Direct injection of whole blood for liquid chromatography/tandem mass spectrometry analysis to support single‐rodent pharmacokinetic studies

Abstract

Mass spectrometric developments in the last decade enable (sub)nanomolar detection of drug compounds in biological matrices in a few microliters of blood. However, the sampling and especially the handling of these small blood volumes is not straightforward. We studied the feasibility of a recently developed 'sorbent sampling technique' to handle these small blood volumes and the application to support pharmacokinetic (PK) screening programs. This technique applies 5-10 microL of blood on a fibrous material packed into a cartridge. Blood samples absorbed on these cartridges are eluted directly, on-line onto a solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE-LC/MS/MS) system. It is shown that the sorbent sampling technique can be applied for a range of drug compounds. In spite of issues with recovery and sample clean-up that need further improvement, the sorbent sampling technique provided similar data as compared to conventional analytics. The technique was successfully applied to derive kinetic data from individual mice, thereby decreasing the number of required mice for a PK study from 21 to 3.

In vivo solid-phase microextraction for single rodent pharmacokinetics studies of carbamazepine and carbamazepine-10,11-epoxide in mice

“…This disadvantage has been addressed by performing studies where all samples are acquired from a single animal by serial blood sample withdrawal, but to date such studies have been difficult to execute in mice due to a limited circulating blood volume (∼1.85 mL/25 g mouse) and difficulties in the handling and sample preparation of the small blood sample volumes. To date, successful serial sampling and sample preparation approaches reported in the literature include the manual withdrawal of a small blood volume (4-10 L) from the tail vein [10,11] or withdrawal of 30 L blood using jugular vein cannulation [12] or lateral saphenous vein puncture [13] followed by plasma protein precipitation, withdrawal of 50-70 L blood using jugular vein cannulation followed by turbulent flow chromatography on 2 L aliquots [14] and sorbent sampling of 5 L volumes of whole blood obtained from the tail vein in combination with protein precipitation and on-line solid-phase extraction [15]. In addition to reduced animal use and increased data accuracy, the advantages of such single rodent PK studies include: (1) a decrease in the amount of compound needed to perform the study which is particularly advantageous in early drug discovery where the quantity of test compound is limited, (2) elimination of the effects of anesthesia from the PK data, (3) the ability to study inter-animal variation, and (4) the opportunity to perform multiple and/or simultaneous pharmacokinetics/pharmacodynamics investigations in mice [10,11,13,16,17].…”

Addressing the analytical throughput challenges in ADME screening using rapid ultra‐performance liquid chromatography/tandem mass spectrometry methodologies

“…Liquid chromatography methodologies deliver improved specificity, but, until the mid‐1990s, they were characterized by low throughput and sensitivity. The combination of LC with ESI‐MS in the late 1980s34 allowed for increased sensitivity and increased selectivity allowing for some compromise in the LC specificity and hence reduced analysis time 35. The development of sub‐2 µm particle technology LC allowed a significant increase in throughput whilst increasing assay performance 36, 37.…”