What Matrix Is It Anyway? Blood to Plasma Partitioning in Drug Discovery and Bioanalysis
Updated: May 8
When I first broke into LC-MS/MS, my research subject matter was focused on agriculture. Natural product analytical chemistry poses their own unique set of challenges, but the matrix is usually very self-explanatory. My first introduction to bioanalytical chemistry was toward the end of my graduate school tenure at the University of Missouri, when my advisor asked if I could develop a method for an analyte in mouse plasma to support a collaboration. At this point, I felt very confident operating our LCMS, but had never worked with plasma, and accepted the challenge without hesitation.
I remember doing a quick online search looking for LC-MS methods in plasma, and to my surprise, there was a vast amount of literature available to guide me. After some time overcoming a very challenging chromatographic hurdle, it was time to develop the extraction in plasma. I returned to the literature, and it seemed the analysis of plasma was almost universally performed for bioanalytical studies, but why? I couldn’t wrap my head around why everyone wasn’t analyzing whole blood.
Fast forward to my first industry role at a CRO, and I’m spending most of my time developing and validating methods for new molecules in plasma. Then the day comes when I’m tasked with developing a method in whole blood for a client’s drug. The curiosity that had the best of me a few years prior resurfaces, and I ask why the matrix is whole blood for this project. Most of my colleagues had no idea, other than what it was what we were asked to do. My curiosity didn’t fade, and I still had a job to do. As I began working with whole blood for the first time in an analytical capacity, I quickly learned why people don’t work with whole blood – it’s messy and difficult to pipette. Whole blood is also very perishable and requires frequent procurement during a project, unlike plasma. At this point, I’m happy to wrap the method validation up and return to plasma, however, my question is left unanswered.
A few years later, I’m more involved in a drug metabolism capacity when another whole blood project comes along. I had developed a strong relationship with this client over a few years and felt this was my opportunity to get my questions answered. The goal of this project was to establish a PK/PD relationship and we would be measuring their drug in plasma, as well as two biomarkers in whole blood: adenosine triphosphate (ATP) and 2,3 diphosphoglyceric acid (DPG). During our initial conversations I asked them WHY do you want to measure ATP and DPG in whole blood?
“These two molecules distribute highly into red blood cells and most likely won’t be detected in plasma.”
All my past experiences came together for a lightbulb moment. The foundation of modern drug development involves the measurement of a drug/metabolite/biomarker in plasma, which at the end of the day is simply a surrogate for systemic circulation. However, plasma is far and away cleaner and easier to work with analytically. The reason this approach works, is that a majority of small molecule drugs have a blood-to-plasma partition ratio that is within close proximity to 1. Therefore, when blood samples are processed to plasma, a very similar concentration will be determined, regardless of the matrix that is analyzed. However, there are some instances, such as these two biomarkers, where one of the matrices would not be suitable for the application.
At Veloxity Labs, with more than 100 years of experience at 20 stints at other CROs, we can provide these services and consult our sponsors in the right direction now. A recent example of this was when we were contracted to develop a method for the quantitation of Rapamycin in plasma. This was a long-term study and samples were already being collected. We had the benefit of working with Rapamycin previously and knew that it distributes highly (>95%) into red blood cells. We suggested the sponsor make the switch from plasma to whole blood and offered to analyze both matrices. Rapamycin concentrations were below the detection limit in plasma and aligned with their expectations in whole blood. The client was thrilled with our guidance and expertise.
As micro sampling continues to emerge as a medium to provide richer datasets and improve the quality of life of patients, the emergence of dried blood analysis increases. There are situations where a microsample of blood needs to be analyzed (oncology, pediatrics) and compared to a historic plasma dataset. This can all be scaled or bridged by understanding the whole blood-to-plasma ratio and applying a correction factor to a dataset to accurately compare concentrations. Bridging plasma to blood and dried blood is a service we routinely do at Veloxity.
Determining the blood-to-plasma partition coefficient is critical early in the development of your therapeutic. This can be easily derived in a quick and reliable way using an in-vitro approach to properly understand the partitioning between the red blood cell and plasma compartments. Otherwise, you run the risk of potentially vastly underestimating the exposure and overestimating the clearance.