Liquid Chromatographic Method Research Articles

Analytical Quality by Design (AQbD) Principles in Development and Validation of Stability‐Indicating RP‐HPLC Method for Simultaneous Estimation of Diltiazem HCl and Eugenol in Nanoethosomes

ABSTRACTThe study developed and validated a robust reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method for a simultaneous estimation of Diltiazem HCl and Eugenol in nanoethosomes using analytical quality by design (AQbD) principles. The optimized method utilized a mobile phase of methanol and 0.1% Orthophosphoric acid (OPA) in a 50:50 % v/v ratio, with a flow rate of 1.0 mL/min and isocratic elution on a Phenomenex Luna C‐18 column (5 µm, 150 mm × 4.6 mm) at 30°C. This setup resulted in retention times of 6.838 min for Diltiazem HCl and 23.135 min for Eugenol. Validation followed International Council for Harmonization (ICH) Q2 (R1) guidelines, demonstrating excellent linearity with correlation coefficients of 0.9982 for Diltiazem HCl and 0.9991 for Eugenol across the 5–25 µg/mL range. The limits of detection (LOD) were 1.341 µg/mL for Diltiazem HCl and 0.960 µg/mL for Eugenol, with limits of quantification (LOQ) at 4.065 and 2.912 µg/mL, respectively. Stability testing under acidic, alkaline, oxidative, thermal, and photolytic conditions adhered to ICH Q1A (R2) and Q1B guidelines. This AQbD‐based method is effective for routine analysis of Diltiazem HCl and Eugenol in bulk and pharmaceutical dosage forms, fully complying with ICH standards.

Determination of Polyphenols in Cigar Tobacco by HPLC

A high performance liquid chromatography (HPLC) method was developed for the determination of neochlorogenic acid, cryptochlorogenic acid and caffeic acid in cigar tobacco. Firstly, the extraction method, extraction liquid concentration and dosage, column temperature and mobile phase flow rate of polyphenols in cigar tobacco were discussed. Then the detection limit, recovery rate, precision and standard curve of three polyphenols were tested. Finally, tobacco samples were tested. The results showed that the optimal extraction conditions were: ultrasonic extraction, extraction time was 30 min, methanol volume concentration was 50%, dosage was 20 mL, the optimal detection conditions were: column temperature was 30 ℃, flow rate was 1.0 mL/min, detection wavelength was 340 nm. The contents of neochlorogenic acid, cryptochlorogenic acid and caffeic acid were in the range of 0~0.202 mg/g, 0~0.331 mg/g and 0.050~0.073 mg/g, respectively, and the linear correlation coefficients of the three polyphenols were ≥0.9991, indicating that the linear correlation of the method was good, and the detection limit was ≤0.066 mg/L. The precision was ≤0.427%, and the recovery was 95.13~96.39%. The results showed that the method had good precision, high accuracy, short analysis time and high detection efficiency, and could be used as an effective method for the detection of polyphenols in cigar tobacco leaves.

RP-HPLC-PDA-Based Simultaneous Estimation of Ramipril and Hydrochlorothiazide in Combined Dosage Forms: A Validated Approach

A rapid, highly sensitive High-Performance Liquid Chromatographic method has been developed for the simultaneous determination of Ramipril (RMP) and Hydrochlorothiazide (HCT) in bulk drug and in tablets. Ramipril (RMP) and Hydrochlorothiazide (HCT) were eluted from an Eclipse plus C18 (250 mm × 4.6 mm I.D.) with particle size 5 µm reversed phase column with a mobile phase consisting of acetonitrile and potassium dihydrogen ortho-phosphate pH 3 (40: 60 v/v) at a flow rate of 1 mL/min with UV detection at 210 nm. The retention time for RMP and HCT was found to be 3.31 ± 0.02 min and 6.64 ± 0.02 min, respectively. The linear response (r2 <0.99) was observed in the range of 2 - 12 μg/mL for RMP and 4 - 24 μg/mL of HCT with low values of limits of detection (LOD) and quantification (LOQ) for both drugs. The method shows good recoveries and intra and inter-day relative standard deviations were less than 1.0%. Validation parameters as specificity, accuracy, ruggedness and robustness were also determined. The proposed method provides accurate and precise quality control tool for routine simultaneous analysis of Ramipril and Hydrochlorothiazide in bulk and in tablet dosage form.

Hydrocarbons in waters and bottom sediments of the coastal areas of the Caucasian sector of the Black Sea (2021–2023)

The current level of aliphatic and polycyclic aromatic hydrocarbons was determined in suspended particulate matter of waters (mainly in surface waters) and in upper layer of bottom sediments of coastal areas in the Caucasian sector of the Black Sea of the Russian Federation (September 2021, May and September 2022, March 2023). IR method was used to determine the aliphatic hydrocarbons, and the method of high performance liquid chromatography was employed for PAHs. The average concentration of aliphatic hydrocarbons was ≈20 μg/L and for polycyclic aromatic hydrocarbons ≈130 ng/L in suspended particulate matter in open surface waters of Gelendzhik and Golubaya bays. Their content naturally increased as they approached the coast. Despite the decrease in hydrocarbon concentrations in surface waters in recent years, the Kerch Strait and Novorossiysk remain the most polluted areas as before. Higher concentrations in the waters of the Taman Peninsula may be caused by the seepage of hydrocarbons from the sedimentary layer. Hydrocarbons are accumulated in bottom sediments, that leads to their rising portion in total organic carbon (e.g., aliphatic hydrocarbons up to 14.2 % in the Tuapse region and 13.1 % in the Novorossiysk region). Along with contamination from anthropogenic sources, natural processes also affect the hydrocarbon levels such as high biological productivity of the area and fluid flows from the sediment layer.

Development and Validation of an HPLC Method for Niraparib Analysis: A Comprehensive Approach.

Background: Niraparib is a significant PARP inhibitor utilized in cancer therapy. This study aims to develop and validate a high-performance liquid chromatography (HPLC) method for the quantification of Niraparib in pharmaceutical formulations and biological samples. Results: A Phenomenex Kinetex XB-C8 column (150 x 4.6 mm, 5 µ) was employed with a mobile phase of 0.1% formic acid in acetonitrile (60:40, % v/v). The analysis was performed at an oven temperature of 30℃ with a flow rate of 0.5 mL/min, and detection was achieved at 240 nm. The method demonstrated excellent linearity (R² = 0.9998) across concentrations from 40 to 60 μg/mL. Validation studies confirmed high accuracy, with a mean recovery of 99.96%. Conclusion: The validated HPLC method is robust and reliable for quantifying Niraparib in various formulations, aiding in the stability assessment and ensuring the efficacy and safety of cancer treatment protocols. This method facilitates informed decisions in pharmaceutical development.

Development and Validation of New Stability Indicating Analytical RPHPLC Method for Simultaneous Estimation of Metformin and Alogliptin.

The current study focuses on developing and validating a reverse phase-high performance liquid chromatography (RP-HPLC) method for quantifying metformin HCL (MET) and alogliptin (ALO) in both dosage and bulk drug forms. ALO and MET in tablet form were determined simultaneously using an RP-HPLC technique. The Reverse Phase (Waters) C18 (4.6 mm x 150 mm; 5 µ) column, a 20-injection loop are the components of the gradient system for the RP manufactured by Agilent Technologies. In the procedure, a 50:50 volumetric combination of methanol and water (0.1% acetic acid) was used as the mobile phase at pH 4.5. The developed approach yielded retention times of 3.856 minutes for MET and 6.302 minutes for ALO. The reliability of the established technique was demonstrated in compliance with the International Council of Harmonization (ICH) standards. In general, all of the metrics including linearity, accuracy, range, and robustness, were found to be within the acceptable ranges. Consequently, the methodology was assessed to be simple, precise, cost-effective, and replicable. Hence, the analysis of MET and ALO in both bulk medication and formulated states can be regularly conducted utilising the procedures specified for the purpose of quality control.

High-Performance Liquid Chromatographic Method Development and Validation for Analysis of Expired and Marketed Doxofylline Tablets.

The objectives of the study were to develop a simple, reliable, sensitive and accurate high-performance liquid chromatography (HPLC) method for doxofylline pure and pharmaceutical dosage forms. HPLC method was developed for the estimation of doxofylline in pure and marketed, recently expired, and 1-year expired tablet formulations. Chromatographic separation of the drug was achieved on a stainless steel column 25 cm × 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm), Cosmosil® C18, column using a mobile phase of Potassium dihydrogen orthophosphate buffer: Acetonitrile pH 5.1 (60:40 v/v) at a flow rate of 1-mL/min. The drug eluted was monitored at 274 nm and the retention time of doxofylline was found to be 3.575 minutes, respectively. The validation of developed methods was done according to ICH Q2 (R1) guidelines. The calibration curve was linear over the range of 5 to 50 µg/mL. The correlation coefficient was found to be 0.9943 for doxofylline. The average retention time was found to be 3.538 minutes. The results were reproducible, showing the effectiveness of the system. The plate number was found to be 4909, which is also within the limit, i.e. ≥ 2000 prescribed in I.P. The dissolution studies of all three marketed, recently expired and 1-year expired formulations were carried out by using 0.01 M HCl and the results showed almost a similar rate of release profile and all three formulations accomplished the drug release within the limit. The developed and validated HPLC method offers a precise, accurate, and efficient analytical tool for the determination of doxofylline in pharmaceutical formulations.

Quantification of Bictegravir and its impurities: A novel RP-UPLC Approach for Development and Validation.

A new ultra-pressure liquid chromatography (UPLC) method was developed for accurately and reliably quantifying impurities in Bictegravir. The method utilized Waters Acquity HSSC18 column (50mm x 2.1mm, 1.8 µ) at room temperature. The mobile phase consists of Acetonitrile and Buffer (0.1 percent formic acid) in 80:20 and pumped at a flow rate of 0.2 mL/min. This optimized method achieves exceptional separation of Bictegravir and its impurities with excellent resolution at 281 nm. The method’s accuracy is demonstrated by a % recovery range for Bictegravir and its impurities between 99.1% and 100.3%. The correlation coefficient (R²) for BIC and its impurities is consistently above 0.999, indicating high linearity. The method’s robustness was confirmed by its stability against variations in flow rate and mobile phase ratio. Additionally, forced degradation studies confirmed the stability of Bictegravir, highlighting the method’s reliability. Overall, developed UPLC method is precise, accurate, robust, and linear, making it suitable for routine analysis of BIC-related substances in quality control laboratories at manufacturing sites during commercial production.

Method Development and Validation for the Simultaneous Estimation of Lobeglitazone Sulphate and Glimepiride by HPLC Method.

A simple, precise, and accurate reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous estimation of lobeglitazone sulfate and glimepiride in bulk and tablet dosage forms. The method involved selecting various chromatographic parameters. Specifically, a new method was developed using a 250 mm × 4.6 mm (HSS) reverse-phase C18 column with 5 µm particle size (Shim-pack C18, 250 × 4.6 mm; 5 µm). The mobile phase consisted of 40 volumes of phosphate buffer (pH 3) and 60 volumes of acetonitrile, with methanol as the diluent, running as an isocratic elution. The flow rate was set at 1.0 mL/min, and UV detection was performed at 254 nm. The injection volume was 2 µL, and the total runtime was 10 minutes. The recoveries for lobeglitazone sulfate were found to be in the range of 101% to 100.6%, while those for glimepiride were in the range of 101.5% to 100%. The method’s accuracy is evident from these results. The inter-day and intra-day precision of the new method were both below the maximum allowable limit (RSD% ≤ 2.0), as per International Council on Harmonisation (Q2 R1) guidelines. The method exhibited a linear response, with correlation coefficient (r2) values of 0.9972% for pioglitazone and 0.998 for glimepiride. The validation parameters, such as accuracy, precision, linearity, specificity, stability in the analytical solution and robustness, met the acceptance criteria. Hence, this method can be effectively used for the simultaneous estimation of lobeglitazone sulfate and glimepiride in both bulk and pharmaceutical dosage forms.

A Sustainable Chromatographic Method for Simultaneous Analysis of Fluoxetine and Olanzapine Antidepressants: Assessment of Greenness with Green Metric Tools .

The combination of Fluoxetine HCL and Olanzapine treats depression in bipolar disorder and patients unresponsive to other antidepressants. The research aims to optimize a sensitive, simple, rapid, reliable, eco-friendly liquid chromatographic method by minimizing feedstock’s, reagents, energy, and waste to protect the environment and conserve resources, additionally assessed the environmental impact of a method using various green metric tools, and applied the method to pharmaceuticals. The separation was conducted using isocratic RP-HPLC with a DAD at 227nm. The mobile phase used was a 40:40:20 (v/v/v) ratios of buffer, acetonitrile, and ethanol. An L1 column (150 x 4.6mm, 5µm) was utilized with a flow rate of 1.5mL/min and injection volume of 5µL. The column oven and auto-sampler were both maintained at 45°C and 25°C respectively. The optimized method was validated as per ICH guidelines and found to be specific, precise, and robust to slight changes in mobile phase flow, column temperature, and buffer pH. Linearity was demonstrated from 0.1-150µg/mL for olanzapine and 0.4-610µg/mL for fluoxetine HCL. The accuracy was tested with recovery ranges of 0.1–157 µg/mL for olanzapine and 0.4–606 µg/mL for fluoxetine HCl in tablet placebo, and 0.1–150 µg/mL for olanzapine and 0.4–605 µg/mL for fluoxetine HCl in capsule placebo, indicating that the method is suitable for both tablet and capsule dosage forms. The method’s greenness was evaluated using AES, NEMI, GAPI, and AGREE tools. The optimized method was successfully applied to pharmaceutical analysis. The newly optimized method found simple, sensitive, rapid, eco-friendly, incorporating green analytical concepts, has been successfully achieved for the identification and quantification of Fluoxetine HCL and Olanzapine present in pharmaceuticals.

QbD-Driven Approach to Cleaning Method Development and Validation for Darunavir Analysis in Oral Films.

The current investigations involve developing a high-performance liquid chromatographic method that’s simple to operate, fast to establish, accurate, precise, and economical through design-enabled methods. A positive experimental design is offered to utilize the central composite design of pH and the mobile phase, two crucial components of the RP-HPLC technology. The chromatographic conditions were optimized with the release of Design Expert software version 13.0. Symmetry C18 column (4.6×150mm, 5µm) was used in the procedure, along with acetonitrile (20:80 v/v) and potassium dihydrogen orthophosphate buffer pH 4.0 as the mobile phase. There was a 0.70 ml/min flow rate. 263 nm is the wavelength of detection. Darunavir’s sharp, resolved peak was seen 2.3 minutes after swabbed samples from the 10*10 cm SS plate were injected. A linear calibration curve with an R2 of 0.9991 was observed in the concentration range of 0.25 to 25µg/ml. For precision %RSD is 1.03,1.48. Accuracy % concentration 50%,100%,150% mean recovery 99.43-100.53.LOD & LOQ 1.12µg/ml, 3.39µg/ml. Robustness was tested with modest modifications to the wavelength and flow rate. Forced degradation studies were carried out and found to be within the limits. the degradation parameters such as Acid, Base, Oxidative, Photolytic, and thermal degradation parameters are according to the ICH guidelines. % Assay was done for oral films and it was found to be within the limits. Thus, the outcomes unequivocally demonstrated that the QbD technique could be effectively used to enhance the HPLC method for the measurement of darunavir in production settings.

Simultaneous multianalyte trace-level quantification of eight genotoxic nitrosamine impurities in valsartan Active Pharmaceutical Ingredient and tablet formulation using UFLC-MS/MS and greenness assessment

Valsartan, a widely prescribed antihypertensive drug, was identified for the presence of carcinogenic nitrosamine impurities in 2018, leading to product recalls worldwide and necessitating sensitive liquid and gas chromatographic methods for trace level detection of nitrosamine impurities. This study developed and validated an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for the quantification of eight genotoxic nitrosamines (NDMA, NDEA, NDPA, NEIPA, NDIPA, NMBA, NDBA, and NMPA) in valsartan drug substance and tablet formulations. The method employed a triple quadrupole analyzer, atmospheric pressure chemical ionization (APCI) ionization source and multiple reaction monitoring (MRM) scan mode for the analysis. Chromatographic separation was achieved on a Phenomenex biphenyl column (150 × 4.6 mm, 3 µm) maintained at 55 °C. The mobile phase consisted of a binary gradient of solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in methanol), with a total run time of 20 min for the sensitive and specific detection of the analytes. Method validation demonstrated excellent linearity, recovery, precision, and sensitivity. The greenness of the developed analytical method was critically assessed through comprehensive evaluations using the GAPI, AGREE, and AES metrics and the results obtained are compared with the reported methods in the literature. This novel UFLC-MS/MS method offers a reliable and sensitive approach for routine quality control analysis of nitrosamine impurities in Valsartan drug substance and tablet formulations.

Gabapentin: An impurity profiling approach with hydrogen peroxide induced thermal oxidation in autoclave

Predicting unconventional pathways in long term stability studies can be very challenging, especially in solid state degradation. This is of vital importance for enhancement in the level of degradation and to further enable the isolation of unknown degradation products. Predicting the mechanism or pathway also help in designing control strategies to minimise the potential chances of impurity formation. In this context, the lagtime of most oxidative degradation mechanisms is extremely important, which allows enough requisite of free radicals etc., one of the unconventional pathways. This is difficult to replicate in degradation studies involving usual solution form. In this investigation, we demonstrate that employing autoclave degradation with hydrogen peroxide conditions provide an enhanced level of an unknown degradation product, which was found to be futile in traditional forced degradation studies. The conditions used probably bypass the lag time. This impurity has been isolated using preparative liquid chromatography (Prep-HPLC) method and spectroscopic techniques like high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance NMR (1H, 13C, DEPT-135, NOESY, APT) for elucidation of the molecular structure. The impurity was identified as a dimer of gabapentin impurity-A (impurity (2,2′-diaza[l,2′-bispiro[4.5]decane]-3,3′-dione) and named it as impurity-U. A plausible mechanism for the formation of isolated impurity is proposed. HPLC methods have been developed and reported for the determination of gabapentin and its related substances using UV detection.

Method Development and Validation of Alcaftadine by UHPLC in Bulk and Dosage Form

This research developed and validated a new, simple sensitive, suitable, economical, accurate, and robust ultra-high performance liquid chromatography (UHPLC) method for determining Alcaftadine in bulk drug and ophthalmic dosage formulation. The separation was performed using an HPLC method with a UV detector and Open lab EZchrome workstation program, Kromasil C18, 250mm X 4.6mm ID, 2.7μm Acetonitrile: 0.05% OPA (60:40%V/V) with a flow rate of 1.0mL/min and detected at 282nm. The developed UHPLC method yielded a suitable retention time for Alcaftadine of 2.20min, which was optimised using a trial-and-error basis. The linearity of the determined method was found a correlation coefficient (r2) of 0.9998 over the concentration range of 2.0-30.0μg/mL. The percentage RSD for the method's precision was found to be less than 2.0percent. The percentage recovery was discovered within the limit 0.259ug/mL and 0.784ug/mL were found to be the LOD and LOQ, respectively. The developed and validated UHPLC system takes less time and cheaply can be used in the industry for routine quality control/analysis of bulk drugs and marketed Alcaftadine products. In present studies, the retention time was less than previously reported. The developed method was validated in terms of specificity, linearity, accuracy, precision and robustness according to the ICH guidelines.

The Use of HPLC-DAD Method in Determining the Quality of Honeys: 5-Hydroxymethylfurfural (HMF) Analysis

The recognition of 5-hydroxymethylfurfural (HMF) content in honeys has been the subject of many scientific studies as it is a significant indicator of the toxicity, its processing method, storage situations and age. In this research, the amount of HMF in 95 different flower and honeydew honey samples obtained from markets, factory sales offices and local beekeepers in Turkiye was determined. High performance liquid chromatography (HPLC) method was employed to recognize HMF contents. Before sample analysis, the standard HMF solutions were prepared at different concentrations and method validation studies were carried out. The HMF content limit value of flower and honeydew honey in the Honey Communiqué (TFC/Communiqué No:2020/7) is stated as 40 mg.kg-1. According to the obtained results, the HMF contents in honey samples were found as 0.00-16.65 mg.kg-1. It was determined that the amounts of HMF were at the highest level in monofloral milk vetch honey (MH_MV) samples coded MH_MV3 and MH_MV4. Therefore, it was concluded that all of the honey samples were compatible with the Honey Communiqué in terms of HMF and had appropriate honey quality values.

Metabolomics and quantitative analysis to determine differences in the geographical origins and species of Chinese dragon's blood.

The aim of this study was to comprehensively analyze the differences in Chinese dragon's blood (CDB), specifically Dracaena cochinchinensis and Dracaena cambodiana, from different geographical origins. Metabolomic analysis of CDB was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A reliable ultrahigh-performance liquid chromatography method with a photodiode array detector (UHPLC-PDA) was developed and applied for the quantitative analysis of 12 phenolic compounds in 51 batches of samples. A total of 1394 metabolites were detected, of which 467 were identified as differentially accumulated metabolites. Multivariate analysis revealed that both origin and species had an effect on the composition of CDB, with greater variation between species. 19 phenolic compounds were selected as quality markers to distinguish D. cochinchinensis (Hdsp) from D. cambodiana (Hdca), and oppositin and spinoflavanone a were identified as quality markers to discriminate D. cochinchinensis samples from Hainan (Hdsp) and Guangxi Provinces (Gdc). Quantitative analysis indicated that four phenolic compounds, including loureirin D, 4H-1-benzopyran-4-one,2,3-dihydro-3,5,7-trihydroxy-3-[(4-methoxyphenyl)methyl]-,(R)-, loureirin B, and pterostilbene, showed significant differences between Gdc and Hdsp. Additionally, five phenolic compounds, namely resveratrol, loureirin D, pinostilbene, 4H-1-benzopyran-4-one,2,3-dihydro-3,5,7-trihydroxy-3-[(4-methoxyphenyl)methyl]-, (R)-, and loureirin B, exhibited significant differences between Hdsp and Hdca. There are significant differences in the quality of CDB from different geographical origins and species, which lays the foundation for the in-depth development and utilization of different sources of CDB.

Characterization, quantitation, and natural variability of phenolics in Rhododendron arboreum leaves of western Himalayan origin using UPLC-PDA and UHPLC-ESI-Q/TOF-MS/MS

Rhododendron arboreum Sm. is very popular in the Indian Himalayan region due to its significant ethnomedicinal importance and potential commercial applications. Apart from the ornamental value derived from its vibrant red flowers, fresh flowers are used to prepare medicinal juices, and dried are utilized as spices. Despite being a rich source of phenolics, these lack identified marker chemicals and validated methods. The present work encompasses the chemical investigation of R. arboreum leaves using UHPLC-ESI-Q/TOF-MS/MS. The chemotypic variability was assessed through a reverse phase ultra-performance liquid chromatography method coupled with a photodiode array detector (RP-UPLC-PDA) chemical signatures of R. arboreum leaves from different altitudes combined with chemometric analysis. The chromatographic method was developed and validated for the quantitative analysis of three vital secondary metabolites, i.e., Isoquercetin or Quercetin-3-O-glucoside (Qg), Reynoutrin or Quercetin-3-O-xyloside (Qx), and Quercitrin or Quercetin-3-O-rhamnoside (Qr)The hyphenation of the method with ESI-Q/TOF-MS/MS has confirmed the distribution of 11 chemicals (nine known and two unknown) in R. arboreum leaves belonging to the phenolic and hydrolyzable tannins class. Samples from higher altitudes >3000 m (above mean sea level) were exceptionally rich in Quercitrin. The chemometric investigation, comprising hierarchical cluster analysis (HCA), principal component analysis (PCA), and partial least square method-discriminant analysis (PLS-DA), successfully categorized thirty-three samples from diverse geographic regions into two distinct categories. This current study provides a validated method for the chemotypic discrimination and quality assessment of the R. arboreum leaves from Uttarakhand, Western Himalayan region of India, and laid the foundation for the comprehensive utilization of these renewable and sustainable resources.

Comparing the Impact of Continuous Endurance and High-intensity Interval Training on Cecal Butyrate and Propionate Levels in Diabetic Rats Induced by High-fat Diet

Background: Dysbiosis and metabolic disorders of the microbiota, often caused by an imbalance in the intestinal microbial composition, are significant issues linked to immobility, obesity, and diabetes. Physical exercise is recognized for its role in managing these symptoms by regulating the composition and metabolites of the intestinal microbiota, thereby improving gut health and overall metabolic function. Objectives: This study aimed to investigate and compare the effects of continuous endurance training (CET) and high-intensity interval training (HIIT) on two key cecal microbiota metabolites, butyrate and propionate, in diabetic rats. Methods: Forty-five male Wistar rats were made diabetic by a high-fat diet and were trained under CET and HIIT exercise protocols. Cecal tissue samples were taken from the rats to evaluate the effect of exercise, and the levels of two microbial metabolites, butyrate and propionate, were measured using the high-performance liquid chromatography (HPLC) method. Results: Among the exercise patterns studied, HIIT significantly improved the concentrations of butyrate and propionate, while CET showed no effect on these metabolites. Conclusions: Our findings suggest that, unlike CET, HIIT may effectively mitigate metabolic disturbances resulting from gut dysbiosis in diabetic patients. However, any definitive conclusion about the effects of CET and HIIT exercises on intestinal microbial metabolites necessitates further comprehensive tests on other metabolites and an examination of additional supporting evidence, such as changes in the composition of the intestinal microbiome.

Separation and identification of oligonucleotides impurities and degradation products by reversed phase ultra-high performance liquid chromatography using phenyl-bonded stationary phases without ion pairs - A step towards sustainability

This manuscript discusses the development of a reversed-phase ultra high-performance liquid chromatography (RP UHPLC) method based on phenyl-bonded stationary phases without ion-pairs for the separation and identification of oligonucleotides. The elimination of ion-pair reagents makes the proposed protocol as more compliant to the principles of green chemistry, compared to the traditional ion-pair reversed-phase liquid chromatography methods (IP RP LC). In detail, three phenyl-based stationary phases were tested, namely a C18/AR (a C18 stationary phase with the addition of aromatic groups), a Phenyl-hexyl, and a Diphenyl. Generally, the retention of oligonucleotides increases with the increase of salt concentration and the decrease of the pH, thus confirming the significant impact of van der Waals interactions, salting-out effect, and π-electrons interactions in the retention mechanism. The highest retention and best peak symmetry were observed for the C18/AR stationary phase, while the lowest retention for the Phenyl-hexyl, with retention influenced by the type of salt in the mobile phase. The obtained methods using C18/AR stationary phases allow for the effective separations of positional isomers and for identifying impurities and degradation products using RP UHPLC Q-TOF-MS in a comparatively short time. The application of RP UHPLC Q-TOF-MS provides reasonable selectivity for the resolution of 33 impurities and two degradation products. Both groups of compounds are mainly 3′N and 5′N-shortmers, but in the case of impurities, modifications of cyclic phosphate and phosphate groups were also identified. Nevertheless, Diphenyl and Phenyl-Hexyl may be applied to separate modified oligonucleotides with higher salt concentrations. The proposed separation methods without ion-pair reagents contribute to a more sustainable approach in oligonucleotide analysis.

Enhanced Detection of Curcumol in Biological Samples via Acetonitrile‐Anhydrous Sodium Sulfate Salt‐Assisted Liquid‐Liquid Microextraction Coupled With High‐Performance Liquid Chromatography

ABSTRACTCurcumol, a significant bioactive molecule, requires accurate detection methods due to its extensive pharmacological properties. This study develops high‐performance liquid chromatography (HPLC) methods to detect curcumol and constructs sample pretreatment techniques using salt‐assisted liquid‐liquid microextraction (SALLME). The optimization process focuses on conditions for acetonitrile (ACN) anhydrous sodium sulfate‐assisted liquid‐liquid microextraction. By adding excess anhydrous sodium sulfate to an ACN aqueous solution, ACN precipitates, forming a saturated saline solution and resulting in a two‐phase system. Curcumol, being highly soluble in ACN, separates into and enriches within the ACN phase, achieving effective matrix separation. SALLME demonstrated an enrichment factor of up to 12.4 for curcumol. The extracted ACN is then injected into an HPLC instrument for quantitative detection and analysis. The method exhibited a good linear range (0.5–10.0 µg/mL) and a low detection limit (0.05 µg/mL). The results indicate that curcumol can be effectively separated from complex matrices using this technique. By optimizing salt concentration and temperature, the HPLC method for determining curcumol in biological samples was successfully established, achieving recoveries between 86.2% and 137.6% in spiked urine and blood samples. Furthermore, this method offers rapid, efficient, and environmentally friendly sample preparation, promising significant applications in pharmacological and clinical settings.