High-performance Liquid Chromatography-tandem Mass Research Articles

Pharmacokinetics of cannabidiol-/cannabidiolic acid-rich hemp oil in juvenile cynomolgus macaques (Macaca fascicularis)

IntroductionCannabinoids are increasingly popular in human and veterinary medicine and have been studied as an alternative treatment for a wide range of disorders. The goal of this study was to perform a pharmacokinetic analysis of oral cannabidiol (CBD)-/cannabidiolic acid (CBDA)-rich hemp oil (CBD/ArHO) in juvenile cynomolgus macaques (Macaca fascicularis).MethodsAfter a 2 mg/kg CBD/ArHO pilot study, 4 and 8 mg/kg direct-to-mouth CBD/ArHO were administered (n = 4 per dose) once daily for 14 days and blood was collected at 0-, 0.5-, 1-, 2-, 4-, 8-, 12-, and 24-h, and on Days 7 and 14, to quantify serum cannabinoid concentrations by high-performance liquid chromatography–tandem mass spectrometry. Serum biochemistries and complete blood counts were performed on Days 0, 1, and 14.ResultsThe maximum mean serum concentration (Cmax) of CBDA was 28.6–36.2 times that of CBD at 4 and 8 mg/kg. At 8 mg/kg, the Cmax of CBD was 1.4 times higher (p = 0.0721), and CBDA was significantly 1.8 times higher (p = 0.0361), than at 4 mg/kg. The maximum mean serum concentration of ∆9-tetrahydrocannabinol (THC) was 4.80 ng/mL at 8 mg/kg. Changes in serum biochemistries and complete blood counts over time were not clinically significant.DiscussionGiven the low serum CBD concentrations, the doses and frequency used in this study may be insufficient for a therapeutic effect of CBD in particular; therefore, clinical studies are needed to determine the therapeutic dose of CBD and CBDA for macaques, which may differ based on the disorder targeted.

Proteomic analyses identify HK1 and ATP5A to be overexpressed in distant metastases of lung adenocarcinomas compared to matched primary tumors

Lung cancer is the leading cause of cancer-related deaths worldwide with lung adenocarcinoma (LUAD) being the most common type. Genomic studies of LUAD have advanced our understanding of its tumor biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD are still insufficiently explored. The prognosis for lung cancer patients is still mostly determined by the stage of disease at the time of diagnosis. Focusing on late-stage metastatic LUAD with poor prognosis, we compared the proteomic profiles of primary tumors and matched distant metastases to identify relevant and potentially druggable differences. We performed high-performance liquid chromatography (HPLC) and electrospray ionization tandem mass spectrometry (ESI–MS/MS) on a total of 38 FFPE (formalin‐fixed and paraffin‐embedded) samples. Using differential expression analysis and unsupervised clustering we identified several proteins that were differentially regulated in metastases compared to matched primary tumors. Selected proteins (HK1, ATP5A, SRI and ARHGDIB) were subjected to validation by immunoblotting. Thereby, significant differential expression could be confirmed for HK1 and ATP5A, both upregulated in metastases compared to matched primary tumors. Our findings give a better understanding of tumor progression and metastatic spreads in LUAD but also demonstrate considerable inter-individual heterogeneity on the proteomic level.

Synthesis of a core-shell magnetic covalent organic framework for the enrichment and detection of aflatoxin in food using HPLC-MS/MS.

A porous magnetic covalent organic framework, Fe3O4@TPBD-TPA (terephthalaldehyde (TPA) , N, N, N', N'-tetrakis(p-aminophenyl)-p-phenylenediamine (TPBD)), was synthesized using the Schiff base reaction under mild reactionconditions. This adsorbent exhibited excellent adsorption performance for aflatoxins. The adsorption capacity of Fe3O4@TPBD-TPA for aflatoxins ranged from 64.4 to 84.4 mg/g. A magnetic solid-phase extraction combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method based on Fe3O4@TPBD-TPA was developed for the efficient determination of four types of aflatoxins in food samples (maize, maize oil, peanut, and peanut oil). The determination coefficients (R2) were ≥0.9972. The method exhibited detection limits ranging from 0.01 to 0.06 μg/kg and spiked recoveries of 80.0 to 113.1%. The intra-day and inter-day precision were less than 6.77%, indicating good repeatability. The adsorbent showed promising prospects for the efficient enrichment of trace amounts of aflatoxins in complex food matrices.

TKI Dose Optimization Based on Therapeutic Drug Monitoring（TDM) with Encouraging Outcomes and Well-Tolerated for Chronic Myeloid Leukaemia Patients

Background Imatinib, nilotinib, and dasatinib are tyrosine kinase inhibitors (TKIs) that have become first-line treatments for Philadelphia chromosome-positive chronic myeloid leukemia (CML). Series studies indicated TKIs had a high therapeutic index, which led to the registration of these medications at fixed doses. Therapeutic drug monitoring (TDM) is a strategy for dosage optimization to provide individual treatment through dose adjustment to improve efficacy or avoid TKI-related adverse events (AEs). Objective We prospectively analyzed the outcome based on trough plasma concentrations (Cmin) to manage TKI dosage optimization during imatinib, nilotinib, and dasatinib therapy in patients with CML by 12 months. The primary efficacy endpoint was the rate of MMR at 12 months, the secondary endpoints included the cumulative rate of significant AEs defined as grade 3/4 fluid retention, all grade pleural effusion and pulmonary hypertension, grade 3/4 bilirubin increased, and grade 3/4 hematological toxicity related to TKIs within the first 12 months of therapy. Methods We examined 204 CML patients treated with imatinib, nilotinib, or dasatinib in the first line for CML-CP. We used high-performance liquid chromatography-tandem mass spectrometry (UPLC/MS-MS) to detect the Cmin of TKIs. Dasatinib or imatinib Cmin (24±3h post-dose) and nilotinib Cmin (12±3 h post-dose) were assessed after 7~10 days of therapy. We set the Cmin of imatinib at 1000~1500 ng/mL and that of nilotinib at 600~800 ng/mL, dasatinib at 1~3 ng/ml, respectively. Results Imatinib group (n = 67), Nilotinib group (n = 72), and Dasatinib group (n = 65), respectively. The initial dose of imatinib was 400mg qd, based on Cmin of imatinib was 900~1100ng/mL, there were 17 patients with dose reduction(1 case with 100mg qd, 5 cases with 200mg qd, 11 cases with 300mg qd) and 2 patients increased dose to 500mg qd; The initial dose of nilotinib was 300mg bid, based on Cmin of imatinib was 600~800ng/mL, there were 21 patients with dose reduction( 4 cases with 150mg bid, 16 cases with 450mg daily, 1 cases with 150mg qd) and 1 patients increased to 400mg bid; the initial dose of dasatinib was 100mg qd, based on Cmin of dasatinib was1~3 ng/ml, there were 32 patients with dose reduction(1 case with 20mg qd, 4 cases with 40mg qd, 20 cases with 50mg qd, 7 cases with 70mg qd ). Among the patients treated with imatinib, dasatinib, and nilotinib, no statistically significant associations were observed between gender, age, ELT scores, and plasma trough concentration (p>0.05). Early molecular reaction (EMR BCR: ABL(IS) <10%) was achieved by 83.6% (95% confidence interval [CI], 74.6%-92.5%) in the imatinib group, 87.7% (95% CI, 80.0%-95.4%) in the nilotinib group, and 83.3% (95% CI, 75.0%-91.7%) in the dasatinib group of patients at 3 months. The incidence of MMR at 12 months was 82.1% (95% CI, 73.1%-93.0%) in the imatinib group, 73.8% (95% CI, 64.6%-84.6%) in the nilotinib group, and 75.0% (95% CI, 65.3%-84.7%) in the dasatinib group, respectively. The cumulative incidence of grade 3/4 hematological toxicity was 7.8% (16/204), all-grade pleural effusion was 23.1% (15/65) in the dasatinib group, grade 3/4 bilirubin increased was 6.9% (5/72) in the nilotinib group, and there were no patients with pulmonary hypertension. Conclusion There are large individual differences in plasma trough concentrations in CML patients taking TKIs, the TKI dosage should be adjusted based on target plasma concentrations to maximize efficacy and minimize the incidence of adverse events.

The presumable effects of hydroxychloroquine and its metabolites in the treatment of systemic lupus erythematosus

ObjectiveHydroxychloroquine (HCQ) is an essential drug in the treatment of systemic lupus erythematosus (SLE). This study aimed to detect the concentrations of HCQ and its metabolites from peripheral blood of SLE patients and to investigate the relationship between those concentrations and SLE disease activity. Methods176 SLE patients treated with HCQ were enrolled in this study. The concentrations of HCQ and its metabolites in their peripheral blood were measured by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Patients’ disease activity was evaluated with the systemic lupus erythematosus disease activity index (SLEDAI). The variables between different concentrations or treatments were statistically analyzed. Linear regression was employed to explore relationships between the concentrations of HCQ and its metabolites with the disease activity. ResultsThe SLEDAI was lower in patients with higher concentrations of HCQ, desethylhydroxychloroquine (DHCQ), and desethylchloroquine (DCQ) (P = 0.024, P = 0.018, and P = 0.003, respectively). There were no significant differences in SLEDAI and the concentrations of HCQ and its metabolites among groups with different treatments (P > 0.05). After adjusting age, gender, disease duration, HCQ dose adjusted to actual body weight, and glucocorticoid (GC) dose, the SLEDAI was negatively correlated with the concentrations of HCQ, DHCQ, DCQ and bisdesethylchloroquine (BDCQ) (P = 0.007, P = 0.011, P = 0.029, and P = 0.008, respectively). After grouping analysis, in patients treated with HCQ and GC, the SLEDAI was negatively correlated with concentrations of HCQ, DHCQ and BDCQ (P = 0.011, P = 0.035, and P = 0.036, respectively). ConclusionsThe concentrations of HCQ and metabolites were correlated with the SLE disease activity after adjusting possible confounding factors, indicating that HCQ and its metabolites might play certain immunoregulatory roles in SLE treatment. Moreover, GC might have a synergistic effect with HCQ. It is helpful in clinical management and follow-up to monitor the concentrations of HCQ and its metabolites in SLE patients.

Extraction of Covid-19 drug (Favipiravir) from plasma samples by yolk-shell mesoporous silica before HPLC-MS/MS determination

In this work, a simple and inexpensive dispersive solid phase extraction method using SiO2 @MCM-41-Co3O4 yolk shell as a sorbent was developed for the extraction of favipiravir from plasma samples. The sorbent was synthesized with a simple and novel method. Optimization of the extraction procedure was performed using one parameter at a time strategy. For selective measurement of favipiravir in real samples, multiple reaction monitoring mode in high-performance liquid chromatography-tandem mass spectrometry was used. The synthesized sorbent presented a high adsorption capacity for favipiravir due to its mesoporous structure and different interactions. After optimization of effective parameters including the amount of sorbent, pH, and adsorption and desorption times, the analytical parameters of the method were evaluated. The developed method exhibited a wide linear range from 0.50 to 1000 μg/L. The detection limit and quantification limit of the method were 0.15 and 0.50 μg/L, respectively. The relative standard deviation of the method was obtained by using intra- and inter-day tests, and in both cases, it was less than 6.0%. Finally, the method was successfully used to measure favipiravir in plasma samples with relative recoveries in the range of 87–105%.

Simultaneous quantification method for multiple antiviral drugs in serum using isotope dilution liquid chromatography–tandem mass spectrometry

We describe the simultaneous quantification of six antiviral drugs in serum based on high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS). The target drugs—hydroxychloroquine, chloroquine, favipiravir, umifenovir, ritonavir, and lopinavir—were extracted and purified from serum with 75 % v/v methanol as the precipitant reagent. The six analytes were clearly separated within 15min using gradient elution and mixed-mode stationary phase. The measurement accuracy and precision were assured by adopting isotopes as internal standards. The optimized measurement procedure was strictly validated in linearity, sensitivity, accuracy, and precision. To confirm the robustness of the method in matrix, the method was additionally applied to various types of serum, namely hyperlipidemic and hyperglycemic serum. The method was then applied to assess the stability of the drugs in serum in order to set sample handling and storage guides for laboratory testing. Lastly, the method was implemented in different LC-MS systems to confirm its applicability across similar equipment commonly used in clinical testing laboratories. The overall results show that the optimized protocol is suitable for the accurate, simultaneous quantification of the six antiviral drugs in serum, and it is anticipated to satisfactorily serve as a reference protocol for the analysis of a wide range of other antiviral drugs for drug monitoring with various purposes.

Chemical investigation of biological trace evidence; toxicological screening of waste residues obtained from DNA extraction processes.

In a forensic scenario, if biological stains are found in very small quantities, these are usually collected for DNA analyses, considered essential for the forensic investigation and thus excluding possible investigations by other forensic disciplines as forensic toxicology.We developed an experimental study to evaluate the feasibility of analyzing DNA extraction residues obtained from DNA extraction procedures to perform toxicological analysis, with the aim to extract both genetic and toxicological information without affecting or compromising the genetic sample and/or DNA extraction.DNA extraction from four blood samples (fortified with 5 molecules of interest with a final concentrations of 1µg/mL, 100ng/mL, 10ng/mL and 5ng/mL, respectively) were analyzed with QIAGEN QIAmp® DNA Mini kit. Three waste residues collected from the DNA extraction were analyzed for the toxicological investigation via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). The analytical investigation revealed that our analytes of interest were detected in two different residues of the DNA extraction procedure, allowing both genetic and toxicological analyses without affecting the DNA identification. At last, the experimental protocol was applied to a hypothetical case, with encouraging results and allowing the identification of our molecules of interest.

Porcupine quills keratin peptides induces G0/G1 cell cycle arrest and apoptosis via p53/p21 pathway and caspase cascade reaction in MCF-7 breast cancer cells.

Porcupine quills, a by-product of porcupine pork, are rich in keratin, which is an excellent source of bioactive peptides. The objective of this study was to investigate the underlying mechanism of anti-proliferation effect of porcupine quills keratin peptides (PQKPs) on MCF-7 cells. Results showed that PQKPs induced MCF-7 cells apoptosis by significantly decreasing the secretion level of anti-apoptosis protein Bcl-2 and increasing the secretion levels of pro-apoptosis proteins Bax, cytochrome c, caspase 9, caspase 3 and PARP. PQKPs also arrested the cell cycle at G0/G1 phase via remarkably reducing the protein levels of CDK4 and enhancing the protein levels of p53 and p21. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis identified nine peptides with molecular weights less than 1000 Da in PQKPs. Molecular docking results showed that TPGPPT and KGPAC identified from PQKPs could bind with p53 mutant and Bcl-2 protein by conventional hydrogen bonds, carbon hydrogen bonds and van der Waals force. Furthermore, the anti-proliferation impact of synthesized peptides (TPGPPT and KGPAC) was shown in MCF-7 cells. These findings indicated that PQKPs suppressed the proliferation of MCF-7 breast cancer cells by triggering apoptosis and G0/G1 cell cycle arrest. Moreover, the outcome of this study will bring fresh insights into the production and application of animal byproducts. © 2023 Society of Chemical Industry.

Quantification of nine bufadienolides of Shexiang Tongxin Dropping Pills in rat plasma and tissues using UPLC-MS/MS and its application to healthy and ischemia-reperfusion rats pharmacokinetic studies

Shexiang Tongxin Dropping Pill (STDP) is a well-known compound preparation used in traditional Chinese medicine for treating cardiovascular diseases. Bufadienolides are the major active compounds of toad venom and are the key to the seven medicinal herbs that comprise STDP. In this study, a reliable and sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantitative determination of nine bufadienolides (bufalin, gamabufotalin, resibufogenin, marinobufagin, arenobufagin, desacetylcinobufagin, telocinobufagin, hellebrigenin, and hellebrigenol) in rat plasma and tissues (heart and liver). The chromatography column used was a Waters ACQUITY UPLC HSS-T3 column with gradient elution using mobile phase consisting of acetonitrile–water (0.1% formic acid added) at a flow rate of 0.25 mL/min. This method passed the methodological validation of plasma and tissues and was successfully applied to pharmacokinetic and tissue distribution studies after oral administration of STDP in healthy and ischemia-reperfusion (I/R) rats. This indicated that most bufadienolides were well absorbed and quickly distributed in the heart and liver. The area-under-the-curve (AUC)(0−t) of most analytes increased in a dose-dependent manner. Moreover, most of the tested components showed lower plasma and higher tissue concentrations in I/R rats than in healthy rats. The above results on the oral pharmacokinetics and tissue distribution may be helpful for the clinical application of STDP.

Carbonized aerogel/ZnO-based dispersive solid phase extraction of daclatasvir and sofosbuvir from biological samples prior to liquid chromatography–tandem mass spectrometry

Daclatasvir and sofosbuvir are antiviral medications utilized in the treatment of chronic hepatitis C virus (HCV) infection. Due to their low therapeutic index, careful monitoring is necessary to ensure that the optimal dose is administered. High-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) is an exceptionally sensitive and specific technique for quantifying these drugs within biological matrices. In this study, we developed a novel dispersive solid-phase extraction method employing a carbonized bio aerogel composite with ZnO for efficient extraction of daclatasvir and sofosbuvir from exhaled breath condensate, urine, and plasma samples. The extracted analytes were subsequently subjected to analysis using HPLC–MS/MS. Optimal parameters including pH adjustment, sorbent quantity variation, and elution solvent selection were fine-tuned to achieve maximum recovery efficiency while ensuring selectivity enhancements. The developed method demonstrated broad linearity ranging between 1.2 and 200 ng/mL along with good precision (relative standard deviations ≤6.2 %) and an acceptable coefficient of determination (r2 ≥0.994). These findings establish our proposed method as suitable for reliable drug quantification in clinical samples.

Bimetallic metal–organic framework as an efficient sorbent for the extraction of tacrolimus and cyclosporine from plasma before HPLC-MS/MS analysis

This study investigated the application of pores-size protocols into metal–organic frameworks that can lead to improved adsorption capacity of sorbents. A selective, fast, and sensitive analytical procedure based on dispersive solid phase extraction using a bimetallic metal organic framework named ZrZnMOF coupled with high-performance liquid chromatography-tandem mass spectrometry was established for the simultaneous quantification of tacrolimus and cyclosporine in plasma. The effects of several parameters, such as the dosage of sorbent, separation and elution times, volume and type of elution solvent, were investigated. Under optimal conditions, high extraction recovery (70 and 89 for tacrolimus and cyclosporine, respectively), low limits of detection (0.15 and 0.31 μg L-1 for tacrolimus and cyclosporine, respectively) and quantification (1.0 and 0.51 μg L-1 for tacrolimus and cyclosporine, respectively) wide linearity (1.0–1000 and 0.51–500 μg L-1 for tacrolimus and cyclosporine, respectively), and acceptable repeatability (n = 5, relative standard deviation less than 5.6 %) were attained. The validated method was successfully applied to quantify tacrolimus and cyclosporine A in plasma samples obtained from different volunteers.

Tear Tacrolimus Levels and Clinical Response After Adjunct Therapy With Cutaneous Application of Tacrolimus 0.1% Over Upper Eyelid Skin in Chronic Vernal Keratoconjunctivitis.

The aim of this study was to evaluate the role of cutaneous application of 0.1% tacrolimus eye ointment over the skin of the upper eyelid in chronic vernal keratoconjunctivitis (VKC). A prospective, longitudinal, noncomparative, open-label clinical study of moderate-to-severe grade steroid-dependent VKC was performed. Study participants were initiated on adjunct therapy of cutaneous application of 0.1% tacrolimus ointment twice daily on the upper eyelid skin. Ocular surface evaluation parameters, meibomian gland imaging, intraocular pressure, visual acuity, and clinical disease severity scoring were performed to assess clinical response at baseline and month 3 of therapy. Tear levels of tacrolimus were measured at month 3 using high-performance liquid chromatography tandem mass spectrometry and correlated with the clinical score. Palpebral form of VKC was observed in 85% of the cases, with positive family history in 5%, atopy in 7.5%, and keratoconus in 11.25%. Clinical assessment revealed improvement in 97.5% patients with discontinuation of concomitant topical steroids in 64% of patients. There were no changes in visual acuity, intraocular pressure, or ocular surface evaluation after therapy. Tacrolimus was detected in the tears of all our study patients after cutaneous application over the upper eyelid skin, proving its bioavailability with mean tear tacrolimus levels of 6.55 ± 21.43 ng/mL. Correlation analysis revealed a moderate negative correlation between the clinical score and tacrolimus concentration (Spearman correlation coefficient: -0.34, P = 0.002). Cutaneous tacrolimus 0.1% ointment over the upper eyelid skin is an efficacious alternative method of application in treatment of VKC, with no resultant ocular irritation.

Study of the pharmacokinetic profile of the medicinal product Gidazepam®, tablets, 50 mg in a bioequivalence study

Aim. The primary aim of this study was to evaluate the pharmacokinetic parameters and confirm the bioequivalence of drugs containing gidazepam, namely Gidazepam® (Valenta Pharm, Russia) and Gidazepam VIC (VIVA Pharm, Republic of Kazakhstan), after a single administration of 1 tablet (50 mg) to healthy volunteers under fasting conditions. The secondary aim was a comparative analysis of safety profiles (adverse events) after a single administration of the studied drugs.Materials and methods. An open, randomized, crossover, two-period comparative study of pharmacokinetics and bioequivalence with adaptive design was conducted in healthy volunteers. Blood sampling was performed 15 minutes before and 20 min, 40 min, 1 h, 2 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h after drug administration. High-performance liquid chromatography-tandem mass spectrometry was used for the evaluation of gidazepam and its metabolite (desalkylgidazepam) concentration with the subsequent calculation of pharmacokinetic parameters.Results. From both formulations, gidazepam was quickly absorbed and biotransformed into an active metabolite. Studied drugs had similar pharmacokinetic profiles, as 90% confidence intervals for the ratio of geometric means for Cmax and AUC(0-72) were within the bioequivalence acceptance range of 80.00–125.00 %. No adverse events were recorded as a result of clinical, laboratory or instrument evaluations during the study.Conclusion. Study drugs are considered bioequivalent and show comparable tolerability after a single administration under fasting conditions.

Modulating intestinal barrier function by sphingosine-1-phosphate receptor 1 specific agonist SEW2871 attenuated ANIT-induced cholestatic hepatitis via the gut-liver axis

Bile acid (BA) homeostasis throughout the enterohepatic circulation system is a guarantee of liver physiological functions. BA circulation disorders is one of the characteristic clinical manifestations of cholestasis, and have a closely relationship with intestinal barrier function, especially ileum. Here, our in vivo and in vitro studies showed that intestinal tight junctions (TJs) were disrupted by α-naphthylisothiocyanate (ANIT), which also down-regulated the protein expression of sphingosine-1-phosphate receptor 1 (S1PR1) in the top of villus of mice ileum. Activating S1PR1 by specific agonist SEW2871 could improve TJs via inhibiting ERK1/2/LKB1/AMPK signaling pathway in the ileum of ANIT-treated mice and ANIT-cultured Caco-2 cells. SEW2871 not only regained ileum TJs by activating S1PR1 in the epithelial cells of ileum mucosa, but also recovered ileum barrier function, which was further verified by the recovered BA homeostasis in mice ileum (content and tissue) by using of high-performance liquid chromatographytandem mass spectrometry (LC-MS/MS). Subsequently, the improved intestinal injury and inflammation further strengthened that SEW2871 modulated intestinal barrier function in ANIT-treated mice. Finally, our data revealed that along with the down-regulated levels of serum lipopolysaccharides (LPS), SEW2871 improved liver function and relieved hepatitis via blocking TLR4/MyD88/NF-kB signaling pathway in ANIT-treated mice. In conclusion, these results demonstrated that activating intestinal S1PR1 by SEW2871 could modulate intestinal barrier function, leading to the improvement of cholestatic hepatitis in ANIT-treated mice via the “gut-liver” axis.

Quantification and characterization of biological activities of glansreginin A in black walnuts (Juglans nigra).

Glansreginin A has been reported to be an indicator of the quality of walnuts (Juglans spp.). However, bioactive properties of glansreginin A have not been adequately explored. In the present study, we quantified concentrations of glansreginin A in black walnuts (Juglans nigra) using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed an array of in vitro bioassays to characterize biological activities (e.g., antibacterial, antioxidant, anticancer capacities) of this compound. Results from HPLC-MS/MS analysis indicated that glansreginin A was presented in all 12 black cultivars examined and its contents were variable among black walnut cultivars, ranged from 6.8mg/kg (Jackson) to 47.0mg/kg (Hay). Glansreginin A possessed moderate antibacterial activities against Gram-positive pathogens (Staphylococcus aureus and Bacillus anthracis). This compound exhibited no antioxidant activities, did not induce the activity of antioxidant response element signaling pathways, and exerted no antiproliferative effects on tumorigenic alveolar epithelial cells and non-tumorigenic lung fibroblast cells.

Determination of amanita peptide and tryptamine toxins in wild mushrooms by high performance liquid chromatography-tandem mass spectrometry

The discovery and identification of mushroom toxins has long been an important area in the fields of toxicology and food safety. Mushrooms are widely favored for their culinary and medicinal value; however, the presence of potentially lethal toxins in some species poses a substantial challenge in ensuring their safe consumption. Therefore, the development of a robust and sensitive analytical method is necessary for accurately identifying the risks associated with mushroom consumption. The study of mushroom toxins, which are characterized by their diversity and substantial variations in chemical structures, present a considerable challenge for achieving precise and high-throughput analysis. To address this issue, the present study employed a robust approach combining a solid-phase extraction (SPE) purification technique with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to establish an analytical method for the detection and quantification of five amatoxins and two tryptamines (psilocybin and bufotenine) present in some mushrooms. Several optimization procedures were undertaken, including optimizing the chromatographic conditions, mass spectrometric parameters, and sample extraction and purification. The procedure involved the extraction of dry mushroom powder with methanol containing 0.3% formic acid, followed by purification using a strong cation exchange cartridge (SCX). The analytes were separated on a T3 chromatographic column (100 mm×2.1 mm, 1.8 μm) using mobile phases of acetonitrile and 5 mmol/L ammonium acetate solution containing 0.1% formic acid. The multiple reaction monitoring (MRM) mode was employed for data acquisition. Amatoxins were quantified using matrix-matched standard calibration curves, whereas isotopic internal standards were used to quantify tryptamine. The results showed that all seven toxins exhibited good linearities (r2>0.99) within the optimized concentration range. The limits of detection (LODs) for bufotenine, psilocybin, and amatoxins were determined as 2.0, 5.0, and 10 μg/kg, respectively, while the limits of quantification (LOQs) were determined as 5.0, 10, and 20 μg/kg, respectively. The LOD and LOQ values further underscore the ability of the method to detect minute quantities of toxins, making it particularly well suited for screening food samples for potential contamination. Using dried shiitake mushroom powder as the matrix, the recoveries of the two tryptamines ranged from 80.6% to 117%, with relative standard deviations (RSDs) ranging from 1.73% to 5.98%, while the recoveries of amatoxins ranged from 71.8% to 115%, with RSDs varying from 2.14% to 9.92% at the three concentration levels. The consistent and satisfactory recoveries of amatoxins and tryptamines demonstrated the ability of this method to accurately quantify the target analytes even in a complex matrix. Comparison with the results of supplementary test method recognized by State Administration for Market Regulation for food (BJS 202008) demonstrated comparable results, indicating no significant differences (p>0.05) in amatoxin contents. The newly developed method is rapid, accurate, precise, meets the required standards, and is suitable for the detection of seven toxins in wild mushrooms. As part of the application of this method, a comprehensive investigation of the distribution of toxins in wild mushrooms from Fujian Province was undertaken. In this study, 59 wild mushroom samples from nine cities were collected in the Fujian province. Species identification was conducted using rDNA-internal transcribed space (rDNA-ITS) molecular barcode technology, which revealed the presence of toxins in the two samples. Notably, one specimen named Amanita fuligineoides contained α-amanitin, β-amanitin, and phalloidin in quantities of 607, 377, and 69.0 mg/kg, respectively. Additionally, another sample, identified as Tricholomataceae, had a psilocybin concentration of 12.6 mg/kg.

Yokukansan, a Traditional Japanese Medicine, Suppresses Stress-Induced Sympathetic Activation via Central Responses in Rats

Objectives: Yokukansan (YKS) is a traditional Japanese Kampo medicine approved by the Ministry of Health, Labour and Welfare of Japan. Recently, in addition to its indications as a drug treatment, YKS has been shown to have ameliorative effects on various psychological and behavioral responses such as anxiety, aggression, and stress responses. Stress exposure activates not only the hypothalamic–pituitary–adrenal axis but also the sympathetic nervous system. Sympathetic activation in brain regions such as the paraventricular hypothalamic nucleus (PVN) stimulates elevation of plasma catecholamine (noradrenaline and adrenaline) levels. We previously reported that various stress-related neuropeptides and several kinds of stressors, such as restraint stress (RS), increase plasma catecholamine and that brain prostanoids and their synthases mediate these responses in rats. In the present study, to determine if YKS treatment can affect stress-induced sympathetic activation, we examined the effects of YKS treatment on the RS-induced elevation of plasma catecholamine levels and related prostanoid production in the PVN of rats. Methods: YKS (1000 mg/10 mL/kg, p.o.) or vehicle was administered to rats daily for 14 days, and all rats were exposed to RS for 60 min on day 14. Before and during stress exposure on day 14, blood samples and PVN dialysates were collected and analyzed by high-performance liquid chromatography and liquid chromatography-ion trap tandem mass spectrometry, respectively. Results: Our results showed that repeated administration of YKS suppressed the RS-induced increase in plasma adrenaline but not noradrenaline. Furthermore, YKS administration also suppressed the RS-induced elevation of both prostaglandin E2 and thromboxane B2 levels in the PVN. In addition, we found that repeated administration of YKS suppressed the RS-induced increase in serotonin, gamma-aminobutyric acid, and acetylcholine in the PVN. Conclusion: Our results suggest that YKS can ameliorate stress-induced sympathetic activation via inhibition of stress responses in the brain.

The Extraction Process, Separation, and Identification of Curcuminoids from Turmeric Curcuma longa.

Turmeric Curcuma longa is a well-known spice with various health benefits, attributed primarily to curcumin. Soxhlet extraction, cold maceration, ultrasound-assisted extraction (UAE), and supercritical fluid extraction were performed, and the content of total phenols, proanthocyanidins, and antioxidants was analysed by UV/VIS spectrophotometry. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was employed to identify and quantify the curcumin content. Supercritical extracts had the highest total phenolic content (538.95 mg GA/100 g material), while the Soxhlet extracts had the highest content of proanthocyanidins (4.77 mg PAC/100 g of material). Extracts obtained by UAE and supercritical extraction have the highest antioxidant potential. Antioxidant activity measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH•) was 64.27% and 1750.32 mg Trolox per g dry weight by 2,2-azinobis(3-ethylbenzothiazoline 6 sulphonic acid) (ABTS+•) for the extract obtained by supercritical extraction. The UAE resulted in the highest amount of curcumin (1.91 mg curcumin/g material). A kinetic study showed that extraction yield in supercritical extracts decreased with increasing temperature and that the content of isolated curcumin was inversely proportional to solvent-to-feed ratio (S/F). The present study has confirmed that turmeric is an excellent source of antioxidants, such as curcumin, that play an important role in reducing cellular stress by neutralising free radicals.

Fusarium bilaiae, a new cryptic species in the Fusarium fujikuroi complex associated with sunflower

ABSTRACT A Fusarium species associated with sunflower based on multilocus genealogy, morphological, physiological, ecological, mating type, and mycotoxin production data is formally described as the newly discovered species Fusarium bilaiae. The F. bilaiae strains formed a genealogically exclusive lineage within the African clade of the F. fujikuroi species complex. Comparison of morphological characteristics of F. bilaiae strains with those of the closely related F. phyllophilum strain NRRL 13617 revealed similarities in the main micromorphology of both species: production of numerous one-celled microconidia in false heads and short chains on monophialides and polyphialides and the absence of macroconidia and sporodochia. There was a slight but significant distinction between the two species when the strains were grown on different agar media, as well as in the shape and width of microconidia. Fusarium bilaiae strains isolated from symptomatic sunflower were not pathogenic to members of the Asteraceae tested; apparently, they live as saprophytes or endophytes in sunflower tissues. A difference between the strains of the two species in the production of mycotoxins was demonstrated with high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) analysis. On autoclaved rice, F. bilaiae did not produce fumonisins and beauvericin but produced moniliformin, whereas F. phyllophilum produced all these mycotoxins. A polymerase chain reaction (PCR) assay specific for mating type alleles identified F. bilaiae as a putative heterothallic species with MAT1-1 and MAT1-2 idiomorphs, but laboratory crosses were unsuccessful. Determining the area and host range of the new endophytic species F. bilaiae is a priority for future research.