Reversed-phase High-performance Liquid Chromatography Research Articles

A Simple, Cost-Effective, and Sustainable Method for Extraction and Quantification of Chebulagic Acid from Terminalia bellirica (Gaertn.) Roxb. Using RP-HPLC: A Comparative Study

Chebulagic acid (CA), a hydrolysable tannin found in Terminalia bellirica (T. bellirica) (Gaertn.) Roxb., has gained prominence as a significant component in Ayurvedic medicine. This study optimized methods for determining CA in T. bellirica, focusing on fruit samples and prioritizing simplicity, cost-effectiveness, less time duration, and sustainability. We evaluated various extraction techniques, including continuous shaking, microwave assisted extraction, and Soxhlet extraction using green solvents. Quantification of CA was conducted using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), with confirmation achieved via UV-Visible (UV-Vis) spectroscopy, Thin Layer Chromatography (TLC), and Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). Results indicated that Soxhlet and microwave significant extraction methods, particularly with ethanol as the green solvent. Soxhlet extraction yielded 49607.73±18.82 μg/mL for fresh fruit and 42952.53±19.09 μg/mL for dry fruit, while microwave extraction resulted in 43627.56±113.48 μg/mL for fresh fruit and 40466.33±130.90 μg/mL for dry fruit respectively. These findings conclude that ethanol as a green solvent in Soxhlet or microwave assisted extraction emerge as most efficient, straightforward, and effective approach for detecting and screening CA in T. bellirica samples.

Evaluation of Quercetin's Bioenhancing Effect on Oral Pharmacokinetics of Rosuvastatin in Wistar Rats Using RP-HPLC Method.

The absolute oral bioavailability of rosuvastatin (RST), a secondgeneration statin, is low i.e. 20% and only 10% is recovered as metabolite N-desmethy l rosuvistatin. Since it is a hydrophilic statin, RST relies on the organic anion transporting polypeptide- 1B1 (OATP-1B1), as the key mechanism for active transport into hepatocytes. Quercetin (QUE) being a bio enhancer and inhibitor of OATP1B1 can augment the bioavailability and pharmacokinetics of RST. The present study includes the development of a simple and validated bioanalytical Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method for the estimation of RST and to study the effect of co-administration of QUE as a bio enhancer on its bioavailability. An analytical column of Kromasil 100, C18 (250 mm × 4.6 mm, 5 μm), was used for chromatographic separationand acetonitrile (ACN): acetic acid buffer pH 3.0 adjusted with glacial acetic acid (55:45 Vol. %) as mobile phase with flow rate 1.0 ml/min monitored at 242 nm. The ACN: methanol (50:50 Vol. %) was employed as the final solvent for extraction. The developed method has been successfully applied in a study on the pharmacokinetics of the drug RST in rats after co-administration of QUE, which was carried out using non-compartmental analysis in order to estimate the blood concentration of the drug. The pharmacokinetics of RST was found to be altered significantly (highest concentration of RST in the blood (Cmax) = 67.3 ng/ml to 122.2 ng/ml) (p < 0.001), area under curve (AUC)0-t (p < 0.0001) and AUC0-inf (p = 0.0005) when co-administered with QUE at 120 min (tmax). The results are in accordance with the fact that QUE increases plasma levels in rats through herb-drug interactions.

Green RP-HPLC method for simultaneous quantification of epigallocatechin-3-gallate and rosmarinic acid in lipid-based nanocarriers and biological fluids: Quality by Design-driven optimization and lean six sigma approach

This study presents the development and validation of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for concurrent quantification of EGCG and RA in prepared lipid-based nanocarriers and biological fluids using a QbD approach, greenness assessment, and Six-Sigma methodology. Initially, variations in critical analytical attributes were identified using the Ishikawa fishbone diagram and Risk Priority Number scores. A Taguchi Orthogonal Array design was employed to screen the critical method parameters influencing method development. Systematic optimization of the RP-HPLC method was subsequently achieved using a Box-Behnken design, with the % organic phase, flow rate, and column temperature as the variables. The responses were retention time, tailing factor, and theoretical plates for both EGCG and RA. Chromatographic separation was accomplished with a methanol and 0.1% formic acid (60:40) isocratic flow system on a Phenomenex Luna C18 column (4.6 × 250 mm, 5 μm) at a 1 mL/min flow rate. The method was rigorously validated according to ICH guidelines. Linearity was observed over a concentration range of 2 to 10 µg/mL for both EGCG and RA, yielding correlation coefficients of 0.998 and 0.999, respectively. The LOD and LOQ were determined to be 0.51 µg/mL and 1.54 µg/mL for EGCG, and 0.35 µg/mL and 1.07 µg/mL for RA. Moreover, the %RSD for all validation parameters were consistently below 2%. Forced degradation studies were conducted under acidic, basic, oxidative, and photolytic conditions to elucidate potential degradation pathways and identify degradation products. The developed method was applied to estimate EGCG and RA in prepared lipid-based nanocarriers and biological fluids like blood plasma and urine. Recovery assays in lipid-based nanocarriers, plasma, and urine samples demonstrated excellent recoveries (96.2–102.1%). The method's greenness was assessed using various tools, and its accuracy was evaluated using Six Sigma methodology. Overall, the developed HPLC method offers a rapid, sensitive, and reliable approach for quantifying EGCG and RA, laying the groundwork for their further investigation as anticancer agents, alone and in combination therapies.

HPLC with parallel ESI-MS and ICP-AES detection as a tool for the speciation studies of phosphovanadotungstates

The presence of different equilibrated chemical species in aqueous solutions of polyoxometalates represents a challenge for their identification, requiring the use of both separation and detection techniques. The use of reversed-phase ion-pair high-performance liquid chromatography (HPLC) in gradient elution mode resulted in optimal separation conditions. The detection was accomplished by parallel hyphenation of the inductively coupled plasma atomic emission spectrometry (ICP-AES) and electrospray ionization mass spectrometry (ESI-MS), which combined the advantages of element and molecule specific detectors. This hyphenation resulted in an unambiguous identification of the products of the synthesis of mixed-addendum Keggin-type phosphovanadotungstates of [PVxW12-xO40](3+x)- (x = 1 – 3) general formula without the isolation of the individual species.

Biogenic Amines: Catalysis, Quality, and Safety Aspects of Food Items Consumed in Saudi Arabia

Introduction: In this study, the identification and quantification of biogenic amines in 45 commonly consumed food samples in Saudi Arabia were carried out. The enzymes responsible for producing these biogenic amines include spermidine (SPD), putrescine (PUT), tryptamine (TRP), tyramine (TYR), and histamine (HIS), which are synthesized through organo-catalytic pathways. Method: The diverse range of samples analyzed encompassed various types of beef, pickle varieties, canned fish, vegetables, chicken varieties, spices, fruits, and salad ingredients. Sample preparation involved the use of dansyl chloride after aqueous extraction, followed by isolation and analysis using reversed-phase HPLC with a UV detector. In five beef samples, mean concentrations of SPD, PUT, TRP, HIS, and TYR were identified as 9.41, 8.98, 155.8, 100.8, and 304.2 mg kg-1, respectively. Canned fish samples exhibited mean concentrations of TRP, PUT, HIS, TYR, and SPD at 71.6, 3.88, 29.2, 2.56, and 2.02 mg kg-1, respectively. Result: Among five pickle samples, mean concentrations of TRP, PUT, HIS, TYR, and SPD were reported as 118.8, 39.12, 35.2, 27.2, and 2.56 mg kg-1, respectively. Chicken samples primarily contained TRP, HIS, and SPD as the identified biogenic amines, with mean concentrations of 87.2, 105.6, and 5.22 mg kg-1, respectively. Fruit samples generally exhibited low levels of all enzymes except for TRP. Conclusion: It was found that vegetables, seasonings, and salad ingredients either had undetectable or low quantities of biogenic amines.

Aedes aegypti control in breeding sites through an insecticidal coating with dual effect: Laboratory trials and safety assessment.

Ground water tanks are known to be preferred Aedes aegypti oviposition places providing opportunities for adult and larvae control. Therefore, a dual-effect insecticidal coating (IC) (alphacypermethrin/ pyriproxyfen) with a slow-release mechanism and safe for users could be applied within Aedes spp. breeding sites, representing a promising option. Bioassays were designed to determine the mortality and sterilizing effects on gravid mosquitoes exposed to IC. The effect of inhibition of emergence was evaluated in eggs, larvae and pupae exposed in different containers. For the water safety assessment concentrations of active ingredients were determined by reverse phase high performance liquid chromatography (RP-HPLC) and the health risk was calculated. The IC applied to the interior walls of water-holding containers showed efficacy against Ae. aegypti in terms of high gravid-female mortality (81% at 24 h, p < 0.01), sterilizing effect (inhibition of oviposition by 63%, p < 0.01) and emergence inhibition (100% in eggs, L3 and L4; 97% in pupae). The offspring rate was reduced [only 0.15 (38/250) new adults emerged per exposed gravid females as against 11.90 per unexposed female (2976/250) at baseline]. Emergence inhibition was recorded up to 12 months and adult mortality >80% up to 6 months. The use of water stored in treated containers, either for washing or drinking, is not expected to pose a health risk to users. IC applied to domestic water containers has dual and complementary action that reduces Ae. aegypti densities (immature and adult stages). This represents baseline information for a cluster randomized efficacy trial in Colombia.

Novel analytical approach for baclofen quantification in rodent plasma.

A simple, accurate, and robust reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantification of Baclofen in rat plasma. The method demonstrated high degree of linearity (r2 = 0.9936) across a concentration range of 10-50 μg/mL. Precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness were evaluated according to ICH guidelines. The LOD and LOQ were found to be 0.076197 and 0.23090 μg/mL, respectively. This method provides an efficient approach for Baclofen quantification in plasma, making it suitable for pharmacokinetic and bioavailability studies. The novelty of this study lies in its optimization for routine use in laboratories, ensuring reproducibility with minimal variations across different conditions and analysts.

Structure-Activity-Relationships of the Stability of six Pentathiepins towards Glutathione: Possible Correlations with Biological Activities.

The biological properties of pentathiepins have been intensively studied in recent years. Although the proposed mechanism of action requires activation by intracellular thiols, the dependence of activity on the stability of pentathiepins towards glutathione (GSH) has not been directly investigated. Here, we determined the structure-related stability of six different pentathiepins with four different scaffolds in the presence of GSH by using reversed-phase high-performance liquid chromatography (RP-HPLC) and UV-vis spectroscopy over a wide range of GSH concentrations. We found significant differences in compound stability depending on the pentathiepin scaffold; these differences were reflected in their cytotoxic activities. However, we found no substantial differences in their inhibition of glutathione peroxidase 1 (GPx-1). While the intact pentathiepin ring is necessary for the antiproliferative activity of pentathiepins, the depletion of intracellular GSH content with dl-buthionine-(S,R)-sulfoximine (BSO) led to a significant increase in cytotoxicity of the tested substances. In view of the increased cytotoxicity following artificial GSH depletion, this calls into question the sole role of GSH in the intracellular activation mechanism.

Scalp condition improvement with botanical extracts possessing chemical and physical antioxidant activity.

Oxidative stress is implicated in scalp and hair health manifesting in several ways, including skin barrier, hair retention, healthy hair appearance and scalp sensation. We previously linked markers of oxidative damage to dandruff and skin barrier impairment and have linked key anti-dandruff technologies to the resolution of these issues. Here we expand the therapeutic space demonstrating many botanical extracts offer protective benefits against ROS stress via both chemical and biological antioxidant mechanisms. Chemical antioxidant activity of 10 botanical extracts was measured using oxygen radical absorbance capacity (ORAC) and biological antioxidant activity was measured using a Nrf-2 cell assay. A three-dimensional human keratinocyte skin equivalent model from MatTek was used to measure efficacy of reducing reactive oxygen species (ROS) from both the botanicals added as a solution and from a shampoo product. A 4-week consumer study with 56 panels was conducted with a leave-on treatment containing a botanical extract. Measurement of the oxidized lipids (9 and 13-Hydroxy-10E 12Z-octadecadienoic acid, HODE) extracted from scalp tape strips was made using gradient reversed-phase high-performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS). We demonstrated invitro that many botanical extracts exert antioxidant activity by quenching radicals in ORAC testing as well as activating biological antioxidant pathways via nrf-2 up-regulation in a cellular reporter system. Furthermore, we demonstrate these antioxidant activities for these botanicals in 3D skin models and ultimately show the reduction of lipid oxidation products in a consumer use context with a rosemary extract. Improved scalp condition can be achieved with incorporation of botanicals having the potential to exert antioxidant activity in hair/scalp care products.

Eco-friendly RP-HPLC approach for simultaneously estimating the promising combination of pentoxifylline and simvastatin in therapeutic potential for breast cancer: Appraisal of greenness, whiteness, and Box–Behnken design

Abstract Breast cancer affects millions of women worldwide. This study explores the potential of combining pentoxifylline (PTX) and simvastatin (SIM) as a treatment for breast cancer. We aimed to develop six sustainability tools using green and white metrics to evaluate the environmental impact of reversed-phase high-performance liquid chromatography (RP-HPLC) methods for analyzing and separating PTX and SIM in their pure forms. The tools include analytical GREEnness, green analytical procedure index, Complexgreen analytical procedure index, analytical greenness metric for sample preparation, blue applicability grade index, and the RGB 12 algorithm. For the separation, we used a Novapack C8 column (15 × 0.46 cm, 5 µm) at 25°C. The injection volume was 5.0 µL, the wavelength was set to 210 nm, and the total runtime was 5 min. We identified optimal chromatographic conditions efficiently using the Box–Behnken design with minimal trials. We investigated the effects of three factors on retention time and resolution: acetonitrile ratio, pH, and flow rate. We used overlay plots with a 60:40 ratio (v/v) of acetonitrile and bi-distilled water to forecast the most effective mobile phase. The calibration curves for PTX and SIM showed a correlation value of over 0.999 within the range of 5–60 µg·mL−1. The recovery rates ranged from 99.9% to 100.2%, indicating high accuracy. Our RP-HPLC technique proves to be reliable and efficient for the simultaneous estimation of multiple anticancer drugs. We evaluated the environmental sustainability of this approach using green and white metrics, and the recommended method has been thoroughly validated according to International Council for Harmonisation guidelines, making it highly reliable for analyzing new formulations.

TLC vs. HPLC: A green comparison for veterinary drug analysis

This work describes two analytical methods for determining lidocaine (LD) and oxytetracycline (OTC) in veterinary dosage form: a traditional Thin-Layer Chromatography (TLC) method and a more environmentally friendly Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) method.Initially, the TLC method was used due to its speed, simplicity, and accuracy. It successfully separated LD and OTC in veterinary dosage forms with a linearity range of 0.5–11.0 µg/band for LD and 4.0–34.0 µg/band for OTC. However, TLC proved to be inadequate for resolving the binary mixture in the presence of toxic lidocaine impurity (2,6-dimethylaniline) (DMA), and was less environmentally friendly.Consequently, an RP-HPLC method was developed using a C18 column and a mobile phase consisting of ethanol and phosphate buffer (pH 7) in a 70:30 ratio, with a flow rate of 1.0 mL/min. This method effectively separated OTC, LD and DMA, using a PDA detector at wavelengths of 272, 232, and 210 nm and with retention times of 1.529, 2.77, and 3.581 min for OTC, DMA, and LD respectively. The RP-HPLC method showed linearity ranges of 270.0–972.0 µg/mL for OTC, 0.5–25.0 µg/mL for DMA, and 5.0–18.0 µg/mL for LD.Both methods were validated for linearity, precision, accuracy, robustness, detection limit, and quantitation limit. The greenness of each method was evaluated using NEMI, Eco-scale, and Agree tools, with results demonstrating the RP-HPLC method as the more environmentally sustainable method.

Assignment of Disulfide Bonds in HNTX-XXI by Double-Enzymatic Digestion and Edman Degradation.

HNTX-XXI, a peptide toxin derived from the venom of the spider Ornithoctonus hainana, comprises a 64-amino-acid protein architecture that notably incorporates eight cysteine residues positioned at positions 2, 10, 14, 16, 17, 23, 36, and 63. The close spatial proximity of Cys16 and Cys17 poses a challenge in resolving their disulfide bridge configurations using standard methodologies. In this study, we introduce an innovative and highly efficient approach for delineating disulfide pairings in peptides containing adjacent cysteines. Our methodology integrates a two-step proteolytic digestion strategy utilizing trypsin and Glu-specific staphylococcal V8 protease coupled with a subsequent round of Edman degradation. This multifaceted approach enables the precise characterization of the disulfide bonds within the peptide. Specifically, targeted proteolysis by trypsin and V8, followed by reversed-phase HPLC separation of the resulting peptides, facilitated the unambiguous identification of disulfide linkages between Cys10-Cys23 and Cys14-Cys63. For the fragment containing the four remaining cysteines, a single cycle of Edman degradation was employed, strategically breaking the peptide bond between the adjacent cysteines. This pivotal step enabled the isolation and analysis of the resulting fragments. Subsequently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was utilized, revealing the presence of two additional disulfide bonds: Cys2-Cys17 and Cys16-Cys36. Collectively, these findings allow for the definitive assignment of the four disulfide linkages in HNTX-XXI as Cys2-Cys17, Cys10-Cys23, Cys14-Cys63, and Cys16-Cys36. This rapid and sensitive methodology represents a significant advancement in the structural characterization of peptide toxins with complex disulfide bond patterns, underscoring its potential for broad application in the field of venom peptide research.

Unlocking the Potential of Isopropanol as an Eco-Friendly Eluent for Large-Scale Fractionation of Fulvic Acids via Preparative Reversed-Phase High-Performance Liquid Chromatography and Multidimensional RP-HPLC: Evaluation of Molecular Diversity and Element Composition.

Humic substances are organic mixtures of extreme complexity, which significantly complicate their analysis by any method. Fractionation into more homogeneous mixtures seems to be almost the only way to overcome these difficulties. Preparative high-performance liquid chromatography (HPLC) provides almost any amounts of substances required for both further fractionation and studies by other methods. For the first time, isopropyl alcohol (IPA) is proposed for these purposes; its advantages are shown by the example of groundwater fulvic acids (FA). IPA is much safer than conventional solvents and elutes the most nonpolar compounds that are nonelutable by methanol and acetonitrile. The isolated fractions differ significantly in their molecular composition, which is confirmed by ultrahigh-resolution mass spectrometry and molecular spectroscopy. Stepwise IPA-gradient HPLC-UV analysis of each preparative fraction demonstrates the possibilities of multidimensional HPLC for FA. The isolated fractions were studied for contents of a broad range of elements, and the relationships between the molecular and trace-element compositions of the fractions were revealed. Our data reveals the existence of numerous and different organometal compounds in FA composition; thus, the proposed approach can be used for a more in-depth study of the mechanisms of element migration in the environment.

Automatic periodical negative air ions reduce postharvest decay and maintain texture and flavor quality of ‘Fuji’ apple during long-term cold storage

The aim of this study was to explore the effect of periodical negative air ions (NAI) on the quality of ‘Fuji’ apple fruit after treatment at 0, 30, 60, and 90 min every 4 weeks. NAI for 60 min decreased weight loss, mitigated decay, and maintained hardness of the apple fruit during the 40 weeks of storage. Treatment of fruits with NAI for 60 min enhanced the apple defense ability against pathogens by maintaining the cell wall integrity and improving the related-enzyme activities as well as accumulation of total phenols and flavonoids. Reverse-phase HPLC and solid-phase microextraction GC–MS analysis showed that NAI treatment for 60 min increased the levels of fructose, sucrose, sorbitol, malic acid and volatiles, such as 2-hexenal, 2-methylbutyl acetate, hexyl acetate, and ethyl butanoate. Taken together, NAI is promising for improving the quality ‘Fuji’ apples during the cold storage.

Composition and Biological Activity of Flavonoid-containing Fractions of an Extract from Gratiola officinalis L.

Gratiola officinalis L. (hedge hyssop), a medicinal plant of the Scrophulariaceae family, has diuretic, purgative, and vermifuge properties. It is used as a herbal tea to treat chronic gastroenteritis, renal colic, jaundice, and intestinal worms. Previously, we have found that an extract from G. officinalis is nontoxic and has antitumor, antioxidant, antimicrobial, antiinflammatory, anticachexic, and other properties. Our aims in this study were to separate the G. officinalis extract into individual fractions, to identify the most biologically active fractions, and to examine the chemical composition of these fractions and their biological activity toward A498 renal carcinoma cells. The G. officinalis extract was fractionated by reversed-phase high-performance liquid chromatography, and each fraction was tested for antitumor activity. The active fractions were characterized by UV-visible electron spectral analysis, circular dichroism analysis, Fourier transform infrared spectroscopy, high-performance liquid chromatography, electrospray ionization tandem mass spectrometry, and nuclear magnetic resonance spectroscopy. Two antitumor-active fractions of a flavonoid nature were isolated and chromatographically purified. On the basis of the nuclear magnetic resonance data, the aglycone fragment of the main component of one fraction was found to be structured as 2-(3,4-dimethoxyphenyl)-7-hydroxychroman-4-one, or 3',4'-dimethoxy-7- hydroxyflavanone. The antitumor effect of the most active fraction containing 7-O-glucoside of apigenin, glycoside 7,3'-di-O-luteolin and trace amounts of eupatilin against renal carcinoma A498 cells was manifested in its cytotoxic, cytostatic, apoptotic and autophagosomal activities. In addition, we found 3-(1-2)-glucoside of soyaspogenol B, which is a pentacyclic triterpenoid in the structure.

Characterization of dark chocolates based on polyphenolic profiles and antioxidant activity.

Dark chocolates were characterized according to geographical origin, cocoa variety, and cocoa content using the methylxanthine and polyphenolic composition and antioxidant activity as the data. The main study objective was to uncover sample patterns and identify possible markers of quality, variety, or origin to deal with authentication or fraud detection issues. In the study, a set of 26 dark chocolates from different varieties (e.g., Criollo, Forastero, and Trinitario) harvested in Africa, America, and Asia was analyzed. The optimized sample treatment consisted of defatting the chocolate (1g of sample with 5mL of cyclohexane for 15min, three times) and then extracting the analytes by sonication with methanol/water 60:40 (v:v) for 15min. The filtered extracts were analyzed by reversed-phase high-performance liquid chromatography with UV and spectrophotometric methods (Folin-Ciocalteu, ferric reducing antioxidant power, and aluminum methods) to determine individual phenolics and overall indexes of antioxidant and flavonoid content. Results from this chocolate set indicated that American samples are richer than African counterparts in alkaloids and phenolics (e.g., 1.7 vs. 1.1mgg-1 caffeine and 14.5 vs. 12.5mgg-1 total flavanols, respectively). Regarding cocoa varieties, Criollo cocoa was richer in bioactive compounds and antioxidant capacity (e.g., 16, 15, and 12mgg-1 total flavanols for Criollo, Forastero, and Trinitario, respectively). These results indicate that the analytes resulted in potential descriptors of varietal or geographical attributes.

Application of Reversed-Phase HPLC Method for the Simultaneous Determination of Lenacapavir and Bictegravir in Tablets Dosage Form

Application of Reversed-Phase HPLC Method for the Simultaneous Determination of Lenacapavir and Bictegravir in Tablets Dosage Form

Study of the Acidic, Basic, and Thermal Degradation Kinetics of Three Antihypertensive Drugs—Individually and in Combination

Background/Objectives: The importance of fixed-dose combinations (FDCs) for the treatment of hypertension is well established. However, from a stability perspective, FDCs present a challenge since the degradation of one active pharmaceutical ingredient (API) can be affected by the presence of another API. The aim of this study was to compare the degradation behaviors and evaluate the degradation kinetics of three antihypertensive drugs, perindopril tert-butylamine (PER), amlodipine besylate (AML), and indapamide (IND). Methods: The degradation processes were studied using the previously developed reverse phase high-performance liquid chromatographic (RP-HPLC) method after exposing each drug individually, as well as the combinations of two/three drugs, to different stress factors, such as light, oxidation, acidic, basic, or neutral pH values at different temperatures. Results: The results show that PER is most unstable under basic conditions and that AML displays a negative, while IND displays a positive effect, on PER stability when combined. AML is most affected by basic conditions and oxidation, and its stability is affected by both drugs positively; IND undergoes extreme photolysis, which is positively affected by AML but negatively by PER. Conclusions: Great care must be taken when formulating FDCs with these three drugs, as well as solutions or oral suspensions adjusted for geriatric or pediatric populations, since the stability of all three drugs is greatly affected by pH conditions, as well as light or oxidation factors and their interactions.

Stability indicating RP-HPLC method development and validation for quantification of impurities in gonadotropin-releasing hormone (Elagolix): Robustness study by quality by design.

The purpose of this research was to establish and validate a reverse phase HPLC method for the determination of Elagolix impurities in pharmaceutical dosage form. Mobile phase A, consisting of 10mM sodium dihydrogen phosphate (pH6.0) and acetonitrile in a 95:5v/v ratio, and mobile phase B, containing 85:10:5v/v/v of acetonitrile, Milli-Q water, and methanol, were used to achieve the method's specificity in the analytical column Kromasil 100-C18 (250 mm × 4.6mm, 5μm). The gradient program includes (%B/Time [min]: 36/0, 36/10, 38/15, 85/55, 85/65, 36/67, and 36/75). The flow rate is 0.8mL/min. The overall run duration is 75.0min, the injection volume is 10.0μL, and the detection is at 210 nm in UV. The samples were subjected to hydrolysis, oxidation, and heat conditions in order to facilitate their forced degradation. The procedure was validated and determined with the standards of ICH guidelines. From the LOQ to a concentration level of 200%, the linearity of the technique was ascertained. An accuracy range of LOQ to 150% was established for the method, and the average recovery was acceptable. Design of experiments, part of the quality by design idea, was used to prove the method's reliability.

Esterase-responsive Self-immolative Prodrugs for the Sustained Delivery of the Anticancer Drug 5-Fluorouracil with Turn-on Fluorescence.

Stimuli-responsive prodrugs of anticancer drugs are advantageous for the selective delivery of drugs to cancer cells with minimized off-target side effects. In the present study, esterase-activatable fluorogenic prodrugs of the chemotherapeutic drug 5-fluorouracil (5-FU) have been rationally designed and synthesized using multi-step organic synthesis. While 5-FU was connected directly with the fluorophore via a C-N bond in the prodrug BJ-50, an additional self-immolative benzylic spacer with a carbonate linker was incorporated in the prodrug BJ-92. Although absorption and emission spectroscopic studies revealed the activation of both the prodrugs by porcine liver esterase (PLE), reverse-phase HPLC studies confirmed the inability of BJ-50 to release the active drug 5-FU. In contrast, a sustained release of 5-FU and Cou-OH was observed from BJ-92 in the presence of PLE. The endogenous esterase-mediated activation of the prodrug BJ-92 was validated by the turn-on fluorescence in HeLa cells and the anti-proliferative activities in A549, HeLa, and HEK-293 cells. Modulation of the expression of a few cancer marker proteins by BJ-92 and 5-FU was studied to evaluate their anticancer activities. As esterases are overexpressed in cancer cells, the prodrug in the present study would be helpful in selectively delivering 5-FU to cancer cells with reduced off-target side-effects.