Fraction Collection Research Articles

Fly ash and sorbent particles agglomeration and removal in system of electrostatic agglomerator and electrostatic precipitator

The paper presents experimental studies on the effect of sodium bicarbonate and calcium hydroxide dry, low temperature sorbent injection on the concentration and size distributions of precipitated particles (fly ash and sorbent particles mixture), fractional collection efficiency, and particles morphology. The measurements were carried out on a lab-scale system comprising unipolar electrostatic agglomerator and electrostatic precipitator. In the experiments presented in this paper, the sorbent was injected upstream of electrostatic agglomerator. Results of SEM studies revealed the process of sorbent and fly ash particles agglomeration. The measurement of resistivity of fly ash and sorbent mixtures have shown that injected sorbent has a large effect on the particles resistivity, when the temperature of dust cake is increasing. In practical situations, too high resistivity can lead to back corona discharge generation, while too low causes the particles re-entrainment; both of these phenomena can reduce the dust collection efficiency of an electrostatic system.

OR30-03 High Resolution Daily Profiles Of Tissue Adrenal Steroids By Portable Automated Collection

Abstract Disclosure: T. Upton: None. E. Zavala: None. P. Methlie: None. M. Oksnes: None. M.A. Grytaas: None. K. Simunkova: None. S. Bensing: None. I. Botusan: None. K. Berinder: None. D.A. Vassiliadi: None. O. Kampe: None. S. Tsagarakis: None. E.S. Husebye: None. G.M. Russell: None. S.L. Lightman: None. Background: Rhythms are intrinsic to endocrine systems, and disruption of these hormone oscillations occurs at very early stages of disease. Since adrenal hormones are secreted with both circadian and ultradian periods, conventional single time point measures provide limited information about rhythmicity, and crucially do not provide information during sleep when many hormones fluctuate from nadir to peak concentrations. If blood sampling is attempted overnight this necessitates admission to a clinical research unit, can be stressful and disturbs normal sleep. Aim: To obtain automated ambulatory 24-hour profiles of adrenal steroids in healthy human subcutaneous tissue to characterize healthy variability without blood sampling. Methods: We used microdialysis, an ambulatory fraction collector and liquid-chromatography mass-spectrometry to obtain 72-point profiles of tissue free adrenal steroids over 24 hours in n=214 healthy volunteers. For validation, we compared tissue against plasma measurements in n=7 inpatient healthy volunteers. We used stationery statistics, time series analyses, and mathematical methods to characterise interindividual and intraindividual variability of these hormones at different times of the day. Results: There were no serious adverse events, and the method was well tolerated, allowing almost all normal activities to continue. In addition to cortisol, we identified daily and ultradian variation in free cortisone, corticosterone, 18-OHF, aldosterone, THF, and aTHF. Rhythmic hormones were highly correlated when measured across the day, especially cortisol with 18OHF (rs = 0.99), cortisol with cortisone (rs = 0.96) and cortisol and 18-OHF with corticosterone (rs = 0.96-0.97). There was a very strong correlation between mean values of cortisol and 18OHF with aldosterone (rs = 0.91-0.93), but aldosterone profiles were characterized by high interindividual variability. Tissue 18-OHF was the most highly correlated with plasma cortisol (rs 0.89, range 0.74—0.97, p<0.001), and 18-OHF phase was advanced with respect to tissue cortisol. The ratio of tissue cortisol:cortisone exhibited a stronger daily rhythm than in plasma, with cortisol concentrations often falling below cortisone in the late afternoon and early part of sleep.Using the data, we computed mathematically defined metrics (“dynamic biomarkers”) stratified by sex, age, and BMI to quantify individual variability of hormone rhythms. Provisional analysis suggests no evidence for an effect of sex on cortisol or 18OHF, but evidence for a small effect of BMI on total 24-hour cortisol, 18OHF, corticosterone, and aldosterone. Conclusions: These data provide, for the first time, an insight into the dynamics of adrenal steroids in tissue in real world setting and will serve normative reference data for establishing novel and improved biomarkers of endocrine disease. Presentation: Sunday, June 18, 2023

SAT296 Ambulatory 24-hour Tissue Microdialysis Of Cortisol And Metabolites, A Tool For Precision Management In Addison’s Disease

Abstract Disclosure: I. Botusan: None. K. Berinder: None. P. Methlie: None. T. Upton: None. E. Zavala: None. K. Simunkova: None. D.A. Vassiliadi: None. I. Marinelli: None. G.M. Russell: None. M.A. Grytaas: None. S. Tsagarakis: None. S.L. Lightman: None. E.S. Husebye: None. O. Kampe: None. M. Oksnes: None. S. Bensing: None. Background: The autoimmune attack on the adrenals in AD (Addison’s disease) results in lack of the vital hormones cortisol and aldosterone, rendering AD patients dependent on life-long hormone replacement therapy. Current therapies cannot replicate the physiological well-tuned, circadian and ultradian hormonal oscillations properly, probably contributing to increased mortality, morbidity and reduced quality of life. No established biomarker for treatment monitoring exists hampering treatment optimization. Ambulatory 24-hour dynamic steroid profiling may represent a novel therapy-monitoring tool, providing new insights in different replacement therapy regimes. Aim: We aimed to assess 24-hour profiles of free cortisol and metabolites in patients with AD on different glucocorticoid replacement therapies (GRT) and compare the results with healthy volunteers (HV). Material and methods: The study includes 41 patients with AD on different GRT regimes (hydrocortisone, cortisone acetate, modified release hydrocortisone) and 214 healthy participants. Twenty minute microdialysis fractions were collected ambulatory from abdominal subcutaneous tissue over 24 hours using a novel and portable microdialysis fraction collector (U-RHYTHM). Steroid hormones were analyzed by ultrasensitive liquid chromatography tandem mass spectroscopy (LC-MS/MS). Dynamic biomarkers were used to define hormone parameters in both healthy and hormone-replaced participants, and to assess the “time in range” for patients on GRT compared with the profiles of healthy controls. Results: Free cortisol and cortisone levels were measurable in all AD patients, while 18- hydroxycortisol, and corticosterone were detected only in a few AD patients suggesting the persistence of residual adrenal function. Lower cortisol levels during night were observed in AD compared with controls. Ambulatory 24-hour dynamic steroid profiling revealed marked interindividual variations partially depending on the type of GRT and dosing-regime. Conclusions: The 24-hour profiles of free cortisol and cortisone differs between patients with AD and healthy volunteers. Ambulatory 24-hour microdialysis samplings may provide a novel dynamic tool to monitor GRT in AD providing opportunity for individual dose adjustment. Presentation: Saturday, June 17, 2023

Development and evaluation of a triangular prism-multiple tubular wet electrostatic precipitator for capturing moxibustion oil fumes

Moxibustion oil fumes (MOF) have caused serious indoor air pollution. To solve the high costs problem existing in the current air purification technology, a triangular prism-multiple tubular wet electrostatic precipitator (WESP) was developed. The discharge characteristics of the modified WESP and the factors affecting its fractional collection efficiency (FCE) were explored in detail, and the collection performance of the modified WESP was further characterized by laboratory-scale tests. The results showed that the flushing water not only effectively cleaned the collection electrodes but also enhanced the corona current. Although increasing the applied voltage and reducing the flue gas velocity can improve the FCE, it was necessary to ensure that the ozone concentration met the emission standards. Specifically, the FCE of the modified WESP reached 43.09 %-97.48 % when the applied voltage was 15.5 kV, the flue gas velocity was 0.9 m·s−1, and the water consumption was 7.93 L· min−1·m−2. Under the optimal operation parameters, the modified WESP had an extremely low pressure drop (23 Pa) and a high clean air delivery rate (43.38 m3·h−1). After 12-h of continuous operation, the FCE of the modified WESP decreased slightly, but the FCE can be effectively recovered by cleaning the discharge electrodes. In conclusion, the modified WESP had the potential to efficiently capture MOF at low cost. Furthermore, the characteristic of MOF particles charged by the combustion process also helped us understand the high FCE of the modified WESP for MOF. Moreover, a modified efficiency equation of the triangular prism-multiple tubular WESP was proposed, and the experimental data were in good agreement with theoretical values. In addition, the modified WESP can provide a viable technical pathway to control oily particles emission.

Using stable carbon isotope ratio analysis to detect adulteration in red yeast rice dietary supplements

Red yeast rice (RYR) is marketed as a dietary supplement because it contains natural 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), including monacolin K. However, there is concern that some RYR supplements may be adulterated with the pharmaceutical drug lovastatin to enhance health claims. We have developed an optimized method to isolate monacolin K/lovastatin from complex RYR dietary supplement matrices to then test for adulteration in RYR supplements using stable carbon isotope (δ13C) analysis. Samples were initially screened for monacolin K/lovastatin using liquid chromatography with mass spectrometric detection (LC-MS). To ensure the extraction process did not affect the measured isotopic values (i.e., isotopic fractionation effects), neat lovastatin standards were spiked into two types of blank RYR matrices (powder and gel). The monacolin K/lovastatin peaks were detected using high performance liquid chromatography with ultraviolet detection (HPLC-UV) and isolated using fraction collection. Residual matrix components were removed from targeted fractions by solid phase extraction (SPE) using graphitized carbon black cartridges. The resulting isolates were then analyzed using elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) to measure δ13C values. The δ13C values of the extracted lovastatin standards were compared to their respective neat lovastatin δ13C values and demonstrated negligible isotopic fractionation effects. Using this optimized clean up method and carbon isotope analysis, thirty-one samples were screened. Eight RYR dietary supplement samples had >0.8 mg/g of monacolin K/lovastatin, our minimum threshold for analyzing samples using this method. Four of these eight samples had δ13C values greater than −28.3‰, a previously proposed cutoff value for natural monacolin K, indicating likely adulteration. Additionally, five RYR powder samples were analyzed as part of a collaborative study using in-house methods from two laboratories and the data shows acceptable agreement in the δ13C values of monacolin K/lovastatin (differences ranging from ±0.02‰ to ±0.76‰). This optimized method represents a robust, reproducible procedure for detecting lovastatin adulteration in dietary supplements with minimal isotopic fractionation.

An open-source smart fraction collector for isocratic preparative liquid chromatography

Preparative liquid chromatography is a technique for separating complex samples or isolating pure compounds from complex extracts. It involves eluting samples through a packed column and selectively collecting or isolating the separated bands in a sequence of fractions. Depending on the column length and the sample complexity, a large number of fractions may be obtained, making fraction collection a laborious and time-consuming process. Manual fraction collection is also tedious, error-prone, less reproducible, and susceptible to contamination. Several commercial and lab-made solutions are available for automated fraction collection, but most systems do not synchronize with the instrument detector and collect fractions at fixed volumes or time intervals. We have assembled a low-cost Arduino-based smart fraction collector that can record the signal from the UV–vis detector of the chromatography instrument and enable the automated selective collection of the targeted bands. The system consists of a robot equipped with position sensors and a 3-way solenoid valve that switches the column effluent between the waste or collection positions. By proper programming, an Arduino board records the detector response and actuates the solenoid valve, the position sensors, and the stepper motors to collect the target chromatographic bands.

A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples

We present a method and a simple system for high-pH RP-LC peptide fractionation of small sample amounts (30–60 µg), at micro-flow rates with micro-liter fraction collection using ammonium bicarbonate as an optimized buffer for system stability and robustness. The method is applicable to targeted mass spectrometry approaches and to in-depth proteomic studies where the amount of sample is limited. Using targeted proteomics with peptide standards, we present the method's analytical parameters, and potential in increasing the detection of low-abundance proteins that are difficult to quantify with direct targeted or global LC-MS analyses. This fractionation system increased peptide signals by up to 18-fold, while maintaining high quantitative precision, with high fractionation reproducibility across varied sample sets. In real applications, it increased the detection of targeted endogenous peptides by two-fold in a 25 cell-cycle-control protein panel, and in-depth MS analyses of nuclear extracts, it allowed the detection of up to 8,896 proteins with 138,417 peptides in 24-concatenated fractions compared to 3,344 proteins with 23,093 peptides without fractionation. In a relevant biological problem of CDK4/6-inhibitors and breast cancer, the method reproduced known information and revealed novel insights, highlighting that it can be successfully applied in studies involving low-abundance proteins and limited samples.•Tested nine high-pH buffer/solvent systems to obtain a robust, effective, and reproducible micro-flow fractionation method which was devoid of commonly encountered LC clogging/pressure issues after months of use.•Peptide enrichment method to improve detection and quantitation of low-abundance proteins in targeted and in-depth proteomic studies.•Can be applied to diverse protein samples where the available amount is limited.

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System.

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

Liquid chromatography on a centrifugal platform for separation and collection of water-soluble dyes

A reversed-phase chromatographic process is developed on a centrifugal platform to separate and collect water-soluble dyes from a mixture. A separation column filled with C18-reversed phase silica gel was used to separate the components from a mixture and the eluate was collected by a series of collecting chambers. The purified components can then be identified and extracted from the collecting chambers. The effects of the silica gel's particle size (7–10, 20–45, and 46–63 µm) and the platform's rotational speed (1000, 1500, 2000 RPM) on the separation and collection efficiency were investigated. Experimental results showed that dye separation could be well performed in the column with smaller-sized silica gels (7–10 µm) under a low rotational speed (1000 RPM). However, for the eluate collection, the high eluent flowrate and long processing time resulted in a convective band-broadening problem in the collecting chambers, which affected the recovery ratio of the dyes. Experimental results showed that the convective band broadening effect can be reduced by reducing the flowrate, shortening the collecting time, and switching the eluent to a different composition. The best recovery ratio of the dyes in the current design can be achieved by using the column with a powder size distribution of 46–63 µm and operating at the rotational speed of 1500 RPM. This platform can process a sample volume of 1 μL and the processing time is about 30 min. Since the only instrument used is a motor, the complete chromatographic process, from separation to fraction collection, can be carried out on a centrifugal platform at a low cost.

Exploration and optimization of extraction, analysis and data normalization strategies for mass spectrometry-based DNA adductome mapping and modeling

Although interest in characterizing DNA damage by means of DNA adductomics has substantially grown, the field of DNA adductomics is still in its infancy, with room for optimization of methods for sample analysis, data processing and DNA adduct identification. In this context, the first objective of this study was to evaluate the use of hydrophilic interaction (HILIC) vs. reversed phase liquid chromatography (RPLC) coupled to high resolution mass spectrometry (HRMS) and thermal acidic vs. enzymatic hydrolysis of DNA followed by DNA adduct purification and enrichment using solid-phase extraction (SPE) or fraction collection for DNA adductome mapping. The second objective was to assess the use of total ion count (TIC) and median intensity (MedI) normalization compared to QC (quality control), iQC (internal QC) and quality control-based robust locally estimated scatterplot smoothing (LOESS) signal correction (QC-RLSC) normalization for processing of the acquired data. The results demonstrate that HILIC compared to RPLC allowed better modeling of the tentative DNA adductome, particularly in combination with thermal acidic hydrolysis and SPE (more valid models, with an average Q2(Y) and R2(Y) of 0.930 and 0.998, respectively). Regarding the need for data normalization and the management of (limited) system instability and signal drift, QC normalization outperformed TIC, MedI, iQC and LOESS normalization. As such, QC normalization can be put forward as the default data normalization strategy. In case of momentous signal drift and/or batch effects however, comparison to other normalization strategies (like e.g. LOESS) is recommended. In future work, further optimization of DNA adductomics may be achieved by merging of HILIC and RPLC datasets and/or application of 2D-LC, as well as the inclusion of Schiff base stabilization and/or fraction collection in the thermal acidic hydrolysis-SPE sample preparation workflow.

High-resolution daily profiles of tissue adrenal steroids by portable automated collection.

Rhythms are intrinsic to endocrine systems, and disruption of these hormone oscillations occurs at very early stages of the disease. Because adrenal hormones are secreted with both circadian and ultradian periods, conventional single-time point measurements provide limited information about rhythmicity and, crucially, do not provide information during sleep, when many hormones fluctuate from nadir to peak concentrations. If blood sampling is attempted overnight, then this necessitates admission to a clinical research unit, can be stressful, and disturbs sleep. To overcome this problem and to measure free hormones within their target tissues, we used microdialysis, an ambulatory fraction collector, and liquid chromatography-tandem mass spectrometry to obtain high-resolution profiles of tissue adrenal steroids over 24 hours in 214 healthy volunteers. For validation, we compared tissue against plasma measurements in a further seven healthy volunteers. Sample collection from subcutaneous tissue was safe, well tolerated, and allowed most normal activities to continue. In addition to cortisol, we identified daily and ultradian variation in free cortisone, corticosterone, 18-hydroxycortisol, aldosterone, tetrahydrocortisol and allo-tetrahydrocortisol, and the presence of dehydroepiandrosterone sulfate. We used mathematical and computational methods to quantify the interindividual variability of hormones at different times of the day and develop "dynamic markers" of normality in healthy individuals stratified by sex, age, and body mass index. Our results provide insight into the dynamics of adrenal steroids in tissue in real-world settings and may serve as a normative reference for biomarkers of endocrine disorders (ULTRADIAN, NCT02934399).

A cell culture system to model pharmacokinetics using adjustable-volume perfused mixing chambers

The pharmacokinetic (PK) profile of a drug is an essential factor in determining its efficacy, yet it is often neglected during in vitro cell culture experiments. Here, we present a system in which standard well plate cultures may be “plugged in” and perfused with PK drug profiles. Timed drug boluses or infusions are passed through a mixing chamber that simulates the PK volume of distribution specific to the desired drug. The user-specified PK drug profile generated by the mixing chamber passes through the incubated well plate culture, exposing cells to in vivo-like PK drug dynamics. The effluent stream from the culture may then optionally be fractionated and collected by a fraction collector. This low-cost system requires no custom parts and perfuses up to six cultures in parallel. This paper demonstrates a range of PK profiles the system can produce using a tracer dye, describes how to find the correct mixing chamber volumes to mimic PK profiles of drugs of interest, and presents a study exploring the effects of differing PK exposure on a model of lymphoma treatment with chemotherapy.

Butyrate modulation of inflammatory responses to lipopolysaccharide in porcine monocytes

Abstract Butyrate is a short-chain fatty acid produced through microbial fermentation of dietary fibers in the lower intestinal tract. Butyrate production is associated with enhanced mucosal immunity, increased barrier integrity, and modulation of immune cells. Butyrate driven immunomodulation occurs through a variety of mechanisms, including histone deacetylase (HDAC) inhibitor activity and inhibition of NF-kB signaling. Butyrate impacts lipopolysaccharide (LPS)-induced cytokine production and gene expression by porcine monocytes in a dose-dependent manner. To investigate the mechanisms driving butyrate modulation of porcine monocyte responses to LPS, peripheral monocytes were cultured with LPS +/− butyrate. After incubation, cells were processed for collection of cytoplasmic and nuclear fractions. HDAC activity in nuclear fractions and NF-kB signaling state (IkBa, p-IkBa, p65, p-p65) in cytoplasmic fractions was assessed. Butyrate did not inhibit HDAC activity after 1 hour (h) but at 24 h, butyrate exposure resulted in inhibition of HDAC activity regardless of LPS exposure, in a dose-dependent manner. Neither the low nor high concentration of butyrate altered IkBa, p-IkBa, p65, or p-p65 status, though p-IkBa and p-p65 status was dependent on LPS exposure. Overall, the impact of butyrate on HDAC activity was time and dose-dependent, and butyrate had little to no effect on NF-kB signaling induced by LPS exposure. Continued investigation is warranted to understand the potential mechanisms driving butyrate modulation of monocyte responses to LPS to enhance abilities to modulate immune status. USDA-ARS CRIS 005

Creation of a versatile automated two-step purification system with increased throughput capacity for preclinical mAb material generation

The ever-increasing speed of biotherapeutic drug discovery has driven the development of automated and high throughput purification capabilities. Typically, purification systems require complex flow paths or third-party components that are not found on a standard fast protein liquid chromatography instrument (FPLC) (e.g., Cytiva's ÄKTA) to enable higher throughput. In early mAb discovery there is often a trade-off between throughput and scale where a high-throughput process requires miniaturized workflows necessitating a sacrifice in the amount of material generated. At the interface of discovery and development, flexible automated systems are required that can perform purifications in a high-throughput manner, while also generating sufficient quantities of preclinical material for biophysical, developability, and preclinical animal studies. In this study we highlight the engineering efforts to generate a highly versatile purification system capable of balancing the purification requirements between throughput, chromatographic versatility, and overall product yields. We incorporated a 150 mL Superloop into an ÄKTA FPLC system to expand our existing purification capabilities. This allowed us to perform a range of automated two-step tandem purifications including primary affinity captures (protein A (ProA)/immobilized metal affinity chromatography (IMAC)/antibody fragment (Fab)) followed by secondary polishing with either size exclusion (SEC) or cation exchange (CEX) chromatography. We also integrated a 96 deep-well plate fraction collector into the ÄKTA FPLC system with purified protein fractions being analyzed by a plate based high performance liquid chromatography instrument (HPLC). This streamlined automated purification workflow allowed us to process up to 14 samples within 24 h, enabling purification of ∼1100 proteins, monoclonal antibodies (mAbs), and mAb related protein scaffolds during a 12-month period. We purified a broad range of cell culture supernatant volumes, between 0.1 and 2 L, with final purification yields up to 2 g. The implementation of this new automated, streamlined protein purification process greatly expanded our sample throughput and purification versatility while also enabling the accelerated production of greater quantities of biotherapeutic candidates for preclinical in vivo animal studies and developability assessment.

Evaluation of alternative gas chromatographic and mass spectrometric behaviour of trimethylsilyl-derivatives of non-hydrolysed sulfated anabolic steroids.

Sulfated metabolites have shown to have potential as long-term markers (LTMs) of anabolic-androgenic steroid (AAS) abuse. The compatibility of gas chromatography-mass spectrometry (GC-MS) with trimethylsilyl (TMS)-derivatives of non-hydrolysed sulfated steroids has been demonstrated, where, after derivatisation, generally, two closely eluting isomers are formed that both have the same molecular ion [M-H2 SO4 ]•+ . Sulfated reference standards are in limited commercial availability, and therefore, the current knowledge of the GC-MS behaviour of these compounds is mainly based on sulfating and analysing the available standard reference material. This procedure can unfortunately not cover all of the current known LTMs as these are often not available as pure substance. Therefore, in theory, some metabolites could be missed as they exhibit alternative behaviour. To investigate the matter, in-house sulfated reference materials that bear resemblance to known sulfated LTMs were analysed on GC-MS in their TMS-derivatised non-hydrolysed state. The (alternative) gas chromatographic and mass spectrometric behaviour was mapped, evaluated and linked to the corresponding steroid structures. Afterwards, using fraction collection, known sulfated LTMs were isolated from excretion urine to confirm the observed findings. The categories that were selected were mono-hydroxy-diones, 17-methyl-3,17-diols and 17-keto-3,16-diols as these are commonly encountered AAS conformations. The ability to predict the GC-MS behaviour of non-hydrolysed sulfated AAS metabolites is the corner stone of finding new metabolites. This knowledge is also essential, for example, for understanding AAS detection analyses, for the mass spectrometric characterization of metabolites of new designer steroids or when one needs to characterize an unknown steroid structure.

Off-line two-dimensional separation involving supercritical fluid chromatography for the characterization of the wastewater from algae hydrothermal liquefaction

An off-line multidimensional method involving liquid chromatography combined with supercritical fluid chromatography was developed for the characterization of the wastewater of hydrothermal liquefaction of microalgae Chlorella sorokiniana. The first dimension consisted of a phenyl hexyl column operated in reversed-phase mode, whereas the second dimension was performed on a diol stationary phase. Optimization of the kinetic parameters of the first and second dimensions were performed, taking into account the fraction collection system. The beneficial effect of working at high flow rate in both dimensions, as well as the need to work with short columns (50 mm) in the second dimension was evidenced. Injection volume was also optimized in both dimensions. The first dimension benefited from on-column focusing, while in the second dimension, untreated water-rich fractions could be injected without peak deformation. The performances of offline LCxSFC were compared to LC-HRMS, SFC-HRMS and LCxLC-HRMS for the analysis of the wastewater. Despite a long analysis time of 3.3h, the off-line separation coupled to high-resolution mass spectrometry exhibited a very large orthogonality with 75 % occupation rate of the separation space, reaching an effective peak capacity of 1050. While other evaluated techniques were faster, one-dimensional techniques failed to separate the numerous isomers while LCxLC exhibited lower orthogonality (45% occupation rate).

Effects of Addition of Tea Polyphenol Palmitate and Process Parameters on the Preparation of High-Purity EPA Ethyl Ester.

High-purity eicosapentaenoic acid (EPA) ethyl ester (EPA-EE) can be produced from an integrated technique consisting of saponification, ethyl esterification, urea complexation, molecular distillation and column separation. In order to improve the purity and inhibit oxidation, tea polyphenol palmitate (TPP) was added before the procedure of ethyl esterification. Furthermore, through the optimization of process parameters, 2:1 (mass ratio of urea to fish oil, g/g), 6 h (crystallization time) and 4:1 (mass ratio of ethyl alcohol to urea, g/g) were found to be the optimum conditions in the procedure of urea complexation. Distillate (fraction collection), 115 °C (distillation temperature) and one stage (the number of stages) were found to be the optimum conditions for the procedure of molecular distillation. With the addition of TPP and the above optimum conditions, high-purity (96.95%) EPA-EE was finally obtained after column separation.

Alterations in Abundance and Compartmentalization of miRNAs in Blood Plasma Extracellular Vesicles and Extracellular Condensates during HIV/SIV Infection and Its Modulation by Antiretroviral Therapy (ART) and Delta-9-Tetrahydrocannabinol (Δ9-THC).

Alterations in Abundance and Compartmentalization of miRNAs in Blood Plasma Extracellular Vesicles and Extracellular Condensates during HIV/SIV Infection and Its Modulation by Antiretroviral Therapy (ART) and Delta-9-Tetrahydrocannabinol (Δ9-THC).

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

A new approach on data extrapolation for mortar dating in the Zagreb Radiocarbon Laboratory

There is no unique way of mortar sample preparation that would always provide the true date. We propose a procedure of 14C age extrapolation from CO2 fractions obtained by sequential dissolution of mortar grain size 32 – 63 μm by phosphoric acid. The collection of CO2 fractions is deduced from kinetics of mortar-hydrolysis curve.The procedure was designed from data obtained from mortars prepared in laboratory and tested on mortar/plaster from three archaeological cases, with confirmed ages. The first fractions gave true result for historical mortars, but for laboratory ones only extrapolated values were true. When certain conditions regarding CO2 fraction collection were met, extrapolated values agreed with the first fractions for the historical mortars. Although the extrapolation procedure seems to eliminate the effect of dead carbon contamination, it is not effective against influence of delayed hardening or restoration.