Being regulated by agencies' guidances, the importance of a robust validated bioanalytical method is crucial as it may impact the validity of the pharmacokinetic data generated. During blood collection and processing, the presence of hemolyzed plasma samples may occur and as a result its impact must be investigated to ensure method robustness. Indeed, hemolyzed samples may affect the analyte recovery efficiency, as well as the chromatography. Furthermore, the stability of an analyte in hemolyzed plasma can be an issue as analyte degradation may occur. In this article we report two case studies where the analyte instability was a result of sample hemolysis. A description of the appropriate actions undertaken for the resolution of the issue will be discussed.
We have shown that monitoring the parent drug [M-H](-) or [M+NH(4)](+) whenever possible could be an easy approach used by bioanalytical scientists to minimize the impact of in-source CID of acylglucuronides to the parent drug.
Eugénie-Raphaëlle Bérubé has obtained a Bachelor of Science in Biochemistry from Université du Québec à Montréal. She previously worked at the St-Lawrence Center of Environment, Canada, conducting biomarker analysis to measure the impact of contaminants on aquatic species. She has been working in the bioanalysis industry for the past 8 years at Algorithme Pharma, a CRO located in Laval, Canada, becoming a scientist in bioanalytical method development for the quantitation of pharmaceuticals in biological fluids. The presence of hemolyzed plasma samples can negatively impact preclinical and clinical sample analysis. During the method development of morphine, post-extracted instability issues were encountered in human hemolyzed plasma when compared with nonhemolyzed plasma (called normal plasma for simplicity). Investigation revealed that the presence of methemoglobin using a high pH reconstitution solution led to degradation of morphine over time. The degradation probably results from radical oxidation of the ionized phenolic group promoted by the presence of methemoglobin. Pseudomorphine, the product of oxidative dimerization of morphine, was observed as one of the degradation products in hemolyzed plasma. This hypothesis was extended to raloxifene, another phenol-containing compound. On the other hand, no instability was detected for drug products bearing a masked phenol group or carboxylic acid functionality. The issue of morphine instability was resolved by using a reconstitution solution at a pH below the pKa of the phenol moiety.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.