HPLC System Research Articles

The kynurenine and serotonin pathway, neopterin and biopterin in depressed children and adolescents: an impact of omega-3 fatty acids, and association with markers related to depressive disorder. A randomized, blinded, prospective study.

https://www.isrctn.com/ISRCTN81655012, identifier ISRCTN81655012.

Determination of free and bound antioxidants in Kamut® wheat by HPLC with triple detector (DAD-CAD-MS)

Kamut® wheat (Triticum turgidum ssp. turanicum), an ancient, underutilized cereal, offers potential health benefits due to its phenolic compounds. This study aimed to investigate the antioxidant potential of Kamut® wheat's free and bound phenolic extracts using an HPLC system equipped with three detectors. The bound extracts, released after alkaline hydrolysis, exhibited higher total phenolic and flavonoid content compared to the free extracts (p < 0.05). The total antioxidant capacity of bound extracts was six-fold greater than in free extracts (p < 0.05). The main antioxidants in free extracts were tyrosine, phenylalanine, tryptophan, and apigenin. In bound extracts, ferulic acid, its dimers and trimer were present. Kamut® wheat exhibited a source of dietary antioxidants and should be considered a potential ingredient for the development of functional foods. Also, the HPLC-triple detector system is effective for in-depth profiling of antioxidant compounds, paving the way for future research on similar grains.

Quantification of Riboflavin and Thiamine in GI (Geographical Indication) Branded Yogurts Collected from Bogura, Bangladesh Using HPLC Equipped with a Fluorescence Detector

Thiamine (vitamin B1) and riboflavin (vitamin B2) are both essential vitamins for human health. Thiamine (vitamin B1) is essential for energy metabolism, nervous system function, carbohydrate metabolism, cardiovascular health, digestion, muscle function, etc. On the other hand, Riboflavin (vitamin B2), which acts as an antioxidant, is essential for energy production, healthy skin, hair and nails, eye health, red blood cell production, nervous system function, etc. Due to its specialty, Bogura's famous yogurt was recognized as a Geographical Indication (GI) on June 26, 2023 by the Directorate of Patents, Designs, and Trademarks (DPDT) under the Ministry of Industry, the Government of the People’s Republic of Bangladesh (Prothom Alo, 2023). The purpose of the study was the quantitative determination of two water-soluble vitamins, thiamine and riboflavin, in the GI branded yogurt and whey (Ghol), collected from Bogura, Bangladesh to ensure health benefits. The methods employed for the quantitative analysis of these vitamins were reversed-phase high performance liquid chromatography (RP-HPLC) equipped with a fluorescence detector. The extraction process involved acid hydrolysis followed by enzymatic hydrolysis with takadiastase enzyme. Chromatographic separation was achieved with a Shimadzu Prominence HPLC system using isocratic elution mode on a C18 column (4.6 mm × 250 mm, 5 μm). The correlation coefficients for thiamine and riboflavin were 0.9975 and 0.9974, respectively. The recovery rates for thiamine and riboflavin were found as 97.70% and 105.40%, respectively. Twenty-two samples were analyzed. Thiamine and riboflavin were found in the range of 0.0647 to 0.1396 mg/100 g and 0.0227 to 0.3749 mg/100 g, respectively. It can be concluded that these yogurts were rich in thiamine and riboflavin. Therefore, these yogurts are beneficial for health. Bangladesh Pharmaceutical Journal 27(1): 59-66, 2024 (January)

Development of the RP-HPLC Method for Simultaneous Determination and Quantification of Artemether and Lumefantrine in Fixed-Dose Combination Pharmaceutical Dosage Forms.

Developing countries face enormous challenges with substandard and falsified antimalarial drugs. One specific issue is the lack of a simple, cost-effective, and robust HPLC method to simultaneously determine and quantify the active pharmaceutical ingredients (APIs) in fixed-dose artemether-lumefantrine pharmaceutical dosage forms. The current study developed a novel, simple, sensitive, precise, accurate, and cost-effective RP-HPLC method for the simultaneous determination and quantification of artemether and lumefantrine in pharmaceutical dosage forms. The HPLC analysis was carried out on an Agilent 1260 Infinity Series HPLC system equipped with an ODS Intersil-C8 (150 × 4.6 mm) 5.0 µm column, by isocratic elution. The mobile phase composition consisted of acetonitrile and 0.05% orthophosphoric acid buffer of pH 3.5 in the ratio of 70 : 30 v/v. The analysis was performed at a 1 mL/min flow rate and a column temperature of 25°C. The total run time was 6 minutes. The detection was done with a variable wavelength detector (VWD) at an isosbestic point wavelength (λ) of 210 nm. The developed method was validated according to the ICH guidelines concerning system suitability, specificity, linearity, accuracy, precision, and robustness. The system suitability of the developed method revealed satisfactory theoretical plates and symmetry factors. The method proved to be specific, with no interference of mobile phase or excipients. The calibration plot exhibited linearity over the concentration range of 275-1925 μg/mL with R2 = 0.9992 for artemether and a range of 150-1050 μg/mL with R2 = 0.9985 for lumefantrine. The accuracy of the method, determined by the recovery study, was 99.79-100.16% for artemether and 99.04-99.50% for lumefantrine. The % RSD values for intraday precision were 0.175 and 0.203, while interday precision values were 0.340 and 0.554 for artemether and lumefantrine, respectively. The method demonstrated robustness when subjected to slight modifications in the flow rate, column temperature, and mobile phase composition. The developed analytical method proved satisfactory as per ICH guidelines and hence can be used for the determination and quantification of artemether and lumefantrine in bulk drug and pharmaceutical dosage forms.

Quantitative mass spectrometry with ¹⁸O labelling as an alternative approach for determining protease activity: an example of trypsin

SCIENTIFIC RELEVANCE. In the quality control of proteolytic enzyme components of medicinal products, the activity of proteases is determined by spectrophotometry, which involves mea­suring the amidase or esterase activity using a synthetic substrate and the proteolytic activity using the Anson method. These methods require special substrates and have low sensitivity; their specificity may be insufficient, which may lead to serious errors. Quantitative mass spectrometry is an alternative approach to protease activity assays, which involves adding an isotope-labelled peptide to hydrolysates of the test enzyme. This approach allows determining the activity of proteases, notably, by the hydrolysis of specific peptide bonds, while simulta­neously confirming the identity and specificity of the test sample. Quantitative mass spectrometry has high sensitivity and does not require special substrates.AIM. This study aimed to investigate the possibility of enzymatic activity assay and enzyme identification by quantitative mass spectrometry with ¹⁸O labelling through an example of trypsin with casein.MATERIALS AND METHODS. The study used trypsin, casein, and H₂¹⁸O (Izotop, Russia). Peptide separation was performed using an Agilent 1100 HPLC system; mass spectra were obtained using a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer. Quantitative mass spectrometry was performed using a standard peptide, which was obtained from casein by tryptic digestion and HPLC purification. For ¹⁸O labelling, the authors dried the peptide and incubated it in H₂¹⁸О water. The quantitative analysis of the product was carried out using MALDI-TOF mass spectrometry. The authors used quantitative mass spectrometry with ¹⁸O labelling to determine enzymatic activity and calculate the Michaelis constant (KM).RESULTS. Following the tryptic digestion of casein, the authors identified the fragments corre­sponding to casein chains. The authors produced the isotope-labelled standard peptide and calculated its concentration using mass spectrometry. The authors determined the rate of casein digestion by trypsin and calculated the KM for trypsin, which was 13.65±0.60 μM. The standard deviation for repeated measurements showed that the mass-spectrometric method had a lower error of measurement than the spectrophotometric method. The sensitivity threshold for the mass-spectrometric method was 0.50±0.08 μM.CONCLUSIONS. The results obtained with trypsin confirm the possibility of enzymatic activity determination by the proposed method of quantitative mass spectrometry with ¹⁸O labelling. According to the sensitivity evaluation results, this method can be used for the simultaneous determination of enzyme activity, identity, and specificity. The proposed mass spectrometry approach is universal, it does not require expensive materials and reagents, and it can be easily adapted to determine the activity of virtually any protease.

High Performance Liquid Chromatography Method for Analysis of Alpha Arbutin and Niacinamide in Facial Whitening Creams

The combination of alpha arbutin and niacinamide is often used in facial whitening creams at relatively small levels so required valid analysis method. This study aimed to develop HPLC method for analyzing alpha arbutin and niacinamide in three brands of whitening creams. The HPLC system developed was a reverse phase type with a C₁₈ column and the mobile phase was a mixture of metanol: aquabidest (20:80, v/v) with a flow rate of 1.0 ml/min and a detection wavelength of 274 nm. The results showed that all parameters met the requirements. The linearity test yielded a correlation value was r= 0.999. The LOD value for alpha arbutin was 0.405 µg/mL and the LOD for niacinamide was 0.398 µg/mL. The LOQ value of alpha arbutin was 1.351 µg/mL, the LOQ for niacinamide was 1.327 µg/mL and the selectivity was good. Precision test results with %RSD ≤ 2% and accuracy tests yielded values in the 99.04-100.97% range. The content of alpha arbutin in brand A was 0.651%, brand B was 0.551%, and brand C was 0.878%. The content of niacinamide in brand A was 0.526%; brand B was 0.680% and brand C was 1.053%. The results of the analysis meet the requirements.

Six bufadienolides derivatives are the main active substance against human colorectal cancer HCT116 cells of Huachansu injection

BackgroundHuachansu injection (HCS) is an aqueous extract preparation of Traditional Chinese Medicine (TCM) toad skin (Bufo melanosticus or B. bufo gargarizans). As a national second-class and self-developed TCM preparation, it is widely used to treat human colorectal cancer (CRC), yet its substantial toxic side effects, such as vascular irritation reaction, drug fever, and allergic reactions, pose a considerable challenge during clinical use. In-depth research on the material basis of HCS' traditional efficacy is essential for developing more effective and less toxic medicine. Identifying the main components responsible for the efficacy of HCS is crucial in determining its material basis and explaining any adverse reactions. PurposeThe purpose of this study is to clarify the main active components of HCS as a growth inhibitory agent against CRC HCT116 cells and to further understand the material basis of its anti-CRC properties. MethodsSulforhodamine B (SRB) assay was used to screen the compound with strong inhibitory activity against HCT116 cells from 44 compounds isolated from aqueous extracts of toad skin (B. melanosticus). The screened compounds as standard and MeOH extracts from HCS were determined by HPLC coupled with triple quadrupole mass spectrometry. The partial MeOH extract was subjected to HPLC system using solvents in a gradient of increasing polarity, yielding 12 fractions in total (HCS1 to HCS12). Besides, the screened active compounds were knocked out from HCS to obtain a fraction as a negative control, and the knocked-out components were collected as one fraction using semi-preparative HPLC. Those fractions were submitted to HPLC-MS/MS or HPLC-DAD system to determine the presence or absence of the screened compounds. In addition, cytotoxicity of those prepared fractions against CRC HCT116 cells was evaluated to determine the effect of the screened compounds in the anti-CRC of HCS. ResultsSix compounds, namely arenobufagin, telocinobufagin, gamabufalin, hellebrigenin, 19-hydroxylbufagin and argentinogenin, were screened for their inhibitory activity (with an IC50 value of less than 400 nM), and were present in HCS at a total-mass-percentage of 1.35×10−3 % in HCS MeOH extract. In addition, those 6 compounds were mainly distributed in HCS8, which also showed the strongest inhibitory activity against HCT116 cells. And the results of multicomponent knockout assay indicated the negative control lost its anti-CRC activity significantly, while the activity can be restored after those 6 compounds were added to the negative control. ConclusionsThose six bufadienolides derivatives are responsible for the anti-CRC HCT116 cells activity of HCS, and are the main components of HCS on anti-CRC. Through this research, we can more clearly understand the main anti-CRC active components in HCS, and the work also lays the foundation for the development of more effective and less toxic medicine from HCS.

A comparative evaluation of the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC) with capillary zone electrophoresis (CZE) assays for the detection of hemoglobin variants and the quantitation of HbA0, A2, E, and F.

Hemoglobinopathies, including thalassemia and hemoglobin (Hb) variants, are common hematological disorders in tropical countries. Accurate and precise separation of hemoglobin types and reliable quantitation are necessary for differential diagnosis of these disorders. We have evaluated the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC; Trinity Biotech, Co. Wicklow, Ireland) to assist in the presumptive diagnosis of thalassemia and Hb variants commonly found in Southeast Asian countries. HbA0, HbA2, HbE, and HbF levels were separated and quantified in 120 blood samples from unrelated adult subjects and compared with those analyzed by capillary zone electrophoresis (CZE; CAPILLARYS™ 2, Sebia, Norcross, GA, US). The Hb analysis patterns of Hb variants obtained from the PR-HPLC system were also compared to those obtained from HPLC (VARIANT II, β-thalassemia Short Program, Bio-Rad, Laboratories, Hercules, CA, US) and CZE systems. The PR-HPLC had excellent precision with a coefficient of variation (CV) for HbA2 quantitation of 3.8 % within-run and 5.2 % between-run. The levels of HbA2/E quantified by the PR-HPLC system correlated well with those of the CZE system (r=0.997). In addition, thalassemia interpretation results obtained from the PR-HPLC and the CZE showed 100 % agreement. Moreover, chromatograms of the PR-HPLC were also comparable to those of VII-HPLC and CAP2-CZE electropherograms. The PR-HPLC system would be applicable to diagnose common forms of thalassemia and Hb variants in Southeast Asia.

Quantification of Arbortristoside-A isolated from Nyctanthes arbor-tristis using HPLC: Method development and pharmaceutical applications

Arbortristoside-A (Arbor-A) is a naturally occurring iridoid glycoside and herbal-based lead molecule with proven medicinal potential. Aiming at the development of an efficient analytical tool for the quantification of Arbor-A in pharmaceutical dosage forms, in the presented work, we developed an economical, fast, and sensitive RP-HPLC-UV method and validated the procedure as per the ICH guidelines, Q2(R1). The chromatographic separation was accomplished under the optimised experimental conditions using an HPLC system with an LC-2010 autosampler, a PDA detector, and a Phenomenex C18 column with the mobile phase composed of a 70:30 (v/v) water-acetonitrile mixture eluting isocratically at a flow rate of 1 mL/min at ambient temperature, and UV detection at 310 nm. Arbor-A showed a sharp peak at the retention time of 5.60 min and exhibited linearity (R2 = 0.9988) with LOD and LOQ of 0.50 μg/mL and 1.50 μg/mL, respectively. The accuracy of the method was 98.33–101.36 % with acceptable intra-day and inter-day precisions as well as robustness (<2% RSD). To ratify the applicability of the presented approach in emerging pharmaceuticals, a nanoformulation loaded with Arbor-A was designed and analysed utilising the provided methodology. The method has also enabled to determine the degradation kinetics of Arbor-A under stress conditions, etcetera, employing forced degradation and short term stability studies.

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

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

α acid fraction from Hop extract exerts an endothelium-derived hyperpolarization vasorelaxant effect through TRPV4 employing the feedforward mechanism of PKCα.

Until now, the beneficial vascular properties of Hop reported in the literature have been mainly attributed to specific compound classes, such as tannins and phenolic acids. However, the potential vascular action of a Hop subfraction containing a high amount of α or β acids remains completely understood. Therefore, this study aims to investigate the vascular effects of the entire Hop extract and to fraction the Hop extract to identify the main bioactive vascular compounds. A pressure myograph was used to perform vascular reactivity studies on mouse resistance arteries. Phytocomplex fractionation was performed on a semi-prep HPLC system and characterized by UHPLC-PDA-MS/MS coupled to mass spectrometry. Western blot analysis was performed to characterize the phosphorylation site enrolled. The entire Hop extract exerts a direct dose-dependent endothelial vascular action. The B1 subfraction, containing a high concentration of α acids, recapitulates the vascular effect of the crude extract. Its vasorelaxant action is mediated by the opening of Transient Receptor Potential Vanilloid type 4 (TRPV4), potentiated by PKCα, and subsequent involvement of endothelial small-conductance calcium-activated potassium channels (SKCa) and intermediate-conductance calcium-activated potassium channels (IKCa) that drives endothelium-dependent hyperpolarization (EDH) through heterocellular myoendothelial gap junctions (MEGJs). This is the first comprehensive investigation of the vascular function of Hop-derived α acids in resistance arteries. Overall, our data suggest that the B1 subfraction from Hop extracts, containing only α acids, has great potential to be translated into the useful armamentarium of natural bioactive compounds with cardiovascular benefits.

Development and validation of a liquid chromatography coupled to a diode array detector (LC-DAD) method for measuring mitotane (DDD) in plasma samples

IntroductionMitotane (o,p’–DDD) is the drug of choice for Adrenocortical Carcinomas (ACC) and its measurement in plasma is essential to control drug administration. ObjectiveTo develop and validate a simple, reliable and straightforward method for mitotane determination in plasma samples. MethodDrug-free plasma samples were collected in potassium-ethylenediamine tetraacetate (K-EDTA) tubes and spiked with 1.0, 2.5, 10.0, 25.0 and 50.0 µg/mL of mitotane (DDD). The p,p’-DDD was used as an Internal Standard (IS) and was added at 25.0 µg/mL concentration to all samples, standards and controls. Samples were submitted to protein precipitation with acetonitrile and then centrifuged. 50 uL of the supernatant was injected into an HPLC system coupled to a Diode Array Detector (DAD). DDD and IS were detected at 230 nm in a 12 min isocratic mode with a solvent mixture of 60 % acetonitrile and 40 % formic acid in water with 0.1 % pump mixed, at 0.6 mL/min flow rate, in a reversed-phase (C18) chromatographic column kept at 28°C. The sensitivity, selectivity, precision, presence of carry-over, recovery and matrix-effect, linearity, and method accuracy were evaluated. ResultsThe present study's method resulted in a symmetrical peak shape and good baseline resolution for DDD (mitotane) and 4,4’-DDD (internal standard) with retention times of 6.0 min, 6.4 mim, respectively, with resolutions higher than 1.0. Endogenous plasma compounds did not interfere with the evaluated peaks when blank plasma and spiked plasma with standards were compared. Linearity was assessed over the range of 1.00–50.00 µg/mL for mitotane (R2 > 0.9987 and a 97.80 %‒105.50 % of extraction efficiency). Analytical sensitivity was 0.98 µg/mL. Functional sensitivity (LOQ) was 1.00 µg/L, intra-assay and inter-assay coefficient of variations were less than 9.98 %, and carry-over was not observed for this method. Recovery ranged from 98.00 % to 117.00 %, linearity ranged from 95.00 % to 119.00 %, and high accuracy of 89.40 % to 105.90 % with no matrix effects or interference was observed for mitotane measurements. Patients' sample results were compared with previous measurements by the GC-MS method with a high correlation (r = 0.88 and bias = -10.20 %). ConclusionDDD determination in plasma samples by the developed and validated method is simple, robust, efficient, and sensitive for therapeutic drug monitoring and dose management to achieve a therapeutic index of mitotane in patients with adrenocortical cancer.

Simultaneously HPLC Analysis of B1, B9 and B12 Vitamins at Trace Levels via Cloud Point Extraction

An enrichment and determination method based on liquid chromatographically analysis and cloud point extraction (CPE) has been developed for trace levels of B vitamins (B1, B9 and B12) in the proposed study. Vitamin molecules were drawn into the non-ionic surfactant phase of Polyethylene Glycol (PEG-6000) in the presence of pH 9.0 medium. The surfactant-rich phase separated by centrifugation and then dissolved with 700 µL of ethanol. The obtained ethanol phase was filtered by 0.45-micron filter prior to the HPLC analysis. All parameters affecting the CPE method such as pH, buffer volume, incubation time, surfactant and electrolyte concentration, solvent for the surfactant-rich phase and its amount have been individually studied and optimized step by step. After the optimization of all parameters of the CPE process, the detection limits of the developed method for B1, B9 and B12 vitamins were calculated as 1.42 ng mL-1, 7.14 ng mL-1 and 14.28 ng mL-1 , respectively. The linear working ranges for three vitamin molecules was obtained in the range of 5.0-500.0 ng mL-1. After CPE procedure, determination of vitamin molecules was carried out by using HPLC system with diode array detector(DAD) at 244 nm for vitamin B1, 285 nm for vitamin B9, and 361 nm for vitamin B12, respectively

HPLC-LED-Induced Fluorescence Analysis of Tear Fluids: An Objective Method for Primary Open Angle Glaucoma Diagnosis

PurposeThe study showcased the application of the lab-assembled HPLC-LED-IF system to analyze proteins in tear fluid samples collected from individuals diagnosed with primary open-angle glaucoma (POAG).MethodsClinical application of the said technique was evaluated by recording chromatograms of tear fluid samples from control and POAG subjects and by analyzing the protein profile using multivariate analysis. The data analysis methods involved are principal component analysis (PCA), Match/No-Match, and artificial neural network (ANN) based binary classification for disease diagnosis.ResultsMahalanobis distance and spectral residual values calculated using a standard calibration set of clinically confirmed POAG samples for the Match/No-Match test gave 86.9% sensitivity and 81.8% specificity. ANN with leaving one out procedure has given 87.1% sensitivity and 81.8% specificity.ConclusionThe results of the study revealed that the utilization of a 278 nm LED excitation in the HPLC system offers good sensitivity for detecting proteins at low concentrations allowing to obtain reliable protein profiles for the diagnosis of POAG.

Development and Validation of an Analytical RP-HPLC Method for Simultaneous Estimation of Losartan and its Active Metabolite (EXP-3174) in Isolated Perfused Rat Liver

Background: The liver perfusion method is frequently used in drug pharmacokinetic studies and the various effects of drugs on liver tissue. The aim of this study was to establish and validate an analytical method using high-performance liquid chromatography to determine the simultaneous concentration of losartan and its active metabolite, EXP-3174, in an isolated perfused rat liver study. Method: An HPLC system with isocratic mode was used. Various chromatographic parameters were adjusted to develop and validate a method for determination of losartan and its active metabolite in liver perfusion media. Results: In this study, losartan and its active metabolite, EXP-3174, were separated using a C18 stationary phase, a mobile phase consisting of acetonitrile: phosphate buffer at a flow rate of 1 mL.min-1, and UV detection at 254 nm. Retention times for losartan and the metabolite were 10 and 16 minutes, respectively. Linearity from 25-250 ng.ml-1 was validated with acceptable accuracy and precision. The LOD and LOQ for losartan were 7.0 and 21.1 ng.ml-1, respectively. The LOD and LOQ for metabolite were 7.4 and 22.4 ng.ml-1, respectively. ChromGate® software was used to acquire and process the data. Conclusions: The optimized and validated technique was effectively used to analyze losartan and its active metabolite in isolated perfused rat liver.

Evaluation of mycotoxins in grains sold in Idah, Ajaka and Ogbogbo areas of Nigeria.

Mycotoxin contamination in grains is a significant concern due to its adverse effects on human and animal health. Understanding the levels and patterns of mycotoxin contamination in different regions and storage conditions is crucial for developing effective control strategies. This study aimed to assess mycotoxin levels in stored and recently harvested grains in three regions (Idah, Ajaka, and Ogbogbo) and investigate the implications for food safety. The study involved the analysis of mycotoxin levels in maize, rice, sorghum, and millet using appropriate mycotoxin extraction method based on the mycotoxins of interest and a suitable HPLC system. The findings revealed the presence of mycotoxins such as aflatoxins (1±0.2-5±0.4 µg kg-1), deoxynivalenol (520±0.8-700±1.2 µg kg-1), zearalenone (200±0.4-370±0.6 µg kg-1), ochratoxins (2±0.2-4±0.3 µg kg-1), and fumonisin (0±0.0-4±0.3 µg kg-1) in both recently harvested and stored grains. Patulin was absent in most of the samples. Variations in mycotoxin levels were observed among different grains and regions, highlighting the need for targeted interventions. The European Commission mycotoxin standards in grains for human consumption are: aflatoxins 4 µg kg-1 in maize, millet and sorghum while rice is 8 µg kg-1, deoxynivalenal 1750 µg kg-1, zearalenone 100 µg kg-1, ochratoxin A 5 µg kg-1 for maize, rice and millet, then 10 µg kg-1 for sorghum. Fumonisin is 4000 µg kg-1 but no level for rice and patulin is 50 µg kg-1 for rice and none for the other grains. This study demonstrates the persistence of mycotoxin contamination in stored grains and the importance of considering specific crop types and geographical locations when addressing mycotoxin contamination. The findings underscore the significance of implementing effective control measures to mitigate mycotoxin contamination and enhance food safety. The study provides valuable insights into mycotoxin contamination and emphasizes the need for comprehensive risk assessment studies and appropriate regulatory measures.

Novel stability indicating HPLC method for the quantification of Nirmatrelvir in bulk drugs

Nirmatrelviris anantiviral drug that acts asa 3C-likeprotease inhibitor. It is used with the combination of Ritonavir for the treatment of COVID-19. The World Health Organization strongly recommended this medicine to moderate and mild COVID patients, which is now the best option for COVID-19 therapeutics against SARS-CoV-2. As a result, there is an urgent need for quick and low-level quantitative analytical techniques for Nirmatrelvir and its products by focusing on the mass-compatible buffer with a shorter run time as the target. Accordingly, we developed a procedure with the following optimized conditions; mobile phase: 0.10 % formic acid in water and acetonitrile (55:45 %v/v), column oven temperature: 45 °C, flow rate: 1.0 mL/min with 15 min run time in isocratic elution mode. The developed procedure is linear and accurate from 0.20 to 500 µg/mL. Forced degradation studies in all solid and liquid degradations were executed to demonstrate the method's stability indicative capacity. To demonstrate the peak purity of the desired compound orthogonally, all degradation samples were then subjected to peak purity analysis using an HPLC PDA (High-performance liquid chromatography photodiode array) detector and LCMS (Liquid chromatography-mass spectrometry) analysis. The literature methods were developed with highly concentrated buffers which cause ion suppression in LCMS and increase backpressure in HPLC systems but the proposed method is conducive and more sensitive (limit of quantitation: 0.20 µg/mL and limitation of detection: 0.0 6 µg/mL) than the reported methods and has a wide linearity range as well. The developed technique has been fully validated by ICH (International Council for Harmonisation) guidelines. Further, it has been evaluated for greenness character using the AGREE (Analytical GREEnness) statistical tool and the obtained score is 0.61 out of 1.

Taxis-mediated bacterial transport and its implication for the cometabolism of pyrene in a model aquifer

One of the main problems in contaminated soils is that many toxic substances, such as PAHs, which are found in areas close to aquifers and groundwater, are difficult to access and degrade via traditional methods of remediation. The use of controlled bacterial mobility through chemotaxis has been shown to be efficient in increasing the dispersion of pollutant-degrading organisms, increasing the biodegradation rates of pollutants. In this study, using percolation columns as model aquifers, the mobilization of the Pseudomonas putida G7 strain to a distant pyrene source was demonstrated using γ-aminobutyric acid and artificial root exudates as strong chemoeffectors. An increase in the biodegradation rates of the pollutant was observed relative to columns in which the tactic effector was not added. The presence of different metabolites was detected via a fraction collector associated with an HPLC system, providing evidence for the cometabolic capacity of strain G7. The use of chemotactic organisms can be an effective approach for the remediation of polluted sediments associated with aquifers and groundwaters, offering new possibilities for the treatment of contaminated aqueous areas.

Systematic pharmacology-based strategy to explore the mechanism of Semen Strychni for treatment of papillary thyroid carcinoma

The aim of the study was to investigated the mechanism of Strychnos nux-vomica L. (Semen Strychni, SS) against papillary carcinoma thyroid (PTC) by combined of network pharmacology and experimental verification. By searching the TCMSP, SEA and SwissTarget Prediction database, the main active ingredients and related targets were obtained. Utilizing Venny 2.1.0 String database and Cytoscape 3.7.2 to screened the intersection target and constructed protein–protein interaction (PPI) network diagram. Using R 4.0.4 software carried out the enrichment analysis of GO and KEGG. HPLC was carried out using LC-20A modular HPLC system to identify the bioactive compound brucine present in SS. Molecular docking was performed using Discovery 2019 software. The inhibition rate was detected by CCK8 method. Western blot was used to detect the expression levels of brucine anti-PTC related pathway proteins. 14 active components were screened out, of which 4 main components showed tight relationship with PTC. SS may play the anti-PTC role by acting on two main pathways (TNF signaling pathway and MAPK signaling pathway) and mediating various biological functions. HPLC analysis revealed that brucine was a suitable marker for standardization of the SS. 4 active components exhibit strong binding energy with core protein. Brucine could significantly reduce the activity of BCPAP cells compared with isobrucine, stigmasterol, (+)-catechin. Brucine may reduce the protein expression levels of IL-6, VEGFA, JUN, TP53, 1L1B, PTGS2, BCL2, CASP3, CASP8, and CASP9 while increase the protein expression levels of BAD, cleaved-CASP3, cleaved-CASP8, and cleaved-CASP9 in BCPAP cells, respectively. The active components of SS against PTC mainly include isobrucine, stigmasterol, (+)-catechin, brucine. Among them, brucine exhibits the strongest anti-PTC activity in BCPAP cells, which may reduce the PTC-related protein expression levels. Therefore, SS may exhibits the anti-PTC activities through multiple targets and pathways.

Enantioselective Analysis of Lactate, Hydroxybutyrates and Malate in Human Physiological Fluids Using a Three-Dimensional HPLC System

Enantioselective Analysis of Lactate, Hydroxybutyrates and Malate in Human Physiological Fluids Using a Three-Dimensional HPLC System