Validation of an HPLC-CAD Method for Determination of Lipid Content in LNP-Encapsulated COVID-19 mRNA Vaccines.

Lipid nanoparticles (LNPs) are widely used as delivery systems for mRNA vaccines. The stability and bilayer fluidity of LNPs are determined by the properties and contents of the various lipids used in the formulation system, and the delivery efficiency of LNPs largely depends on the lipid composition. For the quality control of such vaccines, here we developed and validated an HPLC-CAD method to identify and determine the contents of four lipids in an LNP-encapsulated COVID-19 mRNA vaccine to support lipid analysis for the development of new drugs and vaccines.

Development and Validation of a Novel Stability Indicating Reverse Phase High Performance Liquid Chromatographic Method for Related Substances and Assay Analysis of Molnupiravir Drug Substance and Drug Product

Molnupiravir, a broad-spectrum antiviral is an isopropyl ester prodrug of beta-D-N4-hydroxycytidine. Molnupiravir targets RNA-dependent RNA-polymerase enzyme of the viruses. A new stability-indicating HPLC-method was developed to determine related substances and assay of molnupiravir. Separation was achieved by using Shim-pack GWS C18 column. The method was validated according to current ICH requirements. The calibration plot gave a linear relationship for all known analytes over the concentration range from LOQ to 200%. LOD and LOQ for all known analytes were found in 0.05-0.08 microg mL-1 and 0.12-0.20 microg mL-1, respectively, the mean recovery was found to be 97.79-102.44 %. Study showed that the method, results of robustness, solution stability studies are precise and within the acceptable limits. Molnupiravir was found to degrade in acid, alkali, and oxidative conditions, and was stable in thermal, moisture, and photolytic degradation condition. The method is simple, accurate, precise, and reproducible for routine purity analysis of drug-samples.Copyright © 2022 Indian Drug Manufacturers' Association. All rights reserved.

Method validation for the recovery of the porcine respiratory and reproductive syndrome virus, a potential SARS-CoV-2 surrogate, from stainless steel.

Virus survival on fomites may represent a vehicle for transmission to humans. This study was conducted to optimize and validate a recovery method for the porcine respiratory and reproductive syndrome virus (PRRSV), a potential SARS-CoV-2 surrogate, from stainless steel. Coupons (1.5 × 1.5 cm) inoculated with ca. 7 logs TCID50 of PRRSV were dried for 15 min at room temperature, followed by incubation at 4°C and 35% relative humidity. After 1 h and 24 h, the coupons were processed by four different methods: vortex in DMEM media, vortex in DMEM media with beads, vortex in elution buffer, and shake in elution buffer. The rinsates were processed for titration using the TCID50 method in the MARC-145 cell line. All four methods were equally effective to recover the virus from the soiled SS surfaces (> 79% recovery). The amount of infectious virus recovered after 24 h was similar (P > 0.05) to that recovered after 1 h, indicating that the virus was stable at 4°C for up to 24 h. Using an elution buffer followed by shaking was the least labor-intensive and most economical method. Therefore, this method will be used for future experiments on PRRSV survival and transfer from food-contact surfaces.

Clinical Diagnostic Point-of-Care Molecular Assays for SARS-CoV-2.

Laboratories faced many challenges throughout the COVID-19 pandemic. Point-of-care (POC) SARS-CoV-2 nucleic acid amplification tests (NAATs) provided a key solution to the need for rapid turnaround time in select patient populations and were implemented at the POC but also within laboratories to supplement traditional molecular assays. Clinical Laboratory Improvement Amendments-waived rapid POC SARS-CoV-2 NAATs offer the benefit of reduced educational requirements for operators and can be performed by non-laboratory-trained individuals. However, these methods must be validated to ensure the manufacturer's performance specifications are met and they are found to be fit-for-purpose in the clinical workflows they are implemented.

All-solid-state paper-based potentiometric combined sensor modified with reduced graphene oxide (rGO) and molecularly imprinted polymer for monitoring losartan drug in pharmaceuticals and biological samples.

A cost-effective, highly selective and sensitive paper-based potentiometric combined sensor for losartan potassium drug (LOS) is fabricated, characterized and used for the drug monitoring. The sensor consists of 2 strips of filter paper (20 × 5 mm each) as platform, each imprinted with 4 mm diameter circular spot of carbon. One carbon spot is covered by a reduced graphene oxide (rGO) for use as a substrate for the recognition sensor and the other without rGO is used for the reference electrode. LOS molecularly imprinted drug polymer (MIP) is applied onto the graphene oxide containing strip to act as a drug recognition sensing material and a solid-state polyvinyl butyral (PVB) is applied onto the second carbon spot to act as a reference electrode. Performance characteristics of the combined sensor are examined with chronopotentiometry (CP) and electrochemical impedance spectroscopy (EIS). Increase effect of rGO on the interfacial double-layer capacitance of the sensing membrane and consequently on the potential stability is confirmed. The developed combined sensor (strip cell) displays a Nernstian slope of -58.2 ± 0.3 mV/decade (R2 = 0.9994) over the linear range 8.5 × 10-7 - 6.9 × 10-2 M with a detection limit of 2.7 ± 0.3 × 10-7 M. The sensor shows remarkable selectivity toward various related compounds especially those commonly used by the COVID-19 patients such as paracetamol, ascorbic acid and dextromethorphan. The assay method is validated and proved to be satisfactory for direct potentiometric determination of LOS-K in some pharmaceutical formulations and in spiked human urine samples. An average recovery of 96.3 ± 0.3-98.7 ± 0.6% of the nominal or spiked concentration and a mean relative standard deviation of ±0.6% are obtained. The use of an indicating and a reference electrodes combined into a single flexible disposable paper platform enables applications to a minimum sample volume due to the close proximity of the responsive membrane and the liquid junction. The efficiency of the proposed sensor in complex urine matrix suggests its application in hospitals for rapid diagnosis of overdose patients and for quality control/quality assurance tests in pharmaceutical industry.

Proficiency testing and ring trials.

The validation of diagnostic methods (and the subsequent results generated by a laboratory) are improved through participation in inter-laboratory comparisons (IC), such as proficiency-testing (PT) programmes and other exercises referred to as 'ring tests' or 'ring trials' (RTs). This is a requirement to comply with international quality standards. Validating a method is a continuous process and taking part in ongoing PT programmes supports the management of a method's life cycle, providing continuing assessment of fitness (sometimes referred to as the 'validation retention status'). Proficiency-testing panel designs ensure that the methods used, particularly diagnostic specificity and sensitivity, are suitably challenged. Appraising PT results over time can illustrate whether the laboratory's performance is stable, improving or worsening, and proficiency tests can also highlight variations in the performance of assays. The development of new proficiency tests can support the implementation of novel diagnostics technologies, such as whole genome sequencing and point-of-care testing, and assist in cross-sectoral partnerships focusing on One Health approaches, which are high on the agenda for infectious disease control. For example, the rapid design and distribution of emergency exempted assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) means that these assays were not as rigorously evaluated as assays for established infectious diseases. Therefore, participation in PT programmes for SARS-CoV-2 is essential to understand the performance of these assays. While other mechanisms help to underpin laboratory activities, PT has been, and should remain, an integral part of laboratory quality assurance. Resources must be directed towards increasing and improving the quality of PT (for example, availability and accessibility of suitable biological and reference materials are essential for a PT provider to execute its duties), to support established and novel methods such as genomic and point-of-care tests.

Les procédures de validation des méthodes de diagnostic (et les résultats obtenus par la suite par les laboratoires) peuvent être améliorées en participant à des comparaisons inter-laboratoires, sous forme notamment de programmes d'aptitude inter-laboratoires ou d'autres exercices désignés collectivement sous l'appellation d'essais comparatifs inter-laboratoires (ring trials en anglais). Cette participation constitue une obligation au regard de la conformité avec les normes internationales de qualité. La validation d'une méthode est un processus continu et la participation à des programmes d'aptitude inter-laboratoires offre des garanties quant à la gestion du cycle de vie de la méthode considérée, car elle se traduit par une évaluation continue de l'aptitude à l'emploi du test (également désignée comme la « conservation de son statut de validation ¼). Les panels des essais d'aptitude sont conçus de manière à garantir que les méthodes utilisées font l'objet d'essais appropriés, en particulier concernant leur spécificité et sensibilité diagnostiques. L'appréciation des résultats des essais d'aptitude dans le temps permet d'établir si les performances d'un laboratoire restent stables, s'améliorent ou se dégradent ; les essais d'aptitude mettent également en lumière les éventuelles variations dans les performances d'un essai. La mise au point de nouveaux essais d'aptitude peut encourager l'utilisation de technologies de diagnostic innovantes, par exemple le séquençage du génome entier et les tests utilisables sur le lieu des soins, et faciliter les partenariats intersectoriels axés sur des approches Une seule santé, qui figurent parmi les grandes priorités en matière de lutte contre les maladies infectieuses. Par exemple, la conception et la distribution rapides de tests de détection du coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2) suivant un protocole d'exception imposé par l'urgence signifient que ces tests n'ont pas fait l'objet d'une évaluation aussi rigoureuse que les tests de détection de maladies infectieuses bien établies. Par conséquent, la participation à des programmes d'essais d'aptitude pour le SARS-CoV-2 est essentielle pour déterminer précisément les performances de ces tests. S'il existe d'autres mécanismes permettent d'étayer les activités d'un laboratoire, les essais d'aptitude ont été et doivent continuer à être une partie intégrante de l'assurance qualité des laboratoires. Il convient d'affecter des ressources à l'accroissement du nombre d'essais d'aptitude et à l'amélioration de leur qualité (il est notamment essentiel que les fournisseurs de tests d'aptitude puissent se procurer facilement des matériels biologiques et réactifs de référence appropriés afin de mener à bien leur tâche), de manière à soutenir aussi bien les méthodes classiques que celles qui procèdent d'innovations comme les tests génomiques et ceux utilisables sur le lieu des soins.

La validación de métodos de diagnóstico (y de los subsiguientes resultados obtenidos por un laboratorio) mejora con la participación en procesos de comparación entre laboratorios, como pueden ser los programas de pruebas de competencia u otros procesos denominados globalmente "pruebas interlaboratorios". Se trata de un requisito de obligado cumplimiento según dictan las normas internacionales de calidad. La validación de un método es un proceso permanente y, en este sentido, el hecho de tomar parte en programas continuos de pruebas de competencia ayuda a gestionar un método de prueba durante todo su ciclo de vida, aportando en todo momento una evaluación de su nivel de idoneidad (lo que a veces se denomina también el "estado de retención de la validación"). El diseño de paneles de pruebas de competencia sirve para garantizar que los métodos empleados, y en particular su especificidad y sensibilidad de diagnóstico, sean convenientemente evaluados. El análisis de los resultados de pruebas de competencia a lo largo del tiempo puede indicar si el laboratorio se mantiene en niveles estables de rendimiento o si este mejora o empeora. Las pruebas de competencia también pueden poner de manifiesto variaciones en el rendimiento de un ensayo. La creación de nuevas pruebas de competencia puede contribuir a la implantación de novedosas tecnologías de diagnóstico, como la secuenciación de genoma completo o las pruebas practicadas en el punto de consulta, y ayudar a trabajar en alianzas intersectoriales desde planteamientos en clave de "Una sola salud", extremo este de lo más prioritario en los planes de lucha contra las enfermedades infecciosas. Valga como ejemplo la celeridad con que se han concebido y distribuido ensayos aplicables al coronavirus del síndrome respiratorio agudo severo de tipo 2 (SARS-CoV-2), con las exenciones propias de un procedimiento de urgencia, lo que supone que estos ensayos no hayan pasado por un proceso de evaluación tan riguroso como el que se aplica a patógenos infecciosos más antiguos. Por ello, en el caso concreto del SARS-CoV-2, la participación en programas de pruebas de competencia es fundamental para aprehender el rendimiento que ofrecen estos ensayos. Si bien hay otros mecanismos que ayudan a reforzar el trabajo de laboratorio, las pruebas de competencia han sido, y deben seguir siendo, parte integrante de la garantía de calidad que ofrece un laboratorio. Para potenciar el uso de métodos ya arraigados o novedosos, como puedan ser la genómica o las pruebas en el punto de consulta, es preciso dedicar recursos a la realización de un mayor número de pruebas de competencia de mejor calidad (la existencia de material biológico y de referencia adecuado y la facilidad de acceso a él, por ejemplo, son sendos factores básicos para que un proveedor de pruebas de competencia pueda cumplir su cometido).

Food Safety Assessment: Overview of Metrological Issues and Regulatory Aspects in the European Union

The safety of the food we consume has a direct impact on individual and population health and affects the economic growth of the region where food safety is practised and enhanced. The central goal of the European Commission’s Food Safety policy is to ensure a high level of protection of human health covering the whole supply chain. In recent years, great attention has been paid to food testing and the application of metrological tools to support food safety. The global food market and national and international food safety regulations have created a huge demand for the measurement traceability and comparability of analytical results that are independent of time or space boundaries. This review provides an overview of the European food safety policy and regulation, with a focus on the measurement-related elements of the European Union (EU) food law. It also highlights how the application of analytical techniques, with particular reference to separation approaches, and metrological tools can ensure the control of certain contaminants that nowadays represent the main challenges for food safety (e.g., mycotoxins, nanoparticles, emerging and process contaminants). METROFOOD-RI-Infrastructure for promoting metrology in food and nutrition is therefore described in this context. This European research infrastructure has been developed and is being implemented in the frame of the European Strategy Forum on Research Infrastructures (ESFRI) to support metrology in food and nutrition and establish a strategy allowing reliable and comparable analytical measurements in food across the entire process line, from primary producers to consumers, and making data findable, accessible, interoperable, and reusable (FAIR).

The determination of Sulfobutylether ß-Cyclodextrin Sodium (SBECD) by LC-MS/MS and its application in remdesivir pharmacokinetics study for pediatric patients.

SBECD (Captisol®) with an average degree of substitution of 6.5 sulfobutylether functional groups (SBE = 6.5), is a solubility enhancer for remdesivir (RDV) and a major component in Veklury, which was approved by FDA for the treatment of patients with COVID-19 over 12 years old and weighing over 40 kg who require hospitalization. SBECD is cleared mainly by renal filtration, thus, potential accumulation of SBECD in the human body is a concern for patients dosed with Veklury with compromised renal function. An LC-MS/MS method was developed and validated for specific, accurate, and precise determination of SBECD concentrations in human plasma. In this method, the hexa-substituted species, SBE6, was selected for SBECD quantification, and the mass transition from its dicharged molecular ion [(M-2H)/2]2-, Molecular (parent) Ion (Q1)/Molecular (parent) Ion (Q3) of m/z 974.7/974.7, was selected for quantitative analysis of SBECD. Captisol-G (SBE-γ-CD, SBE = 3) was chosen as the internal standard. With 25 µL of formic-acid-treated sample and with a calibration range of 10.0-1000 µg/mL, the method was validated with respect to pre-established criteria based on regulatory guidelines and was applied to determine SBECD levels in plasma samples collected from pediatric patients during RDV clinical studies.

SARS-CoV-2 wastewater surveillance data can predict hospitalizations and ICU admissions.

We measured SARS-CoV-2 RNA load in raw wastewater in Attica, Greece, by RT-qPCR for the environmental surveillance of COVID-19 for 6 months. The lag between RNA load and pandemic indicators (COVID-19 hospital and intensive care unit (ICU) admissions) was calculated using a grid search. Our results showed that RNA load in raw wastewater is a leading indicator of positive COVID-19 cases, new hospitalization and admission into ICUs by 5, 8 and 9 days, respectively. Modelling techniques based on distributed/fixed lag modelling, linear regression and artificial neural networks were utilized to build relationships between SARS-CoV-2 RNA load in wastewater and pandemic health indicators. SARS-CoV-2 mutation analysis in wastewater during the third pandemic wave revealed that the alpha-variant was dominant. Our results demonstrate that clinical and environmental surveillance data can be combined to create robust models to study the on-going COVID-19 infection dynamics and provide an early warning for increased hospital admissions.

Determination of hydrogen peroxide on N95 masks after sanitization using a colorimetric method.

Hydrogen peroxide is commonly used as a sterilizing agent for medical devices and its use has recently been extended to N95 masks during PPE shortages as a result of the COVID-19 pandemic. The hydrogen peroxide remaining on the masks after sterilization could potentially pose a health hazard to the mask users. In the present study a colorimetric method was optimized for the determination of hydrogen peroxide on N95 masks following chemical sanitizations. The developed analytical method demonstrated an overall recovery of 98% ± 7%. The limit of detection ranged from 0.16 to 0.25 mg/mask, depending on the type of mask. The expanded measurement uncertainty was 13% (at a 95% confidence interval). The sanitization process itself introduced a significant variation in hydrogen peroxide load between masks. The ozone used in the sanitization process had no significant impact on analytical performance. Stamped and printed marks on the mask surfaces could induce biased readings. Hydrogen peroxide decomposes quickly on the mask surfaces so timing of analysis is an important factor in method standardization.•The validation data demonstrated that the in-house method is reliable and fit for the intended purpose, offering a sensitive, simple, rapid, and inexpensive method of residue monitoring.

Validation of LC-MS/MS methods for determination of remdesivir and its metabolites GS-441524 and GS-704277 in acidified human plasma and their application in COVID-19 related clinical studies.

Remdesivir (RDV) is a phosphoramidate prodrug designed to have activity against a broad spectrum of viruses. Following IV administration, RDV is rapidly distributed into cells and tissues and simultaneously metabolized into GS-441524 and GS-704277 in plasma. LC-MS/MS methods were validated for determination of the 3 analytes in human plasma that involved two key aspects to guarantee their precision, accuracy and robustness. First, instability issues of the analytes were overcome by diluted formic acid (FA) treatment of the plasma samples. Secondly, a separate injection for each analyte was performed with different ESI modes and organic gradients to achieve sensitivity and minimize carryover. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a run time of 3.4 min. The calibration ranges were 4-4000, 2-2000, and 2-2000 ng/mL, respectively for RDV, GS-441524 and GS-704277. The intraday and interday precision (%CV) across validation runs at 3 QC levels for all 3 analytes was less than 6.6%, and the accuracy was within ±11.5%. The long-term storage stability in FA-treated plasma was established to be 392, 392 and 257 days at -70 °C, respectively for RDV, GS-441524 and GS-704277. The validated method was successfully applied in COVID-19 related clinical studies.

Validation of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of hydroxychloroquine and metabolites in human whole blood.

Objectives Hydroxychloroquine (HCQ) is an anti-malarial and immunomodulatory drug reported to inhibit the Corona virus, SARS-CoV-2, in vitro. At present there is insufficient evidence from clinical trials to determine the safety and efficacy of HCQ as a treatment for COVID-19. However, since the World Health Organisation declared COVID-19 a pandemic in March 2020, the US Food and Drug Administration issued an Emergency Use Authorisation to allow HCQ and Chloroquine (CQ) to be distributed and used for certain hospitalised patients with COVID-19 and numerous clinical trials are underway around the world, including the UK based RECOVERY trial, with over 1000 volunteers. The validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of HCQ and two of its major metabolites, desethylchloroquine (DCQ) and di-desethylchloroquine (DDCQ), in whole blood is described. Methods Blood samples were deproteinised using acetonitrile. HCQ, DCQ and DDCQ were chromatographically separated on a biphenyl column with gradient elution, at a flow rate of 500 µL/min. The analysis time was 8 min. Results For each analyte linear calibration curves were obtained over the concentration range 50-2000 µg/L, the lower limit of quantification (LLOQ) was 13 µg/L, the inter-assay relative standard deviation (RSD) was <10% at 25, 800 and 1750 µg/L and mean recoveries were 80, 81, 78 and 62% for HCQ, d4-HCQ, DCQ and DDCQ, respectively. Conclusion This method has acceptable analytical performance and is applicable to the therapeutic monitoring of HCQ, evaluating the pharmacokinetics of HCQ in COVID-19 patients and supporting clinical trials.