Assessing the impact of nonspecific binding on oligonucleotide bioanalysis

Aim: Accurate and reliable quantification of oligonucleotides can be difficult, which has led to an increased focus on bioanalytical methods for more robust analyses. Recent advances toward mitigating sample losses on liquid chromatography (LC) systems have produced recovery advantages for oligonucleotide separations. Results & methodology: LC instruments and columns constructed from MP35N metal alloy and stainless steel columns were compared against LC hardware modified with hybrid inorganic-organic silica surfaces. Designed to minimize metal-analyte adsorption, these surfaces demonstrated a 73% increase in 25-mer phosphorothioate oligonucleotide recovery using ion-pairing reversed-phase LC versus standard LC surfaces, most particularly upon initial use. Conclusion: Hybrid silica chromatographic surfaces improve the performance, detection limits and reproducibility of oligonucleotide bioanalytical assays.

The impact of ligand binding based assays critical reagent characterization and storage

Biological critical reagents are the foundation of many bioanalytical methods and often chemically modified or conjugated with various chemical tags. As such, the quality and performance of these methods are inherently tied to the quality and stability of critical reagents. This article will outline recommendations for conjugated critical reagent development and characterization. Examples of the impact of regent quality will be discussed for the two common bioanalytical assays in support of drug development for biotherapeutics. Finally, a brief discussion of conjugated reagent stability and recommendations for storage and testing will be presented.

An alternative approach for bioanalytical assay development for wastewater-based epidemiology of SARS-CoV-2

Wastewater-based epidemiology could be applied to track down SARS-CoV-2 outbreaks at high spatio-temporal resolution and could potentially be used as an early-warning for emergence of SARS-CoV-2 circulation in the general population. Epidemiological surveillance of SARS-CoV-2 could play a role in monitoring the spread of the virus in the population and controlling possible outbreaks. However, sensitive sample preparation and detection methods are necessary to detect trace levels of SARS-CoV-2 RNA in influent wastewater (IWW).Unlike predecessors, method development of a SARS-CoV-2 RNA concentration and detection procedure was performed with IWW samples with high viral SARS-CoV-2 loads (in combination with seeding IWW with a surrogate coronavirus). This is of importance since the SARS-CoV-2 genome in IWW might have already been subject to in-sewer degradation into smaller genome fragments or might be present in a different form (e.g. cell debris,…). Centricon Plus-70 (100 kDa) centrifugal filter devices resulted in the lowest and most reproducible Ct-values for SARS-CoV-2 RNA. Lowering pore sizes did not improve our limit of detection and quantification. Real-time polymerase chain reaction (qPCR) was employed for the amplification of the N1, N2, N3 and E_Sarbeco-gene.This is one of the first studies to apply digital polymerase chain reaction (dPCR) for the detection of SARS-CoV-2 RNA in IWW. Interestingly, qPCR results were comparable with dPCR results suggesting that qPCR is a valid method. In this study, dPCR was also used as a proxy to assess the precision of qPCR. In this light, dPCR showed high variability at low concentration levels (100 copies/µL), indicating that variability in bioanalytical assays for SARS-CoV-2 RNA might be substantial.On average, the N2-gene showed high in-sample stability in IWW for 10 days of storage at 4 °C. Between-sample variability was substantial due to the low native concentrations in IWW. Additionally, the E-gene proved to be less stable compared to the N2-gene and showed higher variability. Freezing the IWW samples resulted in a 10-fold decay of loads of the N2- and E-gene in IWW.Although WBE can already aid in filling some knowledge gaps in the epidemiological surveillance of SARS-CoV-2, future WBE studies should aim to further validate and standardize bioanalytical assays, especially with regards to methodological limitations.HighlightsDevelopment of an analytical procedure for detection of SARS-CoV-2 RNA in wastewaterExtraction recovery was evaluated in influent wastewaterPrecision measured with dPCR used as a proxy for qPCRqPCR of the N2 gene fragment showed high in-sample stability of SARS-CoV-2 on average

Optimisation and validation of a sensitive bioanalytical method for niclosamide

The SARS-CoV-2 pandemic has spread at an unprecedented rate, and repurposing opportunities have been intensively studied with only limited success to date. If successful, repurposing will allow interventions to become more rapidly available than development of new chemical entities. Niclosamide has been proposed as a candidate for repurposing for SARS-CoV-2 based upon the observation that it is amongst the most potent antiviral molecules evaluated in vitro. To investigate the pharmacokinetics of niclosamide, reliable, reproducible and sensitive bioanalytical assays are required. Here, a liquid chromatography tandem mass spectrometry assay is presented which was linear from 31.25-2000 ng/mL (high dynamic range) and 0.78-100 ng/mL (low dynamic range). Accuracy and precision ranged between 97.2% and 112.5%, 100.4% and 110.0%, respectively. The presented assay should have utility in preclinical evaluation of the exposure-response relationship and may be adapted for later evaluation of niclosamide in clinical trials.

Immuno-biosensor on a chip: a self-powered microfluidic-based electrochemical biosensing platform for point-of-care quantification of proteins

The realization of true point-of-care (PoC) systems profoundly relies on integrating the bioanalytical assays into “on-chip” fluid handing platforms, with autonomous performance, reproducible functionality, and capacity in scalable production. Specifically...

A Current Review on Analytical Tools for Determination of New Oral Antidiabetic Drugs, Empagliflozin, Linagliptin and Biguanides in Bulk Materials, Pharmaceuticals & Biological Samples

Worldwide the R & D divisions of Pharma industry are actively involved in the development of new therapeutic agents. These agents may be either new entities or partial structural modification of the existing one. The recent FDA statistics represent that the average number of drug filings are increasing every year in the thrust areas like anti-cancer agents, anti-diabetic, antibiotics, cardio-vascular drugs, respiratory drugs etc. Sodium glucose co-transporter-2(SGLT-2) inhibitors, dipeptidyl peptidase-4 (DPP-4) inhibitors and biguanides are effective oral anti-diabetic agents used in treatment of type 2 Diabetes Mellitus. Therefore, the necessity to explore and compare the existing analytical and bioanalytical assays used for determination of such drugs either single or in combination is crucial. Many methods were reported in the literature for the bio-analysis and analysis of four novel gliptins combinations, empagliflozin-linagliptin, empagliflozin-metformin HCl, linagliptin-metformin HCl, empagliflozin-linagliptin-metformin HCl combination with application on Glyxambi®, Synjardy®, Jentadueto®, Trijardy® XR tablets respectively. Furthermore, this review offered an overview of different methods used for determination of every drug alone as empagliflozin from SGLT-2 inhibitors, linagliptin from DPP-4 inhibitors and metformin from biguanides in a tabulated comparative way. Moreover, the current review emphasizes the most common stability indicating assays to be of interest to the analysts in the area of drug control. This review helps in understanding the further need for the development of analytical methods for the estimation of such drugs.