Impact of Sample Hemolysis on Drug Stability in Regulated Bioanalysis

Abstract

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.

Impact of hemolysis during sample collection: How different is drug concentration in hemolyzed plasma from that of normal plasma?

“…(6). Specifically, assuming a hematocrit value of 0.43 and the drug RBC: plasma concentration ratio of 25, the % of change in plasma concentration should be around 36% at the hemolysis level of 2%.…”

“…Since additional components, which are normally absent in plasma, are released into the plasma during hemolysis, it is therefore very important to evaluate the associated matrix effect, hemolysis matrix effect, during bioanalytical method development and validation to ensure that hemolyzed plasma samples can be accurately analyzed [1][2][3][4][5]. In some cases, it is even necessary to evaluate the impact of hemolysis on drug stability in hemolyzed samples [6].…”

Utilizing Internal Standard Responses to Assess Risk on Reporting Bioanalytical Results from Hemolyzed Samples

“…For example, plasma with 50% of hemolyzed blood was prepared by mixing 7.5 mL of human control plasma with 7.5 mL of lysed human whole blood. Lysed human whole blood was prepared by twice freezing and thawing human whole blood (20). The color of the resulting mixture deepened with increasing percentage of hemolyzed blood, and it is difficult to distinguish beyond 20% of hemolyzed blood based on color, as depicted in Fig.…”

“…Hemolysis can be expressed in terms of the concentration of free hemoglobin in a given plasma sample, in mg/dL, with ∼550 mg/dL corresponding to roughly 2% of hemolyzed blood in the plasma sample (19) and can be determined spectrophotometrically. Another approach is to estimate the extent of hemolysis in terms of the percentage of hemolyzed blood added to the control plasma (20). Since there are other components besides hemoglobin being released during cell lysis and these components can interfere with the mass spectrometric measurement as well, we decided to express the extent of hemolysis in terms of the percentage of hemolyzed blood added to the control plasma in this work.…”

“…Some regulators, for example, in Europe and Brazil, are recommending the evaluation of matrix factor in hemolyzed plasma in their bioanalytical assay validation guidelines (17,18). Analyses of hemolyzed study samples have been much discussed in the bioanalytical field, with a focus on matrix effect, analyte stability in hemolyzed plasma, and Breportability^of the bioanalytical results (19)(20)(21)(22). Bioanalytical laboratories in general conduct formal evaluation of matrix factor, specificity/selectivity in hemolyzed plasma during method validation, usually at a pre-defined level of hemolysis, for example, plasma spiked with hemolyzed blood to a final percentage of 2% (19).…”