Blood to Plasma Ratio

Blood to Plasma Ratio assay protocol

Data from Cyprotex's Blood to Plasma Ratio assay

Figure 1
Comparison of Cyprotex's blood to plasma ratio values (mean ± standard deviation; n=3) with literature values ^2,3.

Figure 2
Graph illustrating the intra-assay reproducibility of the blood to plasma ratio values for the species-specific positive control compounds (mean ± standard deviation; n=3 replicates).

Questions and answers on Blood to Plasma Ratio

Please provide an overview of Cyprotex's Blood to Plasma Ratio assay.

The blood to plasma ratio determines the concentration of the drug in whole blood compared to plasma and provides an indication of drug binding to erythrocytes.

The blood to plasma ratio protocol is adapted from a method by Yu et al., 2005¹. Test compound is spiked into fresh heparinized whole blood, reference red blood cells and reference plasma. Following the incubation period, the whole blood is centrifuged. Both fractions of the whole blood (plasma and red blood cells) are analyzed by LC-MS/MS alongside the reference samples.

The blood to plasma ratio is calculated from the following equation;

Where

Kb/p is the whole blood to plasma partition coefficient
H = hematocrit

Where

	is the red blood cell to plasma partition coefficient
	is the LC-MS/MS response (peak area ratio to an internal standard) for the plasma fraction
	is the LC-MS/MS response (peak area ratio to an internal standard) for the reference plasma
	is the LC-MS/MS response (peak area ratio to an internal standard) for the red blood cell fraction
	is the LC-MS/MS response (peak area ratio to an internal standard) for the reference red blood cells

Why is the blood to plasma ratio important?

Calculation of pharmacokinetic parameters is typically performed by the analysis of drug concentrations in plasma rather than whole blood. Therefore, pharmacokinetic parameters calculated from the plasma data may be misleading if differences exist between concentrations of the drug in the plasma and the red blood cells due to differential binding to a specific component in the blood. At blood to plasma ratios of greater than 1 (usually as a consequence of the drug distributing into the erythrocyte), the plasma clearance significantly overestimates blood clearance and could exceed hepatic blood flow. Blood to plasma ratio can also be used to understand potential hemotoxicity.

Why have you adapted the Yu et al., 2005 method to analyze the red blood cell portion as well as plasma?

By analyzing both red blood cell and plasma fractions, we have found that the data produced are much more robust in terms of reproducibility and the protocol reduces the occurrence of negative values. However, when analyzing red blood cells, the additional issue of plasma trapped between the cells needs to be considered. To minimize this issue, we have increased centrifugation speeds. At these increased centrifugation speeds, we have calculated that the effect of the trapped plasma is negligible when considering the range of values at which we are interested.

How do you lyse the red blood cells before analysis?

Samples are freeze thawed on three occasions to lyse the red blood cells.

What is the positive control used for Cyprotex's Blood to Plasma Ratio assay?

We use methazolamide as the positive control for human blood, chlorthalidone as the positive control for rat and mouse blood and chloroquine as the positive control for dog blood.

At what concentration should I assess my compounds in the blood to plasma ratio assay?

Concentration dependent partitioning within the blood has been reported. This can arise if compounds are highly protein binding or active transport is occurring within the blood and these processes are saturable at higher drug concentrations^1,2. For this reason, it is advised that for later stage in-depth evaluations a number of concentrations are investigated over the entire clinically relevant range for the drug. For earlier stage screening projects, it may be more appropriate to choose a low concentration to establish if blood partitioning is occurring.

What is the difference between the blood to plasma ratio value and the red blood cell partition coefficient?

The blood to plasma ratio (often referred to as Kb/p) is the ratio of the concentration of drug in whole blood (i.e. contains both red blood cells and plasma) to the concentration of drug in plasma, namely C_B/C_P. The red blood cell partition coefficient (often referred to as Ke/p) is the ratio of the concentration of drug in the red blood cells (i.e. not including plasma) to concentration of drug in plasma i.e. C_RBC/C_P.

¹ Yu S et al. (2005) Rapid Commun in Mass Spectrom 19; 250-254
² Hinderling PH (1997) Pharmacol Rev 49(3); 279-295
³ Obach RS (1999) Drug Metab Dispos 27(11); 1350-1359