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Reputable Mentor II
Reputable Mentor II

The discovery phase of pharmaceutical research involves the synthesis and characterization of a large number of new molecular entities (both large and small molecules). The determination of potency through biological assays, and the drug-like properties through drug metabolism and pharmacokinetics (DMPK) are important to deliver a candidate to development. One important parameter determined in discovery is the extent of binding to plasma proteins.


Workflow Overview for Discovery PPB

Plasma-protein binding (PPB) is an early ADME in vitro study that indicates the likelihood of a test compound to bind to proteins in blood plasma. Only the unbound compound is available to act as a drug, making the bound fraction essentially unusable. The basic question to be answered is what percentage of compound is bound to protein in the blood in an in vitro model? 

This is an in vitro study using serum from several species, including human, rat, mouse, or dog as the binding agent. The sample matrices of PPB are the dirtiest of the routine ADME assays and require solvent addition and centrifugation to remove the proteins prior to injection to the LC/MS system. Utilization of on-line sample preparation such as Turboflow™ technology allows for direct injection from the plasma sample matrix, thereby increasing sample throughput and reducing the cost of additional sample prep consumables. The key benefit of the PPB assay is a well supported estimation of the test compound’s affinity to bind protein in the blood without the influence of other in vivo factors present in a live animal such as metabolism and excretion.





A Novel High Throughput Method Using Full Scan HRAM and Online Extraction for Plasma Protein Binding...

Cook K, Dreyer M, et al.
Scientific Poster

Sample Preparation

Sample Preparation Workflow for Discovery PPB

The Rapid Equilibrium Device (RED)™ device holds 48 two-chamber inserts in a Teflon-coated, 96-well plate format. Plasma containing drug was added to one chamber, while buffer was added to the second chamber separated by a semi-permeable membrane (8K MWCO). The samples were incubated at ~37 °C while shaking at 100 rpm for 4 hours. Afterwards, an aliquot from each chamber (200 µL plasma, 300 µL buffer) was removed, and equal amounts of fresh plasma and buffer were added to the respective incubated aliquots.  The protein/buffer mixtures were precipitated using an acidified organic internal standard (ISTD) cocktail solution, thoroughly  mixed, and centrifuged.  The supernatant was then transferred to a 96-well plate for subsequent analysis.


Mass Spectrometry

Mass Spectrometry Workflow for Discovery PPB

A Thermo Scientific Exactive™ benchtop Orbitrap MS, operating in full-scan mode at 25K resolution (4Hz), was used for all data collection. The maximum inject time was 100 ms with an AGC target setting of 1e6.  Generic ion source conditions were as follows: spray voltage of 4kV; vaporizer and capillary temperatures of 550 & 325 °C, respectively; sheath gas pressure of 45 units, and auxiliary gas pressure of 25 units. Prior to sample acquisition, the instrument was calibrated in positive ionization mode using ProteoMass™ LTQ/FT- Hybrid ESI Positive Mode Cal Mix (Sigma Aldrich).  A lock mass was not required.

Data Analysis

Data Analysis Workflow for Discovery PPB

Thermo Scientific QuickCalc software can be utilized for peak detection, integration, and reporting of peak areas. LC/MS data for both the analyte and internal standard are easily reviewed, modified, stored, and reported in a single software suite. Additionally, custom reporting tools are available for the automatic generation of availability constants (log K) after chromatic data review.


Version history
Last update:
‎08-05-2021 08:07 PM
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