Volumetric absorptive microsampling combined with impact-assisted extraction for hematocrit effect free assays.

AIM: Volumetric absorptive microsampling (VAMS) is a recent technology available for sampling and analyzing low blood volume. The present work describes the utilization of VAMS for the quantitation of naproxen and ritonavir in human blood using a novel bead-based impact-assisted extraction (IAE) procedure. RESULTS: Sampling volume accuracy of the VAMS device was independent of the blood hematocrit (HCT) level, however analyte recovery decreased with increasing HCT when extracted using ultrasonication. In contrast, IAE was unaffected by HCT, resulting in quantitative recovery for all levels evaluated. Precision and accuracy batches, as well as matrix effect evaluation, met acceptance criteria. CONCLUSION: The IAE procedure coupled with VAMS is immune to HCT biases affecting sampling volume and recovery.

Severe impact of hemolysis on stability of phenolic compounds.

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.

Impact of sample hemolysis on drug stability in regulated bioanalysis.

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.

A practical approach to reduce interference due to in-source collision-induced dissociation of acylglucuronides in LC-MS/MS.

BACKGROUND: In LC-MS/MS, glucuronide conjugated metabolites may convert back to the parent drug due to in-source collision-induced dissociation (CID). RESULTS: During the bioanalysis of naproxen, it was noticed that naproxen acylglucuronide exhibited intense in-source CID to the naproxen [M+H](+) ion under positive ESI. However, no in-source CID of the acylglucuronide to the naproxen [M+NH(4)](+) adduct was observed. Furthermore, absolutely no in-source CID was detected under negative ESI. This phenomenon was not only observed for naproxen acylglucuronide but for eight other acylglucuronides compounds. CONCLUSION: 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.

Impact of oxcarbazepine sulfate metabolite on incurred sample reanalysis and quantification of oxcarbazepine.

BACKGROUND: Recently, incurred sample reanalysis (ISR) has become a requirement in bioanalysis. The general guidance recommends investigating ISR failure to evaluate the suitability of an analytical method. In the case of acceptable ISR evaluation, there were no precise recommendations for further testing when sporadic values were obtained. RESULTS: The ISR evaluation performed during a bioequivalence study for the anticonvulsant drug oxcarbazepine showed acceptable ISR results, but one particular chromatographic anomaly led to a thorough investigation. The finding of these tests showed that an oxcarbazepine phase II metabolite occasionally co-eluted with the drug and impacted oxcarbazepine's quantitation through in-source conversion. CONCLUSION: This paper demonstrates the necessity of rigorous interpretation of ISR results and close monitoring of all subject sample results.