Table 4.
Protocol for the Human Plasma Protein Binding Assay
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Thaw human plasma | 3 min, 750 rpm | Thawing and centrifugation at room temperature |
| 2 | Pooling compounds | 3 μL | Nine test compounds + one QC (warfarin) |
| 3 | Dilute pools in plasma | 5.6 μL | Add pooled compounds to 792 μL plasma (to 7 μM) |
| 4 | Sample pool aliquots and dilute to start concentration | 400 μL | 400 μL diluted pools +160 μL of predispensed plasma |
| 5 | Sampling (t = 0 h) | 50 μL | Take t = 0 h control samples from the 5 μM deep well plate for the stability and recovery studies |
| 6 | Add samples to RED devices | 300 μL | Place in RED device plasma chambers |
| 7 | Add phosphate buffer at pH 7.4 | 500 μL | Place in surrounding buffer chamber |
| 8 | Sealing | Mounting of breathable seal on RED Devices | |
| 9 | Equilibrate samples | 18 h, 500 rpm | Incubation with shaking at 37°C and 5% CO2 |
| 10 | Generate standard curves | Serial dilution of 7 μM samples | |
| 11 | Store in freezer | 18 h, −20°C | Crash plates |
| 12 | Adjust samples to RT | 15 min | RED devices, Stability & Recovery and crash plate |
| 13 | Centrifugation | 1 min, 1,000 × g | Stability & Recovery and crash plate at RT, Eppendorf 5810R |
| 14 | Buffer sampling | 50 μL | From RED devices buffer chambers to crash plates |
| 15 | Plasma sampling | 50 μL | From RED devices plasma chambers to crash plates |
| 16 | Sampling (t = 18 h) | 50 μL | From Stability & Recovery plate to crash plate |
| 17 | Lidding and vigorous shaking | 30 s | Silicone Cap Mats |
| 18 | Centrifugation | 20 min, 3,000 × g | 4°C, Sigma 6–16K (SIGMA Laborzentrifugen GmbH) |
| 19 | Dilution and transfer | 1:2 + 1:10 | Crash plate content is diluted and transferred to analysis plates (H2O + 0.2% FA) |
| 20 | Plate sealing | ||
| 21 | Assay readout | Peak area | LC/MS, TargetLynx |
| 22 | Data evaluation | fu, recovery, stability (%) | Genedata Screener |
Step Notes
1–4. Compound pooling is achieved in a PP-based microtiter plate (Product No.: 249944; Thermo Fisher Scientific). Pools are mixed and then diluted in plasma predispensed to deep well plates (Product No.: 260252; Thermo Fisher Scientific). A portion of the diluted pools are further diluted to 5 μM in the same deep well plate type (herein referred to as the Stability & Recovery plate), while the remaining content is used for generation of standard curves (see further down in this Table footnote).
5. A t = 0 h control sample for the Stability & Recovery studies is sampled to a crash plate already containing 400 μL of cold IS solution.
6–9. Plasma samples and buffer are added to the RED devices, sealed (Product No.: BEM-1, Diversified Biotech) and incubated on an orbital shaker (Eppendorf) for 18 h in a Galaxy R incubator (Richmond Scientific) alongside the Stability & Recovery plate.
10. Samples (at 7 μM) from Step 2 are serially diluted in plasma to generate a standard curve from 1.4 nM to 7 μM on the Hamilton STARplus. 50 μL of these samples are immediately transferred to the crash plate (see Step 5).
11. Crash plates are stored at −20°C overnight.
12. The Stability & Recovery plate is vortexed before the centrifugation step.
14–16. Sampling is carried out manually with an 8-channel pipette to crash plates already containing 400 μL of cold IS solution and either 50 μL buffer or plasma to ensure matrix matching between samples.
17. Crash plates are lidded using silicone cap mats (Product No.: 276002; Thermo Fisher Scientific) and vortexed.
19. Crash plate content is diluted in two steps, starting with transfer and mixing of 75–75 μL of acidified water in a PP-based microtiter plate (Product No.: 249944; Thermo Fisher Scientific) on the Hamilton STARplus liquid handler. This content is further diluted 20–180 μL of acidified AcN:H2O (1:3, 0.1% FA).
20. Plates are manually sealed with a 96-well cap natural seal (Product No.: 276002; Thermo Fisher Scientific).
21. The samples are analyzed by using a Waters iClass Acquity and Waters Xevo TQS. Chromatograms are evaluated using Waters TargetLynx software.
22. Data evaluation is achieved through Genedata Screener and approved results are reported to our internal database D360.
PP, polypropylene.