Table 1.

Protocol for the Shake-Flask logD7.4 Assay

Step	Parameter	Value	Description
1	Add octanol	500 μL
2	Add compounds and controls	10 μL	10 test compounds (at 10 mM in DMSO) + two QCs (nicardipine and cyclobenzaprine at 5 mM each) are pooled
3	Add buffer	500 μL	Phosphate buffer pH 7.4
4	Vigorous shaking	1 min
5	Equilibration shaking	3 h	Room temperature
6	Centrifugation	30 min, 3,000 × g	Room temperature
7	Octanol sampling	5 μL
8	Removal of Octanol	495 μL
9	Buffer sampling	20 μL
10	Sample dilution series	1:10	Buffer samples are serially diluted (1:10) in acetonitrile/water (1:2, 0.1% FA, 2 nM verapamil—IS solution) in four steps and octanol samples in five steps
11	Plate sealing
12	Assay readout	Peak area	LC/MS, TargetLynx
13	Data evaluation	logD7.4	Genedata Screener

Step Notes

1–3. Octanol, compounds and buffer are added to a 2 mL PP-based deep well plate (Product No.: 278752; Thermo Fisher Scientific) using a Hamilton STARplus. A PP-based microtiter plate (Product No.: 249944; Thermo Fisher Scientific) is used as an intermediate plate for the compound pooling.

4–5. The 2 mL plate is lidded (Product No.: 276002; Thermo Fisher Scientific) and shaken vigorously followed by equilibration under shaking for 3 h.

6. The phases are separated by centrifugation of the 2 mL plate in an Eppendorf 5810R (Eppendorf).

7–10. Sampling and removal of octanol is carried out on the Hamilton STARplus.

11. Plates are manually sealed with a 96-well cap natural seal (Product No.: 276002; Thermo Fisher Scientific).

12. The samples are analyzed by using a Waters iClass Acquity and Waters Xevo TQS. Chromatograms are evaluated using Waters TargetLynx software.

13. Data evaluation is achieved through Genedata Screener and approved results are reported to our internal database D360.

AcN, acetonitrile; DMSO, dimethyl sulfoxide; FA, formic acid; IS, internal standard; LC/MS, liquid chromatography/mass spectrometry; PP, polypropylene; QC, quality control.