Blood Plasma Partitioning Assay

Creative Bioarray provides Plasma Protein Binding Assay to help customers accurately detect the concentration of the drug in whole blood compared to plasma, predicting the binding of the compound to red blood cells.

Why do we need Blood Plasma Partitioning Assay?

  • Information on the partitioning of a pharmaceutically important molecule in red blood cells (RBC) is critical for understanding the compound's pharmacokinetics and pharmacodynamics and is sometimes critical in selecting a therapeutic candidate without missing out.
  • Blood determines the drug concentration ratio in whole blood to plasma, revealing the binding of the drug to red blood cells.
  • The pharmacokinetic parameters computed from plasma data may be misleading if there is a discrepancy in drug concentration between plasma and red blood cells due to different binding to specific components in the blood.
  • When the blood to plasma ratio is greater than 1 (usually the result of the distribution of the drug into the red blood cells), the plasma clearance rate significantly overestimates the blood clearance rate and may exceed the hepatic blood flow. The ratio of blood to plasma can also be used to understand potential blood toxicity.

Brief Protocol

  • The test compound is added to fresh heparinized whole blood, reference red blood cells, and reference plasma.
  • After the incubation period, the whole blood is centrifuged.
  • Two parts of whole blood (plasma and red blood cells) are analyzed by LC-MS/MS together with the reference sample.
  • The following equation calculates the ratio of blood to plasma.

Brief Protocol

KRBC/PL: the partition coefficient of a given drug in red blood cells;

H: hematocrit;
IREFPL and IPL: the LC-MS/MS response (peak area ratio to an internal standard) for the reference plasma and the plasma fraction.

The spiked whole blood sample and spiked plasma reference sample in the red blood cell distribution testFigure 1. The spiked whole blood sample and spiked plasma reference sample in the red blood cell distribution test

CRef: Reference plasma concentration, which is the same as the concentration of the drug in whole blood;
CPL: spike the overall balance of the drug concentration in plasma blood;
CRBC: drug concentration in red blood cells;
VT: Balanced plasma volume of reference plasma
VRBC: Balanced plasma volume of red blood cells
VPL: Balanced plasma volume of whole blood sample

Case Study

Table 1. Inter-run reproducibility of the phenoprobamate RBC partitioning assay

KRBC/PL at individual incubation time (min)
Run #0103060
Run 10.31±0.050.33±0.060.34±0.080.42±0.03
Run 20.31±0.090.33±0.060.33±0.070.26±0.08
Run 30.34±0.080.25±0.020.31±0.040.29±0.04

Effect of incubation time on RBC partitioning.Figure 2. Effect of incubation time on RBC partitioning.

Effect of compound concentration on RBC partitioning (incubation time = 60 min).Figure 3. Effect of compound concentration on RBC partitioning (incubation time = 60 min).

Quotation and Ordering

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  1. Yu, Shaoxia, et al. "A novel liquid chromatography/tandem mass spectrometry based depletion method for measuring red blood cell partitioning of pharmaceutical compounds in drug discovery." Rapid Communications in Mass Spectrometry: An International Journal Devoted to the Rapid Dissemination of UptotheMinute Research in Mass Spectrometry 19.2 (2005): 250-254.

For research use only. Not for any other purpose.