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Given the inherent complexities of bioanalysis, the role of incurred sample reanalysis (ISR) is increasingly appreciated in regulatory bioanalysis. Incurred sample reanalysis has evolved as an integral part of an assay to ensure method reproducibility. The current regulatory ISR guidelines do not provide clarity regarding ISR assessment for chiral drugs comprising enantiomers. Because chiral assays evaluate two enantiomers, there are additional complexities associated with the ISR data generation and interpretation. Based on the current literature, the practices for conducting ISR in chiral methods were reviewed and assessed. While ISR was conducted in chiral methods for both enantiomers using the acceptance criteria prescribed for non-chiral methods, there may be a need to streamline the nuances of ISR data interpretation and define the ISR requirements for chiral methods. The article provides perspectives on the ISR of enantiomeric drugs, including strategy development, by providing various hypothetical scenarios and possible considerations for defining ISR evaluation for chiral assays.

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Aims: AZD7442 is a combination SARS-CoV-2 therapy comprising two co-dosed monoclonal antibodies. Materials & methods: The authors validated a hybrid ligand-binding assay-LC-MS/MS method for pharmacokinetic assessment of AZD7442 in human serum with nominal concentration range of each analyte of 0.300-30.0 µg/ml. Results: Validation results met current regulatory acceptance criteria. The validated method supported three clinical trials that spanned more than 17 months and ≥720 analytical runs (∼30,000 samples and ∼3000 incurred sample reanalyses per analyte). The data generated supported multiple health authority interactions, across the globe. AZD7442 (EVUSHELD) was approved in 12 countries for pre-exposure prophylaxis of COVID-19. Conclusion: The results reported here demonstrate the robust, high-throughput capability of the hybrid ligand-binding assay-LC-MS/MS approach being employed to support-next generation versions of EVUSHELD, AZD3152.

The measurement of antibodies in human body fluids (e.g., blood, serum) has historically been tied to laboratory tests that may face operational limitations, including susceptibility to interference from other blood components and a reliance on unique reagents that can take months to produce. As such, there is a pursuit of alternative analytical methods to more accurately detect and measure antibody drugs from complex matrices. In the method, the authors describe different techniques that once combined were used to capture, separate, filter, fragment and then detect and measure the co-dosed antibody drugs. This method has been validated in accordance with current health authority guidelines and has been used to support three clinical trials that spanned more than 17 months; that is, the validated method was used to analyze nearly 30,000 serum samples from more than 2000 patients. Collectively, the results reported here demonstrate the robustness and high-throughput capability of this analytical approach.

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Introduction: A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for estimation of bedaquiline (BDQ) in human plasma using the deuterated analogue of the analyte, bedaquiline-d6 (BDQ-d6) as the internal standard. Methods: The plasma sample of 50 µL was extracted by liquid-liquid extraction using methyl tertiary butyl ether (MTBE). The separation was achieved on Zodiac C18 (50 x 4.6 mm, 5 µm) column with a mobile phase consisting of methanol and 5 mM ammonium formate in 0.1 % formic acid (w/v) (90:10, v/v) at a flow rate of 1.0 mL/min. Protonated analyte and internal standard were detected on a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) mode. Results: The linearity of the method was established in the concentration range of 5---1800 ng/mL with correlation coefficient, r2 ≥ 0.99. All the validated parameters were found well within the limits. Discussion: The method was applied for the first time to evaluate the pharmacokinetic parameters after single oral dose of BDQ 100 mg under fed conditions in healthy human volunteers, and the results were further authenticated by incurred sample reanalysis.

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A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of itraconazole (ITZ) and hydroxyitraconazole (ITZ-OH) as part of a human pharmacokinetic study of novel tablet formulations. We demonstrated that 100 µL of plasma sample can be used with a protein precipitation extraction by optimizing different composition of acid in organic solvent for the precipitation solvent, giving comparable recovery to more time-consuming liquid-liquid or solid phase extractions. Additionally, we have shown that by monitoring the halogen isotopic peak for ITZ as well as optimizing chromatographic conditions, we are able to avoid carryover and endogenous interferences, allowing for a lower limit of quantification for our study. We validated the method to quantify ITZ and ITZ-OH from 1 to 250 ng/mL in human plasma and applied this to a formulation research clinical study (NCT04035187). This is the first itraconazole study to demonstrate robustness of the assay by performing interference testing of over-the-counter and common co-administered medications. We are also the first publication to perform incurred sample reanalysis (ISR) at the conclusion of a 672 sample clinical study to show reproducibility of assay performance.

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The bioanalysis of drugs that undergo acyl glucuronidation presents an analytical challenge due to poor stability of acyl glucuronide metabolites in biological matrices. The objective of this study was to investigate the impact of back conversion of acyl glucuronide metabolites on drug concentration measurement in bioequivalence (BE) studies submitted to Abbreviated New Drug Applications (ANDAs). The prevalence of several treatments for preventing the back conversion of acyl glucuronide metabolites and the results of incurred sample reanalysis (ISR) were analyzed. In total, 322 ANDAs for 26 drugs known to generate acyl glucuronide metabolites were surveyed. Many studies have applied multiple preventive treatments during the clinical and bioanalytical phases. More than two-thirds (67.2%) of the studies utilized procedures of lowering the temperature for sample collection during clinical phase. Fewer studies have utilized procedures for lowering the pH of plasma samples (12.3%) or adding enzyme inhibitors (4.4%) in the clinical phase. A small fraction (16.9%) validated the pre-study method in the presence of the acyl glucuronide metabolites. The majority (62.2%) of the studies employed the procedure of lowering the pH during the sample extraction process in the bioanalytical phase. Among the studies that had significantly higher (p-value < 0.01 by sign test) ISR results than the corresponding original concentration values, 41 BE studies did not carry out any preventive treatments during the bioanalysis phase, suggesting that back conversion of acyl glucuronide metabolites to parent drugs may be present in these studies. The awareness of appropriate treatments of study samples for possible back-conversions of acyl glucuronide metabolites is expected to assist generic drug applicants in improving the quality of their future applications.

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Aim: To develop a bioanalytical method for quantifying INCB000928 in human saliva. Materials & methods: Human centrifuged saliva and human whole saliva were compared for matrix selection. Protein precipitation extraction and HPLC-MS/MS was used for analysis. Results & conclusion: Nonspecific binding of INCB000928 was reduced in whole versus centrifuged saliva. Whole saliva was a preferred matrix for INCB000928 bioanalytical method validation. Incurred sample reanalysis (ISR) using a successfully validated method failed in a healthy volunteer study because of inhomogeneous INCB000928 concentration across sample tube depths. Individual mixing of sample tubes followed by immediate aliquoting corrected the ISR failure, with 97.2% of repeats passing versus 41.7% for the same ISR samples.

Fibrodysplasia ossificans progressiva (FOP) is a very rare condition where bone forms outside the skeleton (ossification), leading to restricted movement, decreased quality of life and shortened life span. Mutations in a gene called ALK2 have been identified as causing FOP. INCB000928 is a novel drug (to be taken by mouth) which inhibits ALK2 activity and prevents ossification in a laboratory mouse model of FOP. Because monitoring of the levels and efficacy of a drug often requires blood draws, which can be taxing in patients with FOP, this study aimed to develop a method to measure INCB000928 levels in saliva. The authors propose a unique procedure to process saliva samples to ensure accurate, reproducible quantitation of INCB000928 levels in saliva.

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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.

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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.

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Background Plasma renin levels were determined in the academia-driven, EU-funded "Labeling of Enalapril from Neonates up to Adolescents" (LENA) project to evaluate its role in pediatric heart failure. Quality-controlled bioanalysis is crucial to ensure reliable data generation. However, a comprehensive bioanalytical quality control (QC) concept to monitor the method performance within an academic environment was lacking. Methods Thus, a QC concept was designed encompassing regulatory guidance, international recommendations and current scientific discussions. The concept included (1) a system-suitability test, (2) verification of single bioanalytical runs by calibration curve performance and evaluation of QCs, (3) assessment of the inter-run accuracy according to Clinical Laboratory Standards Institute (CLSI) guideline, (4) monitoring of reproducibility by pediatric incurred samples, (5) blank-sample analysis and (6) participation in interlaboratory testing. Results The concept was successfully applied to the academic project. About 11% of single runs were identified as invalid and triggered a re-analysis of unknown samples being included in those runs. The usefulness of the customized inter-run monitoring was demonstrated and proved the good accuracy from the first to the last run. All 147 reanalyzed incurred sample pairs complied with regulatory requirements. Conclusions The regulatory complied QC concept was customized for the demands of academia-driven pediatric trials and contributed to the reliable quantification of 965 pediatric renin samples.

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While the role of plasma renin activity (PRA) in heart failure has been widely studied in adults, comprehensive data on pediatric heart failure remain lacking. This drawback is increasingly being addressed by academic research. Nevertheless, such pediatric investigations are commonly conducted only once due to ethical constraints. Therefore, the quality of bioanalytical data must be ensured to acquire meaningful insights into maturing humoral parameters. However, appropriate post-validation assessment of bioanalytical runs is currently underrepresented by regulatory guidance. Thus, for applications in an academic environment, an easy-to-handle six-step bioanalytical quality control system was designed based on regulatory guidelines (e.g. U.S. Food and Drug Administration) combined with international recommendations (e.g. Clinical and Laboratory Standards Institute) and current scientific discussion. Its applicability to an enzyme-linked immunosorbent assay for determination of PRA was investigated within three pediatric trials of the EU-funded "Labeling of Enalapril in Neonates up to Adolescents" project. This quality control system identified 15 % bioanalytical runs as non-compliant to the predefined specifications and ensured the reliable quantification of 940 pharmacodynamic samples. The inter-run assessment of quality controls was able to demonstrate the comparability of the study results. Furthermore, 86 % of incurred sample reanalysis pairs complied with regulatory requirements (>67 %), thus underlining the long-term reproducibility of the utilized ligand-binding assay. Successful participation in interlaboratory testing confirmed the accuracy of the applied method throughout the entire study period. Further investigations showed no notable differences between the five applied lots of the PRA assay. The applicability of this quality control system was proven in an academic environment and ensured reliable results for PRA over the entire 24-month study period.

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Pharmacokinetic studies are key to evidence-based pharmacotherapy. The reliability of pharmacokinetic parameters is closely related to the quality of bioanalytical data. Bioanalytical method validation is fully described by regulatory guidelines; however, it is conducted just once. To ensure reliability and comparability of clinical data, appropriate quality control systems must be enforced to monitor post-validation bioanalytical runs. While single bioanalytical run evaluation is described in international guidelines, somehow, the long-term reproducibility of the bioanalytical method is unattended; it becomes pivotal with the involvement of pediatric population. Therefore, a customized quality control system was developed that addresses regulatory requirements and encompasses the specific demands of pediatric research. It consisted of continuous multi-parameter assessment, including calibration curves, quality control samples, incurred sample reanalysis, and internal standard data. The recommendations provided by the guidelines were combined with the additional Westgard rules, statistical evaluation, and graphical observations. The applicability of the developed quality control system was investigated by using data from three pediatric clinical trials, where the system was able to identify 16% of all analytical runs as invalid. Using a pooled standard deviation provided a better estimate of long-term reproducibility by calculating the %CV, which ranged from 3.6 to 10.3% at all quality control levels. Irrespective of the difficulties encountered owing to vulnerable pediatric populations, the incurred sample reanalysis fulfilled the regulatory requirement of at least 67%. This quality control approach ensured reliable and comparable results over a whole 31-month duration in relation to pediatric studies.

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Reliable results of pharmacokinetic and toxicokinetic studies are vital for correct decision making during drug discovery and development. Thus, ensuring high quality of bioanalytical methods is of critical importance. Incurred sample reanalysis (ISR)-one of the tools used to validate a method-is included in the bioanalytical regulatory recommendations. The methodology of this test is well established, but the estimation of the sample size is still commented on and contested. We have applied the hypergeometric distribution to evaluate ISR test passing rates in different clinical study sizes. We have tested both fixed rates of the clinical samples-as currently recommended by FDA and EMA-and a fixed number of ISRs. Our study revealed that the passing rate using the current sample size calculation is related to the clinical study size. However, the passing rate is much less dependent on the clinical study size when a fixed number of ISRs is used. Thus, we suggest using a fixed number of ISRs, e.g., 30 samples, for all studies. We found the hypergeometric distribution to be an adequate model for the assessment of similarities in original and repeated data. This model may be further used to optimize the sample size needed for the ISR test as well as to bridge data from different methods. This paper provides a basis to re-consider current ISR recommendations and implement a more statistically rationalized and risk-controlled approach.

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A selective, sensitive and high throughput liquid chromatography - tandem mass spectrometry (LC-MS/MS) method was developed and validated for estimation of eluxadoline in human plasma. Plasma samples of analyte with internal standard (eluxadoline13CD3) were extracted using solid phase extraction on Orochem Panthera Deluxe cartridges. Chromatographic separation was performed on Ace Phenyl column (150 × 4.6 mm, 5 µm), using a mixture of buffer (2 mM ammonium acetate in 0.01% formic acid), acetonitrile and methanol (20:70:10, v/v/v) as mobile phase at a flow rate of 0.8 mL/min. The run time of analyte was 4.0 min. Tandem mass spectrometry, operating in positive ionization and multiple reaction monitoring modes was used for detection of analyte and internal standard. The method was established with a linear dynamic range of 25.0-5000 pg/mL for eluxadoline using 300 µL human plasma. The sample preparation procedure was consistent and reproducible (accuracy, 96.2-106.1%; precision (%CV), 0.8-6.6%), preventing the ex vivo hydrolysis of acyl glucuronide metabolite of eluxadoline to parent drug. The method was applied successfully to a clinical pharmacokinetic study in six healthy South Indian male subjects under fed conditions and the results were authenticated by incurred sample reanalysis.

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Bioanalysis plays a key role during the drug discovery process to generate the pharmacokinetic data to facilitate unbiased evaluation of leads, optimized leads and drug candidates. Such pharmacokinetic data are used to enable key decisions in the drug discovery process. The aim of the work is to put forward a new strategy of performing the incurred sample reanalysis for select small molecule novel chemical entities at different stages of drug discovery prior to candidate selection. Three discovery programs representing hits, leads and optimized lead candidates were selected for the incurred sample reanalysis (ISR) analysis. From each discovery program, two novel chemical entities were selected for the ISR analysis. The time points considered for ISR generally varied among the programs; however, samples coinciding with drug absorption, distribution and elimination were considered in the ISR assessment. With the exception of a single ISR value that gave a high deviation (about 63%), the observed ISR values supported the discovery bioanalytical assays. While the individual bioanalytical laboratory can draw an algorithm for selecting novel chemical entities and fixing the acceptance criteria for the ISR data, it is proposed that the percentage difference between ISR vs. original concentration for 67% of the repeat samples is contained within ±30% for discovery bioanalysis.

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A method for bioanalysis of pentoxifylline in human plasma was developed using liquid chromatography-tandem mass spectrometry, which is simple, specific, and sensitive. Pentoxifylline D5 was used as the internal standard. Employing only 100 µl of human plasma, processing was done with solid-phase extraction technique. The analyte and the internal standard were separated from endogenous components on Ace phenyl column using a mixture of 5 mM ammonium acetate buffer and high performance liquid chromatography grade acetonitrile (60:40, v/v) as mobile phase at a flow rate of 1 ml/min. The linearity of the method was in the range of 3-1200 ng/ml with r2 > 0.99. Positive ion MRM mode was used for the detection of the analyte and the internal standard. The method was validated as per the US Food and Drug Administration guidelines and the results were within the acceptance limits. The proposed method was applied for comparative pharmacokinetic study of pentoxifylline after oral administration of 400 and 600 mg tablets to South Indian male subjects under fed conditions.

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With 10 years of experiences on incurred sample reanalysis (ISR) as an integrated part of regulated bioanalysis, the European Bioanalysis Forum has reflected on the implementation and the use of ISR. Three surveys were conducted in 2016 and 2017 as a revisit of the ISR experiences within European pharmaceutical industry and contract research organizations: has ISR become a tool for postvalidation and process check of a bioanalytical method performance and has ISR become a routine in our laboratories? Do we agree on the interpretation of guidelines/guidance and are we aligned in our approach - among others?

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Incurred sample reanalysis (ISR) is used to ensure the validity and reliability of bioanalytical data. Additionally, ISR results also help identify issues that could influence or bias the data. Overall, based on a decade of experimental data generated at Eli Lilly and Company, ISR failures are few with less than 5% of ISR samples failing to meet acceptance criteria. In a majority of situations, the cause for ISR failures has been 'human-error.' However, there are examples where ISR has helped identify issues related to the stability of the analyte or the ruggedness of the method. As a strategy, it is beneficial to conduct ISR following the completion of a few sample runs, so any potential issues impacting the validity and reliability of the data can be identified and rectified early.

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In this paper, experiences and learnings are shared from the 10-year application of incurred sample reanalysis (ISR) in support of the AstraZeneca small molecule portfolio. The conclusions from including ISR in every clinical bioanalysis study for a period of 5 years, generating ISR data from 550 studies, are shared. Our preclinical ISR approach is described and data generated using capillary microsampling demonstrate confidence in its routine application. The data demonstrate that ISR failures are very rare and the assessment can and should therefore be limited. Dialogue between the bioanalytical teams internally, as well as with the partner contract research organizations, is however critical for a successful bioanalytical method validation and to avoid any ISR failures.

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Aim: A sensitive method to quantify emixustat and its rapidly formed three major deaminated metabolites in human plasma was necessary to determine exposure in clinical trials. Methods: An LC-MS/MS method was validated for accuracy and precision, linearity, carry over, selectivity, recovery, matrix effects, hematocrit effects and stability. Results: A quantitative procedure for the determination of emixustat, ACU-5116, ACU-5124 and ACU-5149 in human plasma over the concentration range of 0.0500/1.00/1.00/1.00-10.0/1000/1000/1000 ng/ml, was successfully validated and has been used to successfully analyze samples in three clinical trials. Incurred sample reanalysis was performed for all four analytes in each study with >92% of the repeat results and original results within 20% of the mean of the two values.