50% ethanol for 30 min. A Waters ACQUITY UPLCBEH C18 column using the 0.1% formic acid water solution and acetonitrile as mobile phase with a gradient elution was applied to the chromatographic separation. The linearity, precision, repeatability, stability, and recovery of the method were within acceptable ranges. Compared with HPLC analysis methods in Chinese Pharmacopoeia and literature, the established method was faster, simpler, more accurate, and more reliable. The method of simultaneous determination of 15 components in YPFG by LC-MS might provide a basis for the study of the bioactive compounds and the improvement of the quality standard of YPFG.

Development of ultra HPLC analytical method for pymetrozine residues in rice

Ultra HPLC-based analytical method analytical method was developed for the analysis of pymetrozine residues in different rice matrices. Chromatography separation was carried out in XR-ODS II: 150mm X 2.0mm, 5µm column in the mobile phase of water and methanol (60:40), and the resultant chromatograms were detected in a Photodiode Array (PDA). The retention time for pymetrozine neat standard and also matrix match standards was arrived at 4.00 min ± 0.5 min. The pymetrozine at the concentrations of 0.01, 0.05, 0.10, 0.25, 0.50, 0.75, and 1.00 µg/ml and the calibration curve of y = 2E+07x + 19188 obtained with a goodness of fit (R²) 0.995. The samples were extracted using the AOAC QuEChERS method with slight modifications. The harvest time residues for the different rice matrices were below the tolerance limit of pymetrozine. The residues of pymetrozine at 7, 15, and 30 days after the rice harvest in paddy seed, single-polished, double-polished rice, bran, and dehulled rice were not detected. From the developed analytical method, the residues of pymetrozine can be detected in different rice matrices.

HPLC Analytical Method Validation for Xanthohumol in Hop (Humulus lupulus) Extract for Its Standardization as a Functional Ingredient

HPLC Analytical Method Validation for Xanthohumol in Hop (<i>Humulus lupulus</i>) Extract for Its Standardization as a Functional Ingredient

Multivariate HPLC system assessment and optimization for traditional Chinese medicine: a case study of Gastrodia elata.

The development of HPLC analytical methods for traditional Chinese medicine is intricate and time-consuming, influenced by factors such as column wear, solvent purity, and instrumental settings. A comprehensive evaluation of the HPLC system is crucial to mitigate potential variability and ensure the reliability of data. This is especially important given the complex and synergistic nature of the chemical components in traditional Chinese medicine, necessitating a multivariate measurement system analysis (MSA) to assess multiple correlated quality characteristics effectively. This study introduced a multivariate MSA method based on weighted principal components (WPC) to evaluate the HPLC system for the determination of metabolites in Gastrodia elata. By integrating multiple principal components and assigning weights according to their eigenvalues, the WPC method significantly enhanced both accuracy and robustness. It demonstrated a repeatability and reproducibility (% R&R) of 26.43% and a number of distinct categories (ndc) index of 5, confirming the system's acceptability. A full factorial experimental design was employed to identify key performance factors, leading to the recommendation to use five reference solutions for the standard curve and to triple sample preparations for improved precision and accuracy. Monte Carlo simulations confirmed the reliability of the system, showing % R&R and ndc values that follow a normal distribution, ranging from 19% to 22% and 6.07 to 7.38, respectively. Chromatographic conditions were optimized using a Box-Behnken experimental design. Subsequent validation experiments verified the method's high accuracy and reliability, with all relative standard deviation values for analytical precision, repeatability, and stability below 5%. The method also exhibited high recovery rates, exceeding 91% across three concentration levels, with RSD values under 4%. In conclusion, the application of a WPC-based multivariate MSA enabled a detailed evaluation of the HPLC system, ensuring accurate and reliable measurement of quality attributes. This method exemplified a scientifically rigorous approach for developing analytical methods in traditional Chinese medicine, enhancing both precision and reliability.

Central composite design aided optimization and validation of developed an eco-friendly HPLC method for the quantification of Lenalidomide loaded mesoporous silica nanoparticles

Lenalidomide (LND) was encapsulated within mesoporous silica nanoparticles (MSNs), and we developed a reverse-phase (RP)-HPLC analytical method to quantitate the LND content in the formulation. Through a multivariate Central composite design (CCD), we systematically optimized key chromatographic parameters, including the flow rate, sample injection volume, and organic phase ratio. We evaluated the responses of retention time, peak area, and theoretical plate. Our enhanced chromatographic approach employed a Spherisorb ODS C18 column. To investigate the suitableness of the mobile phase for isocratic elution, we adhered to International Conference on Harmonisation Q2(R1) standards. Suggesting the optimality of a methanol and ammonium acetate buffer combination (pH 5.5, adjusted with 1% v/v glacial acetic acid and ammonia solution). This validated RP-HPLC analytical method exhibited specificity for LND even in the presence of the matrix of MSNs. The fabricated MSNs were confirmed by evaluating the surface morphology of the formulation. We successfully applied the developed RP-HPLC method to quantify the amount of LND entrapped and to determine the drug loaded in the MSNs formulation. The % EE for LND in MSNs was found to be 76.66% and % DL for LND in MSNs was found to be 1 4.00%, respectively. The novelty of the Design of expert-based method development is that it reduces the number of trials, thereby reducing solvent wastage and is environmentally friendly, scoring eight green, six yellow, and one red.