Elution Technique Research Articles

Resistance Genes and Mortality in Carbapenem-resistant Klebsiella pneumoniae Bacteremias: Effects of the COVID-19 Pandemic

Emerging carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) (CRKP) bacteremias are presenting significant public health risks due to limited treatment options and increased mortality. K. pneumoniae isolates exhibit carbapenem resistance rates that vary from 25% to 50% throughout the European continent, including our country. To assess the characteristics of CRKP bacteremia, a condition that has recently demonstrated an increasing prevalence in our center. We sought to ascertain the resistance rates of isolated strains to antibiotics other than carbapenems, identify the responsible carbapenemase genes, evaluate the efficacy of antibiotics, determine mortality rates, explore clonality among strains, and investigate the influence of the COVID-19 pandemic on all these factors. Retrospective observational study. This study included patients aged 18 and older who had experienced meropenem-resistant K. pneumoniae bacteremia. Meropenem resistance was confirmed by employing the Kirby-Bauer disk diffusion method. Meropenem minimum inhibitory concentration (MIC) levels were determined using the gradient test, while colistin MIC levels were ascertained using the disk elution technique. Carbapenemase genes were evaluated via colony polymerase chain reaction (PCR), and clonality analysis was performed using the arbitrarily primed PCR technique. The study comprised 230 patients, with a mean age of 63.1 ± 15.9 years, of whom 58.7% were male. Oxacillinase-48 (OXA-48) was detected in 74.8% of the patients, New Delhi metallo-beta-lactamase (NDM) in 12.6%, OXA-48 + NDM in 7.8%, and KPC in 4.8%. The 14-day and 30-day mortality rates were 57% and 69.6%, respectively. Multivariate analysis of the 30-day mortality revealed several crucial factors, including bacteremia development in the intensive care unit, the occurrence of bacteremia during the COVID-19 pandemic, polymicrobial bacteremia, the use of indwelling intravenous catheters, a platelet count of ≤ 140,000/μl, procalcitonin levels of ≥ 6 μg/l, and a Charlson comorbidity score ≥ 3. Notably, the OXA-48 and KPC genes were upregulated significantly during the COVID-19 pandemic, while the NDM gene groups were downregulated. Additionally, both 14-day and 30-day mortality rates increased significantly. In this study, the most prevalent carbapenemase gene was OXA-48; however, there has been a recent increase in KPC genes. No dominant epidemic strain was identified through clonality analysis. The clustering rate was 68% before the pandemic, increasing to 85.7% during the pandemic. The significance of infection control measures is underscored by the rise in both clustering and mortality rates during the COVID-19 pandemic.

Evaluation of the Greenness, Whiteness, and blueness profiles of stability indicating HPLC method for determination of Denaverine hydrochloride and benzyl alcohol in pharmaceuticals

Recently, there has been important emphasis in pharmaceutical analysis on the stability of drug formulations and the development of appropriate stability-indicating studies. In this regard, a comprehensive stability-illustrating RP-HPLC approach has been established and validated for the assay of denaverine hydrochloride (DEN) and benzyl alcohol (BNZ) in Sensiblex® Injectable Solution.Different stress conditions were applied to both components, following the guidelines of the International Conference on Harmonization (ICH). Using the isocratic elution technique, DEN, BNZ, and their degradation products were effectively isolated and quantified on an Inertsil C8 column at room temperature. The mobile phase consisted of acetonitrile: water: orthophosphoric acid (80:20:0.1, by volume), and the flow speed was maintained at 0.8 mL/min. Detection was performed at 220 nm using a diode array detector, with concentration ranges of 10.00–30.00 and 5.00–15.00 μg/mL for DEN and BNZ, respectively. The total analysis time was less than five minutes, establishing this novel chromatographic method as a priority for routine quality control analysis of both drugs. The inclusion of acetonitrile was necessary for achieving adequate resolution. The environmental impact was evaluated using three approaches: Green Analytical Procedure Index (GAPI), Analytical Eco-Scale, and Analytical GREEnness metric (AGREE). Additionally, White Analytical Chemistry (WAC) was employed to evaluate the quality (R), ecological impact (G), and economic viability (B) of the developed approach.Additionally, the newly presented Blue Applicability Grade Index (BAGI) metric was also assessed. The final Eco-scale score of 85 and the AGREE score of 0.65 indicate the environmentally friendly nature of the new chromatographic method. The BAGI final score was 82.5, while the overall score of the WAC tool was 91.8, highlighting the method's practicality and utility. DEN exhibits the highest degradation (24%) in acidic conditions, whereas BNZ is particularly susceptible to oxidative degradation, with a degradation percentage of 35%. New four degradation products were formed, and the two investigated compounds were efficiently separated and analyzed in their presence. Further identification of degradation products for DEN and BNZ is strongly recommended, employing a mass spectrophotometric detector. Notably, the novel method is accurate, reliable, economical, and time-efficient. In conclusion, it can be effectively employed for routine quality control and stability assessment of DEN and BNZ in both their pure form and pharmaceutical formulations.

HPLC METHOD FOR THE DETERMINATION OF SOME ANTIBIOTIC RESIDUES IN DIFFERENT HOSPITALS WASTEWATER IN BAGHDAD CITY, IRAQ

Antibiotics present the greatest threat to soil and aquatic ecosystems among the different therapeutic groups of medicines (which include prescription drugs and treatments for cancer). The strongest drugs, antibiotics, have been utilized to stop the growth of microorganisms or eradicate them. Using high-performance liquid chromatography technology with fluorescence detection, the amounts of levofloxacin and tetracycline in the wastewater from three hospitals (Medical City, Al-Kindi, and Al-Yarmouk) were determined. Levofloxacin and tetracycline were chosen in this study because they are the most important water pollutants. These antibiotic residues were separated and measured using a gradient elution technique on a reverse-phase C18 column. A combination of methanol and deionized water made up the mobile phase system. For a 20-minute analysis period, the detector's excitation and emission wavelengths were set to 310 and 420 nm, respectively. Cleaning the SPE cartridge came after the extraction using pH 4 citrate buffer. Excellent linear ties (R2 > 0.9998) were seen in the calibration curves for levofloxacin and tetracycline at concentrations between 10 and 40 µg/ml. The limits of detection and quantification for levofloxacin and tetracycline were determined to be 0.61 µg/ml, 2.04 µg/ml, and 0.46 µg/ml and 1.54 µg/ml, respectively. The analysis of residues of antibiotics in various wastewater samples was successfully applied using the suggested methodology. The findings demonstrate the presence of tetracycline and levofloxacin in all wastewater samples. However, Al-Kindi had greater concentrations of them than Medical City and Al-Yarmouk. The proposed technique can be applied to a range of medical products in different wastewater sources, such as hospitals and industrial settings. For citation: Ala’a Abdullwahid Jasim, Wasan Abdulwahid Jasim, Jasim M. S. Jamur HPLC method for the determination of some antibiotic residues in different hospitals wastewater in Baghdad city, Iraq. ChemChemTech [Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol.]. 2024. V. 67. N 6. P. 21-28. DOI: 10.6060/ivkkt.20246706.7035.

TANDEM MASS SPECTROMETRIC METHOD FOR THE TRACE LEVEL DETERMINATION OF 2-AMINOPYRIDINE: A POTENTIAL GENOTOXIC IMPURITY IN TENOXICAM API

Objective: This study aimed to develop a highly sensitive method for the determination of the genotoxic impurity 2-amino pyridine in Tenoxicam, employing hyphenated techniques. Methods: The determination of 2-amino pyridine was carried out using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in Selected Ion Monitoring mode (SIM). A LiChrospher RP-18 (100×4.6 mm) 5.0 µm column was utilized for the separation. A gradient elution technique was employed with acetonitrile (mobile phase A) and 0.01M ammonium acetate buffer (mobile phase B) in varying ratios. The gradient program (T/%B) was set as 0/5, 2.50/15, 5.00/30, 10.00/50, 15.00/95, 20.00/95. The developed method was validated according to the International Conference on Harmonization guidelines. Results: The limits of detection (LOD) and quantification (LOQ) for 2-amino pyridine were found to be 0.09 ppm and 0.3 ppm, respectively. The method demonstrated accuracy within the range of 89.1% to 106.6% for the analyte. The method's linearity was confirmed through a six-point calibration graph spanning 6 ppm to 75 ppm, corresponding to a concentration of 20 mg/ml of Tenoxicam. Conclusion: Developed hyphenated LC-MS/MS method presented in this study offers a highly sensitive and accurate means for the determination of the genotoxic impurity 2-amino pyridine in Tenoxicam. With validated LOD and LOQ values, as well as demonstrated accuracy, this method proves to be a robust quality control tool suitable for the quantitation of 2-amino pyridine at very low concentrations in the pharmaceutical compound Tenoxicam.

Antidiabetic Ligand Based Reverse Docking Studies on Isolated Phytoconstituents from Stem Bark Chloroform Extract of Strychnos nux‐vomica

AbstractStrychnos nux‐vomica (S.N) also known as poisonous nut, Dog button, Kuchla whose seeds and barks have various components that are used in folklore and traditional medicines in different countries. Stem bark of SN is collected from local area and extracted using chloroform by soxhlation process and isolated the phytoconstituents from the chloroform extract using column chromatography method by gradient elution technique. Six compounds are isolated from the column out of which four compounds are found to be novel from the S.N. Obtained phytoconstituents are checked for in silico studies using reverse docking process with the help of Schrodinger software and the docking score indicates that the phytoconstituents are found to have very good antidiabetic action.

Mass Spectrometry-Compatible Elution Technique Enables an Improved Mucin-Selective Enrichment Strategy to Probe the Mucinome.

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and disease-driven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to a solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, the elution step required boiling in SDS, thus necessitating an in-gel digest with trypsin. Here, we introduce innovative elution conditions amenable to mucinase digestion and downstream analysis using mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing the glycopeptide signal. We then benchmarked this technique against different O-glycan binding moieties for their ability to enrich mucins from various cell lines and human serum. Overall, the new method outperformed our previous procedure and all of the other enrichment techniques tested. This allowed for the effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.

Enhancement of the antitumor effect of cisplatin and ginsenoside Rg3 by encapsulation in polylactic-co-glycolic acid nanoparticles

Purpose: To develop dual-loaded polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) with two chemotherapeutic agents, i.e., cisplatin (DDP) and 20(R)-ginsenoside Rg3(Rg3), and to evaluate the drug release profiles and synergistic inhibitory effects of the nanoparticles on lung cancer A549 cells.Methods: The dual-laden PLGA NPs were synthesized using modified emulsion and solvent vaporization procedures. Drug loading (DL) and efficacy of co-encapsulation (EE) were determined with a modification of column elution technique. The cytotoxic and inhibitory effects of individual and combined drugs on A549 lung cancer cells were evaluated using MTT assay.Results: A sustained pattern of drug release was shown by PLGA/DDP, PLGA/Rg3, and PLGA/DDP+Rg3. The formulated PLGA and unbound drugs exerted dose- and time-reliant cytotoxic effects on A549 cells. At all concentrations tested, drugs encapsulated in PLGA nanoparticles were more effective in killing the cells than the free drugs (p < 0.05). In particular, PLGA/DDP + Rg3 formulation produced a synergistic inhibitory effect on the proliferation of A549 cells.Conclusion: Single- and dual-load PLGA groups display variabilities in profiles of drug release and in vitro cytotoxic effect. Furthermore, dual-load PLGA group produces significantly enhanced treatment effect. These results suggest that PLGA formulations hold great potential for future drug delivery.

Development of a new staining protocol for the Kleihauer–Betke test to facilitate the reading of difficult cases

BackgroundThe Kleihauer–Betke (KB) test allows the detection of fetal red blood cells (containing fetal hemoglobin, HbF) in the maternal blood to identify and quantify potential fetal-maternal hemorrhages. In certain cases, detecting fetal red blood cells with conventional staining is difficult. False-positive results or overestimation of the quantity of fetal red blood cells may occur in cases of maternal hemoglobinopathy. In this study, we developed a new staining protocol to facilitate the reading of difficult smears and improve the precision of the quantification of fetal red blood cells; we also analyzed the performance of this new method. This study assessed blood samples with and without hemoglobin abnormalities, which present difficulties when interpreting the KB test.MethodsThe new staining formula is based on an improved elution technique and the use of a different stain instead of hematoxylin. To test this staining method, 16 samples from patients with abnormal hemoglobin electrophoresis and 14 samples from patients with normal hemoglobin electrophoresis were analyzed using the KB test with the classical staining method and the new staining method. In addition, a second series was prepared using the same samples spiked with fetal red blood cells from newborn blood, to compare the accuracy of the two methods in identifying fetal red blood cells.ResultsIn the 60 slides analyzed with both staining methods, we found that the new technique improved the accuracy from 78 to 85%; lowered the coefficient of variation between the operators, which decreased from 20.7% to 12.7%; increased the specificity in our population from 56 to 70%; and decreased the number of false-positive cases by 30%.ConclusionsWe successfully developed a new staining technique that facilitates the reading of difficult slides and improves the specificity of the detection of fetal red blood cells. This technique is recommended as a secondary method to use before sending the sample for additional exploration.

Prevalence and Characterisation of Irregular RBC Antibodies in Antenatal Females from a Tertiary Care Center in Northern India: A Cross-sectional Study

Introduction: Haemolytic Disease of the Foetus and Newborn (HDFN) is widely known to be caused by the presence of irregular Red Blood Cell (RBC) antibodies in pregnant females. To prevent HDFN, it is crucial to identify these antibodies in pregnant women. Aim: To evaluate the prevalence of irregular red cell antibodies in pregnant women in a tertiary care hospital. Materials and Methods: A cross-sectional retrospective study was conducted in the Department of Transfusion Medicine at Indraprastha Apollo New Delhi, from November 2009 to December 2017. A total of 8,217 prenatal females were examined for irregular red cell antibodies. Antibody screening and blood grouping were performed. The Capture-R ready screen was used to confirm screen-positive tests. Adsorption, elution, and other advanced techniques were utilised as needed. Data was collected from patient records and entered into a Microsoft excel spreadsheet. The statistical analysis was conducted using Statistical Package for Social Sciences (SPSS) software version 14.0 (USA). Statistical tests, including the Chi-squared and Fisher’s exact test, were performed, with a p-value less than 0.05 considered statistically significant. Results: Out of the 8,217 prenatal females examined, 105 (1.27%) had positive screening results with the four cell panel. Red cell alloimmunisation was observed in groups that were RhD negative (n=93/105; 88.57%) and positive (n=12/105; 11.42%). The most prevalent alloantibody among RhD negative individuals was alloantibody anti-D (n= 83/93; 89.24%), followed by alloantibody anti C+ anti D (n=13/93; 13.97%). Anti-D was the most prevalent alloantibody overall (n=83/105; 79.04%). Conclusion: Alloimmunisation rates were notably high among women lacking the Rh D antigen. Individuals with a history of adverse obstetric outcomes showed a statistically significant association with alloimmunisation. Therefore, it is recommended to screen such individuals for alloantibodies to enable early detection and improve the management of HDFN.

Development and Validation of a Bioanalytical Method for Estimating Ellagic Acid in Wistar Rat Plasma by RP-HPLC Using a Gradient Elution Technique

Abstract Background: Ellagic acid (EA) is a natural polyphenolic compound recognized for bioactive and pharmacologic properties that is found abundantly in various plant groups, particularly eudicotyledons. In the current study a novel, reliable, and cost-effective bioanalytical method was developed for quantifying EA in Wistar rat plasma using RP-HPLC. Methods: Zorbax SB C18 (5μ, 4.6150 mm) and Ascentis C18 (5μ, 1004.6 mm) columns were used in the stationary phase, while the mobile phase consisted of water with 0.1 % formic acid (A) and acetonitrile (ACN) with 0.08% formic acid (B). Results: Optimized parameters were as follows: temperature, 30°C; flow rate, 1.0 ml/min; and PDA detectors, 254 nm. The method exhibited high linearity (r2 = 0.9993) between 5 and 300 μg/ml. Precision, both intra- and inter-day, was within acceptable limits (relative standard deviation <2%), and the mean recovery was 99.73%. The LOD and LOQ were 1.564 ± 0.026 μg/ml and 5.015 ± 0.025 μg/ml, respectively. Stability tests, including short- and long-term evaluations, demonstrated stability under various conditions. Conclusion: The developed method met the necessary criteria and holds promise for application in clinical laboratories for assessing EA levels, either alone or with analytes.

Experimental investigation and comparative environmental impact analysis of conventional and naturally occurring kinetic hydrate inhibitors in offshore environments using toxicity and bioconcentration tools

In this present study, an in vivo experiment was carried out to determine the tendencies of a naturally occurring high-phenolic compound red-onion skin extract (HPC-ROSE) and conventional N-Vinylcaprolactam (N-VCAP) kinetic hydrate inhibitors (KHIs) to bio-accumulate in offshore environment through the analysis of their uptake and depuration kinetics by a Fish-Chemical Model using Cyprinus carpio marine invertebrate. This test was performed in the laboratory in a flow through experimental set up according to the guidelines by [49] (Fish-chemical exposure test). The uptake duration was 10 days with a depuration period of twenty days. The inhibitor toxicity test was evaluated by determining the 50 % lethal concentration (LC50) as highlighted by OSPAR Commission protocols. The solubility of the inhibitors was determined by a column elution technique. Similarly, a proton-enabled nuclear magnetic resonance (1HNMR) technique was used to measure the partitioning characteristics of the inhibitors in an immiscible mixture of octanol and water (Kow) by 1HNMR spectroscopy using a benchtop low-field NMR spectrometer. OSPAR Commission and ASTM standard protocols were used for the laboratory investigation of the static sediment toxicity tests. A reference chemical substance of known BCF and solubility (Ethyl Acetate, EtOAc) was used as control to check the experimental progression. The stock solution was prepared by solid phase desorption mechanisms. The HPC-ROSE does not constitute environmentally significant risk to aquatic life owing to its lower BCF values between 215 L/kg - 251.5L/Kg, Log-Kow of 1.2 and higher lethal concentration of 25140 mg/L. Unlike the NVCAP which is very toxic with lower lethal concentration of 1280 mg/L and higher BCF values in the range of 442.5L/Kg to 485 L/kg with Log-Kow of 1.5. Furthermore, Nuclear magnetic resonance (1HNMR) is a simple and reliable method of estimating partition coefficient characteristics (Kow) because the obtained Log-Kow values showed good agreements with that of shake flask and high-performance liquid chromatography techniques. Also, the chemical concentration in water has an inverse relationship with uptake rate constant (K1) and steady-state bioconcentration factor (BCFss). Finally, the theoretically estimated BCFs were higher than the steady-state (BCFss) values that were obtained from laboratory experiments for the different inhibitor samples and at all concentrations. This is due to the route by which Log-Kow was estimated since most of the empirical models are usually one-factor model consisting of partition coefficient and BCF.

Mass Spectrometry Based Detection of Naturally Expressed FVIII in Liver Sinusoidal-like Endothelial Cells from Healthy Donor and HA Patients with Nonsense Mutations

MS Background: Liver sinusoidal endothelial cells (LSEC) are unique endothelial cells and secretion and expression of FVIII is one of their key functions. Pluripotent stem cells from healthy donors can be a source of generating organ-specific endothelial cells in vitro. However, maintaining the specification of LSEC markers remains a challenge. Currently, EBs with a combination of AMP activation,TGF-ß inhibition,and hypoxia are a state of the art to derive LSEC-like EC from IPS. A 2D monolayer-based approach shows potential in culturing these ECs and in maintaining their correct specification (Fig. 1).IPSc-differentiated LSEC were analyzed for the presence of LSEC-specific markers FCGR2b, Stab2 & F8 by rtPCR. Our data shows that day 10 angioblasts mark an increase of venous markers COUP-TF and NT5E under low VEGF-A conditions. Upon LSEC induction LSEC markers are upregulated compared to HUVEC:FCGR2b(2x), Stab2(37x), and F8(7x) (not shown). Flow cytometry(FC) analyses depict a KDR +CD56 + phenotype for mesoderm on day 4 ,a venous CD31 +CD34 +CD184 lowCD73 high phenotype for day 6 angioblast and a CD34 +FCGR2b +Stab2 +LYVE1 + LSEC-LC population successfully maintained until Day 15 (not shown).ELISA measurements (Fig. 2) using a FVIII-A3 specific biotinylated nanobody detects FVIII in LSEC-LC from healthy donor, HA-patient with I22I and in both high risk inhibitor nonsense mutations R1960X and R2228X. These preliminary results warranted the confirmation of detection as well as the need to decipher the differences in the FVIII protein among the healthy donor and patients with HA. Aim: Detection of endogenous FVIII protein in healthy donors and patients using immunofluorescence(IF). Detection of FVIII protein using mass spectrometry (MS) Methods: IF was conducted using a monoclonal antibody (GMA8021) and the difference in protein levels was measured using the mean fluorescence intensity (MFI) values. An immunoprecipitation-based method to detect the natural FVIII protein using MS was developed. Initially, an ultracentrifugation step eliminated the background proteome which allowed for the antibody (SAF8C) based capture of FVIII using a column-based elution technique. The eluted sample was trypsinized and detected via nano-LC coupled to LC/MS Orbitrap. Results: The MFI of the healthy donor is 2.8x, 2.5x, 2.31x, 2x and 1.3x higher than F8KO, HUVEC, HA-patient samples R2228X, R1960X and I22I,respectively. Healthy donor shows the presence of 5 FVIII peptides. Patient sample with mutation I22I shows the presence of 1 FVIII peptide. Patient sample with nonsense mutation R1960X showed 2 FVIII peptides while patient sample with R2228X was unable to show any FVIII peptides. Conclusion: While FVIII detection in multiple overexpressed models of HA have been established, our current LSEC model is a milestone in detecting the natural, native state of the FVIII protein. This LSEC-LC model provides a 7-fold higher F8 expression compared to HUVEC in vitro system. FC analysis shows the right phenotype for modeling IPSc into LSEC. The additional presence of LSEC-specific receptors and confirmation of FVIII in the healthy donor and patients with nonsense mutations enables a more native and precise disease modeling for HA research and therapy.

DEL variants: review of molecular mechanisms, clinical consequences and molecular testing strategy.

Patients with DEL phenotype, a D variant with a low number of D antigens per red blood cell, are routinely typed as RhD-negative in serology testing and are detectable only by adsorption and elution techniques or molecular methods. DEL is of clinical importance worldwide, as indicated by its genotype-phenotype discrepancies among different populations and its potential to cause anti-D alloimmunization when DEL phenotype individuals are inadvertently managed as RhD-negative. This narrative review summarized the DEL alleles causing DEL phenotype and the underlying mechanisms. The clinical consequences and current molecular testing approach were discussed to manage the transfusion needs of patients and donors with DEL phenotype.

Catch and release: Gold adsorption and sorbent electrochemical regeneration

Wastewater generated from e-waste leaching is rich in precious metals including gold, silver and platinum. Conventional precipitation and solvent extraction are chemically intensive separations with concerning environmental externalities. Sorbents, in particular carbon nanotubes, have low chemical consumption, and have shown promise for gold adsorption due to their high specific surface area and chemical functionalization potential. However, regenerating sorbents used to adsorb Au is hazardous requiring strong acids. Herein, we delineate the effect of various functional groups on the sidewalls of multiwall carbon nanotubes (MWCNTs) on gold adsorption, and we introduced an acid-free electrochemical technique for Au elution from MWCNTs. Pristine MWCNTs (P-MWCNTs), carboxylic functionalised MWCNTs (COOH-MWCNTs) and amide functionalised MWCNTs (NH2-MWCNTs) were compared for their affinity for Au adsorption from acidic AuCl4−solutions mimicking acidic e-waste leachate. Au adsorption affinity onto MWCNTs followed the order of P-MWCNT > NH2-MWCNT > COOH-MWCNTs. Au elution from Au-saturated MWCNTs was subsequently achieved up to 65%, using acid-free electrochemical desorption in neutral aqueous brine. The Au electro-desorption was shown to have a direct relationship with both the applied current and the mass of the Au adsorbed on the MWCNTs. This study demonstrates enhanced adsorption-based preconcentration of gold and acid-free regeneration of sorbents.

Theoretical Study of Non-Isothermal Gradient Elution Liquid Chromatography.

A two-component model of gradient elution chromatography is investigated to theoretically study the effects of simultaneous variations in temperature and solvent strength on the retention behaviors of elution profiles in thermally insulated liquid chromatographic columns. The gradient elution technique is based on the gradual increase or decrease in eluent strength during the chromatographic operation by varying the composition of the mobile phase. The enthalpy of adsorption is primarily responsible for internal temperature variations inside the column, as heat adsorbs during the adsorption process and releases in the desorption phase. Both types of variations change the propagation speeds of moving pulses inside the column which can lead to better separation of the components and a reduction in the recycling time for the next injection. The equilibrium dispersive model (EDM) coupled with the energy balance equation for temperature and transport equation for the volume fraction of the solvent is utilized to simulate this complex process. The resulting nonlinear model equations are approximated by applying a semi-discrete second-order finite volume scheme. The numerical solutions are used to study the impact of a gradient starting and ending times, volume-fraction of the solvent, solvent strength parameter, the slope of gradient, enthalpy of adsorption, injection temperature, and the ratio of specific heats on the concentration profiles.

P403 The adsorption crossmatch cells and elution (AXE) technique to identify true HLA specific antibodies

P403 The adsorption crossmatch cells and elution (AXE) technique to identify true HLA specific antibodies

A NOVEL RP-HPLC GRADIENT ELUTION TECHNIQUE FOR BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATING GALLIC ACID IN WISTAR RAT PLASMA

Objective: Present study aimed to develop and validate a novel, unique, simple, quick, cost-effective, sensitive, specific, accurate, precise, rugged, and robust bioanalytical method for the quantification of gallic acid in rat plasma by reverse phase high-performance liquid chromatography (RP-HPLC) using gradient elution technique. Methods: The stationary phase was a Zorbax SB C18 5 µ (4.6*150) mm column, with the mobile phase being water with 0.1 percent formic acid (A): acetonitrile (ACN) with 0.08 percent formic acid (B). Gradient chromatographic method was used throughout this experiment from the point of view of the estimation of gallic acid from herbal formulations when present along with other phytoconstituents. So at the gradient method, all the present phytoconstituents has cleared off from the column and no any strongly adsorption of phytoconstituents occurred. The experiment was carried out at a flow rate of 1.0 ml/min at 30 °C utilising PDA detectors at 271 nm. The proposed method was validated for different parameters. Results: The approach was found to be linear in the concentration range of 0.5-100 µg/ml, with a r2 of 0.9998. There was not observed any interference of co-eluting peaks of endogenous compounds from the biological matrix at the same retention time (Rt) of gallic acid. The RSD (%) of intra and interday precision was found to be within acceptable limit. The overall % mean recovery was found to be 99.97%. LOD and LOQ were found to be 0.1 and 0.5 μg/ml, respectively. In terms of fluctuation in essential parameters and operating settings, the devised bioanalytical approach was shown to be rugged and resilient. Short-term, long-term, autosampler, bench-top, and freeze-thaw stability experiments revealed that gallic acid is stable. Conclusion: The developed method described in this report was found to be well within an acceptable range. Hence, in the future, this method can be used successfully for the estimation of gallic acid alone or in combination with another analyte or marker present in bulk or an extract containing various phytoconstituents in pharmacokinetic, bioequivalence, and therapeutic drug monitoring studies in clinical laboratories.

A Comparison of Absorption Inhibition and Absorption Elution Techniques for Detection of ABO Blood Groups in Saliva in Meitei Population: A Cross Sectional Study

The ABO blood group system is the significant element for forensic serological examination of blood and body fluids. ABO blood groups are the primary, most common, conspicuous, and easily detectable groups. These blood group specific antigens are abundantly present in many other bodily secretions such as sweat, semen and even saliva. Many studies have detected the presence of ABO blood groups in saliva by using both the absorption inhibition and absorption elution methods. This study of blood group ABO antigens in saliva was carried out using both the techniques in a tertiary care centre in Imphal, Manipur. Using absorption elution method, out of the 90 secretors subjects, positive results were obtained in 82 (91.1%) subjects and using absorption inhibition method, positive results were detected in 71 (78.9%) subjects. On statistical analysis, the mean age in completed years of the participants is calculated as 22.12 years with a standard deviation of 3.04 years. Further, comparative analysis showed that the positive rate of absorption elution is more than that of absorption inhibition. Therefore, absorption elution method remains an important tool for determination of blood group of an individual from saliva and it is an aid in forensic identification and in solving medico-legal cases.

Development and Validation of a Novel Bioanalytical Method for Estimating Epigallocatechin 3 Gallate in Wistar Rat Plasma by RP-HPLC Employing Gradient Elution Techniques

Development and Validation of a Novel Bioanalytical Method for Estimating Epigallocatechin 3 Gallate in Wistar Rat Plasma by RP-HPLC Employing Gradient Elution Techniques

Process optimization and kinetics for ultrasonication assisted extraction of phospholipids from ghee residue

AbstractGhee is a major Indian dairy product with a pleasant flavor. During its manufacture, ghee residue is obtained as the by‐product, which consists of substantial quantity of phospholipids. Phospholipids exhibit emulsifying properties and offer some health benefits. The present study aimed to optimize the process parameters for the extraction of phospholipids from ghee residue (GR) using ultrasonication. The GR was subjected to a series of pre‐treatments before ultrasonication. The hot water treatment led to reduction in nonpolar lipids and improved polar lipids (35.47% fat basis). Taguchi orthogonal array design was employed for optimization of ultrasound power, treatment temperature, time and solvent:solid (S:S) ratio with phospholipid content and antioxidant activity as the response variables. The phospholipid content ranged between 18.54% and 23.89% and antioxidant activity of the extracted samples varied from 47.01% to 50.64% expressed as radical scavenging activity by DPPH assay, while for the untreated GR, the PL and antioxidant activity were 4.98% and 32.75%, respectively. The power level of 80%, 80°C treatment temperature, S:S ratio of 15 and sonication time of 4 min. resulted in maximum extraction of phospholipids (24.12%). The yield of phospholipids was quantified as 13.1% and 8.88% by two elution techniques using solid phase extraction methods. The predicted and experimental values for the responses were in good agreement with R2 value of 0.98. Power and temperature were found to be significant (p < 0.01) in influencing phospholipids yield. For antioxidant activity of the extract, S:S ratio exerted significant effect (p < 0.01). The experimental data were fit in four defined empirical models for understanding the extraction kinetics. Based on the R2 values, the models explained the kinetics in the following order: Parabolic model>Power law>Peleg's model>Elovich's model.Practical applicationsPresently, ghee residue is often discarded as waste or used as animal feed by most of the ghee manufacturing dairy plants. Few studies were conducted to incorporate ghee residue as an ingredient in different food products. However, the present study opens new avenues for utilization of this by‐product for extraction of high value bioactive components, that is, phospholipids, with the help of novel technology; namely ultrasonication. The phospholipids have techno‐functional properties as an emulsifier. One of the classes of phospholipids, that is, sphingomyelin has unique health benefits, therefore this extract will also have scope as a nutraceutical. Thus, there is great practical utility for the present findings. In addition, for the scaling up of extraction techniques, the understanding of mass transfer kinetics is very vital and the same had been studied in the present investigation. Thus, the findings of this study are helpful in determining the ideal ultrasound‐assisted extraction conditions for extraction of phospholipids from ghee residue. Kinetics of extraction will help to explore opportunities for scale‐up of the process.