Abstract
When compared with biological samples in other matrices (plasma, urine, etc.) that are typically seen in bioanalytical applications, whole blood samples present unique challenges in method development, because of the viscous nature of blood and complexity of its constituents. In this article, we have developed and validated a series of quantitative bioanalytical methods for the determination of a pharmaceutical compound, Compound A, and its phosphate metabolite from whole blood matrices using liquid chromatography tandem mass spectrometry. All methods employed a simple protein precipitation procedure that was automated in 96-well format. The methods were subjected to vigorous tests in precision, accuracy, matrix effect, reproducibility, and robustness. Monolithic chromatography was used to improve sample throughput in one of the methods. The results also demonstrated that proper sample preparation procedures, such as sample transfer and lysing of blood cells prior to the extraction, are key to reproducible results for pharmacokinetic parameter determination.
Highlights
In pharmaceutical discovery and development, biological fluids such as plasma, serum, whole blood, and urine are most commonly analyzed for pharmacokinetic parameter evaluation
Simple sample preparation techniques like protein precipitation (PPT) have been used in the whole blood analysis, whole blood analysis typically calls for more labor-intensive treatment such as liquid-liquid extraction (LLE) or solid-phase extraction (SPE)
Various extraction approaches were experimented for optimum recovery and automation feasibility
Summary
In pharmaceutical discovery and development, biological fluids such as plasma, serum, whole blood, and urine are most commonly analyzed for pharmacokinetic parameter evaluation. Simple sample preparation techniques like protein precipitation (PPT) have been used in the whole blood analysis, whole blood analysis typically calls for more labor-intensive treatment such as liquid-liquid extraction (LLE) or solid-phase extraction (SPE). A method for the determination of indapamide in human whole blood has been developed with a sensitivity of 0.5 ng/mL as the lower limit of quantification (LLOQ). While most of whole blood methods reported to date employed manual extraction, 96-well techniques have been developed for whole blood analysis. A 96-well based LC-MS/MS method was developed and validated for the determination of N-methyl-4-isoleucine-cyclosporin (NIM811) over the concentration range of 1–2500 ng/mL in human whole blood using a 0.05 mL sample volume [7]. Compound A is a prodrug and has an active metabolite in phosphate form
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