Whole Blood Stability in Quantitative Bioanalysis

Abstract

Establishing stability at all stages of a sample’s lifespan is a critical part of performing regulated bioanalysis. For plasma assays, this includes the duration between when blood is drawn and when that blood is centrifuged to produce plasma. Here, we provide a discussion of current regulatory expectations around whole blood stability testing for LC–MS plasma assays, as well as the two primary experimental approaches utilized to assess whole blood stability. Next, we interrogated a large dataset of validated methods (1076 methods, the vast majority of which were for measurement of small molecules) to assess the correlation between whole blood and plasma stability profiles, finding them to be highly correlated. Finally, we summarize unique case studies; we have encountered during WB stability testing which offer lessons that may be broadly applicable.

ICH M10 guideline - a harmonized global approach to bioanalysis

“…Krew świeżą można definiować jako przechowywaną w lodówce nie dłużej niż 7 dni. Mrożenie krwi negatywnie wpływa na jej właściwości powodując znaczącą hemolizę [26]. Ledvina (2019) Kolejnym zagadnieniem wymagającym wyjaśnienia jest postępowanie w sytuacji, kiedy wykazana zostanie niestabilność leków podawanych jednocześnie.…”

“…Mrożenie krwi negatywnie wpływa na jej właściwości powodując znaczącą hemolizę [26]. Ledvina (2019) Kolejnym zagadnieniem wymagającym wyjaśnienia jest postępowanie w sytuacji, kiedy wykazana zostanie niestabilność leków podawanych jednocześnie. Ponadto, warto byłoby rozważyć wykorzystanie danych literaturowych o stabilności każdego z poszczególnych analitów osobno w obliczu braku solidnych dowodów naukowych, które wskazywałyby, że na stabilność jednego analitu w matrycy ma pozytywny lub negatywny wpływ obecność innego analitu [25].…”

Perspectives on updates, clarifications and controversies in chromatographic assay guidance for bioanalytical method validation from major regulatory agencies and organizations

“…In these cases, one should be cognizant of an analyte’s propensity to partition into red blood cells – any assessment of stability must ensure full distribution of analytes into the cellular fraction, typically done as a short equilibration time before the whole blood is processed to plasma. (Ledvina, Ewles, Pang, & Cape, 2019; van Teijlingen et al, 2012).…”

“…The EMA does not mention it by name, but states that careful attention should be paid to ensure that concentrations are representative of those at the time of sampling.The strongest statement about WBS is made in the ICH M10 draft guidance by calling for inclusion of such data in the validation report. We consider WBS critical in cases where the analyte structure implies liability, such as ester pro-drugs, N-oxides or hydroxamic acids, although we also acknowledge that the correlation between whole blood and plasma stability is strong(Freisleben et al, 2011;Ledvina, Ewles, Pang, & Cape, 2019). WBS can be assessed by the comparison of spiked whole blood samples with and without a controlled storage condition and time, via the analysis of whole blood, processed plasma or serum, depending on the assay compatibility.…”

Stability Issues in Bioanalysis

“…The reagents used in routine laboratory experiments can have significant impact on the stability of test samples. Finally, Cape and her colleagues discussed the current approaches, challenges and regulatory aspects around the measurement of stability in whole blood [4].…”