Plasma Protein Binding Assay

　　1. Establishment of compound methods

　　1.1 Establish the mass spectrometry method: first prepare a 1 μM concentration solution and a 500 nM concentration solution (including internal standard) for use; determine the molecular ion peak of the compound according to the molecular weight of the sample, and optimize the mass spectrometry parameters;

　　1.2 Establishment of liquid phase method: Use gradient elution method, select appropriate mobile phase, chromatographic column and elution gradient, and perform gradient elution of compounds.

　　2. Preparation for the experiment

　　2.1 Prepare PB solution: 0.5M potassium dihydrogen phosphate solution, MW=136.09g/mol: Add 13.609g of potassium dihydrogen phosphate powder to 200mL of pure water until it is clear, and store it in a 4°C refrigerator for later use; 0.5M Dipotassium hydrogen phosphate solution, MW=228.23 g/mol: Add 13.609g of dipotassium hydrogen phosphate powder to 200 mL of pure water until it is clear, and store it in a 4°C refrigerator for later use;

　　2.2 Mix 41 mL of 0.5 M potassium dihydrogen phosphate solution and 159 mL of 0.5 M potassium dihydrogen phosphate solution, and quantify to 1000 mL with pure water until clear. Store in a 4°C refrigerator for later use;

　　2.3 The test compound and the positive control compound need to be prepared into a 2mM solution and set aside for use;

　　2.4 Prepare 20% ethanol solution and set aside.

　　3. Experimental procedures

　　3.1 First turn on the air bath shaker, set it to 37℃, and the rotation speed is 150rpm/min; write the paper record of the 96-well plate;

　　3.2 Calculate the number of semipermeable membranes required based on the amount of the experiment, and soak the semipermeable membrane in pure water for 60 minutes; after the soaking time in pure water is up, pour out the waste liquid, separate the dialysis membrane, and soak it in 20% ethanol. 20min; after the 20% ethanol soaking time is up, pour out the waste liquid, and finally soak in PB solution for 20min;

　　3.3 Dilute the 2mM stock solution to 0.4mM solution with DMSO solution and set aside;

　　3.4 Use different blank matrices to dilute the 0.4mM solution to 2μM solution, mix well and set aside;

　　3.5 Install the soaked semipermeable membrane on the HTDialysis device; after the above work is completed, start adding samples. Each well of Dialysis has two sides. Add 150 μL of 2 μM solution to the upper side, and add 150 μL of Blank PB solution to the other side (three parallel samples are required for each sample);

　　3.6 Place the loaded HTDialysis in a preheated air bath and incubate it for 6 hours;

　　3.7 Acetonitrile precipitation

　　3.7.1 According to the written paper record of the 96-well plate, add samples to the corresponding well positions. C0 (T0) is to take out 50 μL and 50 μL Blank PB solution in the matrix of 2 μM solution, and then add 400 μL acetonitrile precipitation containing the internal standard (three parallel samples need to be done);

　　3.7.2 After reaching the incubation time, take out 50 μL of sample from the plasma end and add it to the 96-well plate, then add 50 μL of blank PB buffer solution as the binding end, take out 50 μL of the 96-well plate from the PB solution end after equilibrium dialysis, and then add it to the 96-well plate. 50 μL of blank matrix was mixed. Add 400 μL of acetonitrile protein precipitate containing inner edge to the 96-well plate;

　　3.8 Centrifuge for 20 minutes at 4°C and 4000 rpm/min. Finally, take 150 μL of supernatant and reconstitute it with 150 μL of 0.1% formic acid water;

　　3.9 Perform sample analysis in an LC MS/MS instrument.

　　4. Experimental results

　　4.1 Data analysis

　　4.1.1 Fu%= 100 * Cbuffer / Cplasma

　　4.1.2 Recovery rate = (Cbuffer + Cplasma)/C0 x100, where Cbuffer is the concentration in the test buffer after dialysis; Cplasma is the concentration in the plasma after measurement; C0 is the concentration of the plasma solution before dialysis.

　　4.1.3 Note: Recovery should be 100%. If the recovery deviates from 100%, it may indicate binding to the dialysis equipment, degradation in plasma or solubility issues.