Bench Top Research Articles

First report of ergot caused by Claviceps clavispora in switchgrass (Panicum virgatum L.) in Georgia, USA.

Switchgrass (Panicum virgatum L.) is a North American grass species with biofuel potential. Claviceps spp. is known to infect the florets of various grass species, initially characterized by a sticky honeydew exudate and later as sclerotium replacing the seed in the infected ovary (Tanaka et al 2023). Since 2019, from July to October, switchgrass panicles in Georgia have been observed with honeydew and black sclerotia. The disease was first noted on some of the 285 accessions of the genome-wide association study (GWAS) panels at the University of Georgia's Iron Horse Farm in Watkinsville and Gibbs Farm in Tifton, GA. In Watkinsville, GA, ergot incidence was 5%, 6%, 65% and 54% in 2019, 2020, 2021 and 2022, respectively. Symptomatic panicles with honeydew and sclerotia were collected in 2021 (sample Scl) and 2023 (samples Cla_M and ATH20cl) from Watkinsville, GA. Under microcopy, panicles with honeydew symptoms had mycelium and conidia (9 μm long [range of 5-13 μm] and 4 μm wide [3-5 μm]) consistent with Claviceps spp. (Tooley et al. 2001). Sclerotia were 1.5 mm long (range 1-3 mm). Sclerotia were surface sterilized for 3 minutes in 5% NaOCl, followed by 70% ethanol then rinsed three times in distilled water. Sterilized sclerotia were plated on potato dextrose agar and placed on bench top with a 12-hour day/night cycle at room temperature (22°C) (Singh 1976). After 2 months, the sclerotia produced sterile apothecia. For molecular identification, genomic DNA was extracted from three honeydew samples following the protocol of Doyle and Doyle (1987). The internal transcribed spacer (ITS) region and RNA polymerase second largest subunit (RPB2) gene were amplified using ITS4/ITS5 (White et al. 1990) and 5F2/7CR (Liu et al. 1999) primer sets. The ITS region of ATH20cl, Cla_M, and Scl (GenBank nos. PP546317- PP546319) showed 92.83-97.48% identity to C. clavispora (NR_163506.1). The RPB2 region of ATH20cl, Cla_M, and Scl (GenBank nos. PP573916- PP573918) showed 97.46-97.72% identity to C. clavispora (LT216566.1). The maximum likelihood tree constructed in MEGA-X (Kumar et al. 2018) using concatenated ITS (539 bp) and RPB2 (792 bp) gene sequences from this study and eleven reference sequences from Tanaka et al. (2023), revealed close relatedness of ATH20cl, Cla_M, and Scl to C. clavispora under section Pusillae. The pathogenicity test for samples ATH20cl and Cla_M was conducted in the greenhouse on switchgrass cultivar 'Alamo' grown in injection molded pots containing Sungro professional growing mix. Three replicates plants at reproductive (R3) growth stage were inoculated by immersing panicles in 105 spores/ml suspension for 5 minutes and bagged for 3 days (Tooley et al. 2001). Control plants were immersed in distilled water. Honeydew symptoms and sclerotia appeared within 7- and 90-days post-inoculation, respectively, whereas control plants remained symptom-free. The honeydew collected from the infected Alamo panicles were reconfirmed to produce similar spores to those collected from the field. This is the first report of ergot caused by C. clavispora in switchgrass. Ergot was also observed since 2019 on some accessions from the same GWAS panel planted at the University of Tennessee Plant Sciences Farm in Knoxville, TN. This information will be beneficial to determine the effect of ergot on biomass production and seed quality of switchgrass in the southeastern U.S.

Neural net analysis of NMR spectra from strongly-coupled spin systems

Extracting parameters such as chemical shifts and coupling constants from proton NMR spectra is often a first step in using spectra for compound identification and structure determination. This can become challenging when scalar couplings between protons are comparable in size to chemical shift differences (strongly coupled), as is often the case with low-field (bench top) spectrometers. Here we explore the potential utility of AI methods, in particular neural networks, for extracting parameters from low-field spectra. Rather than seeking large experimental sets of spectra for training a network, we chose quantum mechanical simulation of sets, something that is possible with modern software packages and computer resources. We show that application of a network trained on 2-D J-resolved spectra and applied to a spectrum of iduronic acid, shows some promise, but also meets with some obstacles. We suggest that these may be overcome with improved pulse sequences and more extensive simulations.

Abstract TP214: In-Silico versus In-Vitro Evaluation of a New Stent-Retriever Design: A Novel Approach to Pre-Clinical Device Development

Introduction: In-vitro evaluation (analysis in a bench top flow model) is the gold standard for pre-clinical investigation of devices being developed for endovascular thrombectomy (EVT). In-silico evaluation (computer simulated analysis in a virtual stroke model) has the potential to test and optimize large number of stent-retriever design variations in a relatively time and cost-effective manner. We aim to validate the utility of in-silico evaluation when compared with in-vitro evaluation for the SuperNova Stent-retriever. Methods: In-silico analysis was performed using a virtual thrombectomy model, built using data from a fine-grained finite-element model, to estimate the probability of successful recanalization. Neurovascular anatomy of the virtual model was built based on digital subtraction angiography of an in-vitro model (Sim Agility, Mentice, Inc). Gravity Medical Technology’s SuperNova Stent-retriever was the device under investigation. Multiple thrombectomy scenarios were defined and validation analysis was performed. Results: We defined multiple thrombectomy scenarios including 1 cm red blood clot in M1 artery, 2 cm bifurcation M1-M2 red blood clot (Y-shaped clot), 1 cm white blood clot in M1 artery, and 1 cm white blood clot in superior M2 artery. The figure below demonstrates a pictorial representation of the first scenario- 1 cm red blood clot in M1 artery followed by mechanical thrombectomy simulation using the SuperNova stent-retriever. Per our in-vitro analysis (10 thrombectomy experiments), the rate of first pass effect and the rate of complete recanalization after a maximum of 3 passes using SuperNova stent-retriever was 50% and 90%, respectively. Preliminary data suggests 97% concordance. Conclusion: In-silico analysis has the potential to improve and expedite pre-clinical thrombectomy device development. Further studies are required to better understand the scope and potential of this technology.

Ionome mapping and amino acid metabolome profiling of Phaseolus vulgaris L. seeds imbibed with computationally informed phytoengineered copper sulphide nanoparticles

This study reports the effects of a computationally informed and avocado-seed mediated Phyto engineered CuS nanoparticles as fertilizing agent on the ionome and amino acid metabolome of Pinto bean seeds using both bench top and ion beam analytical techniques. Physico-chemical analysis of the Phyto engineered nanoparticles with scanning-electron microscopy, transmission electron microscopy, X-ray diffraction, and Fourier Transform Infrared Spectroscopy confirmed the presence of CuS nanoparticles. Molecular dynamics simulations to investigate the interaction of some active phytocompounds in avocado seeds that act as reducing agents with the nano-digenite further showed that 4-hydroxybenzoic acid had a higher affinity for interacting with the nanoparticle’s surface than other active compounds. Seeds treated with the digenite nanoparticles exhibited a unique ionome distribution pattern as determined with external beam proton-induced X-ray emission, with hotspots of Cu and S appearing in the hilum and micropyle area that indicated a possible uptake mechanism via the seed coat. The nano-digenite also triggered a plant stress response by slightly altering seed amino acid metabolism. Ultimately, the nano-digenite may have important implications as a seed protective or nutritive agent as advised by its unique distribution pattern and effect on amino acid metabolism.Graphical abstract

Stability Indicating an LC-MS/MS Method Development and Validation for the Quantification of Capmatinib in Human Plasma

A linear and accurate liquid chromatographic tandem mass spectrometric (LC-MS/MS) technique for the quantitation of capmatinib in plasma was developed and subjected for validation. Canagliflogin was employed as an internal standard (IS). Extraction of plasma was executed utilizing 5 mL of ethyl acetate solvent. Analysis was performed Zorbax ODS (50 mm × 2.1 mm x 3 μ) stationary phase at room conditions and a movable solvent composition of 0.1% HCOOH, acetonitrile and methyl alcohol (15:50:35). The flowing rate of the movable phase was 0.4 mL/min. Drug and IS were identified in positive mode of ionization with electrospray mode to get the mass transitions of (m/z): Capmatinib, 413.15/386.14 and canagliflogin (IS), 445.15/267.12. The correlation between capmatinib concentrations and their respective peak proportions to canagliflogin was a straight line over 0.2 to 3200 ng/mL concentration levels. Intra day and inter-day precisions were ≤5.18% for capmatinib. Inter and intra day bias were within the range of −4.17 to 4.53%. The mean measured extraction recovery of capmatinib was 99.23%. Recovery of IS was 98.31%. Capmatinib was subjected for long-term, freeze thaw, bench top, short-term stability, auto-sampler, dry extract, and stock solution stability at low QC and high QC levels and it was stable at all these conditions. The established technique can be utilized to regularly quantitfy of capmatinib in plasma samples in industries, forensic labs, and clinical research organizations.

Investigating best practice for specimen preparation for biological testing of root canal sealers

IntroductionBiological characterization of root canal sealers is important as it assesses the ability of the root canal sealer to exert antimicrobial properties thus avoiding treatment failures caused by microbial challenge and also assess the cytotoxic effect on the periapical tissues. Assessment of the biological testing of root canal sealers necessitates the sterilisation of the materials prior to evaluation. This study aims to analyse the influence of various sterilisation techniques conducted prior to biological testing on the microstructure and surface properties of endodontic sealers. Assessment of the initial microbial contamination on the material was also undertaken. MethodsFour commercial sealers were investigated. The sealers were either prepared in a laminar flow cabinet or on a laboratory bench top under ambient conditions. Each group was further divided into 5 groups (n = 3) based on the sterilization technique:1) ethanol-10 mins, 2) ultraviolet-1 h, 3) ethanol-10 mins + ultraviolet-1 h, 4) autoclave, and 5) no sterilisation (control). Microbial levels in the materials were assessed by plate streaking technique. The materials were characterized by scanning electron microscopy and energy dispersive spectroscopy, and Fourier transform infrared spectroscopy, before and after sterilisation, to assess any changes in microstructure and chemical composition. ResultsAll the materials did not exhibit contamination when prepared in laminar flow chamber in sterile conditions compared with sealers prepared on the bench top. Three of the commercial materials showed changes in microstructure while one (TotalFill) was not affected by the sterilisation. AH Plus and BioRoot RCS exhibited alterations in water and alcohol peaks in FT-IR while the single syringe sealers (TotalFill and BioRoot Flow) showed no changes. ConclusionsSterilisation methods cause physical and chemical alterations to sealers. Material preparation should be performed in a laminar flow cabinet and a test for sterility should be performed prior to any biological testing being undertaken. If the materials are not sterile, assessment of the effects of the sterilization methods is recommended.

Development and Evaluation/Verification of a Fully Automated Test Platform for the Rapid Detection of Cyclospora cayetanensis in Produce Matrices.

Cyclosporiasis, caused by the coccidian parasite Cyclospora cayetanensis, has emerged as an increasing global public health concern, with the incidence of laboratory-confirmed domestically acquired cases in the US exceeding 10,000 since 2018. A recently published qPCR assay (Mit1C) based on a mitochondrial target gene showed high specificity and good sensitivity for the detection of C. cayetanensis in fresh produce. The present study shows the integration and verification of the same mitochondrial target into a fully automated and streamlined platform that performs DNA isolation, PCR, hybridization, results visualization, and reporting of results to simplify and reduce hands-on time for the detection of this parasite. By using the same primer sets for both the target of interest (i.e., Mit1C) and the internal assay control (IAC), we were able to rapidly migrate the previously developed Mit1C qPCR assay into the more streamlined and automated format Rheonix C. cayetanensisTM Assay. Once the best conditions for detection were optimized and the migration to the fully automated format was completed, we compared the performance of the automated platform against the original "bench top" Mit1C qPCR assay. The automated Rheonix C. cayetanensis Assay achieved equivalent performance characteristics as the original assay, including the same performance for both inclusion and exclusion panels, and it was able to detect as low as 5 C. cayetanensis oocysts in fresh produce while significantly reducing hands-on time. We expect that the streamlined assay can be used as a tool for outbreak and/or surveillance activities to detect the presence of C. cayetanensis in produce samples.

Abstract 249: In‐silico Versus In‐Vitro Evaluation of a New Stent‐Retriever Design: A Novel Approach to Pre‐clinical Development

Introduction In‐vitro evaluation (analysis in a bench top flow model) is the gold standard and a regulatory requirement for pre‐clinical investigation of devices being developed for endovascular thrombectomy (EVT). In‐silico evaluation (computer simulated analysis in a virtual stroke model) has the potential to test and optimize large number of stent‐retriever design variations in a relatively time and cost‐effective manner. Further, new design concepts can be tested across multiple anatomical scenarios. We aim to validate the utility of in‐silico evaluation when compared with in‐vitro evaluation for the SuperNova Stent‐retriever. Methods In‐silico analysis was performed using a virtual thrombectomy model, built using data from a fine‐grained finite‐element model, to estimate the probability of successful recanalization and emboli in new/distal territory. Neurovascular anatomy of the virtual model was built based on digital subtraction angiography of an in‐vitro model (Sim Agility, Mentice, Inc). Gravity Medical Technology’s SuperNova Stent‐retriever was the device under investigation‐ physical device for in‐vitro analysis and virtual replica for in‐silico analysis (built using finite element analysis incorporating various physical metrics of the stent‐retriever). Experiments performed for in‐vitro analysis were replicated for in‐silico analysis. Multiple thrombectomy scenarios were defined and validation analysis was performed. Data was analyzed using SPSS 23 (IBM, Armonk, NY) Results We defined multiple thrombectomy scenarios including 1 cm red blood clot in M1 artery, 2 cm bifurcation M1‐M2 red blood clot (Y‐shaped clot), 1 cm white blood clot in M1 artery, and 1 cm white blood clot in superior M2 artery. The figure below demonstrates a pictorial representation of the first scenario‐ 1 cm red blood clot in M1 artery followed by mechanical thrombectomy simulation using the SuperNova stent‐retriever. Per our in‐vitro analysis (10 thrombectomy experiments), the rate of first pass effect and the rate of complete recanalization after a maximum of 3 passes using SuperNova stent‐retriever was 50% and 90%, respectively. Complete data from in‐silico analysis is being generated and will be presented at the conference. Preliminary data suggests high degree of concordance. Conclusion In‐silico analysis has the potential to improve and expedite pre‐clinical thrombectomy device development. Further studies are required to better understand the scope and potential of this technology.

B-178 Clinical Routine Ultrasensitive LC-MS/MS Method Development for Analysis of 17β-Estradiol in Human Serum

Abstract Background The increasing clinical demand for the measurement of circulating 17β-Estradiol at very low concentrations consolidates the need of highly sensitive and specific analytical methodologies. These assays are suitable in situations such as antiestrogen therapy monitoring (use of aromatase inhibitor in breast cancer treatment); diagnosis of suspect inherited sex-steroid metabolic disorders; diagnosis of precocious and delayed puberty in female; hormone replacement therapy in postmenopausal women; and estrogen deficiency in men. Time-consuming methods and lack of sensitivity are common in the quantification of 17β-Estradiol in serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The strategy of chemical derivatization is common for analysis of estrogens because it enhances specificity and sensitivity, but this approach becomes laborious in the sample preparation process. For the purpose of solving these analytical challenges, the aim of this work was developing a reliable, sensitive and low-cost LC-MS/MS method without derivatization for clinical routine analysis. Methods Isotope-labeled internal standard 17β-Estradiol-D5 were added to calibrators, quality controls (QC) and serum samples before a liquid-liquid extraction with the organic solvent Diethyl Ether. After reconstitution, the extracts were injected into a Biphenyl column 50 × 2.1 mm, 2,6 µm, chromatographic separated by solvent gradient (Ammonium Fluoride in Water – Acetonitrile) and detected in positive electrospray ionization mode. The analytical method was validated using reference material grade standards and spiked QCs in charcoal stripped pool patient serum at 4 levels by the assessment of linearity, limit of quantification (LoQ), precision inter and intra-assay, accuracy, carryover, method comparison, cross-reactivity and stability (freeze-thawing cycles, bench top, postoperative and stock solution). Results The method has been validated with the analytical measurement range of 2–500 pg/mL. It was successfully optimized to achieve low levels of 17β-Estradiol as proposed with limit of quantitation of 2 pg/mL. Within-run and between-run precision (coefficient of variation) study yielded 1.3%–7.2% and accuracy within 95.7%–112.1%. No carryover, matrix effect and significant cross-reactivity was observed. Estradiol was found to be stable in serum samples under 3 freeze/thawing cycles, 4 h at ambient temperature and 72 h after extraction procedure in auto-injector. The stability of stock solutions was tested in −20 °C freezer and remained stable for 1 month. Method comparison between two reference laboratories (LC-MS/MS method) was performed and the results showed acceptable data for regression statistics (slope (a) = 0.9184; intercept (b) = −0.1576), t-test statistics (bias = 8.5%) and difference plot. Conclusion An ultrasensitive and derivatization-free LC-MS/MS method was developed and it may be beneficial for patients with endocrine disorders associated with estradiol levels.

Detection of common adulterants in olive oils by bench top 60 MHz 1H NMR with partial least squares regression

Olive oil is the oil obtained from the fruit of the olive tree, Olea europaea L., and is commonly used in cooking, cosmetics, and soaps. The high value and demand for olive oil products has led to some producers adding cheaper edible non-olive oils to olive oil to increase their profitability. Many of these adulterant oils have similar taste profiles to extra virgin olive oil; however, these can cause allergic reactions in individuals with sensitivities to the non-olive oil. Given the implications of olive oil adulteration, there is a demand for methods to identify and quantify adulterant oils in olive oil samples. This work explores the utility of low-field benchtop nuclear magnetic resonance (NMR) with a novel chemometrics workflow for the quantification of non-olive oils in authentic extra virgin olive oil samples. The measurements were obtained using a 60 MHz 1H NMR. The icoshift algorithm was utilized for alignment, and partial least squares regression models of the entire NMR spectrum were used for quantification. Here, 17 different varieties of edible non-olive oils were investigated. The models were highly linear, accurate, and precise. Most of these adulterant oils were detectable below 10% v/v in extra virgin olive oil samples.

Stability Indicating RP-HPLC Method for Quantitative Estimation of Lenalidomide and Its Impurities in Solid Dosage Form

The main aim and objectives of the research are to develop an effective, sensitive, economical, and simple reverse-phase HPLC method developed for determining and quantifying Lenalidomide impurities in Lenalidomide solid dosage formulations. The lack of research work and no compendial methods available for estimating this drug influenced the current research investigation to give a simple, sensitive, rapid, precise, accurate and robust gradient high-performance liquid chromatographic method for the determination and quantification of Lenalidomide and its impurities. Samples are analyzed using reverse phase (RP-HPLC) using stationary phase an Inertsil ODS-3V (150 x 4.6 mm, 3μm), and the mobile phase consists of two channels A and B. channel-A: pH 3.0 phosphate buffer and Channel-B: Acetonitrile: water (90:10 v/v) in the proportion of gradient elution. The flow rate is 1.0 mL/min. The column temperature was maintained at 40°C, and the sample cooler temperature was maintained at 5°C, injection volume of 20 μL, and wavelength of 210 nm. The developed HPLC method was validated concerning specificity, and the chromatograms were recorded for blank, placebo, standard, sample, and spiked sample solutions of Lenalidomide and its related substances. Specificity studies reveal that the peaks are well separated from each other. For precision, the results were found to be within acceptable limits. The limit of detection (LOD) and limit of quantitation (LOQ) for impurity-A 0.1124 μg/mL and 0.0371μg/mL, Impurity-B 0.2247μg/mL and 0.0742μg/mL, respectively. The linearity results for Lenalidomide and all the impurities in the specified concentration range are satisfactory, with a correlation coefficient greater than 0.99. The accuracy studies were shown as % recovery for Lenalidomide and its impurities at the specification level; the results obtained were within limits. Solution stability parameter was established; standard, sample, and spiked sample solutions are stable up to 48 hrs on a bench top at the refrigerator. Filter validation parameters were established, and the filtered spiked sample solutions are compatible with both 0.45 μm PVDF & 0.45 μm Nylon filters.

Development and Validation of Stability Indicating RP-HPLC Method for Quantitative Estimation of Enzalutamide in Enzalutamide Capsules Dosage Form

Objectives: A precise, accurate and selective stability-indicating reverse phase high performance liquid chromatographic assay method has been developed for the quantitative estimation of Enzalutamide in Enzalutamide capsules dosage form. Materials and Methods: The separation was achieved by using a stationary phase Waters X-Bridge Shield RP18 (150 x 4.6 mm, 3.5μ) and the mobile phase consisted of perchloric acid buffer and acetonitrile in the proportion of (20:80 volume/volume). The run velocity was 1.2 mL/min. Enzalutamide was identified using UV detector at the wavelength of 210 nm. Column oven temperature 25°C and sample cooler temperature 25°C and infused quantity 20 μL, run time was 15 min. Results: As there is no meddling flanked by blank and placebo at the retention time of Enzalutamide. Degradation study results were shown significant degradation was observed in acid and oxidation (peroxide) stress condition. Hence it can be concluded that Enzalutamide is sensitive to acid and oxidation. To obtain system precision, a study was conducted with six replicate injections. %RSD was estimated from the peak areas of Enzalutamide establish to be 0.55% correspondingly. The relative standard deviation for method exactitude was establish to be 0.55%. The suggested HPLC technique was linear over the range of 100.6-301.8 μg/mL, the correlation coefficient was establish to be 0.9999. The accuracy studies were shown as % recovery for Enzalutamide 50% to150% level. The limit of % recovered revealed is in the assortment of 98 and 102% and the consequences obtained were establish to be within the limits. Hence the technique was establish to be accurate. The solution steadiness of the standard and samples are stable upto 48 hr on a bench top and refrigerator (2-8°C). The method is robust for changes like flow rate and column oven temperature. Performed the filter validation for sample solution 0.45 μm PVDF and 0.45 μm Nylon filterers are suitable for filtration. The method has validated as per ICH guidelines and all the validation parameters are satisfy the ICH Q2 specification acceptance limits. Conclusion: The developed method was validated for an assortment of parameters as per ICH guidelines like accuracy, precision, linearity, specificity, system suitability, solution stability and robustness. The consequences obtained were within the acceptance criteria. So, it can be concluded that the urbanized technique is simple, precise, cost-effective, eco-friendly, and safe and can be successfully employed for the routine analysis of Enzalutamide in bulk and pharmaceutical dosage forms.

A literature review of bench top and pilot lead corrosion assessment studies.

Bench top and pilot lead corrosion studies are gaining more interest, considering revisions and upcoming improvements to the Lead and Copper Rule. This literature review identified studies ranging from simpler month(s)-long bench top dump-and-fill stagnant water tests (coupon tests/standing pipe tests) to more complicated year(s)-long intermittent flow pilot studies (recirculating pipe loops/once through pipe rigs). With increasing complexity in design and operation, studies more closely approximated real plumbing conditions (e.g., by incorporating harvested lead pipes and intermittent flow regimes) at increased cost, footprint, and duration. Comparison of bench top and pilot designs (in terms of lead test piece age/dimensions/configuration/replicates, study duration, sample collection, and other factors) can assist drinking water utilities, consultants, academics, and others to select a design that matches their needs and constraints. No matter the choice, surrogate systems cannot replace actual system water testing and are best complemented by other corrosion assessment tools.

Steady-State Free Precession sequences for high and low field NMR spectroscopy in solution: Challenges and opportunities

The receptivity of NMR spectroscopy is low when compared to other techniques. Historically, increasing the strength of the static magnetic field has been the major approach to increase NMR sensitivity. In recent years several polarization transfer protocols have been used to enhance the signal-to-noise ratio (SNR), although they require special accessories and/or sample preparation. In this paper, we consider both the challenges and opportunities of steady-state free precession (SSFP) pulse sequences as a simple and efficient alternative to enhance SNR, in standard high-resolution and benchtop low-resolution NMR spectrometers. The maximum gain in these sequences is obtained with the shortest time between the pulses (Tp). However, when Tp<T2, the SSFP signal contains FID and echo components which lead to phase, intensity, and truncation artifacts on spectra obtained by Fast Fourier transform (FT). Several phase alternation SSFP sequences were used to cancel the echo component and minimize these problems in the FT spectra. Krylov base diagonalization method (KBDM) was used to eliminate the phase and truncation problems in spectra acquired by SSFP pulse sequences and can be a viable alternative to FT. The experiments were performed in high and low resolution (bench top) NMR spectrometers and significant enhancements in SNR of low receptivity nuclei such as 13C and 15N could be achieved. The SSFP sequences were also shown to enhance SNR in nuclei with high receptivity such as 19F and 31P, in very dilute samples, as is common in environmental and biological samples.

Development and Validation of Stability Indicating RP-HPLC Method for Quantitative Estimation of Tofacitinib in Tofacitinib Tablets Dosage Form

Aim: The main intention of the research work is to develop an effective, sensitive, economical and uncomplicated reverse phase HPLC technique for the estimation of Tofacitinib in Tofacitinib tablets dosage form.&#x0D; Study Design: HPLC based quantification studies.&#x0D; Place and Duration of Study: Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, between August 2022 and November 2022.&#x0D; Methodology: Estimation of Tofacitinib in Tofacitinib tablets dosage form. The separation was achieved by using a stationary phase Kromasil C18 (150 x 4.6 mm, 5µ) and the mobile phase consisted of pH 4.0 phosphate buffer and acetonitrile in the ratio of (80:20 volume/volume). The flow rate was 1.5 mL/min. Tofacitinib was detected using UV detector at the wavelength of 215 nm. Column temperature 25°C and sample temperature 25°C and injection volume 20µL, run time was 20 minutes.&#x0D; Results: As there is no meddling between blank and placebo at the retention time of Tofacitinib. Degradation study results were shown significant degradation was observed in alkali (base) stress condition. Hence it can be concluded that Tofacitinib is sensitive to alkali. To obtain system exactitude, a study was conducted with six replicate injections. %RSD was estimated from the peak areas of Tofacitinib found to be 0.16% respectively. The relative standard deviation for method exactitude was found to be 5 mg and 10 mg is 0.26% and 0.75%. The proposed HPLC technique was linear over the range of 24.88-74.64 µg/mL, the correlation coefficient was found to be 1.0000. The accuracy studies were shown as % recovery for Tofacitinib 50% to150% level. The limit of % recovered shown is in the range of 98 and 102% and the results obtained were found to be within the limits. Hence the method was found to be accurate. The solution stability of the standard and samples are stable upto 48 hrs on a bench top and refrigerator (2-8°C). The technique is robust for changes like flow rate, column oven temperature, pH variation and the organic phase of the mobile phase. Performed the filter validation for sample solution 0.45 µm PVDF filterers are suitable for filtration.&#x0D; The technique has authenticated as per ICH instructions and all the validation parameters are satisfy the ICH Q2 specification acceptance limits.&#x0D; Conclusion: The urbanized technique was validated for various parameters as per ICH guidelines like accuracy, precision, linearity, specificity, system suitability, solution stability and robustness. The results obtained were within the acceptance criteria. So, it can be concluded that the developed method is simple, precise, cost-effective, eco-friendly, and safe and can be successfully employed for the routine analysis of Tofacitinib in bulk and pharmaceutical dosage forms.

A CASE (Computer-Assisted Structure Elucidation) for Bench-Top NMR Systems in the Undergraduate Laboratory for De Novo Structure Determination: How Well Can We Do?

The recent popularity of benchtop (BT) NMR systems has prompted its applications in undergraduate laboratories around the world. Owing to their low maintenance cost, due to the lack of a superconducting magnetic core, and simple operation, these BT NMR systems can fulfill many of the learning objectives outlined in the undergraduate organic chemistry curricula. With a variety of BT NMR systems currently available (e.g., 43, 60, 80, and 100 MHz), it can be overwhelming for instructors to determine which system is appropriate for their needs. When used as a structure elucidation tool, the focus is often placed solely on solving chemical structures, prompting the eventual question of the magnetic field strength requirements for de novo structure elucidation. To answer this question, two artificial intelligence (AI) software packages, namely Structural Elucidator (v.2020.1.2) from ACD/Laboratories and Mnova Structure Elucidation (v 14.2.3) from Mestrelab Research, were used. These software provide an unbiased, yet separate, metric to gauge the effect of magnetic field strength on the accuracy of the determined structures. For comparison purposes, results from these two BT magnetic field strengths will be compared to those obtained from a high field NMR (500 MHz) spectrometer, providing a complete overview of the advances, as well as limitations in current BT systems for undergraduate education. In addition, the spectral data presented in this work can be used as a practical example in class to illustrate the effect of spectral resolution on the accuracy of determined structures, which is fundamental to understanding structure elucidation within organic chemistry.

Validated liquid chromatography-tandem mass spectrometry method for simultaneous quantitation of bendamustine and copanlisib in mouse plasma: Application to a pharmacokinetic study in mice.

In this study, we report the development and validation of an LC-tandem mass spectrometry method for the simultaneous quantitation of bendamustine and copanlisib in mouse plasma as per the US FDA regulatory guidelines. The sample processing involves extraction of bendamustine and copanlisib along with internal standard (IS; warfarin) from 50 μL mouse plasma using a liquid-liquid extraction method. The chromatographic separation of bendamustine, copanlisib and the IS was achieved on an Atlantis dC18 column using an isocratic mobile phase (5 mM ammonium acetate:methanol, 20:80 v/v). Bendamustine, copanlisib and the IS eluted at 0.88, 1.39 and 0.74 min, respectively, with a total run time of 2.5 min. The calibration curve ranged from 3.99-2996 and 4.33-3248 ng/mL for bendamustine and copanlisib, respectively. Inter- and intra-day precision and accuracy, stability in processed samples and upon storage, dilution integrity and incurred sample reanalysis were investigated for both the analytes. The intra- and inter-day precisions were in the ranges of 2.01%-5.05% and 2.74%-6.13% and 1.98%-7.64 and 8.62%-9.04% for bendamustine and copanlisib, respectively. Stability studies showed that both analytes were stable on bench top for 6 h, in auto-sampler for 24 and at -80°C for 30 days. The validated method was successfully applied to a pharmacokinetic study in mice.

V12-01 NEW BIPOLAR RADIOFREQUENCY SEALING TOOL FOR TRANSDERMAL VASECTOMY IN THE RABBIT MODEL: A PARADIGM SHIFT IN VASECTOMY TECHNIQUE?

You have accessJournal of UrologyCME1 May 2022V12-01 NEW BIPOLAR RADIOFREQUENCY SEALING TOOL FOR TRANSDERMAL VASECTOMY IN THE RABBIT MODEL: A PARADIGM SHIFT IN VASECTOMY TECHNIQUE? Gerard Henry, Ryan Griggs, Michel Labrecque, Joel Marmar, and Tariq Hakky Gerard HenryGerard Henry More articles by this author , Ryan GriggsRyan Griggs More articles by this author , Michel LabrecqueMichel Labrecque More articles by this author , Joel MarmarJoel Marmar More articles by this author , and Tariq HakkyTariq Hakky More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002638.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bipolar Radiofrequency (RF) sealing tools have been shown to be successful in the treatment of many areas in the human body. RF has been utilized in the sealing of larger blood vessels and in nerve pain ablation in humans, showing good results for years. Urology has used RF ablation percutaneously for treating renal tumors for years. We evaluate the utilization of a new bipolar RF sealing tool for transdermal vasectomy in the rabbit model following AUA guidelines. METHODS: Two groups of 5 male New Zealand white rabbits were given IV sedation and local anesthetic and subsequent transdermal bilateral partial vasectomy using a high lateral scrotal approach. We isolated the vas with a novel insulated clamp fitting the U-shaped electrode of the sealing tool (Figure 1). In Group 1 (n=5), the device was set at 60W for 20s; however, this may differ across species. In the second group (n=5) the size of the U-shaped tool jaw was reduced and the device was set at 40 and 60W using thermal sensors. Impedance alarms shut off energy at a predetermined level. Wound healing, complications, survival, postmortem biopsies, and histology were evaluated. A doe rabbit was used with semen collections & semen analyses preformed on all male rabbits. RESULTS: We sacrificed 4 of the 5 (80%) of Group 1 rabbits due to poor wound healing secondary to excessive thermal damage. All 5/5 (100%) of Group 2 rabbits had good tissue healing and survived the procedure. The impedance alarm went off at about 5 seconds of sealing time and the device energy shut off in all Group 2 procedures. All rabbits had a negative semen analysis and on histology had occluded vas deferens. In bench top testing, we found that a 5 second interval is optimal for vas ligation. (Figure 1)Bench top testing revealed that thin short jaws (50 degree Celsius) created lower nearby tissue temperature than wide long jaws (77 degree Celsius). CONCLUSIONS: Transdermal bipolar RF sealing successfully occludes the vas deferens in rabbit vasectomy. The jaw size and sealing time using an impedance alarm system appear to be important for wound healing. This novel technique may decrease vasectomy time and morbidity. Canine studies are underway and analysis is pending. Further studies and FDA approval are needed before its application in humans. Source of Funding: Signati © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e967 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gerard Henry More articles by this author Ryan Griggs More articles by this author Michel Labrecque More articles by this author Joel Marmar More articles by this author Tariq Hakky More articles by this author Expand All Advertisement PDF DownloadLoading ...

New Bipolar Radiofrequency Sealing tool for Transdermal Vasectomy in the Rabbit Model: a Paradigm Shift in Vasectomy Technique?

ABSTRACT Introduction Bipolar Radiofrequency (RF) sealing tools have been shown to be successful in the treatment of many areas in the human body. RF has been utilized in the sealing of larger blood vessels and in nerve pain ablation in humans, showing good results for years. Urology has used RF ablation percutaneously for treating renal tumors for years. Objective We evaluate the utilization of a new bipolar RF sealing tool for transdermal vasectomy in the rabbit model. Methods Two groups of 5 male New Zealand white rabbits were given IV sedation and local anesthetic and subsequent transdermal bilateral vasectomy using a high lateral scrotal approach. We isolated the vas with a novel insulated clamp, fitting the U-shaped electrode of the sealing tool (Figure 1) around the clamp. In Group 1 (n=5), the device was set at 60 watts for 20 seconds. In the second group (n=5), the size of the U-shaped tool jaw was reduced and the device was set at 40 and 60 watts using impedance sensing to automatically shut off energy at a predetermined level. Wound healing, complications, survival, postmortem biopsies, and histology were evaluated. A doe rabbit was used with semen collections &amp; semen analyses preformed on all male rabbits. Results We sacrificed 4 of the 5 (80%) of Group 1 rabbits due to poor wound healing due to excessive thermal damage. All 5/5 (100%) of Group 2 rabbits had good tissue healing and survived the procedure. At about 5 seconds of sealing, the impedance sensing automatically shut off power to the device in all Group 2 procedures. All rabbits had a negative semen analysis and on histology had occluded vas deferens. In bench top testing, we found that a 5 second interval is optimal for vas ligation. (Figure 1) Bench top testing revealed that thin short jaws created lower nearby tissue temperature (50 degree Celsius) than wide long jaws (77 degree Celsius). Conclusions Transdermal bipolar radiofrequency sealing successfully occludes the vas deferens in rabbit vasectomy. The jaw size and sealing time using an impedance alarm system appear to be important for wound healing. This novel technique may decrease vasectomy time and morbidity. Further studies are needed before its application in humans. Disclosure Yes, this is sponsored by industry/sponsor: Signati Clarification: Industry initiated, executed and funded study Any of the authors act as a consultant, employee or shareholder of an industry for: Signati

A low-cost, highly functional, emergency use ventilator for the COVID-19 crisis.

Respiratory failure complicates most critically ill patients with COVID-19 and is characterized by heterogeneous pulmonary parenchymal involvement, profound hypoxemia and pulmonary vascular injury. The high incidence of COVID-19 related respiratory failure has exposed critical shortages in the supply of mechanical ventilators, and providers with the necessary skills to treat. Traditional mass-produced ventilators rely on an internal compressor and mixer to moderate and control the gas mixture delivered to a patient. However, the current emergency has energized the pursuit of alternative designs, enabling greater flexibility in supply chain, manufacturing, storage, and maintenance considerations. To achieve this, we hypothesized that using the medical gasses and flow interruption strategy would allow for a high performance, low cost, functional ventilator. A low-cost ventilator designed and built-in accordance with the Emergency Use guidance from the US Food and Drug Administration (FDA) is presented wherein pressurized medical grade gases enter the ventilator and time limited flow interruption determines the ventilator rate and tidal volume. This simple strategy obviates the need for many components needed in traditional ventilators, thereby dramatically shortening the time from storage to clinical deployment, increasing reliability, while still providing life-saving ventilatory support. The overall design philosophy and its applicability in this new crisis is described, followed by both bench top and animal testing results used to confirm the precision, safety and reliability of this low cost and novel approach to mechanical ventilation. The ventilator meets and exceeds the critical requirements included in the FDA emergency use guidelines. The ventilator has received emergency use authorization from the FDA.