High-performance Liquid Chromatography System Research Articles

Caffeic acid phenethyl ester (CAPE) Chitosan capped ZnO nanoparticles: Preparation, characterization, and its potential for the treatment of prostate cancer

The synthesis of zinc oxide nanoparticles/chitosan (ZnONPs/CS) formulation loaded with Caffeic acid phenethyl ester (CAPE) was performed to evaluate its prostate cancer treatment efficiency within the scope of this research. It has been hypothesized that a dual active materials delivery system containing ZnO and CAPE loaded Chitosan (CS) nanoparticles has better bioavailability compared to single one against to cancer cells. ZnONPs were synthesized between 45 and 60 nm particle sizes and then they were capped with CS biodegradable polymer prior to load with CAPE bioactive molecule. ZnONPs/CS-CAPE system was characterized by using Fourier Transform Infrared (FTIR) for structural elucidation, Scanning Electron Microscope (SEM) for particle size determination, High Performance Liquid Chromatography (HPLC) system for determination of CAPE amount. 131I CAPE and 131I ZnONPs/CS-CAPE labeled by the Iodogen method with 131I were used in-vitro cell culture experiments. Cell viabilities (%) of CAPE and ZnONPs/CS-CAPE were examined using Cell Counting Kit-8 assay on PC-3 (human adenocarcinoma prostate), LnCaP (human carcinoma prostate), and RWPE-1 (human normal prostate). IC50 values of ZnONPs /CS -CAPE on all cells were found 2-fold lower than neat CAPE. Based on the FTIR data, the most significant spectral changes (lipid, protein, nucleic acids, glycogen) were monitored for the PC-3 and LnCaP cancer cells incubated with ZnONPs/CS-CAPE samples while being exposed to neat CAPE molecules caused small cellular changes when compared to RWPE-1 healthy cell lines.

Linear solvent strength model on porous graphitic carbon stationary phase using high temperature liquid chromatographic method for allopurinol related substances analysis

A high-performance liquid chromatography (HPLC) method was developed for the analysis of Allopurinol and its Ph.Eur. impurities using a porous graphitic carbon (PGC) stationary phase. Retention behavior of solutes was studied across a wide temperature range (30–90 °C) and various gradient times (5–20 min). Analysis of the data revealed distinct retention mechanisms between reversed-phase and PGC phases. However, it was proved that the retention of Allopurinol and its Ph.Eur. impurities on PGC stationary phase can be effectively modeled using the linear solvent strength (LSS) theory. This allows for the utilization of LSS-based method development software to optimize methods under these conditions. By using commercial chromatographic modeling software, separation of Allopurinol and Ph.Eur. impurities was optimized within a large design space. At the optimized operating conditions (pH = 2.0, tG = 6 min, T = 60 °C), all solutes were separated within 6 min with baseline resolution. Comparison between predicted and experimentally measured chromatograms further confirmed the applicability of LSS theory in developing analytical methods for PGC-based HPLC systems. The presented approach offers a general framework for method development on PGC phases.

Synthesis of Magnetic Attapulgite Nanoparticles Via a Novel Surface Covalent Reaction Method and its Application in the Magnetic Solid Phase Extraction.

In this study, the attapulgite nanoparticle was immobilized on the surface of magnetic nanoparticle Fe3O4 via a novel surface covalent reaction method for the magnetic solid phase extraction (MSPE) for the first time. The surface covalent reaction method has the advantages of controllable steps, and can make the magnetic attapulgite nanoparticle (MANP) have good homogeneity and high stability. Field emission scanning electron microscopy, equipped with an energy dispersive spectrometer, Nitrogen adsorption BET, X-ray diffraction and Fourier transform infrared spectroscopy were applied to characterize the prepared MANP, confirming that the attapulgite nanoparticle could be effectively immobilized on the surface of magnetic nanoparticle Fe3O4 via covalent reactions. Under optimal conditions of the MSPE experiment based on the MANP, the limits of detection were found to be 10ng/mL for melamine and 3ng/mL for cyromazine with a relative standard deviation < 10% by a high-performance liquid chromatography system. Meanwhile, 0.1mg/mL melamine in milk and 0.1mg/mL cyromazine in cucumber can also be detected according to our MSPE procedure. More importantly, the MANP still has good magnetism and enrichment efficiency after several decades of use. These results showed that the MANP prepared by our method is a kind of promising material for the MSPE.

Enantioselective three-dimensional high-performance liquid chromatographic determination of amino acids in the Hayabusa2 returned samples from the asteroid Ryugu

The chirality of amino acids in extraterrestrial materials may provide an insight into the origin of the essential l-enantiopure amino acids in the terrestrial biosphere. In 2020, the Hayabusa2 mission succeeded in bringing back surface materials from the C-type asteroid (162173) Ryugu to the Earth. Amino acids were one of the targeted organic molecules to be studied in the Ryugu samples. To analyze the various structural isomers of amino acids, which were expected to be present, from the limited amount of the returned samples, the development of a highly-sensitive and selective analytical method was necessary. In the present study, a three-dimensional high-performance liquid chromatography (3D-HPLC) system has been developed for the enantioselective determination of five proteinogenic and three non-proteinogenic amino acids in the Ryugu samples, in which amino acids in the sample were separated by reversed-phase, anion-exchange and enantioselective columns after the fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiozole. The applicability of the analytical system to the extraterrestrial samples was evaluated by analyzing several types of carbonaceous meteorites before applying the system to the Ryugu samples. In the analysis of the Ryugu samples, all of the target amino acids were successfully determined quantitatively. Non-proteinogenic amino acids including 2-amino-n-butyric acid, isovaline and norvaline, rarely present in the terrestrial environment, were found as almost racemic mixtures with 47.1 to 55.2 %l.

Quartz Crystal Microbalance as a Holistic Detector for Quantifying Complex Organic Matrices during Liquid Chromatography: 1. Coupling, Characterization, and Validation.

A matrix in highly complex samples can cause adverse effects on the trace analysis of targeted organic compounds. A suitable separation of the target analyte(s) and matrix before the instrumental analysis is often a vital step for which chromatographic cleanup methods remain one of the most frequently used strategies, particularly high-performance liquid chromatography (HPLC). The lack of a simple real-time detection technique that can quantify the entirety of the matrix during this step, especially with gradient solvents, renders optimization of the cleanup challenging. This paper, along with a companion one, explores the possibilities and limitations of quartz crystal microbalance (QCM) dry-mass sensing for quantifying complex organic matrices during gradient HPLC. To this end, this work coupled a QCM and a microfluidic spray dryer with a commercial HPLC system using a flow splitter and developed a calibration and data processing strategy. The system was characterized in terms of detection and quantification limits, with LOD = 4.3-15 mg/L and LOQ = 16-52 mg/L, respectively, for different eluent compositions. Validation of natural organic matter in an environmental sample against offline total organic carbon analysis confirmed the approach's feasibility, with an absolute recovery of 103 ± 10%. Our findings suggest that QCM dry-mass sensing could serve as a valuable tool for analysts routinely employing HPLC cleanup methods, offering potential benefits across various analytical fields.

Assessment of Portuguese fitness centers: Bridging the knowledge gap on harmful microbial contamination with focus on fungi

The lack of knowledge regarding the extent of microbial contamination in Portuguese fitness centers (FC) puts attendees and athletes at risk for bioaerosol exposure. This study intends to characterize microbial contamination in Portuguese FC by passive sampling methods: electrostatic dust collectors (EDC) (N = 39), settled dust (N = 8), vacuum filters (N = 8), and used cleaning mops (N = 12). The obtained extracts were plated in selective culture media for fungi and bacteria. Filters, EDC, and mop samples' extracts were also screened for antifungal resistance and used for the molecular detection of the selected Aspergillus sections. The detection of mycotoxins was conducted using a high-performance liquid chromatograph (HPLC) system and to determine the cytotoxicity of microbial contaminants recovered by passive sampling, HepG2 (human liver carcinoma) and A549 (human alveolar epithelial) cells were employed. The results reinforce the use of passive sampling methods to identify the most critical areas and identify environmental factors that influence microbial contamination, namely having a swimming pool. The cardio fitness area presented the highest median value of total bacteria (TSA: 9.69 × 102 CFU m−2.day−1) and Gram-negative bacteria (VRBA: 1.23 CFU m−2.day−1), while for fungi it was the open space area, with 1.86 × 101 CFU m−2.day−1. Aspergillus sp. was present in EDC and in filters used to collect settled dust. Reduced azole susceptibility was observed in filters and EDC (on ICZ and VCZ), and in mops (on ICZ). Fumonisin B2 was the only mycotoxin detected and it was present in all sampling matrixes except settled dust. High and moderate cytotoxicity was obtained, suggesting that A549 cells were more sensitive to samples’ contaminants. The observed widespread of critical toxigenic fungal species with clinical relevance, such as Aspergillus section Fumigati, as well as Fumonisin B2 emphasizes the importance of frequent and effective cleaning procedures while using shared mops appeared as a vehicle of cross-contamination.

Impurity profile analysis of amphotericin B using on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Amphotericin B (AmB) is a polyene-macrolide antimicrobial drug with a broad antibacterial spectrum and remarkable efficacy against deep fungal infections. It binds to ergosterol on the fungal cell membrane and alters its permeability, thereby destroying the membrane. AmB is a multicomponent antimicrobial medication that contains a wide range of impurities, rendering quality analysis extremely difficult. In the current Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3), high performance liquid chromatography (HPLC) is applied to examine related substances in AmB. However, this technique presents a number of issues. For instance, the mobile phases used in the HPLC method described in both references contain nonvolatile inorganic salts, which cannot be coupled with a mass spectrometry (MS) detector. In addition, because the mobile phases used have a low pH, the component/impurities of AmB drug can easily be degraded or interconverted during the analytical process, leading to reduced analytical accuracy. Therefore, the accuracy and sensitivity of this method must be improved. In this study, a method based on on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (2D HPLC-Q TOF/MS) was developed to analyze the impurity profile of AmB in accordance with the Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3). The method combines on-line dilution and a multiple-capture HPLC system to achieve the efficient separation of AmB component/impurities. It also resolves the issue of poor solvent compatibility in 2D HPLC, increases the analytical flux, enhances the automation capability, reduces the mutual conversion of AmB and its impurities during the analytical process, and increases the detection sensitivity of the method. MS was also used to determine the structural inference of unstable components and impurities. An XBridge Shield C18 column (250 mm×4.6 mm, 3 μm) was used for first-dimensional-liquid chromatography with gradient elution using methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (10∶30∶60, v/v/v, pH 4.7) as mobile phase A and methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (12∶68∶20, v/v/v, pH 3.9) as mobile phase B. An Xtimate C8 column (10 mm×2.1 mm, 5 μm) was used as the trap column, and trapping and desalting were performed using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v). An Xtimate C8 column (250 mm×2.1 mm, 5 μm) was used for second-dimensional-liquid chromatography with gradient elution using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v) and 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (5∶95, v/v) as mobile phases. The data were collected in positive-ion mode. In this study, the structures of six impurities in amphotericin B were inferred, according to the fragmentation, the MS and MS2 spectra of each impurity. The developed method can be used to quickly and sensitively analyze the impurity profile of AmB. Furthermore, the research results on impurity profiles can be applied to guide improvements in AmB production.

Defensin-like peptides from Capsicum chinense induce increased ROS, loss of mitochondrial functionality, and reduced growth of the fungus Colletotrichum scovillei.

In the present study, we identified and characterized two defensin-like peptides in an antifungal fraction obtained from Capsicum chinense pepper fruits and inhibited the growth of Colletotrichum scovillei, which causes anthracnose. AMPs were extracted from the pericarp of C. chinense peppers and subjected to ion exchange, molecular exclusion, and reversed-phase in a high-performance liquid chromatography system. We investigated the endogenous increase in reactive oxygen species (ROS), the loss of mitochondrial functioning, and the ultrastructure of hyphae. The peptides obtained from the G3 fraction through molecular exclusion chromatography were subsequently fractionated using reverse-phase chromatography, resulting in the isolation of fractions F1, F2, F3, F4, and F5. The F1-Fraction suppressed C. scovillei growth by 90, 70.4, and 44% at 100, 50, and 25 μg mL-1, respectively. At 24 h, the IC50 and minimum inhibitory concentration were 21.5 μg mL-1 and 200 μg mL-1, respectively. We found an increase in ROS, which may have resulted in an oxidative burst, loss of mitochondrial functioning, and cytoplasm retraction, as well as an increase in autophagic vacuoles. MS/MS analysis of the F1-Fraction indicated the presence of two defensin-like proteins, and we were able to identify the expression of three defensin sequences in our C. chinense fruit extract. The F1-Fraction was also found to inhibit the activity of insect α-amylases. In summary, the F1-Fraction of C. chinense exhibits antifungal activity against a major pepper pathogen that causes anthracnose. These defensin-like compounds are promising prospects for further research into antifungal and insecticide biotechnology applications. © 2024 Society of Chemical Industry.

Cross-column density functional theory-based quantitative structure-retention relationship model development powered by machine learning.

Quantitative structure-retention relationship (QSRR) modeling has emerged as an efficient alternative to predict analyte retention times using molecular descriptors. However, most reported QSRR models are column-specific, requiring separate models for each high-performance liquid chromatography (HPLC) system. This study evaluates the potential of machine learning (ML) algorithms and quantum mechanical (QM) descriptors to develop QSRR models that can predict retention times across three different reversed-phase HPLC columns under varying conditions. Four machine learning methods-partial least squares (PLS) regression, ridge regression (RR), random forest (RF), and gradient boosting (GB)-were compared on a dataset of 360 retention times for 15 aromatic analytes. Molecular descriptors were calculated using density functional theory (DFT). Column characteristics like particle size and pore size and experimental conditions like temperature and gradient time were additionally used as descriptors. Results showed that the GB-QSRR model demonstrated the best predictive performance, with Q2 of 0.989 and root mean square error of prediction (RMSEP) of 0.749min on the test set. Feature analysis revealed that solvation energy (SE), HOMO-LUMO energy gap (∆E HOMO-LUMO), total dipole moment (Mtot), and global hardness (η) are among the most influential predictors for retention time prediction, indicating the significance of electrostatic interactions and hydrophobicity. Our findings underscore the efficiency of ensemble methods, GB and RF models employing non-linear learners, in capturing local variations in retention times across diverse experimental setups. This study emphasizes the potential of cross-column QSRR modeling and highlights the utility of ML models in optimizing chromatographic analysis.

Antioxidant Activities and Phenolic Composition of Sarcococca saligna Leaves

Abstract: Sarcococca saligna (D. DON) Muel is a vital plant with several steroidal alkaloids and is effective against syphilis, liver diseases, gastrointestinal tract disease, infections, and inflammation. The present study aimed to investigate the in vitro antioxidant activities and phenolic profile of S. saligna. High-performance liquid chromatography (HPLC) analysis has shown the presence of twelve phenolics namely kaempferol-3-O-glucoside, proanthocyanidin B1, quercetin-3-malonylglucoside- 7-glucoside, kaempferol-3-O-sophorotioside, Isorhamnetin-3-O-glucoside-7-Orhamnoside, kaempferolhexoside, 4-O-Caffeoyl-5-O-p-coumaroylquinic acid, 3,4-Di-O-caffeoyl-5- O-feruloylquinic acid, 3,4-Di-O-feruloyl-5-O-caffeoylquinic acid, flavogalloyl-HHDP-gluconic acid (lagerstannin B), 3,4-di-O-caffeoylquinic acid and kaempferol-3-(caffeoyldiglucoside)-7-glucoside. The hot water extract showed the highest anti-lipid peroxidative activities. The IC50 value for 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was 71.5 ± 2.1 μg/mL. The extract could chelate the iron and possess reducing activities on phosphomolybenum assay. It is concluded that extracts of S. saligna are rich in antioxidants and contain essential phytochemicals. objective: The present study was aimed to investigate thein vitro antioxidant activities and phenolic profile of S. saligna. HPLC analysis has shown the presence of twelve phenolics namely kaempferol-3-O-glucoside, proanthocyanidin B1, quercetin-3-malonyl-glucoside-7-glucoside, kaempferol-3-O-sophorotioside,Isorhamnetin-3-O glucoside-7-O-rhamnoside,kaempferolhexoside, 4-O-Caffeoyl-5-O-p-coumaroylquinic acid, 3,4-Di-O-caffeoyl-5-O-feruloylquinic acid, 3,4-Di-O-feruloyl-5-O-caffeoylquinic acid, flavogalloyl-HHDP-gluconic acid (lagerstannin B), 3,4-di-O-caffeoylquinic acid and kaempferol-3-(caffeoyldiglucoside)-7-glucoside. method: The chemical used were 1,10-phenathroline, Thibarbituric acid, TRIS-HCl, amominium molybdate, Iron, sodium nitroprusside. HPLC standards were purchased from Sigma-Aldrich. All the reagents were of analytical grade. Double beam spectrophotometer was used for UV-VIS spectrophotometery. The HPLC system used was Agilent 1260 Infinity HPLC system which consists of quaternary pump, degasser, auto-sampler and diode array detector (DAD). conclusion: The results have shown that S. saligna antioxidant rich extract can be effectively utilized in food and pharmaceutical industries with additional income. However, more detailed understanding of utilization of extract against degenerative diseases is required. There is also need to find out the safety and bioavailability of the plant extract for its use as nutraceutical and as a plant products.

Metabolite profiling of triterpene saponins from different red beetroot cultivars using ultra-high performance liquid chromatography high-resolution mass spectrometry

In recent years, there has been increasing interest in the use of red beets as a valuable source of bioactive compounds. Although saponins may be responsible for the pharmacological and nutraceutical activities of red beets, information on the saponin composition of various red beet cultivars is very limited, and the saponin profiles remain to be fully elucidated. Here, we profiled triterpene saponins from the flesh and skin of different red beetroot cultivars, whose names of Polish cv. are Bonel, Chrobry and Nochowski. In addition, the total triterpene saponin contents in cultivars Bonel, Chrobry, Nochowski and in their various root parts were determined. Triterpene saponins isolated from the skin and flesh of different red beetroot cultivars were qualitatively profiled using two high-performance liquid chromatography systems (HPLC-ESI-IT MS and UPLC-HESI-Q-Orbitrap MS). The total triterpene saponin contents in different cultivars were analyzed using high-performance liquid chromatography-tandem mass spectrometry. In total, 40 triterpene saponins were tentatively identified, and among them, 5 compounds were potentially new saponins, not previously reported in red beets. These saponins are new to red beets and were reported in B. vulgaris for the first time. The analyzed cultivars were different in terms of their total saponin content. The results of our study indicated that the variation in the composition of saponins depended on the red beet cultivars and part of the roots. Higher concentrations of these compounds were found in the skins than in the flesh of roots. We developed an analytical strategy to discover and identify triterpene saponins from B. vulgaris L. using a combination of techniques to detect the comprehensive fragmentation pathways of the saponins present at low concentrations in plant material.

Paediatric orodispersible lozenges produced by moulding process: quality and stability control

Abstract Objectives Active pharmaceutical ingredients for paediatric patients are formulated at hospital pharmacy services and compounding pharmacies when they are not commercially available. This work evaluated the quality and stability of lozenges, an orodispersible dosage form, elaborated by a simple and low-cost moulding process. Methods The lozenges were formulated with 10 mg of hydrochlorothiazide as an active pharmaceutical ingredient and a commercial excipient suitable for paediatric use (Natatroche®). A High-Performance Liquid Chromatography system was used to quantify the active pharmaceutical ingredient from pure and extracted from dosage form samples. The quality of the lozenges was checked by the European Pharmacopeia tests indicated for oromucosal preparations, and additional quality tests specified for orodispersible and dispersible tablets. The stability of this dosage form was evaluated by the International Conference of Harmonization indications. Results The chromatographic method could quantify the active pharmaceutical ingredient extracted from excipient matrix. This formulation met uniformity of dosage units test for oromucosal dosage forms and, in addition, met the disintegration time test for orodispersible tablets and the fineness of dispersion test for dispersible tablets. Hydrochlorothiazide in lozenges was stable for 60 days when stored at 5 °C. Conclusions Moulded hydrochlorothiazide tablets are quick-to-prepare dosage forms that can be prepared in a simple, low-cost manufacturing process. They are safe and stable, disintegrate quickly in the mouth, and are therefore a recommended option for children, or other patients with swallowing problems.

Stability and bioactivities evaluation of analytical grade C-phycocyanin during the storage of Spirulina platensis powder.

C-phycocyanin (C-PC) is a natural high-value blue phycobiliprotein from Spirulina platensis, which has wide biological applications in food, pharmaceutical, and cosmetics. However, the freshness of S. platensis powder (SPP) materials and C-PC purification play critical roles in evaluating the stability and bioactivities of C-PC, which severely affect its commercial application. This study investigated the effect of spray-dried SPP freshness on the biofunctional activities of analytical grade C-PC (AGC-PC). The yield of AGC-PC extracted from spray-dried SPP could reach 101.88mg/g (75% recovery ratio) after purification by reversed phase high-performance liquid chromatography (RP-HPLC) system. The half-life period (t1/2 ) of AGC-PC stability at 60°C and 8000 lux light could remain 171.70min and 176.11h within 6 months storage of spray-dried SPP. The emulsifying activity index (EAI)and foaming capacity (FC) of AGC-PC from fresh-dried SPP showed maximum values of 68.64 m2 /g and 252.9%, respectively. The EC50 of AGC-PC from fresh spray-dried SPP on 2,2-diphenyl-1-picrylhydrazyl (DPPH)and 2,2'-azinobis(3-ethylbenzothiazoline -6-sulfonic acid (ABTS+·) scavenging activity could reach 63.76 and 92.93mg/L, respectively. The EC50 of AGC-PC from fresh spray-dried SPP on proteinase inhibition and anti-lipoxygenase activity were 302.96 and 178.8mg/L, respectively. The stability and biofunctional activities of AGC-PC remained stable within 6 months storage of SPP, and then rapidly decreased after 9 months storage due to the disintegration of the trimeric (αβ)3 and hexameric (αβ)6 forms of C-PC. It is concluded that the optimal storage period of SPP for preparation of AGC-PC in commercial use should be less than 6 months. PRACTICAL APPLICATION: The C-phycocyanin (C-PC) from dried Spirulina platensis powder (SPP) has been widely applied in food nutritional, florescent markers, pharmaceuticals, cosmetics, etc, due to its blue color, fluorescence, and antioxidant properties. However, the effect of dried SPP freshness on the stability and functional activity of C-PC has been rarely reported. This study found that the thermostability, photostability, emulsifying, antioxidant, and anti-inflammatory activities of analytical grade C-PC (AGC-PC) significantly decreased after 6 months storage of SPP. Based on investigations, we have proposed that the suitable storage time of dried SPP for preparation of AGC-PC in commercial application should be within 6 months.

DEVELOPMENT AND VALIDATION OF A NEW RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION AND QUANTIFICATION OF SOFOSBUVIR AND DACLATASVIR

Background: Sofosbuvir (SOF) and daclatasvir (DCV) are usually prescribed in combination for the treatment of hepatitis C worldwide. Objectives: In the present study a novel, simple, rapid, and cost-effective reversed-phase high performance liquid chromatography (HPLC) method was developed, optimised, and validated for simultaneous analysis of sofosbuvir and daclatasvir in human plasma as well as in solid dosage forms. Methodology: The chromatographic analyses were carried out using Shimadzu HPLC gradient system (LC20), while the other HPLC system used for the peak purity and repeated validation studies was Agilent 1260 Infinity fitted with diode array detector. The column used was C18 (150 × 4.6 mm; 5 µm), from Agela Technologies. The mobile phase consisted of acetonitrile and 0.02 M potassium Hexafluorophosphate buffer (42:58, v/v) at pH 2.7, pumped at the rate of 1.3 mL min−1. The limit of detection and limit of quantification were determined using dilution method (RSD/m formula). Results: SOF and DCV combination therapy is significant for the treatment of hepatitis C. Development of this RP-HPLC method for the simultaneous determination and quantification of SOF and DCV is a part of an extensive project of novel formulation development (fixed dose combination tablets of SOF and DCV). This method is successfully applied for raw materials as well as in combination tablets. This method may also be applied for the quantification of sofosbuvir and daclatasvir in blood samples of patients taking these two antivirals for hepatitis C treatment. Conclusion: The optimized method was successfully applied for the determination of sofosbuvir and daclatasvir in single run without any interference. Hence, the developed method is quite appropriate for the simultaneous determination of SOF and DCV in solid dosage form as well as in human plasma for clinical research studies.

Two patients with α-chain hemoglobin variant Hb Q-Iran detected by measuring hemoglobin A1c using the variant mode of the HA-8180V HPLC analyzer.

Hemoglobin variants are often discovered when hemoglobin A1c (HbA1c) levels measured with a high-performance liquid chromatography (HPLC) system in fast mode are found to be low. The HA-8180V HPLC analyzer by Arkray offers two measurement modes: fast mode (FM) and variant mode (VM). Two Japanese patients with α-chain variant Hb Q-Iran detected incidentally after analyses with the HA-8180V in VM showed an abnormal peak, are presented. The first patient was a man in his 70s, and the second patient was a man in his 50s. Both were non-diabetic, but their results from HbA1c measurement in VM showed an abnormal peak. The VM-HbA1c, FM-HbA1c, and HbA1c measured by enzymatic assay and glycated albumin levels of the two patients were all within the reference ranges. They were diagnosed as having Hb Q-Iran (α2-75Asp → His) by globin gene analysis. It is difficult to detect α-chain hemoglobin variants based on abnormal FM-HbA1c levels, but measuring HbA1c in VM is useful for efficiently detecting hemoglobin variants.

Simultaneous determination of six nitroaromatic compounds and three anions in environmental matrices using a liquid chromatography-ion chromatography coupled system

Nitroaromatic compounds are used extensively in various fields such as dyes, pesticides, spices, pharmaceuticals, and explosives. However, the residual raw materials of these compounds accumulate in the environment and pose serious risks to human health. Chronic exposure to low concentrations of nitroaromatic compounds can cause anemia, cancer, and organ damage. Currently, Fenton oxidation and natural bioremediation are the processes most often used to eliminate nitroaromatic compounds from environmental water and soil. According to previous research, the presence of inorganic anions such as chloride, nitrite, and nitrate ions in the environmental matrix exerts an inhibitory effect on the biodegradation of nitroaromatic compounds. Furthermore, high nitrate levels in drinking water can lead to the production of nitrosamine carcinogens, which affect ecological safety and human health, in water bodies. Thus, the simultaneous determination of nitroaromatic compounds and chloride, nitrite, and nitrate ions in environmental soil and water matrices is critical for selecting appropriate nitroaromatic compound degradation methods and monitoring surface water quality. Traditional detection methods require two sample pretreatment steps and two instrumental analytical techniques to determine nitroaromatic compounds and inorganic anions in environmental matrices; moreover, these methods are time consuming, labor intensive, and error prone. Therefore, in this study, a method that combines high performance liquid chromatography (HPLC) and ion chromatography (IC) was developed to simultaneously detect nitroaromatic compounds and anions in environmental matrices. In this method, sample enrichment was achieved through bulk injection and enrichment column collection, which greatly simplified the pretreatment process. The HPLC instrument was connected to the IC instrument using two six-way valves and an enrichment column. The system operation can be divided into four stages: (A) sample loading to the quantitative ring, (B) separation of nitroaromatic compounds and anions, (C) enrichment of anions in an AG20 column, and (D) simultaneous determination of nitroaromatic compounds and anions by HPLC and IC, respectively. The time of the anions flowing out of the C18 column was determined by directly connecting the C18 column to a conductivity detector. Based on the retention times of the anions, the switching time of the six-way valve was optimized to ensure that the anions completely entered the IC column, thereby ensuring the accuracy of the method. During the chromatographic analysis stage, nitroaromatic compounds were separated and analyzed by HPLC system with a mobile phase composed of potassium phosphate buffer (pH 7.0) and acetonitrile (60∶40, v/v) at a flow rate of 1.0 mL/min; in the IC system, the anions were separated and analyzed using a 20 mmol/L sodium hydroxide aqueous solution as the mobile phase under a suppression current of 50 mA. Both anions and nitroaromatic compounds exhibited strong linear correlations within certain concentration ranges, with correlation coefficients greater than 0.993. The recoveries of the nitroaromatic compounds and anions ranged from 88.20% to 105.38% at three spiked levels, with relative standard deviations ranging from 2.0% to 11.5%. The contents of six nitroaromatic compounds and three anions in five surface water and five soil samples were determined using the developed method. Although no nitroaromatic compounds were detected in these samples, the three anions were detected at contents ranging from 0.41 to 55.3 mg/L in surface water samples, and 0.56 to 30.2 mg/kg in soil samples. Methodological validation and actual sample detection demonstrated that the proposed method has a high degree of automation, simple operation, good repeatability, high accuracy, wide applicability, and high sensitivity. Thus, this method is suitable for the rapid determination of chloride, nitrite, nitrate ions and nitroaromatic compounds in soil and water and can be extended to the simultaneous determination of inorganic ions and organic matters in other samples.

Evaluation of antioxidant properties and cytotoxicity of brown algae (nizamuddinia zanardinii) in uterine (hela) and pancreatic cancer cell lines (paca-2).

Pancreatic cancer and cervical cancer are among the most common cancers. Brown algae have anti-inflammatory, anti-cancer, anti-fungal, antioxidant, and immune-boosting properties. This study investigated the antioxidant properties and the effect of brown algae extract on pancreatic and uterine cancer cells. In this study, Cervical (Hela) and pancreas (Paca-2) cancer cell lines were examined. The algae materials were extracted by sequential maceration method and amount of fucoxanthin content in the sample was determined by using High Performance Liquid Chromatography (HPLC) system. The cytotoxic effect of different concentrations of brown algae was measured by the MTT assay. All statistical calculations for comparing IC50 were analyzed using Graph Pad Prism software. the algal sample contained an average of 102.52 ± 0.12μg of fucoxanthin per 100g. IC50 for 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide free radical scavenging activity for methanolic extract was 2.02 and 11.98 ± 0.13 respectively. Brown algae in all fractions inhibited cell growth and survival. In Hela cell lines, the methanolic extract was the most effective inhibitor, while in Paca cell lines, hexane and methanolic extracts were particularly potent. The methanolic extract was more toxic than other fractions on Hela and Paca cell lines. This study highlights brown algae extracts strong anticancer effects on uterine and pancreatic cancer cells, suggesting its potential as a natural anticancer drug. Different fractions of the extract showed superior apoptotic and cytotoxic effects, with higher concentrations leading to increased apoptotic effects and reduced survival rates of cancer cells.

Rapid detection of glycosylated hemoglobin levels by a microchip liquid chromatography system in gradient elution mode

BackgroundThe determination of glycosylated hemoglobin (HbA1c) is crucial for diabetes diagnosis and can provide more substantial results than the simple measurement of glycemia. While there is a lack of simple methods for the determination of HbA1c using a point-of-care test (POCT) compared to glycemia measurement. In particular, high-performance liquid chromatography (HPLC) is considered the current gold standard for determining HbA1c levels. However, commercial HPLC systems usually have some sort of disadvantages such as bulky size, high-cost and need for qualified operators. Therefore, there is an urgent demand to develop a portable, and fast HbA1c detection system consuming fewer reagents. ResultsWe present a novel microchip that integrates a micromixer, passive injector, packed column and detection cell. The integrated microchip, in which all the microstructures were formed in the CNC machining center through micro-milling, is small in size (30 mm × 70 mm × 10 mm), and can withstand 1600 psi of liquid pressure. The integrated design is beneficial to reduce the band broadening caused by dead volume. Based on the microchip, a microchip liquid chromatography (LC) system was built and applied to the analysis of HbA1c. The separation conditions of HbA1c in blood calibrator samples were optimized using the microchip LC system. Samples containing four levels of HbA1c were completely separated within 2 min in optimal gradient conditions, with an inaccuracy (<3.2 %), a coefficient of variation (c.v. < 2.1 %) and a correlation coefficient (R2 = 0.993), indicating excellent separation efficiency and reproducibility. SignificanceThe POCT of HbA1c is critical for diabetes diagnosis. The microchip chromatography system was developed for HbA1c determination, which contains an integrated microchip and works under a gradient elution. It surpasses existing chip technology in terms of separation performance and detection speed, providing a competitive advantage for POCT of HbA1c. It is considered one important step for realizing efficient portable systems for timely and accurate diabetes diagnosis.

D -Alanine Affects the Circadian Clock to Regulate Glucose Metabolism in the Kidney.

Key Points d-Alanine affects the circadian clock to regulate gluconeogenesis in the kidney. d-Alanine itself has a clear intrinsic circadian rhythm, which is regulated by urinary excretion, and acts on the circadian rhythm. d-Alanine is a signal activator for circadian rhythm and gluconeogenesis through circadian transcriptional network. Background The aberrant glucose circadian rhythm is associated with the pathogenesis of diabetes. Similar to glucose metabolism in the kidney and liver, d-alanine, a rare enantiomer of alanine, shows circadian alteration, although the effect of d-alanine on glucose metabolism has not been explored. Here, we show that d-alanine acts on the circadian clock and affects glucose metabolism in the kidney. Methods The blood and urinary levels of d-alanine in mice were measured using two-dimensional high-performance liquid chromatography system. Metabolic effects of d-alanine were analyzed in mice and in primary culture of kidney proximal tubular cells from mice. Behavioral and gene expression analyses of circadian rhythm were performed using mice bred under constant darkness. Results d-Alanine levels in blood exhibited a clear intrinsic circadian rhythm. Since this rhythm was regulated by the kidney through urinary excretion, we examined the effect of d-alanine on the kidney. In the kidney, d-alanine induced the expressions of genes involved in gluconeogenesis and circadian rhythm. Treatment of d-alanine mediated glucose production in mice. Ex vivo glucose production assay demonstrated that the treatment of d-alanine induced glucose production in primary culture of kidney proximal tubular cells, where d-amino acids are known to be reabsorbed, but not in that of liver cells. Gluconeogenetic effect of d-alanine has an intraday variation, and this effect was in part mediated through circadian transcriptional network. Under constant darkness, treatment of d-alanine normalized the circadian cycle of behavior and kidney gene expressions. Conclusions d-Alanine induces gluconeogenesis in the kidney and adjusts the period of the circadian clock. Normalization of circadian cycle by d-alanine may provide the therapeutic options for life style–related diseases and shift workers.

First Report of the Joint Exposure to Glyphosate and Glufosinate of a Male Population in the Province of Córdoba (Argentina).

Despite potential health implications, data on the presence of Glyphosate (GLY) and other non-GLY herbicides in human matrices remain scarce. This study aimed to develop a simple and cost-effective methodology for detecting and quantifying GLY, its primary biodegradation product; aminomethylphosphonic acid (AMPA); and glufosinate (GLU) in plasma and urine of environmentally and occupationally exposed populations from the province of Córdoba (Argentina). Different alternatives of pre-treatment, derivatization with FMOC-Cl, solid phase extraction, and final sample conditioning steps were evaluated to improve the quantification of the herbicides by a high-performance liquid chromatography system coupled to a triple-quadrupole mass spectrometer. Recoveries ranged from 39 to 84% in both matrices, while limits of quantification were 3, 1, and 0.3 ng/mL and 3.6, 5.1, and 0.3 ng/mL for AMPA, GLY, and GLU in plasma and urine, respectively. In plasma samples, GLY was the most frequently detected analyte (32%), followed by GLU (10%). In urine samples, GLU was the most frequently detected herbicide (13%), followed by GLY (6%). No differences between group or matrix correlations were found. This study is the first report of GLU in human biological matrices and should be used to establish baseline values for future surveillance systems.