Plasma Extracts Research Articles

Introduction of an Innovative approach for Bioanalytical Method Development and Validation of Febuxostat by using LC-ESI-MS/MS in Human Plasma

The aim of the present work is to develop and validate an accurate, sensitive, rapid, precise, and simple bioanalytical method for estimation of Febuxostat in human plasma by using LC-ESI-MS/MS. The method was developed by gradient elution technique with the combination of the mobile phase as 0.1% Formic Acid in Milli-Q water and 0.1% Formic Acid in Acetonitrile at a flow rate of 0.5ml/min. The Analyte and IS (Tolbutamide) were separated by a C18 Phenomenex Kinetex (50x3mm, 5µ) column. The chromatographic run time was 7.0 minutes. The plasma extraction was done by a simple protein precipitation technique (PPT). The LOD and LLOQ were found to be 6.25ng/ml and 125ng/ml, respectively. The extraction recovery of the drug from plasma was more than 90%. The validation parameters were found within the range, as mentioned by USFDA and EMA guidelines.

Single Quadrupole Multiple Fragment Ion Monitoring Quantitative Mass Spectrometry.

Single quadrupole mass spectrometry (MS) with enhanced in-source multiple fragment ion monitoring was designed to perform high sensitivity quantitative mass analyses. Enhanced in-source fragmentation amplifies fragmentation from traditional soft electrospray ionization producing fragment ions that have been found to be identical to those generated in tandem MS. We have combined enhanced in-source fragmentation data with criteria established by the European Union Commission Directive 2002/657/EC for electron ionization single quadrupole quantitative analysis to perform quantitative analyses. These experiments were performed on multiple types of complex samples that included a mixture of 50 standards, as well as cell and plasma extracts. The dynamic range for these quantitative analyses was comparable to triple quadrupole multiple reaction monitoring (MRM) analyses at up to 5 orders of magnitude with the cell and plasma extracts showing similar matrix effects across both platforms. Amino acid and fatty acid measurements performed from certified NIST 1950 plasma with isotopically labeled standards demonstrated accuracy in the range of 91-110% for the amino acids, 76-129% for the fatty acids, and good precision (coefficient of variation <10%). To enhance specificity, a newly developed correlated ion monitoring algorithm was designed to facilitate these analyses. This algorithm autonomously processes, aligns, filters, and compiles multiple ions within one chromatogram enabling both precursor and in-source fragment ions to be correlated within a single chromatogram, also enabling the detection of coeluting species based on precursor and fragment ion ratios. Single quadrupole instrumentation can provide MRM level quantitative performance by monitoring/correlating precursor and fragment ions facilitating high sensitivity analysis on existing single quadrupole instrumentation that are generally inexpensive, easy to operate, and technically less complex.

Prevalence of occult hepatitis C virus infection in beta-thalassemia major patients in Ahvaz, Iran.

Occult hepatitis C virus infection (OCI) is defined by the presence of HCV RNA in peripheral blood mononuclear cells (PBMCs) and liver tissue cells despite the absence of HCV RNA in plasma. Currently, OCI is classified into two types: seropositive OCI (anti-HCV positive and serum HCV RNA negative) and seronegative OCI (anti-HCV and serum HCV RNA negative). Beta-thalassemia is described as a blood disorder that decreases the synthesis of hemoglobin. Repeated blood transfusion is the standard treatment for patients with beta-thalassemia major (BTM), and this increases the risk of exposure to infectious agents. The aim of this study was to investigate the prevalence of OCI among BTM patients. Plasma and PBMCs were collected from 90 BTM patients who were referred to Shafa Hospital in the city of Ahvaz and were screened for HCV antibody using a commercial ELISA kit as the first step. Next, nested RT-PCR was performed on extracts of plasma and PBMCs. HCV RNA from positive PBMCs was sequenced, the sequences were aligned, and a phylogenetic tree was constructed to determine their relationship to reference sequences retrieved from the GenBank database. Seventy-nine out of 90 patients (87.8%) were negative for HCV Ab (seronegative), while 11 patients (12.2%) were seropositive. HCV RNA was found in PBMCs of four patients (66.7%) who were negative for HCV Ab (seronegative) and two patients (33.3%) who were positive for HCV Ab (seropositive). HCV RNA was not detected in plasma samples from these six patients. Six out of 90 BTM patients (6.7%) had OCI. HCV genotyping revealed that all six patients were infected with HCV subtype 3a. We found a high frequency of OCI in BTM patients, which warrants more attention, considering the importance of this infection. Further studies are needed to determine the actual prevalence of OCI in BTM patients in Iran.

Hydroxychloroquine serum concentration in coronavirus disease 2019 (COVID-19) patients: a retrospective study

Algeria has adopted a therapeutic protocol using hydroxychloroquine (HCQ) as a first-line treatment for patients with coronavirus disease 2019 (COVID-19). The administration of HCQ must be accompanied by appropriate cardiac and therapeutic pharmacological monitoring to avoid side effects and toxicity. This work is a retrospective descriptive study aiming to estimate the HCQ levels of COVID-19 patients. HCQ concentrations in blood samples from COVID-19 patients were determined using high performance liquid chromatography with diode array detector (HPLC-DAD) after liquid-liquid extraction of plasma. The coefficient of determination (r 2) of this method was 0.9999. The limits of quantification and detection were 0.05 mg/L and 0.02 mg/L, respectively, with coefficients of variance less than 6%. Patient monitoring was performed following recommendations for the therapeutic pharmacological monitoring of HCQ in patients treated for SARS-CoV-2 (COVID-19) infection validated by the coordinated action (AC43) of the National Agency for Research on AIDS and Viral Hepatitis (ANRS) and Therapeutic Pharmacological Monitoring and Personalization of Treatments (STP-PT) group. A total of 267 blood samples from 240 patients were analyzed, 49% for males and 51% for females. More than a third of the patients were 60–80 years old. The majority (68%) of the HCQ concentrations were between 0.1 and 1 mg/L. Additionally, more than 20% were between 0.05 and 0.1 mg/L, while only 7.5% were less than 0.05mg/L and 1.5% were greater than 1 mg/L. In summary, patients with COVID-19 showed a good tolerance to HCQ in the majority of cases. Key Points HCQ was proposed to be a promised treatement for COVID-19. A reverse phase HPLC-DAD method was validated for HCQ concentration determination in plasma. Patients hospitalized for COVID-19 were monitored according to the guideline established by the French National Team AC43 of the ANRS and the STP-PT group. Patients showed a very good tolerance to HCQ with no observed cases of toxicity.

Prolonged efavirenz exposure reduces peripheral oxytocin and vasopressin comparable to known drugs of addiction in male Sprague Dawley rats.

IntroductionSeveral drugs of abuse (DOA) are capable of modulating neurohypophysial hormones, such as oxytocin (OT) and vasopressin (VP), potentially resulting in the development of psychological abnormalities, such as cognitive dysfunction, psychoses, and affective disorders. Efavirenz (EFV), widely used in Africa and globally to treat HIV, induces diverse neuropsychiatric side effects while its abuse has become a global concern. The actions of EFV may involve neurohypophysial system (NS) disruption like that of known DOA. This study investigated whether sub-chronic EFV exposure, at a previously-determined rewarding dose, alters peripheral OT and VP levels versus that of a control, ∆9-tetrahydrocannabinol (∆9-THC), methamphetamine (MA) and cocaine.Materials and methodsTo simulate the conditions under which reward-driven behavior had previously been established for EFV, male Sprague Dawley rats (n = 16/exposure) received intraperitoneal vehicle (control) or drug administration across an alternating sixteen-day dosing protocol. Control administration (saline/olive oil; 0.2 ml) occurred on odd-numbered and drug administration (EFV: 5 mg/kg, ∆9-THC: 0.75 mg/kg, MA: 1 mg/kg, or cocaine: 20 mg/kg) on even-numbered days followed by euthanasia, trunk blood collection and plasma extraction for neuropeptide assay. Effect of drug exposure on peripheral OT and VP levels was assessed versus controls and quantified using specific ELISA kits. Statistical significance was determined by Kruskal-Wallis ANOVA, with p < 0.05. Ethics approval: NWU-00291-17-A5.ResultsDelta-9-THC reduced OT and VP plasma levels (p < 0.0001, p = 0.0141; respectively), cocaine reduced plasma OT (p = 0.0023), while MA reduced plasma VP levels (p = 0.0001), all versus control. EFV reduced OT and VP plasma levels (p < 0.0001; OT and VP) versus control, and similar to ∆9-THC.ConclusionEFV markedly affects the NS in significantly reducing both plasma OT and VP equivalent to DOA. Importantly, EFV has distinct effects on peripheral OT and VP levels when assessed within the context of drug dependence. The data highlights a possible new mechanism underlying previously documented EFV-induced effects in rats, and whereby EFV may induce neuropsychiatric adverse effects clinically; also providing a deeper understanding of the suggested abuse-potential of EFV.

The network pharmacology integrated with pharmacokinetics to clarify the pharmacological mechanism of absorbed components from Viticis fructus extract

Ethnopharmacological relevanceViticis fructus (VF) has been widely used in alleviating the swelling and pain, owning to its pharmacologically active components including agnuside, 10-O-vanilloylaucubin, luteolin and casticin. Aim of the studyThe pharmacokinetic profiles of the absorbed components from aqueous and ethanolic extracts of VF in rat plasma were performed, and explored the molecular mechanisms of absorbed components via network pharmacology. Materials and methodsUltra-performance liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS) was employed to identify the absorbed components from rat plasma. Liquid-liquid extraction with ethyl acetate was used to purify the plasma samples. Plasma pharmacokinetics parameters of the components absorbed were analyzed after oral administration of both extracts. Network pharmacology was used to predict the biological functions and potential signaling pathways of VF. The anti-cancer effects of VF extract and absorbed components have been confirmed by in vitro experiments. ResultsThe method was very sensitive with lower limit of quantification (LLOQ) of 1.0, 2.5, 0.2 and 0.5 ng/mL for agnuside, 10-O-vanilloylaucubin, luteolin and casticin, respectively. With the exception of 10-O-vanilloylaucubin which was not detected in the ethanolic extract of VF, all other components were detected in both extracts in plasma. The pharmacokinetic parameters of the four components from rat plasma were significantly different between the two extracts. According to the results of network pharmacology, the absorption components of VF are enriched in 32 key pathways, and 15 pathways are related to cancer. Ultimately, the anti-cancer effects, as well as the signaling pathways of VF ethanolic extract and absorbed components were verified by in vitro experiments. ConclusionThe optimized, sensitive and validated UHPLC-MS/MS method was successfully applied for the plasma pharmacokinetics comparison analysis of the two VF extracts. The combination of network pharmacology and pharmacokinetics provides a useful method to elucidate the biological effects and molecular mechanism of the absorbed components of VF.

Cytokine and interleukin profile in patients with headache and COVID-19: A pilot, CASE-control, study on 104 patients

BackgroundThe presence of headache during the acute phase of COVID-19 could be associated with the innate response and the cytokine release. We aim to compare the cytokine and interleukin profile in hospitalized COVID-19 patients at the moment of admission with and without headache during the course of the disease.MethodsAn observational analytic study with a case control design was performed. Hospitalized patients from a tertiary hospital with confirmed COVID-19 disease were included. Patients were classified into the headache or the control group depending on whether they presented headache not better accounted for by another headache disorder other than acute headache attributed to systemic viral infection. Several demographic and clinical variables were studies in both groups. We determined the plasmatic levels of 45 different cytokines and interleukins from the first hospitalization plasma extraction in both groups.ResultsOne hundred and four patients were included in the study, aged 67.4 (12.8), 43.3% female. Among them, 29 (27.9%) had headache. Patients with headache were younger (61.8 vs. 69.5 years, p = 0.005) and had higher frequency of fever (96.6 vs. 78.7%, p = 0.036) and anosmia (48.3% vs. 22.7%, p = 0.016). In the comparison of the crude median values of cytokines, many cytokines were different between both groups. In the comparison of the central and dispersion parameters between the two groups, GROa, IL-10, IL1RA, IL-21, IL-22 remained statistically significant. After adjusting the values for age, sex, baseline situation and COVID-19 severity, IL-10 remained statistically significant (3.3 vs. 2.2 ng/dL, p = 0.042), with a trend towards significance in IL-23 (11.9 vs. 8.6 ng/dL, p = 0.082) and PIGF1 (1621.8 vs. 110.6 ng/dL, p = 0.071).ConclusionsThe higher levels of IL-10 -an anti-inflammatory cytokine- found in our sample in patients with headache may be explained as a counteract of cytokine release, reflecting a more intense immune response in these patients.

Blood Plasma Separation: Enhanced Diamagnetic Repulsion of Blood Cells Enables Versatile Plasma Separation for Biomarker Analysis in Blood (Small 23/2021)

In article 2100797, Joo H. Kang, and co-authors present a new blood plasma extraction device using augmented diamagnetic repulsion of blood cells by supplementing superparamagnetic iron oxide nanoparticles to whole blood and demonstrate its utility for various biomarker analysis, such as prostate-specific antigen, platelets, exosomes, and nucleic acids, in whole blood without prior blood plasma separation.

Bile Salt and FGF19 Signaling in the Early Phase of Human Liver Regeneration.

The involvement of bile salt–fibroblast growth factor 19 (FGF19) signaling in human liver regeneration (LR) is not well studied. Therefore, we studied aspects of bile salt–FGF19 signaling shortly after liver resection in patients. We compared plasma bile salt and FGF19 levels in arterial, portal and hepatic venous blood, calculated venous‐arterial differences (ΔVA), and determined hepatic transcript levels on two intra‐operative time points: before (< 1 hour) and immediately after (> 2‐3 hours) liver resection (i.e., following surgery). Postoperative bile salt and FGF19 levels were assessed on days 1, 2, and 3. LR was studied by computed tomography (CT)–liver volumetry. Following surgery, the liver, arterial, and portal bile salt levels were elevated (P < 0.05). Furthermore, an increased amount of bile salts was released in portal blood and extracted by the remnant liver (P < 0.05). Postoperatively, bile salt levels were elevated from day 1 onward (P < 0.001). For FGF19, intra‐operative or postoperative changes of ΔVA or plasma levels were not observed. The bile salt–homeostatic regulator farnesoid X receptor (FXR) was markedly up‐regulated following surgery (P < 0.001). Cell‐cycle re‐entry priming factors (interleukin 6 [IL‐6], signal transducer and activator of transcription 3 [STAT3], and cJUN) were up‐regulated following surgery and were positively correlated with FXR expression (P < 0.05). Postoperative hyperbilirubinemia was preceded by postsurgery low FXR and high Na+/Taurocholate cotransporting polypeptide (NTCP) expression in the remnant liver coupled with higher liver bile salt content (P < 0.05). Finally, bile salt levels on postoperative day 1 were an independent predictor of LR (P < 0.05). Conclusion: Systemic, portal, and liver bile salt levels are rapidly elevated after liver resection. Postoperative bile salts were positively associated with liver volume gain. In the studied time frame, FGF19 levels remained unaltered, suggesting that FGF19 plays a minor role in human LR. These findings indicate a more relevant role of bile salts in human LR.

Disruption of Estrogenic and Androgenic Bioactivities in Human Fetuses Exposed to Maternal Smoking

Endocrine disruptors (EDs) interfere with hormonal signalling and, given that multiple developmental processes are hormone-driven, the prenatal period is a window of increased sensitivity. Maternal smoking is a real-life model of in utero exposure to a complex mixture of EDs. Cigarette smoke contains of >7,000 pollutants, including polycyclic aromatic hydrocarbons (PAHs), which are AhR ligands and cross-talk with the estrogen receptor (ER) system. Prenatal exposure to cigarette smoke is associated with adverse outcomes, including intrauterine growth restriction and increased risk of metabolic syndrome later in life. We aimed to evaluate ED effects associated with smoke exposure in human fetuses. Fetal tissues (plasma, n=48; placenta, n=30; liver, n=29) from elective terminations of normally progressing pregnancies, ranging from 10 to 20 gestation weeks, were collected (SAFeR and FEGO studies: REC 15/NS/0123, REC 04/S0802/21). PAHs and PAH-like compounds were extracted from placenta and fetal liver. Bioactivity levels in plasma, placenta and liver extracts were determined using ER and androgen receptor (AR) transactivation reporter gene assays. PAH burden was evaluated using the AhR-responsive DRhp-CALUX assay. Smoke exposure was associated with a 1.3-fold increase in plasma estrogenic activity. The developmental trajectory of androgenic activity was altered in plasma of smoke-exposed fetuses, with significant anti-androgenic activity in older fetuses (>16 weeks of gestation). In males, plasma androgenic activity was positively associated with testes weight and anogenital distance. In contrast, placentas from smoking mothers had significantly increased androgenic potential. Furthermore, AhR-like activity was 2.9-fold higher in smoke-exposed placentas compared to controls, and 2.3-fold higher in female compared to male fetal livers. Overall, all bioactivity levels were higher in placentas compared to fetal liver. Prenatal exposure to cigarette smoke is associated with higher placental AhR activation, indicative of increased xenotoxicants burden. We also report that smoke-exposed fetuses showed increased circulating estrogenic activity and disrupted androgenic potential, across 10-20 weeks of gestation, in both fetal plasma and placenta. This demonstrates that EDs present in cigarette smoke are able to interfere with hormonal signalling and alter dynamic endocrine activity profiles, which are critical to ensure appropriate, sex-specific, development. These ED effects are likely to disturb placental function and reprogramme fetal development and thus impacting on life-long health.

The engineering design of quarter size negative beam source for the comprehensive research facility for fusion technology

A high-power radio frequency (RF) beam source is a key component for Negative Neutral Beam Injector (NNBI) in ASIPP. According to the technical route planning of CRAFT-NNBI beam source project, ASIPP started the development of quarter-size RF negative source and corresponding test facility. The inner diameter of the faraday shield (FS) of RF driver is 240 mm, the outer diameter of the accelerator is 1 m, with three-layer grids extraction system, which comprises the plasma grid (PG), extraction grid (EG) and ground grid (GG). The size of PG is 512 mm × 396 mm × 9 mm, with two rows of 16 × 6 apertures made of pure molybdenum. The structure design of RF negative beam source and the manufacturing process of key components are presented, with emphasis on the manufacturing process of FS as well as grids. Furthermore, the fluid-thermal-structural analysis of PG, the mechanical analysis and material bonding strength test of the main insulation structure of beam source, as well as the preliminary layout of the latest test facility are presented.

Targeted and Untargeted Liver Metabolomics Demonstrate Key Differences of Perchloric acid and Acetonitrile Isopropanol Water Extraction Through Gas Chromatography Mass Spectrometry

In this study, untargeted metabolic profiling by Gas Chromatography Mass Spectrometry (GC-MS) was leveraged to identify differences between Perchloric acid extraction (PCA) and 3:3:2 acetonitrile/isopropanol/water (IPA) extraction for liver metabolomics research. Perchloric acid extraction (PCA), a common means of precipitating excess protein through acidification, has been used in physiology literature for decades and is considered as the standard method for extracting water soluble metabolites. However, PCA extraction commonly requires tedious pH neutralization with potassium hydroxide (KOH) and subsequent desalting. While IPA extraction can also precipitate protein it is pH neutral and bypasses the need for neutralization and is more amenable for high throughput sampling. Several studies have demonstrated that IPA has a high extraction efficiency amongst other organic solvent based extractions such as 50:50 acetonitrile water and 80% methanol water in mammalian fecal extracts [Yang, Y., Yin, Y., Chen, X. et al Scientific Reports. 2019, 9(12017)] and plasma extracts [Jiye, A. et al. 2005 Anal. Chem. 77, 8086–8094] for GC-MS. Similarly, plasma extractions for Nuclear Magnetic Resonances (NMR) have demonstrated that 50:50 acetonitrile water extractions, a variant of organic extraction similar to IPA, are reproducible and are capable of eliminating excess protein from extracts while preserving low concentration metabolites [Daykin et al. Analytical Biochemistry 2002, 304, 220 –230]. In this study we expand the current knowledge of IPA versus PCA extractions in the field of liver metabolomics. Liver is a potential target for study, due to the increasing prevalence of Non-Alcoholic Fatty liver disease (NAFLD) and Non-alcoholic Steatohepatitis (NASH), which now affects a combined 80-100 million Americans [Perumpail et al. World J Gastroenterology. 2017, 23(47), 8263]. Due to the multifaceted nature of NAFLD approaches including NMR, mass spectrometry and various extraction variants have aimed to identify metabolic changes that would shed light on the mechanism of pathogenesis. To achieve a relevant background for analyzing the suitability of PCA or IPA for liver metabolomics in NAFLD, we extracted ex vivo C57/BLKS perfused mouse livers under fed, fasted and pooled conditions. We utilize C57/BLKS mice as they are prone to type 2 diabetes, atherosclerosis and obesity, all of which are significant cofactors for NAFLD development. Through our untargeted metabolomics panel several previously unidentified metabolites were assessed by principle components analysis and determined to be similar (Figure I). From our targeted metabolomics panel of extracted standards we also observed that several metabolites are largely similar in extraction efficiency with only the ketone 3-hydroxybutyrate having a higher extraction efficiency by PCA rather than IPA (Figure I). In conclusion, the extraction efficiency and statistical comparison of PCA and IPA extractions for GC-MS metabolomics assessments demonstrate their suitability and equivalence for all but a few metabolites.

The effect of combaine administration of L-arginine and omega-3 polyunsaturated fatty acids on the spectrum of free amino acids and biogen amines in hyppocampus of rats undergoing subtotal cerebral ischemia

The aim of this study was to estimate the changes in the pool of free amino acids and their derivatives in hippocampus of rats undergoing subtotal cerebral ischemia (SCI) and treated with L-arginine and omega-3 polyunsaturated fatty acids (PUFA). Experiment was held on 18 rats: 12 animals were undergoing bilateral filament occlusion of arteries carotid, 6 of them L-arginine and omega-3 PUFA was administrated. The drug omega-3 PUFA "Omegamed" (at a dose of 5 g/kg of body weight) was injected intragastrically during the week preceding the simulation of SIMG. L-arginine (at a dose of 100 mg/kg body weight) was injected intravenously just before ligation of the common carotid arteries. The analyses of free amino acids and their derivates levels in the blood plasma extracts were carried out by reversed phase HPLC. In the hippocampus of rats with SIGM, there was an increase in the levels of histidine, 3-methylhistidine, glutamine, α--aminobutyrate, isoleucine, leucine, valine, as well as a decrease in the levels of threonine, tyrosine, and α--aminoadipic acid. Administration of L-arginine and omega-3 PUFAs prevented ischemia-induced disruption of threonine, histidine, glutamine, α--aminobutyrate, α--aminoadipic acid levels, and also had a corrective effect on the serotonin system of the hippocampus.

A prepared platelet-rich plasma extract, namely Self-Growth Colony, inhibits melanogenesis by down-regulating microphthalmia-associated transcription factor in skin melanocyte.

During melanogenesis, melanocytes produce melanin through enzymatic reactions. Microphthalmia-associated transcription factor (MITF) is a major regulator in controlling the expressions of melanogenic enzymes tyrosinase (TYR), tyrosine-related protein-1 (TRP1), and dopachrome tautomerase (DCT). Self-Growth Colony (SGC) is prepared from human platelet-rich plasma (PRP) having an enrichment of growth factors, and which has claimed skin regeneration function. In this study, we aim to identify and investigate the novel role of SGC in skin melanogenesis. MTT assay was performed to determine the cytotoxicity of applied SGC. Melanin assay was adopted to quantify the intracellular melanin after SGC treatment. Promoter-driven luciferase assay, real-time PCR, and Western blotting were performed to determine the expressions of melanogenic enzymes and MITF in SGC-treated cultured Melan-A cells, being treated with or without UV induction. Ex vivo mouse skin was treated with SGC, and then was subjected to Western blotting and histochemical staining. We identified that SGC inhibited melanogenesis in cultured melanocytes and ex vivo mouse skin. The phenomena were attributed to a reduction of MITF expression, which subsequently down-regulated the melanogenic enzymes, that is, TYR, TRP1, and DCT. Moreover, ERK signaling was activated in the SGC-inhibited melanogenesis. The findings suggest that SGC extracting from human blood can be a safe and potential agent in promoting skin whitening.

Mass spectrometric identification and quantification of the antibiotic clavulanic acid in broiler chicken plasma and meat as a necessary analytical tool in finding ways to increase the effectiveness of currently used antibiotics in the treatment of broiler chickens

Clavulanic acid is a molecule with antimicrobial effect used in several livestock species treatment. Its inclusion in the treatment of infectious diseases of broilers requires determination of pharmacokinetic and pharmacodynamic parameters in order to determine the appropriate dosage for broilers and ensure safety of chicken products for human health. The present study describes the optimisation of analytical LC-MS/MS method for identification and quantification of clavulanic acid in broiler chicken plasma and meat. The limit of detection and the limit of quantification for the developed method were 3.09 μg·L−1 and 10.21 μg·L−1 for plasma and 2.57 μg·kg−1 and 8.47 μg·kg−1 for meat. The recoveries of the developed plasma and tissue extraction procedure were > 105.7% and > 95.6%, respectively. The achieved coefficient of variation of within-run precision ranged from 2.8 to 10.9% for plasma and from 6.5 to 8.5% for meat. The pharmacokinetic experiment was performed in 112 Ross broiler chickens assigned into time interval groups ranging from 10 min to 24 h in accredited animal facilities. Administered dose of clavulanic acid was 2.5 mg·kg−1 according to the manufacturer’s recommendations. The pharmacokinetic parameters obtained from the experiment are as follows: Cmax = 1.82 ± 0.91 mg·L−1, Tmax = 0.25 h, T1/2 = 0.87 h, Kel = 0.80 ± 0.04 h−1, AUC0-∞ = 2.17 mg·h ·L−1.

Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics.

To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole-genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP, n = 7) and using quantitative PCR (Q-PCR, n = 116). The size profile in healthy individuals was remarkably homogenous when using DSP sequencing or SSP sequencing. CfDNA size profile had a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes, and chromatin of higher molecular size (>1000 bp) can be estimated as 67.5% to 80%, 9.4% to 11.5%, and 8.5% to 21.0%, respectively. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97%–98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between metastatic colorectal cancer patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content.

Evaluation of Pharmacokinetics and Metabolism of Phosphorothioate Antisense Oligonucleotide G3139 in Rat by Capillary Electrophoresis with Laser-Induced Fluorescence.

A sensitive and specific capillary electrophoresis with laser-induced fluorescence (CE-LIF) and a simple extraction process was developed to simultaneously detect G3139 and its metabolites as a model of antisense oligonucleotides (ASOs). This method has shown excellent linearity within the tested concentration range for G3139 and its metabolites, with a detection limit of 3.0 pM and a recovery of >84.2%. Based on our developed plasma extraction method, we have evaluated the pharmacokinetics and metabolites from rat plasma after intravenous administration of G3139 at 0.76 mg/kg. The results showed that G3139 and its metabolites were successfully simultaneously detected and analyzed through a single run using CE-LIF with baseline separation until the 30-h test sampling time point. The half-life of G3139 and its metabolites was observed at 31 and 68 h, respectively. This study may provide an effective analytical method for the pharmacokinetic and metabolite evaluation required to develop ASOs to treat a variety of diseases.

Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Vardenafil and Its Application of Bioequivalence.

A simple, rapid, and sensitive method of liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of vardenafil in rabbit plasma. A simple protein precipitation method with ice-cold acetonitrile was used for plasma extraction. The mass transitions m/z 489⟶151 and m/z 390⟶169 were used to measure vardenafil and tadalafil (internal standard), respectively, with a total assay run time of 6 min. The limit of detection was 0.2 ng/mL. The assay was reproducible with intra-assay and interassay precision ranging 1.17%–9.17% and 1.31%–5.86%, respectively. There was also good intra-assay and interassay accuracy between 89.3%–105.3% and 94%–102% of the expected value, respectively. The linearity range was 0.5–60 ng/mL in rabbit plasma (r2 ≥ 0.99). The measured AUC from 0 to 24 h (AUC0 − 24t) for the test and reference formulations were 174.38 ± 95.91 and 176.45 ± 76.88, respectively. For the test, Cmax and Tmax were 75.36 ± 59.53 ng/mL and 1.42 ± 0.19 h, whereas, for the reference, these were 58.22 ± 36.11 ng/mL and 2.04 ± 0.33 h, respectively. The test formulation achieved a slightly lower AUC0 − 24t value (p > 0.05), higher Cmax values (p > 0.05), faster Tmax (p < 0.05), and almost equal bioavailability compared with the reference formulation.

Gold nanoclusters-based paper sensor for the visualized detection of nephrotoxic aristolochic acids

Aristolochic acids (AAs), the main compounds of Aristolochia plants, have aroused increasing attention due to their irreversible nephrotoxicity, carcinogenicity and mutagenicity. Common approaches for AAs monitoring usually need expensive equipment, specially trained manipulators and time-consuming procedures. Thus, it is desirable to develop a simple, fast and easy to use approach for AAs detection. Herein, we reported a fluorescent approach to detect AAs by using the lysozyme-functionalized Au nanoclusters (Lys-AuNCs) as probe. The probe shows excellent tolerance to environment temperature and pH, and it is proved to work successfully for AAs detection in real samples (serum, plasma, urine and herbal extracts). Compared with high-performance liquid chromatography method (HPLC), fluorescence method can be finished within 1 min with a recovery of 117.49 %. With the help of the visible fluorescence as signal, a paper sensor is further prepared for a direct visual monitoring of AAs. It also exhibits good stability in various conditions. After the addition of AA-I, a concentration-dependent fluorescence signal on paper was observed by naked eyes under a UV lamp. The developed paper-based fluorescent sensor provides a portable and simple method for AAs detection in herbal products and environment.

Conceptual design of a beam source for negative neutral beam injector of CRAFT facility

The Comprehensive Research Facility for Fusion Technology (CRAFT) is a large scientific device that is preferentially deployed in China's "Thirteenth Five-Year Plan" for the construction of major national science and technology infrastructures. A beam source of negative neutral beam injector (NNBI) is required for the CRAFT device with the beam energy of 400 keV, neutral beam power of 2 MW, and beam duration of 100 s. The beam source is the key component of NNBI system. A radio frequency (RF) based negative beam source was designed for the CRAFT NNBI system. It contains a large plasma generator and a negative ion accelerator. The plasma generator contains a large expansion chamber and four RF drivers which arrange in a vertical column (1 × 4). The accelerator has a similar design to ITER. Consider the 400 keV beam energy, the accelerator was designed with four layers of electrodes, which are plasma grid, extraction grid, acceleration grid, and ground grid. The detailed structures of negative beam source were designed initially with the RF plasma generator and the negative ion accelerator.