Development Of Analytical Methods Research Articles

Application of a Heuristic Framework for Multilevel Interventions to Eliminate the Impact of Unjust Social Processes and Other HarmfulSocial Determinants of Health.

There is consensus about the importance of developing a strong cadre of effective multilevel interventions to eliminate the impacts of unjust social processes, such as structural racism and other harmfulsocial determinants of health (SDOH), on health inequities in the USA. However, the available cadre of rigorously evaluated evidence-based interventions for SDOH mitigation remains underdeveloped relative to the magnitude of historic and current health inequities. The proposed manuscript addresses this gap in two ways: first, by introducing a heuristic framework to inform decisions in multilevel intervention development, study design, and selection of analytic methods and, second, by providing a roadmap for future applications of the framework in multilevel intervention research through an exemplar application using the ongoing NIH-funded evaluation study of the Nurse-Community-Family Partnership (NCFP) intervention. NCFP leverages individual, family, institutional, and system factors to shape COVID-19 mitigation outcomes at the individual and household levels. NCFP takes an approach informed by the heuristic framework to addressing and mitigating unjust social processes and other harmful SDOH. We discuss the application of a two-arm parallel explanatory group randomized trial to evaluate the efficacy of NCFP in improving the primary (COVID-19 testing uptake) and secondary (adoption of COVID-19 control measures, COVID-19 vaccine uptake, mutual aid capacity, etc.) outcomes at the individual and household levels. The analysis approach relies on random-intercept models, and we calculate the variance partitioning coefficient to estimate the extent to which household- and individual-level variables contribute to the outcome, allowing examination of NCFP effects at multiple levels.

Development of a High-Throughput Analytical Method for Antimicrobials in Wastewater Using an Automated Pipetting and Solid-Phase Extraction System

Antimicrobial resistance (AMR) has emerged and spread globally. Recent studies have also reported the presence of antimicrobials in a wide variety of aquatic environments. Conducting a nationwide monitoring survey of AMR in the environment to elucidate its status and to assess its impact on ecosystems and human health is of social importance. In this study, we developed a novel high-throughput analysis (HTA) system based on a 96-well plate solid-phase extraction (SPE), using automated pipetting and an SPE pre-treatment system. The effectiveness of the system as an HTA for antimicrobials in environmental water was verified by comparing it with a conventional manual analytical system in a domestic hospital over a period of two years and four months. The results of the manual analysis and HTA using a combination of automated pipetting and SPE systems were generally consistent, and no statistically significant difference was observed (p > 0.05) between the two systems. The agreement ratios between the measured concentrations based on the conventional and HTA methods were positively correlated with a correlation coefficient of r = 0.99. These results indicate that HTA, which combines automated pipetting and an SPE pre-treatment system for rapid, high-volume analysis, can be used as an effective approach for understanding the environmental contamination of antimicrobials at multiple sites. To the best of our knowledge, this is the first report to present the accuracy and agreement between concentrations based on a manual analysis and those measured using HTA in hospital wastewater. These findings contribute to a comprehensive understanding of antimicrobials in aquatic environments and assess the ecological and human health risks associated with antimicrobials and antimicrobial-resistant bacteria to maintain the safety of aquatic environments.

Development of an innovative analytical method for forensic detection of cocaine, antidepressants, and metabolites in postmortem blood using magnetic nanoparticles.

Cocaine and antidepressants rank high globally in substance consumption, emphasizing their impact on public health. The determination of these compounds and related substances in biological samples is crucial for forensic toxicology. This study focused on developing an innovative analytical method for the determination of cocaine, antidepressants, and their related metabolites in postmortem blood samples, using unmodified commercial Fe3O4 nanoparticles as a sorbent for dispersive magnetic solid-phase extraction (m-d-SPE), coupled with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. An aliquot of 100 µL of whole blood and 5 µL of the internal standard pool were added to 30mg of nanoparticles. The nanoparticles were separated from the sample using a neodymium magnet inserted into a 3D-printed microtube rack. The liquid was then discarded, followed by desorption with 300 µL of 1/1/1 acetonitrile/methanol/ethyl acetate. The sample was vortexed and separated, and 1.5 µL of the organic supernatant was injected into the LC-MS/MS. The method was acceptably validated and successfully applied to 263 postmortem blood samples. All samples evaluated in this study were positive for at least one substance. The most frequent analyte was benzoylecgonine, followed by cocaine and cocaethylene. The most common antidepressants encountered in the analyzed samples were citalopram and fluoxetine, followed by fluoxetine's metabolite norfluoxetine. This study describes the first report of this sorbent in postmortem blood analysis, demonstrating satisfactory results for linearity, precision, accuracy, and selectivity for all compounds. The method's applicability was confirmed, establishing it as an efficient and sustainable alternative to traditional techniques for forensic casework.

Professor Carla Bottoli, an enthusiastic scientific researcher, kindly spoke to BrJAC

Carla Beatriz Grespan Bottoli holds a degree in Industrial Chemistry from the Federal University of Santa Maria (1996), as well as a Ph.D. in Chemistry (2002), and a postdoctoral degree in Chemistry (2002-2004), both from the State University of Campinas (Unicamp). In 2005, she undertook an internship at the Institute of Nuclear Sciences of the National Autonomous University of Mexico (UNAM) to study radiolysis in vitamins using gamma radiation from a source of Cobalt 60. In 2017, she presented her habilitation thesis (associate professor's thesis) at Unicamp. Currently, she is Associate Professor and Postgraduate Advisor at the Institute of Chemistry at Unicamp (IQ-Unicamp), where she works in the area of analytical chemistry, with emphasis on separation methods, mainly liquid chromatography. Prof. Bottoli coordinates the NovaCrom – Liquid Chromatography Laboratory (https://novacromlab.wixsite.com/novacrom), which is a research group that has been working on the development of new monolithic stationary phases for capillary liquid chromatography. Research is also focused on the development of analytical methods that employ liquid chromatography coupled with mass spectrometry to study the translocation of pesticides in plants such as coconut, palm, and soybean. Between 2019 and 2021, Prof. Bottoli was an associate coordinator of the Research Commission of the IQ-Unicamp and is currently a member of the Research Integrity Commission at Unicamp. She is a member of the Brazilian Chemical Society and a researcher at the National Institute of Science and Technology in Bioanalytics (INCTBio). Carla Bottoli is the mother of Murilo, 13 and Mateus, 10.

Analytical Method Development and Validation of a UV‐Spectrophotometric Technique for Pioglitazone HCL Estimation in Bulk and Polymeric Nanoparticles

AbstractThe type 2 diabetes drug pioglitazone is encapsulated in polymeric (Chitosan/PEG blended PLGA) nanoparticles utilizing solvent evaporation methods. The current research aimed to develop and validate an accessible, accurate, fast, and commercial UV spectrophotometric approach for assessing Pioglitazone in bulk and polymeric nanoparticles. In the estimation method that involves a pH 7.4 phosphate buffer, the maximum absorbance of Pioglitazone is reported to be 269 nm. With a correlation coefficient of 0.999, the drug exhibits linearity in the concentration series of 5–60 µg mL‐1. The proposed technique is validated as per ICH guidelines for linearity, accuracy, precision, and ruggedness. When the proposed technology is used in nano‐formulation, the percent amount of drug estimated to be 99.35% agreed well with the label claim. Recovery experiments at three distinct levels, 50%, 75%, and 100% are used to test the method's accuracy. The recovery rate is estimated to be between 98.91% and 99.11%. The low percent relative standard deviation (RSD) value indicates the method's accuracy and reproducibility. The method's repeatability, intraday, and interday precision are all examined. The approach is precise if the percent RSD value is less than 2. With the assistance of two analysts, the ruggedness of the suggested method is investigated. For the scheduled examination of Pioglitazone in bulk and polymeric nanoparticles, the above process has been developed and validated and proved to be a quick and cost‐effective quality‐control tool in pharmaceuticals.

A novel albumin-based supramolecular biosensor for ratiometric on-site determination of buprofezin

Buprofezin (BUP) is an effective insecticide against Homopteran and Thysanoptera pests. However, exposure to BUP may result in several harmful effects on the non-target organism including human body, such as hepatotoxicity and DNA damage. Therefore, development of a reliable analytical method for BUP holds paramount importance. This study presents a novel albumin-based supramolecular biosensor, DPP@ALB, designed for the sensitive detection of BUP in environmental matrices, including water, soil, and real food samples. The features of this biosensor include a fast response, high sensitivity, and visually detectable fluorescence color change, enabling on-site detection of BUP based on the portable paper strips and 3D-printed miniaturized testing system. Overcoming challenges associated with the low chemical reactivity of BUP, this supramolecular biosensor emerges as the very first fluorescent sensor for efficient and reliable monitoring of BUP with applications in broader areas of environmental analysis and food safety.

Natural flavonols as probes for direct determination of borax: From conventional fluorescence analysis to paper-based smartphone sensing

Borax is strictly regulated in the food processing and pharmaceutical industry due to its physiological toxicity, and the development of a direct analytical method is essential for effectively monitoring the borax abuse. In this work, the fluorescence properties of flavonoids, including flavones, isoflavones and flavonols, were systematically investigated from aqueous to borax solutions, and it was found that the weak intrinsic fluorescence of flavonols could be pervasively sensitized by borax. A natural flavonol, morin, was subsequently chosen as a representative probe to develop a turn-on fluorescence sensing method for borax analysis, which achieved a linear response spanning four orders of magnitude with a detection limit of 1.07 μM (0.22 μg mL−1 in terms of Na2B4O7 content). Furthermore, a smartphone-assisted paper-based test device was designed and constructed by 3D printing technology. Using morin-impregnated test strips as the carrier, the borax could be visually detected by the RGB signals of the captured images, with a detection limit of 0.13 mM (27.05 μg mL−1 for Na2B4O7). Combining ion exchange treatment for food samples and sodium periodate oxidation for drug samples, the developed methods were successfully applied for the direct analysis of borax in various products with the recoveries of 86.9–106.3% for traditional fluorescence analysis and 82.7–108.8% for smartphone-assisted fluorescence sensing. The fluorescence property of the morin-borax system was studied using time-dependent density functional theory, and the sensing mechanism was discussed in conjunction with experimental research.

Quantification of Quaternary Ammonium Compounds by Liquid Chromatography-Mass Spectrometry: Minimizing Losses from the Field to the Laboratory

Quaternary Ammonium Compounds (QACs) are widely used in household, medical and industrial settings. As a consequence, they are ubiquitously found in the environment. Although significant efforts have been put into the development of sensitive and reproducible analytical methods, much less effort has been dedicated to the monitoring of QACs upon sample storage and sample preparation. Here we studied the effect of storage, concentration, and extraction procedures on the concentrations of QACs in samples. Thirteen QACs selected amongst benzalkonium compounds (BACs), dialkyldimethylammonium compounds (DADMACs) and alkyltrimethylammonium compounds (ATMACs) were quantified in aqueous and solid samples using LC-MS/MS. Most QACs adsorbed on container walls could be recovered using a short washing step with MeOH containing 2% v/v formic acid. Concentrations of QACs from aqueous solutions using solid phase extraction (SPE) with Strata-X cartridges and elution with acidified MeOH utilized to wash the emptied containers gave highly satisfactory recoveries (101-111%). Good recoveries (89-116%) were also obtained when extracting a spiked organic-rich synthetic soil using accelerated solvent extraction (ASE) with acidified MeOH at low solid/solvent ratio (0.4 g/20 mL). Applying the recommended methodologies to real samples collected from a Canadian wastewater treatment plant (WWTP) gave QAC concentrations in the ranges of 0.01-30 µg/L, < 1.2 µg/L, and 0.05-27 mg/kg for the influent, effluent and biosolids samples, respectively.

A review: Exploratory analysis of recent advancement in green analytical chemistry application

It is always a concern to ensure personnel and environment safety in the field of chemistry which has caused to development of green analytical chemistry methods. Green chemistry aims to create an eco-friendly environment in laboratories by using various analytical methods/strategies to reduce the use of toxic solvents which are harmful to humans and the environment.  It is a way that protect the environment by using green solvents and methods. Green analytical chemistry is a rapid analytical technique that describes the separation, identification, and quantification of an analyte in drugs, environments, and humans. Various green methodologies such as automation, miniaturization, precipitations, and passivation are utilized in the recovery of solvents and reagents. Green analytical chemistry aims to create an eco-friendly environment in the laboratories by using various analytical methods/strategies to reduce the use of toxic solvents which are harmful to the environment/humans as well as to decrease the amount of waste generated. In this review, we explore different green solvents that can replace other toxic solvents used during extraction processes. In this review, the various extraction methods and analytical techniques used to analyze different components have been discussed.

The Quality by design Approach for Analytical Method Development and Validation of the RP-HPLC Method for Estimation of Quercetin in Pure, Marketed, and Cream Formulation

ABSTRACT: A simple, precise reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the flavonoid quercetin, isolated from Tridax procumbens L. The method was validated by using Phenomenex C18 (250 x 4.6mm i.d) Column. A simple, cost effective mobile phase consisting of (ACN and 10 m/moL Phosphate buffer as mobile phase in proportion of 50:50 v/v ) pH 3, 1.0 ml/min Flow rate at 370nm by using UV Visible detector. The retention time of Quercetin was found to be 3.392 minutes. The Quercetin linearity range was found to be 05 to 25 μg/mL. The accuracy and precision of commercially available preparations and in-house cream formulations were investigated using a one-way ANNOVA test. The Percentage recovery of both formulations was found to be 99.83%, 99.88%, 99.82% and 98.92%, 98.18%, 98.86%. Robustness of analytical method was studied by using 2/3 full factorial design by using Design expert software. The Percentage assay and % RSD of marketed capsule and in house cream was found to be 98.38 %, 97.40% and 0.05273, 0.02053 respectively. The Limit of Quantitation and Limit of detection were found to be 0.9053μg/mL and 2.5435μg/mL respectively. The development method of quercetin is simple, accurate, precise, sensitive, and robust

Development of a dispersive liquid-liquid microextraction method for the evaluation of maternal-fetal exposure to cocaine employing human umbilical cord tissue.

Illicit drug use is a serious and complex public health problem, not only due to the severity of the health damage but also to the social implications, such as marginalization and drug trafficking. Currently, cocaine (COC) is among the most abused drugs worldwide with about 22 million users. Drug abuse has also been found in women during the pregnancy period, which has shed light on a new group for epidemiology. The diagnosis of COC use in these cases usually depends largely on the mother's reports, which in several cases omit or deny consumption. Therefore, considering physical-chemical methods of sample preparation and exposure biomarkers, the development of analytic toxicological methods can help to confirm drug use during pregnancy. Thus, the objective of the present work was to develop an analytical method based on dispersive liquid-liquid microextraction (DLLME) for the determination of COC analytes, using umbilical cord tissue as an alternative biological matrix, and detection by gas chromatography coupled to mass spectrometry (GC-MS). Therefore, after optimization, the DLLME method was fully validated for quantification of COC, benzoylecgonine (BZE), cocaethylene (CE), ecgonine (ECG), ecgonine methyl ester (EME) and norcocaine (NorCOC). The limits of detection were between 15 and 25 ng/g, the limits of quantification (LOQ) were 30 ng/g for ECG, and 25 ng/g for the other analytes. Linearity ranged from the LOQ to 1,000 ng/g. Coefficients of variation for intra-assay precision were <18.5%, inter-assay was <8.75%, and bias was <16.4% for all controls. The developed method was applied in 10 suspected positive samples, based on the mother's report and maternal urine screening and confirmation. COC, BZE, ECG, and EME were quantified in 4 umbilical cords with concentrations that ranged from 39.6 to 420.5 ng/g.

Development of Analytical Method for the Quantitation of Monoclonal Antibodies Solutions via Raman Spectroscopy: The Case of Bevacizumab

Personalized dosages of monoclonal antibodies are being used more regularly to treat various diseases, rendering their quantitation more essential than ever for the right dose administration to the patients. A promising alternative, which overcomes the obstacles of the well-established chromatographic techniques regarding the quantification of biopharmaceuticals, is Raman spectroscopy. This study aimed to develop and validate a novel analytical method for the quantitation of bevacizumab in solutions via Raman spectroscopy. For this purpose, a droplet of the solution was left to dry on a highly reflective carrier and a home-made apparatus was employed for rotation of the sample. Hence, each recorded Raman spectrum was the average of the signal acquired simultaneously from multiple points on a circular circumference. The method was validated, and the detection limit of the antibody was found to be 1.06 mg/mL. Bevacizumab was found to be highly distributed at the formed coffee ring of the dried droplet, though this was a function of solution concentration. Finally, Raman spectra at different distances on the coffee ring were obtained from the four quarters. The lowest bevacizumab detection limit was found at a distance of 75 μm from the external side of the coffee ring and it was determined to be equal to 0.53 mg/mL.

Analytical Method Development and Validation Of Stability Indicating RP-HPLC Method For Assay and Related Substances of Paracetamol and Caffeine Effervescent Tablets: Review

he ongoing, interdependent tasks related to the departments of research and development, quality control, and quality assurance are the creation and validation of analytical methods. Analytical methods are essential for risk management and equivalency assessments. It aids in the development of acceptability standards unique to a product and the consistency of outcomes. Validations establish whether the analytical process is appropriate for the goal for which it was designed. A review of the literature indicates that paracetamol and caffeine can be determined individually or in combination with other medications using analytical techniques based on UV spectroscopy, RP-HPLC, and HPTLC. The parameters were checked for correctness, precision, robustness, and other aspects of analytical validation in accordance with ICH guidelines. The discovered techniques can be applied to the bulk and tablet dosage form analysis of paracetamol and caffeine in effervescent form since they are straight forward, sensitive, and repeatable. The review also outlines the applicability and constraints of numerous published analytical techniques for paracetamol and caffeine analysis. Stability studies have to be conducted on time and in compliance with the standards issued by the World Health Organization, International Conference of Harmonization, and other bodies. The capacity of a pharmaceutical product to meet the physical, chemical, microbiological, toxicological, protective, and informational requirements of a specific formulation in a particular container-closure system is known as its stability. It also ensures that a pharmaceutical product's effectiveness, safety, and performance will be maintained for the duration of its shelf life, which is seen as a need for approval and acceptability. The requirement for ongoing quality and purity monitoring of medications and products gave rise to various stability test techniques. This review covers the various drug substance stability categories as well as the applicability of various techniques for drug substances that are stable, the applicability of various methods used to test the pharmaceutical product's stability, guidelines released to test the pharmaceutical product's stability, protocols for stability testing that outline the essential elements of a well-managed and regulated stability test, and other aspects of stability are all covered in this review. The researcher working on paracetamol and caffeine in effervescent form will greatly benefit from this thorough review study.

Development and application of a high-performance liquid chromatography diode-array detection (HPLC–DAD) method for the simultaneous quantification of phenolic compounds in the aerial part of Glehnia littoralis

Glehnia littoralis, a medicinal herb employed in traditional practices for alleviating fatigue, cough, and a dry throat, is recognized for its beneficial properties due to a diverse array of active compounds found in its extracts. For example, the G. littoralis roots (Radix Glehniae) mainly contain coumarins and phenolic acids, serving as the primary focus of this study. Despite the widespread use of the tools in various industries and the development of multiple analytical methods for their examination, the edible aerial parts have industrial potential, and there is currently no analytical method available to identify their key components. In this study, a high-performance liquid chromatography method combined with diode array detection (HPLC–DAD) was developed to simultaneously detect 16 phenolic compounds previously reported to be present in the edible aerial parts of G. littoralis. The proposed approach included using gradient elution to change the solvent system from water/acetonitrile to water/methanol. Furthermore, the method validation was conducted, assessing its linearity, limit of detection, limit of quantification, precision, accuracy, and recovery, all of which demonstrated satisfactory results. Subsequently, the developed method was applied to quantify the phenolic compounds in various G. littoralis samples obtained from different organs, solvent extraction processes, and processing methods. Moreover, the online HPLC-ABTS (2,2ʹ-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assay was used to evaluate the antioxidant capacities of individual constituents, identifying four important antioxidants and estimate the overall antioxidant capacity of the G. littoralis extract.

Working hours and health - key research topics in the past and future.

This paper discusses the past and present highlights of working hours and health research and identifies key research needs for the future. We analyzed over 220 original articles and reviews on working hours and health in the Scandinavian Journal of Work, Environment & Health published during the last 50 years. Key publications from other journals were also included. The majority of identified articles focussed on the effects of shift and night work, with fewer studying long and reduced working hours and work time control. We observed a transition from small-scale experimental and intensive field studies to large-scale epidemiological studies utilizing precise exposure assessment, reflecting the recent emergence of register-based datasets and the development of analytic methods and alternative study designs for randomized controlled designs. The cumulative findings provide convincing evidence that shift work and long working hours, which are often associated with night work and insufficient recovery, increase the risk of poor sleep and fatigue, sickness absence, occupational injuries, and several chronic health conditions such as cardiovascular diseases and cancer. The observed risks are strongly modified by individual and work-related factors. Although the observed health risks of shift work and long working hours are mostly low or moderate, the widespread prevalence of exposure and the hazardousness of the many associated potential outcomes makes such working time arrangements major occupational health risks. Further research is needed to identify exposure-response associations, especially in relation to the chronic health effects, and to elucidate underlying pathways and effective personalized intervention strategies.

Development of ultra HPLC analytical method for pymetrozine residues in rice

Ultra HPLC-based analytical method analytical method was developed for the analysis of pymetrozine residues in different rice matrices. Chromatography separation was carried out in XR-ODS II: 150mm X 2.0mm, 5µm column in the mobile phase of water and methanol (60:40), and the resultant chromatograms were detected in a Photodiode Array (PDA). The retention time for pymetrozine neat standard and also matrix match standards was arrived at 4.00 min ± 0.5 min. The pymetrozine at the concentrations of 0.01, 0.05, 0.10, 0.25, 0.50, 0.75, and 1.00 µg/ml and the calibration curve of y = 2E+07x + 19188 obtained with a goodness of fit (R²) 0.995. The samples were extracted using the AOAC QuEChERS method with slight modifications. The harvest time residues for the different rice matrices were below the tolerance limit of pymetrozine. The residues of pymetrozine at 7, 15, and 30 days after the rice harvest in paddy seed, single-polished, double-polished rice, bran, and dehulled rice were not detected. From the developed analytical method, the residues of pymetrozine can be detected in different rice matrices.

Novel analytical method development and validation for simultaneous estimation of curcumin, ascorbic acid and salicylic acid in bulk and its pharmaceutical formulation by RP-HPLC

Novel analytical method development and validation for simultaneous estimation of curcumin, ascorbic acid and salicylic acid in bulk and its pharmaceutical formulation by RP-HPLC

Synthesis, structural characterization, analytical profiling, and application to authentic samples of synthesized Phase I metabolites of the synthetic cannabinoid 5F-MDMB-PICA.

5F-MDMB-PICA, an indole-type synthetic cannabinoid (SC), was classified illicit globally in 2020. Although the extensive metabolism of 5F-MDMB-PICA in the human body warrants the development of robust analytical methods for metabolite detection and quantification, a current lack of reference standards for characteristic metabolites hinders such method creation. This work described the synthesis of 18 reference standards for 5F-MDMB-PICA and its possible Phase I metabolites, including three hydroxylated positional isomers R14 to R16. All the compounds were systematic characterized via nuclear magnetic resonance, Fourier transform infrared spectroscopy, and high-resolution mass spectrometry. Furthermore, two methods were developed for the simultaneous detection of all standards using liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. By comparison with authentic samples, R17 was identified as a suitable urine biomarker for 5F-MDMB-PICA uptake.

Analytical quality by design based on knowledge organization: A case study of developing an ultrahigh-performance liquid chromatography method for the detection of phenolic compounds.

Despite numerous successful cases, there are still some challenges in using analytical quality by design (AQbD) for the development of analytical methods. Knowledge organization helps to enhance the objectivity of risk assessment, reduce the number of preliminary exploratory experiments, identify potential critical method parameters (CMPs) and their scope. In the present study, we aimed to develop a simple, rapid, and robust analytical method for detecting phenolic compounds in Xiaochaihu capsule intermediates utilizing knowledge organization. Knowledge organization and AQbD were combined to obtain the initial analytical conditions through knowledge collection, extraction, reorganization, and analysis. The quantitative relationship between critical method attributes (CMAs) and CMPs was then established by a definitive screening design. The method operable design region was calculated using an exhaustive Monte Carlo approach based on the probability of reaching the standard. Robustness investigation and methodological validation were finally performed. Analytical target profiles, CMAs, potential CMPs, and initial analytical conditions were initially identified, and the optimized ranges of operating parameters were obtained. A UHPLC method was successfully established for the analysis of phenolic compounds in ginger-ginger pinellia percolate, and the method validation outcomes were also satisfactory. The developed method can be a reliable means to detect the phenolic compounds of Xiaochaihu capsule intermediates. Knowledge organization provides a new approach for making better use of prior knowledge, significantly enhancing the efficiency of analytical method development. The approach is versatile and can be similarly applied to the development of other methods.

Anion exchange HPLC monitoring of mRNA in vitro transcription reactions to support mRNA manufacturing process development.

mRNA technology has recently demonstrated the ability to significantly change the timeline for developing and delivering a new vaccine from years to months. The potential of mRNA technology for rapid vaccine development has recently been highlighted by the successful development and approval of two mRNA vaccines for COVID-19. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, e.g., treatments for cancer, metabolic disorders, cardiovascular conditions, and autoimmune diseases. There is currently significant demand for the development of improved manufacturing processes for the production of mRNA therapeutics in an effort to increase their yield and quality. The development of suitable analytical methods for the analysis of mRNA therapeutics is critical to underpin manufacturing development and the characterisation of the drug product and drug substance. In this study we have developed a high-throughput, high-performance liquid chromatography (HPLC) workflow for the rapid analysis of mRNA generated using in vitro transcription (IVT). We have optimised anion exchange (AEX) HPLC for the analysis of mRNA directly from IVT. Chromatography was performed in under 6min enabling separation of all of the key components in the IVT, including nucleoside triphosphates (NTPs), Cap analogue, plasmid DNA and mRNA product. Moreover, baseline separation of the NTPs was achieved, which facilitates accurate quantification of each NTP such that their consumption may be determined during IVT reactions. Furthermore, the HPLC method was used to rapidly assess the purification of the mRNA product, including removal of NTPs/Cap analogue and other contaminants after downstream purification, including solid phase extraction (SPE), oligo deoxythymidine (oligo-dT) affinity chromatography and tangential flow filtration (TFF). Using the developed method excellent precision was obtained with calibration curves for an external mRNA standard and NTPs giving correlation coefficients of 0.98 and 1.0 respectively. Intra- and inter-day studies on retention time stability of NTPs, showed a relative standard deviation ≤ 0.3% and ≤1.5% respectively. The mRNA retention time variability was ≤0.13%. This method was then utilised to monitor the progress of an IVT reaction for the production of Covid spike protein (C-Spike) mRNA to measure the increasing yield of mRNA alongside the consumption of NTPs during the reaction.