Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients

Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1–20.3), and intermediate precision ≤ 12.1% (95% CI 9.2–17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.

Quantification of abemaciclib and metabolites: evolution of bioanalytical methods supporting a novel oncolytic agent

Aim: Bioanalytical methods undergo many revisions and modifications throughout drug development to meet the objectives of the study and development program. Results: Validated LC–MS/MS methodology used to quantify abemaciclib and four metabolites in human plasma is described. The method, initially validated to support the first-in-human study, was successfully modified to include additional metabolites as in vitro and in vivo information about the activity and abundance of human metabolites became available. Consistent performance of the method over time was demonstrated by an incurred sample reanalysis passing rate exceeding 95%, across clinical studies. An overview of the numerous methods involved during the development of abemaciclib, including the quantification of drugs evaluated as combination regimens and used as substrates during drug–drug interaction studies, is presented. Conclusion: Robust bioanalytical methods need to be designed with the flexibility required to support the evolving study objectives associated with registration and post-registration trials.

Validation and clinical application of dried blood spot assay for quantitative assessment of edoxaban in healthy adults

Aim: Dried blood spot (DBS) is a sampling approach that offers several advantages over plasma and whole blood (WB) sampling, but several factors, such as hematocrit and temperature, can adversely affect quantitation. Methodology & results: In an open-label, three-way crossover study in healthy subjects, we explored the correlation between DBS, WB and plasma samples, and between DBS samples from finger-prick and venipuncture blood for measuring edoxaban and its metabolite M-4 using LC–MS/MS. The methods were validated comprehensively. The incurred sample reanalysis experiments demonstrated quantitation reproducibility in all three matrices. Overall, there was a good correlation (near perfect concordance for edoxaban) among plasma, WB and DBS measurements. M-4 concentrations in DBS and WB were lower than in plasma. Conclusion: These results indicate using DBS may be used as an alternative methodology to measure edoxaban pharmacokinetics.

Evaluation of correlation between bioanalytical methods

Bioanalytical methods evolve throughout clinical development timelines, resulting in the need for establishing equivalency or correlation between different methods to enable comparison of data across different studies. This is accomplished by the conduct of cross validations and correlative studies to compare and describe the relationship. The incurred sample reanalysis acceptance criterion seems to be adopted universally for cross validations and correlative studies; however, this does not identify any trends or biases between the two methods (datasets) being compared. Presented here are graphing approaches suitable for comparing two methods and describing equivalence or correlation. This article aims to generate awareness on graphing techniques that can be adopted during cross validations and correlative studies.

Development and validation of bioanalytical methods to support investigations of AZD9496 in the clinic

Aim: AZD9496 is an oral nonsteroidal, potent and selective antagonist and degrader of ER-α. Two major active metabolites (M3 and M5 as diastereomers) were identified in humans. Methodology/results: Multianalyte, sensitive LC–MS/MS method in human plasma was developed and validated that overcame the challenges encountered. The method demonstrated acceptable precision, accuracy and selectivity for AZD9496 and two major metabolites. Incurred sample reanalysis was acceptable from evaluation in clinical studies, indicating adequate reproducibility. In addition, a urine method for AZD9496 was also developed and validated. Conclusion: Robust and sensitive LC–MS/MS assays for the quantitation of AZD9496 and two diastereomeric metabolites in human plasma and AZD9496 in human urine have been validated and successfully applied to clinical studies.

A comprehensive quality control system suitable for academic research: application in a pediatric study

Aim: Clinical research in pediatrics is progressively initiated by academia. As the reliability of pharmacodynamic measures is closely linked to the quality of bioanalytical data, bioanalytical quality assurance is crucial. However, clear guidance on comprehensive bioanalytical quality monitoring in the academic environment is lacking. Methods & results: By applying regulatory guidelines, international recommendations and scientific discussions, a five-step quality control system for monitoring the bioanalysis of aldosterone by immunoassay was developed. It comprised performance qualification, calibration curve evaluation, analysis of the intra- and inter-run performance via quality control samples, incurred sample reanalysis and external quality assessment by interlaboratory testing. A total of 55 out of 70 runs were qualified for the quantification of aldosterone in the study sample enabling the evaluation of 954 pediatric samples and demonstrating reliability over the 29-month bioanalysis period. Conclusion: The bioanalytical quality control system successfully monitored the aldosterone assay performance and proved its applicability in the academic environment.

Simultaneous Bioanalysis of Prodrug Oseltamivir and its Metabolite Oseltamivir Carboxylic Acid in Human Plasma by LC/MS/MS Method and its Application to Disposition Kinetics

Introduction: A selective, sensitive, precise and rapid analytical method using liquid chromatography- tandem mass spectrometry (LC/MS/MS) for simultaneous determination of oseltamivir and oseltamivir carboxylic acid in plasma has been developed and validated, using oseltamivir-D5 and oseltamivir acid-D3 as internal standards. Methods: The analytes were extracted from 300μL of human plasma using solid phase extraction technique. A mixture of methanol and 0.1% formic acid (60:40, v/v) was used as mobile phase at a flow rate of 0.7mL/min, to separate the analytes on Zorbax SB-C18 (50x4.6mm, 3.5μm) analytical column. Results: The calibration curves obtained were linear over the concentration ranges of 0.5-200ng/mL and 2.0-800ng/mL for oseltamivir and oseltamivir carboxylic acid respectively. A run time of 2.5min makes it possible to analyze more than 350 plasma samples in a day, thereby increasing the productivity. Conclusion: The present method was applied successfully to a clinical pharmacokinetic study in South Indian male subjects with 75mg oseltamivir phosphate capsule under fasting conditions and the results were authenticated by incurred sample reanalysis.

An Ultrasensitive LC-APPI-MS/MS Method for Simultaneous Determination of Ciclesonide and Active Metabolite Desisobutyryl-Ciclesonide in Human Serum and Its Application to a Clinical Study

Background The development of more efficient drug delivery devices for ciclesonide inhalation products requires an ultrasensitive bioanalytical method to measure systematic exposure of ciclesonide (CIC) and its active metabolite desisobutyryl-ciclesonide (des-CIC) in humans. Method Serum sample was extracted with 1-chlorobutane. A reversed-phase liquid chromatography coupled with atmospheric pressure photoionization–tandem mass spectrometry (LC-APPI-MS/MS) method was used for quantification of 1–500 pg/mL for both analytes in a 0.500-mL serum. The analysis time was 4.7 min/injection. CIC-d11 and des-CIC-d11 were used as the internal standards. Results Calibration curves showed good linearity (r2 > 0.99) for both analytes. This novel method was precise and accurate with interassay precision and accuracy of all within 9.6% CV and ± 4.0% bias for regular QC samples. Extraction recovery was approximately 85% for both analytes. Serum samples are stable for 3 freeze–thaw cycles, 24 h at bench top, and up to 706 days at both −20 °C and −70 °C. This method was successfully used to support a pharmacokinetic (PK) comparison between the inhalation suspensions and an inhalation aerosol of ciclesonide in healthy participants. The method robustness was also supported by the good incurred sample reanalysis reproducibility. Conclusion APPI, a highly selective and sensitive ionization source, made possible for quantifying CIC and des-CIC with a lower limit of quantification (LLOQ) of 1 pg/mL in human serum by LC-MS/MS. A 10-fold sensitivity improvement from the most sensitive reported method (LLOQ, 10 pg/mL) is essential to fully characterize the PK profiles of CIC and des-CIC in support of the clinical development of the ciclesonide-related medications for patients.