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Gambierdiscus spp. are epi-benthic dinoflagellates that have been associated with ciguatera poisoning. These microalgae can have complex secondary metabolite profiles including ciguatoxins, maitotoxins, and gambierones, with varying compositions and toxicities across species and strains. Given this chemical diversity there is a need to develop selective and sensitive methods for secondary metabolite profiling. In this study, we used a cultured Caribbean strain of Gambierdiscus silvae to develop sample preparation and analysis strategies for characterizing vic-diol containing secondary metabolites. A pooled cellular extract was first screened by liquid chromatography–high–resolution mass spectrometry (LC–HRMS) for ciguatoxin-related compounds, which resulted in the confirmation of gambierone (1) and tentative identification of 29-methylgambierone (3). Treatment of the extract with periodate confirmed that the gambierones each contained one reactive vic-diol, which was exploited for the development of a selective extraction procedure using m-aminophenylboronic acid gel and the non-aqueous binding solvent chloroform. Using this non-traditional boronate affinity procedure, LC-HRMS also revealed the presence of additional sulfated polycyclic ethers in the gambierone-containing vic-diol fraction, while pigments and other contaminants were removed. The developed tools could be applied to screen collections of Gambierdiscus and other benthic algae to provide additional chemical characterization of gambierone-related compounds. The selective extraction procedure may also prove useful as a step in the isolation of these sulfated polyethers for structural, toxicological and biotransformation studies.

This study was conducted to determine the phenolic compounds that may be utilized as chemotaxonomic study to identify some phenolic compounds concentration that are useful for pharmaceutical purposes from 5 taxa belongs to the Brassicaceae family which are; Cardaria draba (L.) Desv., Lepidum aucheri Boiss., Sisymbrium irio L., Sisymbrium officinale (L.) Scop., and Sisymbrium septulatum DC. Leaves powder of these taxa were collected and analyzed by High-performance liquid chromatography (HPLC). The results identified 9 phenolic compounds, chlorogenic acid, cinnamic acid, ferulic acid, genstic acid, kaempferol, p-coumaric acid, protocatechuic acid, sinapic acid, and vanillic acid. The results showed that the genus Lepidium aucheri had the highest concentration of 4 phenolic compounds, cinnamic acid, genstic acid, p-coumaric acid, and sinapic acid with concentration of (37.2, 80.79, 62.91, and 37.10µg/ ml) respectively. On the other hand, the genus Cardaria draba showed the lowest concentration of 3 phenolic compounds, cinnamic acid, p- coumaric acid, and protocatechuic acid, and doesn't have 2 phenolic compounds, genstic acid and kaempferol. This variation in the phenolic concentrations might be due to the genotype and plant growth stage differences. The results of this study provide a source for bioactive compounds to future studies. http://dx.doi.org/10.25130/tjps.23.2018.027

A particular attention should be paid to isolation, characterization, and assessment of bioactive flavonoids, especially for anti-arthritic activity. The slurry of adsorbent (silica gel; 60–120 lattice) was blended in n-hexane to form a uniform slurry, and this was then employed as a fixed-stage adsorbent. Butanolic solution (10 g) was dissolved in the minimum volume of butanol, and then adsorbed on silica gel (60–120 mesh) while still wet. The wet powder was then dried, and the separated liquid phase was collected and applied to the column to try to isolate possible phytoconstituents. A whole plate full of fractions was monitored simultaneously on a TLC plate with a mixture of chloroform:methanol:water (68:30:02) as the solvent system. Six fractions (F1-F6) were generated after the colours of F1 and F2 were mixed next to each other, and after Rf was discovered in F3. A separate group of six animals was created for the survey, which was made up of five treatment groups and one control group. It was concluded that yellow crystalline solid luteolin was obtained as a result, with the Shinoda test confirming that it was a flavonoid. The absorption of luteolin was discovered to be in an aqueous alkaline solution. There were five different resonances, occurring at different frequencies, in the 400 MHz 1H NMR spectrum: 7.00 (d, J = 13.5 Hz, 2H), 6.73 (s, 1H), 6.56 (s, 1H), 6.18 (s, 1H), 6.05 (s, 1H), 4.78 (s, 1H), 4.24 (s, 1H), 3.81 (s, 1H) (s, 1H). Inhibiting arthritic inflammation at doses of 40 mg/kg (luteolin) was significant (P

The strain Bacillus oceanisediminis H2, which was isolated from tannery processing industrial wastelands, grown on biosurfactant production media supplemented with glucose as carbon source and urea as nitrogen source was successful in the production of a crude biosurfactant. The crude biosurfactant was purified using silica-gel column chromatography and thin layer chromatography (TLC), which was then lyophilized for further analysis. TLC of both the crude and purified biosurfactant showed similar Rf values of 0.678 (of one of the spots) and 0.614 respectively. The solid purified product was then subjected to FTIR and LC-MS analysis which confirmed that the purified biosurfactant is surfactin by nature. The crude biosurfactant produced has the ability to reduce surface tension to 46.909 mN/m from 72 mN/m as that of water.

SARS-CoV-2 infection spawns from an asymptomatic condition to a fatal disease. Age, comorbidities, and several blood biomarkers are associated with infection outcome. We searched for biomarkers by untargeted and targeted proteomic analysis of saliva, a source of viral particles and host proteins.Saliva samples from 19 asymptomatic and 16 symptomatic SARS-CoV-2 infected subjects, and 20 controls were analyzed by LC-MS/MS for untargeted peptidomic (flow through of 10 kDa filter) and proteomic (trypsin digestion of filter retained proteins) profiling.Peptides from 53 salivary proteins were identified. ADF was detected only in controls, while IL1RA only in infected subjects. PRPs, DSC2, FABP5, his-1, IL1RA, PRH1, STATH, SMR3B, ANXA1, MUC7, ACTN4, IGKV1-33 and TGM3 were significantly different between asymptomatic and symptomatic subjects. Retained proteins were 117, being 11 highly different between asymptomatic and symptomatic (fold change ≥2 or ≤-2). After validation by LC-MS/MS-SRM (selected reaction monitoring analysis), the most significant discriminant proteins at PCA were IL1RA, CYSTB, S100A8, S100A9, CA6, and FABP5.The differentially abundant proteins involved in innate immunity (S100 proteins), taste (CA6 and cystatins), and viral binding to the host (FABP5), appear to be of interest for use as potential biomarkers and drugs targets.

Twenty four ethanol leaf and stem bark extracts of 17 indigenous plants were examined for their phytochemical composition, antimicrobial and antioxidant properties. Phytochemical compositions were analysed with GC-MS while antimicrobial activities on Staphylococcus aureus and Pseudomonas aeruginosa were investigated by the agar well diffusion method. The antioxidant activities were determined with Ferric reducing antioxidant power (FRAP), total phenolic content (TPC) and 2, 2,-dihenyl-1-picryhydazyl (DPPH) radical scavenging assays. The antibacterial activity was more towards the gram positive S. aureus than the gram negative P. aeruginosa for all the plant extracts. A wide range of phenolic concentrations among the aqueous plant extracts which varied from 28.04 to 500.26mg GAE per gram were observed. Inhibition percentages of DPPH ranged from 19.13 to 95.77% showing effectiveness in radical scavenging. GC-MS characterization of the plant extracts showed a total of 18 components including alkaloids, flavonoids, phenols, saponins, terpenoids, steroids and glycosides. Irvingia gabonensis leaf (IGL) extract and Tamarind stem bark (TSB) exhibited excellent ferric reducing abilities of 2.11 and 1.56 respectively while Voucanga Africana leaf (VCA) extract indicated the lowest ferric reducing power of 0.50. Extracts of IGL and TSB exhibited the highest antioxidant capacities and therefore could be the main sources of natural antioxidant. An important relationship between total phenolic content was observed showing that the major contributor to the antioxidant properties were phenolic compounds.

BackgroundEnvironmental water samples are increasingly recognised as an important reservoir of antimicrobial resistance (AMR) genes. Polymerase chain reaction (PCR) and next generation sequencing (NGS) offer a potentially inclusive surveillance platform for a wide range of AMR genes. However, molecular methods are dependent upon the extraction of DNA of high yield and quality. Current options for DNA extraction from complex environmental matrices for downstream molecular applications are either expensive or low yielding. We present here a novel magnetic bead-based DNA extraction method, for the detection of antimicrobial resistance genes (ARGs) from river water in Malawi, named MagnaExtract. MethodsMagnaExtract involves initial filtration of 250ml freshwater, followed by an overnight incubation of the filter in 15ml buffered peptone water (BPW), common procedure in microbiology laboratories. 200{micro}l is then taken for a boil (95{degrees}C) and spin step and mixed with magnetic beads to bind DNA. Following washes with ethanol, the DNA is eluted in nuclease-free water. To determine the effectiveness of this method, 98 freshwater samples were collected from two rivers in Southern Malawi, and DNA was isolated using the MagnaExtract method, two commercial Qiagen (Germany) kits; PowerWater and DNeasy Blood and tissue, alongside a boil and spin of BPW, and a boil and spin from bacterial isolate grown on agar media. All samples were screened with a high-resolution melt (HRM) PCR panel previously validated for the detection of third generation cephalosporin and carbapenem ARGs. We compared the DNA yield obtained using all extraction methods, as well as the identification of each ARG. ResultsDNA yield using MagnaExtract was statistically greater than both boil and spin methods and DNeasy Blood & Tissue (Qiagen, Germany). DNA yield was slightly lower than using PowerWater (Qiagen) but the difference was not statistically significant. MagnaExtract was the only method to identify ARGs in all 98 water samples compared with PowerWater (n=82), DNeasy (n=95) boilate of BPW (n=75) and boilate of bacterial isolate (n=87). The most commonly detected ARG was OXA-48 (n=93). In addition, we found overnight incubation in non-selective enrichment broth (BPW) to promote the growth of bacteria harbouring extended spectrum beta lactamase (ESBL) genes and reduction in the detection of carbapenemase genes. ConclusionThe MagnaExtract approach offers a simple, affordable, high yielding DNA extraction method for the detection of ARGs isolated from river water samples.

An analytical procedure has been introduced to enable a study of the excretion of free bisphenol A (BPA), total BPA and its main metabolite bisphenol A glucuronide (BPA-GLUC). In the experiment, in which 100 μg/kg b. w. BPA was administered daily to one Istrian Pramenka sheep for 5 days with consecutive urine and feces samples being taken, BPA and total BPA were determined in samples using high-performance liquid chromatography (HPLC) with fluorescence detection. Because of their good recovery, precision, and sensitivity, the methods have also proved applicable to further ecotoxicological studies of free BPA, BPA-GLUC and total BPA. The results were subsequently compared with reported field studies of BPA in livestock excreta.

In healthcare role of biological analyte like uric acid plays a crucial role. Among various antioxidants, uric acid is significant antioxidant that helps in regulating blood-pressure and also reduces the antioxidant stress as well. In this proposed research work different concentration of uric acid were taken for detection using optical absorbance based spectroscopy. After the characterization it was found that as the concentration of uric acid increases, absorbance associated with peak absorbing wavelength also increases. The concentration of uric acid was taken 2, 4 and 8 mg/dl respectively for testing and absorbance found to be 0.75, 1.8 and 2.2 respectively. The sensitivity of device found to be 1 mg/dl.

Aim and Objective: A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining hypaphorine, a potential agent for treating osteoclast- based bone loss, was developed and validated in rat plasma. Materials and Methods: Plasma samples were pretreated by the protein precipitation. Chromatographic separation was performed using an Inertsil ODS-3 column (50 mm × 4.6 mm, 5 μm). The mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile in a gradient mode at a flow rate of 0.5 mL/min. The transitions from protonated precursor ion [M + H]+ to the particular daughter ion were acquired using selected reaction monitoring (SRM). The mass transitions of hypaphorine and IS were found to be 247 → 188 and m/z 219 → 188, respectively. The method was validated in terms of selectivity, linearity, accuracy and precision, extraction recovery and matrix effect, stability, and carryover. Results: It showed good linearity over the range of 1-2000 ng/mL (R2 = 0.9978). The intra-batch accuracy was within 93.95-105.81%, and the precision was within 4.92-11.53%. The inter-batch accuracy was within 96.18-100.39% with a precision of 6.22-11.23%. The extraction recovery and matrix factors were acceptable. Conclusion: The simple and rapid method was successfully applied to the pharmacokinetic study in rats following oral administration of hypaphorine at the doses of 0.5, 1.5, and 4.5 mg/kg.

Background: The fruit pulp decoction of Crescentia cujete, commonly known as calabash, is traditionally used for the treatment of several respiratory diseases and is available as syrup formulations. Unfortunately, there is no detailed investigation on analytical methods for warranting the quality of these products. Aim and objective: To develop and validate an appropriate analytical method for the simultaneous quantification of transcinnamic acid, 4-hydroxybenzoic acid, verbascoside and 6-epi-aucubin in the decoction and commercial cough syrups of Crescentia cujete fruit. Materials and methods: A reversed-phase ultra-high-performance liquid chromatographic method coupled to a diode array detector (UPLC-DAD) was validated following the ICH guidelines. The chromatographic analysis was performed using a C18 column, the mobile phase system consisted of water and acetonitrile containing 0.1% formic acid, and UV chromatograms were recorded from 200 to 400 nm. Results: A new UPLC-DAD method was validated for the simultaneous quantification of trans-cinnamic acid, 4- hydroxybenzoic acid, verbascoside and 6-epi-aucubin in calabash-derived products. After successful validation, this method was applied for the quantification of the selected chemical markers in an in-house decoction and three commercial cough syrups. Among the selected chemical markers, 6-epi-aucubin was the main compound in the calabash decoction, while trans-cinnamic acid and 4-hydroxybenzoic acid were the major compounds in the commercial products. Verbascoside and 6-epi-aucubin were below the limit of quantification in all syrup samples. Conclusion: The proposed method was successfully applied for the analysis of three commercial syrup formulations and can be useful for standardization and quality control of raw and pharmaceutical calabash preparations.

Abstract: Inhibition of cholinesterases is a common strategy for the treatment of several disorders, especially Alzheimer´s disease. In vitro assays represent a critical step towards identifying molecules with potential anticholinesterase effect. This study aimed at providing a comprehensive review of the methodologies used in vitro for the anticholinesterase activity based on the spectrophotometry of Ellman’s method. This work used two databases (PubMed and ScienceDirect) to search for original articles and selected publications between 1961 and 2019, which reported in vitro spectrophotometry assays for anticholinesterase activity. After the search process and the selection of publications, the final sample consisted of 146 articles published in several journals submitted by researchers from different countries. Although the studies analyzed in this work are all within the same conception of in vitro tests based on Ellman’s method, one can observe a wide divergence in the origin and concentration of enzyme, the choice and pH of the buffer, the concentration of the substrate, the sample diluent, incubation time, temperature, and time of the spectrophotometric reading interval. There is no consensus in the methodology of studies with in vitro tests for anticholinesterase assessment. The methodological variations related to kinetic parameters may interfere in the characterization of cholinesterase inhibitors.

Neuron–specific enolase (NSE) is a promising small–cell lung cancer (SCLC) biomarker composed of αγ and γγ isozyme dimers. As the conventional immunoassays are prone to interferences and cannot differentiate between the isozymes, we developed a multiplex immunoaffinity (IA) liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the quantification of NSEα and NSEγ in human serum. A calibrator was prepared by performing cold denaturation of recombinantly expressed αα and γγ enolase dimers to induce a new dimer equilibrium that was determined to be approximately 1αγ:1γγ:1αα. Selective sample purification was achieved by performing IA extraction using an antibody specific towards NSEγ. The isolated αγ and γγ dimers were denatured and trypsin digested to allow quantification of the selected signature peptides and their corresponding isotopically labelled peptide internal standard. The obtained linear dynamic ranges were determined to be 1.5–56 ng/mL and 0.64–167 ng/mL for NSEα and NSEγ (R2 = 0.88 and 0.97 respectively). Validation of the assay showed acceptable accuracy and precision for NSEα and NSEγ. The method was successfully applied to patient serum in which both isozymes were detected. Compared to the conventional immunoassay, substantially lower total NSE concentrations were measured in IA LC–MS/MS. With this multiplex IA LC-MS/MS assay, the clinical value of quantifying the individual isozymes can be explored. In addition, together with the calibrator described here, it may be applied to standardize NSE immunoassays across different platforms.

Human papillomavirus (HPV) vaccines based on HPV L1 virus-like particles (VLPs) are already licensed but not accessible worldwide. About 38.0 million people were living with HIV in 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against both viruses are an urgent need. In this study, the HIV-1 P18I10 CTL peptide from the V3 loop of HIV-1 gp120 glycoprotein was inserted into the HPV16 L1 protein to construct chimeric HPV:HIV (L1:P18I10) VLPs. Instead of the traditional baculovirus expression vector/insect cell (BEVS/IC) system, we established an alternative mammalian 293F cell-based expression system using cost-effective polyethylenimine-mediated transfection for L1:P18I10 protein production. Compared with conventional ultracentrifugation, we optimized a novel chromatographic purification method which could significantly increase L1:P18I10 VLP recovery (~56%). Chimeric L1:P18I10 VLPs purified from both methods were capable of self-assembling to integral particles and shared similar biophysical and morphological properties. After BALB/c mice immunization with 293F cell-derived and chromatography-purified L1:P18I10 VLPs, almost the same titer of anti-L1 IgG (p = 0.6409) was observed as Gardasil anti-HPV vaccine-immunized mice. Significant titers of anti-P18I10 binding antibodies (p < 0.01%) and P18I10-specific IFN-γ secreting splenocytes (p = 0.0002) were detected in L1:P18I10 VLP-immunized mice in comparison with licensed Gardasil-9 HPV vaccine. Furthermore, we demonstrated that insertion of HIV-1 P18I10 peptide into HPV16 L1 capsid protein did not affect the induction in anti-L1 antibodies. All in all, we expected that the mammalian cell expression system and chromatographic purification methods could be time-saving, cost-effective, scalable platforms to engineer bivalent VLP-based vaccines against HPV and HIV-1

Highlights • Supported liquid extraction is easy to use as visual phase separation isn't needed. • Automated sample preparation allows fast sample process and subsequent analysis. • Automated liquid handling coupled with LC-MS/MS as high-throughput method. • Washout gradient in LC method removes strongly retaining compounds from the column. • Sensitive method of 1.08/1.41 ng/ml for amodiaquine/desethylamodiaquine., Amodiaquine is a drug used for treatment of malaria and is often used in combination with artesunate in areas where malaria parasites are still susceptible to amodiaquine. Liquid chromatography tandem-mass spectrometry was used to quantify amodiaquine and its active metabolite, desethylamodiaquine, in plasma samples. A low sample volume of 100 µl, and high-throughput extraction technique using a supported liquid extraction (SLE+) technique on an automated liquid handler platform for faster sample processing are some of the advantages of this method. Separation of amodiaquine from desethylamodiaquine was achieved using a reversed phase Zorbax SB-CN 50 mm × 4.6 mm, I.D. 3.5 µm column with acetonitrile and 20 mM ammonium formate with 1% formic acid pH ~ 2.6 (15–85, v/v) as mobile phase. The absolute recoveries of amodiaquine and desethylamodiaquine were 66% to 76%, and their isotope label internal standard were in the range of 73% to 85%. Validation results of the developed method demonstrated intra-batch and inter-batch precisions within the acceptance criteria range of ± 15.0%. There were no matrix or carry-over effects observed. The lower limit of quantification was 1.08 ng/ml for amodiaquine and 1.41 ng/ml for desethylamodiaquine. The method showed robust and accurate performance with high sensitivity. Thus, the validated method was successfully implemented and applied in the evaluation of a clinical trial where participants received artemether–lumefantrine plus amodiaquine twice daily for three days (amodiaquine dose of 10 mg base/kg/day).

Remimazolam besylate is an ultra-short-acting benzodiazepine derivative recently approved in Japan for general anaesthesia. However, less attention has been paid to the compatibility of remimazolam with infusion solutions, and the mechanism underlying the incompatibility remains unknown. The patient was a 65-year-old man who underwent a high tibial osteotomy. After the induction of general anaesthesia using remimazolam solution (5 mg/mL), we noticed precipitate completely blocking the lumen of the intravenous tube connected to a Ringer’s acetate Physio140 drip. The mixture of remimazolam solution (5 mg/mL) with Physio140 solution immediately resulted in the formation of substantial precipitate. Nuclear magnetic resonance analysis revealed that the precipitate was remimazolam. Ultraviolet spectrophotometry revealed that the mixture of remimazolam solution with higher ratios of Physio140 resulted in significantly lower solubility, concomitant with an increase in pH. It would be important to consider the remimazolam concentration and infusion solution pH to avoid the production of precipitates.

In search of novel potential drug candidates that could be used as treatments or prophylactics for memory impairment, an aporphine alkaloid magnoflorine (MAG) isolated from the root of Berberis vulgaris was proven to exhibit beneficial anti-amnestic properties. Its effects on immunoreactivity to parvalbumin in the mouse hippocampus were assessed together with a study on its safety and concentration in the brain and plasma. For this purpose, four experimental groups were created: the MAG10 group—treated with 10 mg MAG/kg b.w. i.p., the MAG20 group—treated with 20 mg MAG/kg b.w. i.p., the MAG50 group—treated with 50 mg MAG/kg b.w. i.p., and a control group—injected with saline i.p. at a volume corresponding to their weight. Our results indicated that the hippocampal fields CA1–CA3 were characterized by an elevated number of parvalbumin-immunoreactive neurons (PV-IR) and nerve fibers in mice at the doses of 10 and 20 mg/kg b.w. (i.p.). No significant changes to the levels of IL-1β, IL-6 or TNF-α were observed for the above two doses; however, the administration of 50 mg/kg b.w. i.p. caused a statistically significant elevation of IL-6, IL-1beta plasma levels and an insignificant raise in the TNF-alpha value. The HPLC–MS analysis showed that the alkaloid’s content in the brain structures in the group treated with 50 mg/kg b.w. did not increase proportionally with the administered dose. The obtained results show that MAG is able to influence the immunoreactivity to PV-IR in hippocampal neurons and might act as a neuroprotective compound.

Firefly flashes are well-known visual signals used by these insects to find, identify, and choose mates. However, many firefly species have lost the ability to produce light as adults. These “unlighted” species generally lack developed adult light organs, are diurnal rather than nocturnal, and are believed to use volatile pheromones acting over a distance to locate mates. While cuticular hydrocarbons, which may function in mate recognition at close range, have been examined for a handful of the over 2000 extant firefly species, no volatile pheromone has ever been identified. In this study, using coupled gas chromatography - electroantennographic detection, we detected a single female-emitted compound that elicited antennal responses from wild-caught male winter fireflies, Photinus corruscus. The compound was identified as (1S)-exo-3-hydroxycamphor (hydroxycamphor). In field trials at two sites across the species’ eastern North American range, large numbers of male P. corruscus were attracted to synthesized hydroxycamphor, verifying its function as a volatile sex attractant pheromone. Males spent more time in contact with lures treated with synthesized hydroxycamphor than those treated with solvent only in laboratory two-choice assays. Further, using single sensillum recordings, we characterized a pheromone-sensitive odorant receptor neuron in a specific olfactory sensillum on male P. corruscus antennae and demonstrated its sensitivity to hydroxycamphor. Thus, this study has identified the first volatile pheromone and its corresponding sensory neuron for any firefly species, and provides a tool for monitoring P. corruscus populations for conservation and further inquiry into the chemical and cellular bases for sexual communication among fireflies.

A simple and efficient dithizone-functionalized solid-phase extraction (SPE) procedure, online coupled with high-performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry, was developed for the first time for enrichment and determination of ultra-trace mercury (Hg) species (inorganic divalent Hg (Hg(II)), methylmercury (CH3Hg(II)) and ethylmercury (C2H5Hg(II)) in cereals and environmental samples. In the proposed method, functionalization of the commercial C18 column with dithizone, enrichment, and elution of the above Hg species can be completed online with the developed SPE device. A simple solution of 2-mercaptoethanol (1% (V/V)) could be used as an eluent for both the SPE and HPLC separation of Hg species, significantly simplifying the method and instrumentation. The online SPE method was optimized by varying dithizone dose, 2-mercaptoethanol concentration, and sample volume. In addition, the effect of pH, coexisting interfering ions, and salt effect on the enrichment was also discussed. Under the optimized conditions, the detection limits of Hg species for 5 mL water sample were 0.15 ng/L for Hg(II), 0.07 ng/L for CH3Hg(II), and 0.04 ng/L for C2H5Hg(II) with recoveries in the range of 85%–100%. The developed dithizone-functionalized C18 SPE column can be reused after a single functionalization, which significantly simplifies the enrichment step. Moreover, the stability of Hg species enriched on the SPE column demonstrated its suitability for field sampling of Hg species for later laboratory analysis. This environment-friendly method offers a robust tool to detect ultra-trace Hg species in cereals and environmental samples.

Background: Frankincense is a resin secreted by the Boswellia tree. It is used in perfumery, aromatherapy, skincare, and traditional Chinese medicine. However, all Boswellia species are under threat owing to habitat loss and overexploitation. As a result, the market is getting flooded with counterfeit frankincense products. Objective: This study aims to establish a high-throughput method to screen and identify the authenticity of commercial frankincense products. We report, for the first time, a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based method for rapid and high-throughput screening of frankincense samples. Methods: MALDI-TOF MS, HPLC, thin-layer chromatography (TLC), and in vitro anti-inflammatory activity assay were used to examine the frankincense samples. Results: Well-resolved peaks of frankincense triterpenoids in the spectra were observed in the crude extract of commercial samples, including α-boswellic acids (αBAs), β-boswellic acids (βBAs), 11-keto-β-boswellic acids (KBAs), acetyl-11-keto-β-boswellic acids (AKBAs), and their esters. These compounds can be used as indicators for determining the authenticity of frankincense. Conclusion: Unlike LC–MS, which is a time-consuming and expensive method, and TLC, which requires a reference sample, our inexpensive, rapid high-throughput identification method based on MALDI-TOF MS is ideal for large-scale screening of frankincense samples sold in the market.

Ginger (Zingiber officinale Rosco) belonging to the family Zingiberaceae is one of the world’s most important spices and produces a pungent, aromatic rhizome that is valuable all over the world. Qualitative and quantitative analysis of 6-gingerol in different parts (in vivo and in vitro) of Zingiber officinale using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) have been performed. Data of TLC showed spots having identical Rf value (0.15), according to the synthetic standards of 6-gingerol in all samples extract. 6-gingerol was detected in all extracts of different parts of ginger derived from in vivo and in vitro culture conditions. Quantitative determination of 6-gingerol using HPLC technique was carried out. Comparing with the peaks of 6-gingerol in synthetic standards, in vivo rhizomes and in vitro cultures of different ginger parts was showed similar UV spectra characteristics. The quantity of 6-gingerol in rhizomes (in vivo and in vitro) and in vitro microrhizomes (45.37; 42.64; 28.11 mg/g respectively), were showed a higher value than that of in vitro calli, shoots and roots (7.89; 7.46; 6.40 mg/g respectively).

At present, the cyanide gold extraction process is still the main technology for gold production. Generated cyanide tailings containing highly toxic substances exhibit potential environmental risks. These tailings are in urgent need of purification treatment, especially after being classified as hazardous waste. In this study, the impacts of elution methods, operating time, tailings/water ratios, reagent types on the elution rates of cyanide were investigated. Furthermore, the composite elution method developed in this research was extended for engineering. Results showed that the optimum elution conditions were determined to be: stirring elution, tailings/water ratio (M/V; 1:1) and operating time (10-20 min). Besides, 4 reagents (sodium dodecyl benzene sulfonate, cyclodextrin, sodium silicate and calcium hydroxide) were selected from four categories of 21 reagents for further composite elution. The cyanide elution rate was the highest (90.7%±0.1%) while the molar ratio of these 4 reagents was 5:2:2:1. Moreover, the combination of reagent elution and positive pressure filtration improved the elution efficiency of cyanide (92.6%±0.8%). And the cyanide content in the toxic leaching solution was lower than the standard value (5.0 mg/L). Furthermore, the composite elution method developed in this study was also extended for engineering. The concentration of cyanide in the leachate was5.0 mg/L, and was stable during 189 days of detection. Notably, the effluent can be reused directly, or reused after further treatment. The zero discharge of effluents and solid wastes was realized in the processes. The above results provided supports for the engineering treatment of cyanide tailings.

A new methodology was proposed to determine the dispersive component of the surface energy ${\gamma}_s^d$ of a solid taking into account the effect of the temperature on the surface area of n-alkanes, methylene group (${a}_{- CH2-}$) and polar molecules, thus defeating the method used by Dorris–Gray Schultz et al. We determined the correct ${\gamma}_s^d$ of the surface energy, the specific free energy, enthalpy and entropy of adsorption of polar molecules as well as the acid base constants of silica particles with an excellent accuracy. We confirmed the dependence of the dispersive component of the surface energy on the variations of the surface areas of organic molecules used in IGC technique at infinite dilution. The specific properties of interactions of silica particles were determined. The new proposed model took into account this thermal effect. Obtained results proved that the other used IGC methods gave inaccurate values of the specific parameters of silica surface, except for the vapor pressure method that led to excellent results of the specific free energy, enthalpy and entropy of adsorption, and the acid–base constants of the silica particles.

BACKGROUND: Periprosthetic joint infection is a major complication of total joint arthroplasty, with treatment requiring a two-stage exchange procedure and 6 weeks of systemic antibiotics. However, depending on the infection site, intravenous delivery of antibiotics like vancomycin (VCM) can have poor tissue transferability, thus reducing their therapeutic effect. OBJECTIVE: This study demonstrates the 24-week in vivo release profile and antibacterial activity of VCM from calcium phosphate cement impregnated with VCM (CPC/VCM) and compares them with those from polymethylmethacrylate impregnated with VCM (PMMA/VCM). METHODS: Rats were implanted with the test specimens between the fascia and quadriceps. After implantation for 24 weeks, the test specimens were removed and residual VCM was extracted to calculate the concentration of VCM released into rat tissues. We also examined the antibacterial activity of releasable VCM from the removed test specimens by placing them directly onto the surface of agar. RESULTS: CPC/VCM released greater concentrations of VCM for a longer period of time within the 24 weeks than PMMA/VCM. Moreover, CPC/VCM released 1.4 to 26.1-fold more VCM than PMMA/VCM. Using Staphylococcus aureus, antibacterial activity was logarithmically correlated with VCM concentration across the entire concentration range tested (12.5–800 μg/mL). While the area within which inhibition was observed—the inhibition zone—for both CPC/VCM and PMMA/VCM formed and gradually shrank with time after implantation, that for CPC/VCM was significantly larger than that for PMMA/VCM in each week after implantation. CONCLUSION: CPC/VCM releases greater amounts of VCM with antibacterial activity for longer periods of time than PMMA/VCM, suggesting that CPC is effective for facilitating the release of antibiotics for local action in patients with established postoperative infection.

Pyrethroids, organophosphorus pesticides and fipronil have been listed as priority chemi-cals in human biomonitoring studies because of their wide use and potential health effects in humans. The determination of 13 pesticides, including pyrethroids (deltamethrin, cyper-methrin, permethrin, cyfluthrin, bifenthrin), organophosphorus (chlorpyrifos, chlorpyrifos-methyl, and malathion), fipronil, neonicotinoids (imidacloprid, acetamiprid and thiacloprid) and triazole (prothioconazole), together with 13 corresponding metabolites in human urine samples was achieved by solid-phase extraction and analysis by liquid chromatography coupled to tandem mass spectrometry. All targeted compounds, except malathion dicar-boxylic acid, were measured with a mean within-accuracy ( n = 5) of 71%-114% (RSD: 1%-14%) and between-run ( n = 15) accuracy of 80%-118% (RSD: 2%-14%). Limits of quantitation of the targeted analytes ranged from 0.1 to 16 pg/mL. The detection result of urine samples from 25 volunteers indicated that the detection frequencies of 3,5,6-trichloro-2-pyridinol (median: 448 pg/mL), 6-chloropyridine-3-carboxylic acid (median: 193 pg/mL), 2-methyl-3-phenylbenzoic acid (median: 181 pg/mL), 3-phenoxybenzoic acid (median: 99 pg/mL), 2-isopropyl-6-methyl-4-pyrimidinol (median: 77 pg/mL), cyfluthrin (median: 59 pg/mL), cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (cis-DCCA, median: 53 pg/mL), trans-DCCA (median: 25 pg/mL), prothioconazole (median: 21 pg/mL), imidacloprid (median: 7 pg/mL), and prothioconazole-desthio (median: 1 pg/mL) were > 50%. The obtained results show that the validated method is suitable for the human biomonitoring of these current-use pesticides and their metabolites. (c) 2021 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Ovalbumin (OVA) is a high nutritious protein, but the poor emulsibility limited its application. The present study glycosylated OVA with dextran (Dex) by controlled wetheating (60–90 °C for 3 h). Temperature was an inductive factor for glycosylation degree (DG and browning intensity), and higher temperature could accelerate the reaction. Variations in molecular structure of OVA were analyzed by SDS-PAGE, FTIR, fluorescence spectroscopy and UV spectroscopy, which verified successes in the generation of glycoconjugate with more flexible structure. Emulsifying activity index (EAI) and emulsion stability index (ESI) for the emulsion of OVA-Dex glycoconjugates were significantly enhanced with the increasing of glycosylation temperature. Moreover, confocal laser scanning results revealed that the emulsion exhibited smaller size and more uniform distribution, and slower transmission profiles were checked by LUMiSizer centrifugal analysis as well, confirming the emulsibility improvement of OVA. Thus, controlled glycosylation reaction is an available method to improve the emulsifying properties of OVA.

A single-step and rapid chromatographic method-based purification of Gracilaria corticata (J. Agardh) R-phycoerythrin (R-PE) was attained using polyacrylamide gel electrophoresis (PAGE) technique without affecting structural integrity. The purified R-PE had a characteristic UV–Vis spectrum with three absorbance maxima at 496, 535, and 565 nm, and fluorescence at 575 nm. R-PE was obtained with a purity index of 4.2 and a recovery yield of 44.3%. SDS-PAGE analysis exhibited three sub-units i.e., 18, 21, and 31 kDa, which corresponds to α, β, and γ, respectively. This report's purification process was considered less time-consuming and could be efficiently applied to purify phycobiliproteins. The purified R-PE showed optimal stability up to 6 h at pH 7.0 when exposed to light (3000 lx), while the temperature at which the maximum stability was retained was at 20 °C. The cellular imaging property of R-PE was effectively implemented to evaluate its credentials without affecting the cell proliferation of Vero and Hep-2 cell lines with the higher IC50 concentrations in vitro. Under fluorescence microscopy and flow cytometry analysis, purified R-PE displayed the characteristic affinity towards cell imaging functions in preliminary in vitro studies.

Pseudomonas stutzeri MTCC 101 was explored as new bacterial strain for secreting anticancer L-methioninase. The partitioning study of this enzyme was further performed using Aqueous two-phase system (ATPS) and Triple phase system. Polyethylene glycol (PEG 4000) and magnesium sulfate (MgSO4) was selected as suitable ATPS system for its partitioning with higher partition coefficient (1.92) and 80% yield. Triple phase partition system (TPP) using n-butanol: ammonium sulphate showed its lower degree of yield (51.6%). ATPS phase components of PEG 4000 and MgSO4 was supplemented with its production media for highly recovery of L-methioninase. The culture condition (pH, temperature, inoculum volume, incubation period and agitation speed) during extractive fermentative production of L-methioninase was optimized using Response surface methodology (RSM). This model showed good adequacy with high value of coefficient of determination (R2) as 99.62%. The experiment was validated under statistically optimized culture condition with higher value of yield as 94.909%. This improved rate of yield of L-methioninase during extractive fermentative condition at optimized cultute conditions were significant finding for this research work. This extractive fermentative approach could be used as low cost-effective downstream processing techniques for its large-scale production in industry.

The phytochemical-rich extract obtained from black jamun pulp were encapsulated using vibrating dripping extrusion technique. The utilisation of alginate (AL) with four variations of core-shell material comprising gum Arabic (AL-GA), guar gum (AL-GG), pectin (AL-P) and xanthan gum (AL-X) was engaged to form calcium-alginate based lyophilised jamun extract encapsulated beads. It resulted that among four variations, lyophilised alginate with AL-GG based encapsulated jamun extract filled beads have better physicochemical characteristics and 95% encapsulation efficiency. The results revealed the morphological comparison of each variation. The release behaviour of AL-GG based beads has a higher release of total phenolics (TPC) and total anthocyanin content (TAC). The release kinetics model involving Ritger-Peppas and Higuchi model were applied for release TPC and TAC of all variations of beads. The Ritger-Peppas model was found best suitable in terms of average R2 (0.965) and lowest χ2 (0.0039). The release kinetics study showed that AL-GA based beads followed by AL-GG could also be the best suitable in release behaviour using simulated gastrointestinal fluids at 140–160 min. Overall, results shown the encapsulated Jamun beads have the best agro-industrial efficacy in form of phytochemical compounds based microparticles, holding decent antioxidant potential.

Filtration membranes are widely applied in medical fields. However, these membranes are challenged by bacterial contamination in hospitals, which increases the risk of nosocomial infections. Thus, it is significant to develop antibacterial filtration membranes. In this work, an oxidated dextran (ODex)-based antibacterial coating was designed and constructed on microfiltration (MF) membranes and melt-blown fabrics. Polyhexamethylene guanidine (PHMG) was synthesized as an antibacterial agent, and was fixed by ODex onto filtration membranes. The functionalized MF membranes increased the filtration efficiency for E. coli from 20.9% to 99.9%, and improved the absorption ratio for endotoxin by 59.1%, while the water flow rate still remained as high as 5255 L (h m2)−1. Furthermore, the trapped bacteria were inactivated by the antibacterial coating. For the melt-blown fabrics, the aerosol filtration efficiency was increased from 74.6% to 81.0%, and the antibacterial efficiency was promoted to 92.0%. The present work developed a facile and universal antibacterial functionalization strategy for filtration membranes, which provided a new method for the design and development of various novel antibacterial filtration materials in the medical field.

Background The measurement of plasma concentrations of retinol binding protein is a component of nutritional assessment in neonatal intensive care. However, serial testing in newborns is hampered by the limited amount of blood that can be sampled. Limitations are most severe with preterm infants, for whom close monitoring may be most important. Methods We developed an assay to quantify retinol binding protein using trypsin digestion and liquid chromatography-tandem mass spectrometry, which requires a serum or plasma volume of 5 µl. Additionally, we validated the method according to current recommendations and performed comparison with a standard nephelometry platform in clinical use. Results The assay demonstrated linearity from below 1 mg/dL (0.48 µM) to more than 20 mg/dL (9.7 µM), and an imprecision of 11.8% at 0.43 mg/dL (0.21 µM). The distribution of results observed with the new method was different when compared with nephelometry. Conclusion Liquid chromatography-tandem mass spectrometry facilitated testing a smaller sample volume, thereby increasing the ability to monitor key nutritional markers in premature infants. The differences in results compared with a commercially-available nephelometric assay revealed questionable results for lower concentrations by immunoassay.

Alternative liposome surface coatings for PEGylation to evade the immune system, particularly the complement system, have garnered significant interest. We previously reported poly(2-methacryloyloxyethyl phosphorylcholine) (MPC)-based lipids (PMPC-lipids) and investigated the surface modification of liposomes. In this study, we synthesize PMPC-lipids with polymerization degrees of 10 (MPC10-lipid), 20 (MPC20-lipid), 50 (MPC50-lipid), and 100 (MPC100-lipid), and coated liposomes with 1, 5, or 10 mol% PMPC-lipids (PMPC-liposomes). Non-modified and PEGylated liposomes are used as controls. We investigate the liposome size, surface charge, polydispersity index, and adsorption of plasma proteins to the liposomes post incubation in human plasma containing N,N,N',N'-ethylenediamine tetraacetic acid (EDTA) or lepirudin by some methods such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and automated capillary western blot, with emphasis on the binding of complement protein C3. It is shown that the coating of liposome PMPC-lipids can suppress protein adsorption more effectively with an increase in the molecular weight and molar ratio (1-10 mol%). Apolipoprotein A-I is detected on PMPC-liposomes with a higher molecular weight and higher molar ratio of PMPC-lipids, whereas α2-macroglobulin is detected on non-modified, PEGylated, and PMPC-liposomes with a shorter polymer chain. In addition, a correlation is shown among the PMPC molecular weight, molar ratio, and C3 binding. The MPC10-lipid cannot inhibit C3 binding efficiently, whereas surface modifications with 10 mol% MPC20-lipid and 5 mol% and 10 mol% MPC50-lipid suppress both total protein and C3 binding. Hence, liposome modification with PMPC-lipids can be a possible strategy for avoiding complement activation.

The hydrophilic interaction chromatography (HILIC) technique outperforms other methods in terms of precision and sensitivity. The development and optimization of a HILIC method for validating a separation method for simvastatin estimation in pharmaceutical formulations necessitated determining the optimal mobile phase, buffer concentration, and pH value. The proposed HILIC methods using two columns demonstrated high precision (RSD less than 1%) and a robust linear relationship between the calibration graph and simvastatin concentration ranges of 0.05–4 µg mL−1 with a coefficient of determination (R2) of 0.9996 and 0.9994 for simvastatin. The HILIC methods provided excellent validated LOD (0.023 and 0.015 µg mL−1) and LOQ (0.069–0.045 µg mL−1) values for simvastatin. The HILIC approach was successfully used to estimate simvastatin in commercial pharmaceutical tablets, resulting in an excellent simvastatin recovery > 99%. Using statistical tests, the findings of the HILIC method were compared to the British pharmacopoeia protocol for simvastatin drug. There was no difference in accuracy between the methods.

Polyphenols display potential functional activities especially for rosmarinic acid (RosA) and caffeic acid (CafA). In this study, two different methods including ultrasonic-assisted ethanol extraction (60%) and ultrasound-assisted cellulase (≥ 15000 U/g, 2%) hydrolysis were applied for the extraction of total phenolics from 44 species of Perilla frutescens. Folin-Ciocalteu method detection showed that the content of total phenolics extracted by cellulase hydrolysis was the highest and attained to 28.00 mgGAE/gextracts for ZB1. Continuously, the extracts were purified by XDA-8 macroporous resin and medium-pressure liquid chromatography (MPLC), and the content of total phenolics improved to 66.62 mgGAE/gextract. High-performance liquid chromatography (HPLC) assay showed that the total polyphenols were mainly composed of gallic acid, caffeic acid, rosmarinic acid, luteolin and apigenin. Besides, a sequential XDA-8 macroporous resin combined with high-speed counter-current chromatography (HSCCC)/MPLC system was established for simultaneously isolation and preparation of RosA (purity 98.29%) and CafA (purity 97.01%) from the extracts. Further, antibacterial activities of the total polyphenols were evaluated by disc diffusion method and scanning electron microscope (SEM) observation. The results verified that the total polyphenols had effective antibacterial capacity on three kinds of bacteria including E. coli, S. aureus, and B. subtilis in a concentration-dependent manner. All these results demonstrated that the ultrasound-assisted cellulase hydrolysis extraction of total polyphenols and proposed three-step separation of RosA and CafA had a high yield and good purity, and they exhibited an effective antibacterial ability.

Aim: We aimed to establish and validate a simple and sensitive UPLC–MS/MS method for the determination of UNC1999, a dual inhibitor against EZH1 and EZH2 in plasma samples. Materials & methods: UNC1999 in rat plasma was processed with protein precipitation method and then separated on a C18column and detected under positive ionization mode. The method presented good linearity over the range of 1.0–2000 ng/ml with good accuracy and precision. UNC1999 was absorbed slowly and achieved a maximum concentration of 118.8 ± 12.0 ng/ml 1.5 h after oral administration. Conclusion: The method provides a favorable character in selectivity, linearity, accuracy, precision, recovery, matrix effects and stabilities and was suitable for describing the pharmacokinetic profile of UNC1999.

Aim: To develop a new sensitive RP-HPLC method for simultaneous estimation of 5-fluorouracil (5-FU) and sonidegib (SDG). Materials & methods: Analytical and bioanalytical methods for simultaneous quantification of 5-FU and SDG in bulk, nanoformulations and in rat plasma were developed and validated using a gradient elution technique. Results: Separation of the analytes was effected on a Luna® C18 LC column using a mobile mixture comprising acetonitrile and acidified water. 5-FU and SDG were extracted from plasma matrix using liquid–liquid extraction. The applicability of the method was verified through single-dose oral pharmacokinetic study in Wistar rats. Conclusion: The developed methods allow a specific, sensitive and steady analytical procedure for the simultaneous estimation of 5-FU and SDG in nanoformulations and biological matrix.

Recently, interest has increased in natural metabolites compounds from plants for industrial and pharmaceutical use rather than synthetic compounds. The pomegranate peels are one of the mostly thrown away in general and considered not so useful. Using different chromatographic techniques, extracted, separated, cleaned, and defined two flavonoids’ compounds as a result of these experiments were obtained the following compounds which are quercetin-3-methoxy glucoside and quercetin-3′-O-β-glucopyranoside. The structures of these compounds were identified by using, UV and NMR. The quantitative estimation of total flavonoids, total alkaloids, total saponins, and total tannins showed presence in a good amount in pomegranate peel. The phytochemical reactions of pure extract and the corresponding sub-fractions of pomegranate peel showed the presence of target groups of secondary metabolites in crude and ethyl acetate fractions.

Aim: In the theme of quantitative LC–MS bioanalysis of oligonucleotides free of ion-pairing, a 22-mer RNA oligonucleotide took center stage. The focus was on a unique polar-based retention scheme to produce a high-recovery extraction presenting a high-performance alternative extraction means, also there was the opportunity to involve hydrophilic-interaction liquid chromatography and contemporary high-resolution MS as the end point. Results: Original LC–MS methodology was developed for the oligonucleotide and the performance was robust for both nominal and accurate mass detection, the latter affording 10× improvement in sensitivity and 4000-fold linear dynamic range, 500 pM to 2000 nM. Conclusion: A novel means of solid-phase extraction is exhibited within a robust pair-free methodology, reaching pM sensitivity with the demonstrably beneficial accurate mass platform.

Aim: Ruthenium-labeled antibodies are commonly used detection reagents in bioanalysis assays and must be characterized to ensure quality. The aim of this work was to develop a method to determine the concentration and incorporation ratio (the degree of labeling [DOL]) of ruthenium-labeled antibodies by UV/VIS spectroscopy. Materials & methods: Free SULFO-TAG compound was scanned using UV/VIS and showed an absorbance peak at 292 nm. In contrast, antibodies demonstrate UV absorbance at 280 nm. After experimentally determining the extinction coefficients at 280 and 292 nm of free ruthenium and antibody, we generated a formula based on the Beer–Lambert law that calculates both concentration and DOL of these ruthenium-labeled antibodies. Conclusion: The concentration and DOL values determined by our method were comparable to those determined from bicinchoninic acid and LC/MS for the same reagents. This method creates a faster and more accessible reagent characterization process that uses far less reagent than the more traditional alternatives.

For patients who are suffering from liver dysfunction or metabolic obstruction, excessive bilirubin (BIL) in their bodies may cause jaundice with irreversible cerebral injury. Traditional exchange transfusion and photodynamic therapy pose a risk of serious adverse reactions or limited curative effects. Therefore, as a generally used treatment, hemoperfusion (HP) purifies patients' blood with solid adsorbents. However, the development of clinical BIL absorbents is greatly impeded by low selectivity and unsatisfactory blood compatibility. Herein, inspired by oviparity, we propose BIL-imprinted poly(acrylic acid-co-sodium p-styrenesulfonate)-reduced graphene oxide (PAA-SS-rGO@BIL) hydrogel beads as BIL adsorbents via self-sacrificing micro-reactors. In the micro-reactors, cross-linked polymerization is achieved and a solidified gel is formed. The received hydrogel beads show outstanding selective adsorption capabilities toward BIL due to the recognition sites, and π-π and hydrophobic interactions. Such hydrogel beads possess superior blood compatibility owing to their bioinspired heparin-mimicking gel structure. Simulated BIL selective adsorption experiments in vitro demonstrate that the BIL concentrations in the plasma of a patient with severe jaundice can be restored to a moderate level within 3 hours. Therefore, hydrogel beads offer new options for clinical BIL adsorption.

This study includes investigation and evaluation of the factors that affect the preparation and characterization of nanobinary ethosmal dispersion for flufenamic acid in comparison to liposomal dispersion to optimize its efficiency and produce prolong action. It is involved in the preparation of eighteen formulas of nanoethosomal dispersion and two conventional liposomal dispersions which are optimized by utilizing different methods of preparation and different concentrations of ethanol, propylene glycol (PG), phosphatidylcholine (PC), and cholesterol. The dispersions were characterized morphologically by transmission electronic microscopic (TEM), physical appearance, pH, ʓ-potential, particle size, polydispersity index (PDI), encapsulation efficiency percentage (EE%) and in vitro drug release. The selected optimized nanoethosomal formula (F8) demonstrated pH (6.53), EE% (85.24%), particle size (226.3 nm), PDI (0.010), ʓ-potential (-93.43 mV), sustained release of 94.52% after 24 h and exhibited acceptable stability for 4 weeks preferentially at refrigerated conditions. These results significantly outperformed the classical liposomal dispersion (F1), which demonstrated EE% (70.00%), vesicles size (635.4 nm), PDI (0.347), ʓ-potential (-9.76 mV) and in vitro release of 55.41% after 24 h' demonstrated the potential influence of ethanol and PG on improving the in vitro characterization of the vesicles dispersions. This work succeeded in preparing a well optimized and stable nanoethosomal dispersion for flufenamic acid that can be utilized to prepare a suitable topical preparation to penetrate deep into the epidermal and dermal tissues, giving its action up to 24 h upon single administration.

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte’s pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS., Deutsche Forschungsgemeinschaft, Bundesministerium für Forschung und Technologie

In this study, a monoclonal antibody (mAb) specific to forchlorfenuron (CPPU) with high sensitivity and specificity was produced and designated (9G9). To detect CPPU in cucumber samples, an indirect enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold nanobead immunochromatographic test strip (CGN-ICTS) were established using 9G9. The half-maximal inhibitory concentration (IC50) and the LOD for the developed ic-ELISA were determined to be 0.19 ng/mL and 0.04 ng/mL in the sample dilution buffer, respectively. The results indicate that the sensitivity of the antibodies prepared in this study (9G9 mAb) was higher than those reported in the previous literature. On the other hand, in order to achieve rapid and accurate detection of CPPU, CGN-ICTS is indispensable. The IC50 and the LOD for the CGN-ICTS were determined to be 27 ng/mL and 6.1 ng/mL. The average recoveries of the CGN-ICTS ranged from 68 to 82%. The CGN-ICTS and ic-ELISA quantitative results were all confirmed by liquid chromatography—tandem mass spectrometry (LC-MS/MS) with 84–92% recoveries, which indicated the methods developed herein are appropriate for detecting CPPU in cucumber. The CGN-ICTS method is capable of both qualitative and semiquantitative analysis of CPPU, which makes it a suitable alternative complex instrument method for on-site detection of CPPU in cucumber samples since it does not require specialized equipment.

Levetiracetam (LEV) is a broad spectrum antiseizure medication that is used in various seizure types. There is evidence that therapeutic drug monitoring (TDM) of LEV is of value in selected patient populations, therefore determination of LEV plasma concentrations is essential. Herein we developed and validated a simple, reproducible, and practical method for the quantification of LEV concentrations in human plasma samples using high performance liquid chromatography (HPLC). Plasma samples (0.3 mL) deproteinization was done using acetonitrile. HPLC chromatographic separation of plasma samples was accomplished by reversed phase C18 column. The mobile phase constituted water and acetonitrile (90:10, v/v) ran at flow rate of 1 mL/min. Signal acquisition was conducted at a wavelength of 192 nm. Calibration curves showed excellent linearity (Correlation coefficient r2 > 0.99) over a concentration range of 3–80 μg/mL. Both inter and intraday assay accuracy and precision were less than 8% (except for the lowest limit of quantification was within 20%). Elution time was 15 min. The developed method excluded the use of buffers and utilized small volumes of plasma samples with simple mobile phase composition. Therefore, our method could be practically applied to routine TDM.

Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield and productivity while consuming less co-solvent.

Crude β-D-galactosidase from Lactobacillus leichmannii 313 was used to synthesise galacto-oligosaccharides (GOS) from lactose. Using an initial lactose concentration of 170 g/L, the yield of GOS was 52 g/L (i.e. 62% lactose conversion) after 2 h of enzymatic reaction. The highest number of unique individual GOS were generated after 48 h of reaction. ESI-MS and HPAEC analysis of the GOS enriched fractions obtained from 48 h mixtures showed the presence of sugars with degrees of polymerization (DP) ranging from 2 to 6. Further analysis (by NMR spectroscopy) of GOS in fraction 3 (containing ~ 96% w/w GOS) showed non-reducing terminals that were mostly Galp; and reducing ends that were →4)-Glcp, and →6)-Glcp. The most abundant internal linkages in fraction 3 were deduced to be →3)-Galp-(1→, and →6)-Galp-(1→. The predominance of β-D-(1→3, or 6) linkages in GOS fractions suggests that β-D-galactosidase from LL313 is suitable for generating unique GOS from lactose.

Mass-spectrometry-based proteomics is increasingly employed to monitor purification processes or to detect critical host cell proteins in the final drug substance. This approach is inherently unbiased and can be used to identify individual host cell proteins without prior knowledge. In process development for the purification of new biopharmaceuticals, such as protein subunit vaccines, a broader knowledge of the host cell proteome could promote a more rational process design. Proteomics can establish qualitative and quantitative information on the complete host cell proteome before purification (i.e., protein abundances and physicochemical properties). Such information allows for a more rational design of the purification strategy and accelerates purification process development. In this study, we present an extensive proteomic characterisation of two E. coli host cell strains widely employed in academia and industry to produce therapeutic proteins, BLR and HMS174. The established database contains the observed abundance of each identified protein, information relating to their hydrophobicity, the isoelectric point, molecular weight, and toxicity. These physicochemical properties were plotted on proteome property maps to showcase the selection of suitable purification strategies. Furthermore, sequence alignment allowed integration of subunit information and occurrences of post-translational modifications from the well-studied E. coli K12 strain.

Helium is commonly used as a carrier gas in gas chromatography-tandem mass spectrometry (GC-MS/MS); however, there are growing concerns regarding its global shortage and the resulting limited supply and high cost. Using nitrogen as an alternative carrier gas in GC-MS/MS with the widely used electron ionisation (EI) technique leads to a significantly lower sensitivity; thus, in this study, we explored the use of atmospheric-pressure chemical ionisation (APCI) as the ionisation method and examined the applicability of GC-(APCI)MS/MS with nitrogen gas for the determination of pesticide residues. GC-(APCI)MS/MS using nitrogen provided slightly wider peaks, and poorer isomeric separation compared to those using helium under identical conditions; however, the peak intensities were comparable. GC-(APCI)MS/MS using nitrogen was validated for 166 pesticides in green tea at a spiking level of 0.01 mg/kg and was compared with the conventional GC-(EI)MS/MS using helium gas. Except dimethomorph and resmethrin, GC-(APCI)MS/MS showed satisfactory results that were comparable to those of GC-(EI)MS/MS for most compounds, with trueness in the range of 73%–95% and relative standard deviations of