Dec 09, 2024
Shake-Flask Aqueous Solubility assay (Kinetic solubility)
- Dmytro Lesyk1,
- Yurii Kheilik1
- 1Enamine Ltd
- ASAP Discovery
Protocol Citation: Dmytro Lesyk, Yurii Kheilik 2024. Shake-Flask Aqueous Solubility assay (Kinetic solubility). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8y41l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 14, 2024
Last Modified: December 09, 2024
Protocol Integer ID: 106073
Keywords: Kinetic solubility method, Shake-flask method, UV-Vis, LC-MS/MS detection, Drug discovery process, solubility, ADMET
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Abstract
Determining compound solubility is an essential tool for the early stages of the drug discovery process, as well as for lead optimization. Low solubility can lead to unpredictable and unreliable results during in vitro testing, thereby increasing the development costs. Solubility issues at the later stages of the drug discovery may lead to poor bioavailability, underestimated toxicity, and other obstacles, lowering the chances of a given drug candidate for success.
Typically, for early-stage drug discovery, the kinetic solubility method is used, as it is fast and well-suited for the HTS format. In this case, solid compounds are first dissolved in DMSO, and then linear serial dilutions of each compound are added to an aqueous buffer and observed for precipitate formation when the compound is not completely soluble. For better precision, the solution can be subjected to high-speed centrifugation or filtration using special solubility filter plates and then the compound concentration is measured in the saturated solution directly by UV or LC-MS/MS using separately built calibration curves. Measurements were performed by the shake-flask method with UV-Vis or LC-MS/MS detection.
Materials
Equipment
- Water purification system Millipore Milli-Q Gradient A10 (Sartorius Arium™ Mini)
- Thermomixer R Block, 1.5 mL (Eppendorf, Germany)
- Matrix Multichannel Electronic Pipette 2-125 µL, 5-250 µL, 15-1250 µL (Thermo Scientific, USA)
- SpectraMax Paradigm™ Reader (Multi-Mode Detection Platform)
- MS/MS detector API 3000 PE with TurboIonSpray Electrospray module (PE Sciex, USA)
- Multi-Well Plate Vacuum Manifold (Pall Corporation, USA)
- Vacuum pump (Millipore, USA)
Material
Phosphate buffered saline, pH 7.4 (Sigma-Aldrich, USA)Phosphate buffered saline powder, pH 7.4, for preparing 1 L solutions Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3813
Acetonitrile Chromasolv, gradient grade, for HPLC, ≥99.9% (Sigma-Aldrich, USA)AcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34851
DMSO (Sigma-Aldrich, USA)
Costar 96 Well Assay Blocks (Corning, USA)Costar 96-Well Microplate, Deep Well, Sterile, V-Bottom, 2 mL; 25/CsCorningCatalog #3960
MultiScreen HTS 96 Well Filter Plates (Millipore)
UV-Star® 96 Well Microplate (Greiner Bio-One)
Multichannel pipettors 1-30 µL, 2-125 µL, 30-850 µL (Thermo Scientific)
Flex-Tubes Microcentrifuge Tubes, 1.5mL (Eppendorf, Germany)Eppendorf® Flex-Tubes Microcentrifuge TubesMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP022364120
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Preparation of auxiliary solutions
Preparation of auxiliary solutions
2w
2w
1 L PBS: 0.01 Molarity (M) Phosphate buffer
In a bottle for reagents with a cap of 1 L capacity, place:
• contents of 1 L sachet with phosphate buffer,
• 1 L of water and mix thoroughly and filter through a membrane filter with a pore diameter of 0.45 µm .
Note
The shelf life of the solution is 2 weeks when stored at 5 °C .
100 mL A solution (AcN:PBS, 50:50, v/v):
In a bottle for reagents with a cap of 100 mL capacity, place:
• 50 mL of AcN,
• 50 mL of PBS and mix thoroughly.
Note
The shelf life of the solution is 1 month when stored at Room temperature .
100 mL B solution (AcN:PBS:DMSO, 49:49:2, v/v/v):
In a bottle for reagents with a cap of 100 mL capacity, place:
• 49 mL of AcN,
• 49 mL of PBS,
• 2 mL of DMSO and mix thoroughly.
Note
The shelf life of the solution is 1 month when stored at Room temperature .
100 mL C solution (AcN:DMSO, 98:2, v/v):
In a bottle for reagents with a cap of 100 mL capacity, place:
• 98 mL of AcN,
• 2 mL of DMSO and mix thoroughly.
Note
The shelf life of the solution is 3 months when stored at Room temperature .
Preparation of 20 mM stock compound
Prepare 20 millimolar (mM) stock solutions of test substances in DMSO. As a rule, 50 µL of solution is enough.
Sample preparation
Sample preparation
2h
2h
Incubation of 400 micromolar (µM) solution
In Matrix Storage tubes (1.4 mL), add 490 µL of buffer, followed by 10 µL of stock solution of the test and control substances. Prepare two incubation mixtures for each substance to ensure reproducibility. Place the tubes in a thermomixer. Set the thermomixer to 850 rpm and incubate for 02:00:00 .
2h
Preparation of Calibration Solutions
Using Matrix Storage tubes (1.4 mL) with 20 µL of stock solutions, prepare calibration solutions according to Table 1. Mix the solutions by pipetting 5 times. To prevent evaporation, cover the tubes with strip caps.
| A | B | C | D | E | F | G | |
| Calibration standard/solution (µL) | Concentration (µM) | ||||||
| 400* | 200 | 100 | 50 | 25 | 10 | ||
| A solution | 490 | ||||||
| B solution | 250 | 250 | 250 | 250 | 150 | ||
| 20 mМ DMSO stock | 10 | ||||||
| 400 µM | 250 | ||||||
| 200 µM | 250 | ||||||
| 100 µM | 250 | ||||||
| 50 µM | 250 | ||||||
| 25 µM | 100 | ||||||
| 10 µM | |||||||
Table 1
*Note: The 400µM standard is not used in the measurements.
For the incubation mixture, add 250 µL of Solution C (well A11-12) into the tube. See Figure 1 for the arrangement of tubes in the rack.
Figure 1
Filtration of the Incubation Mixture
Place the Deep Well Plate in the manifold and close the manifold.
Place the filtration plate on top.
Transfer 290 µL of the incubation mixture into the filtration plate, cover with a plastic lid.
Turn on the vacuum pump and gradually adjust the manifold valve to 0.2 atm.
After filtration, close the manifold valve, turn off the vacuum pump, and transfer 250 µL of the filtrate into the tube with Solution C (well A8 in Figure 1).
Mix by pipetting 5 times.
Preparation for Measurement
Transfer 200 µL of the solutions from the tubes (in duplicates) to a UV-Star® plate, following the layout in Figure 2.
Figure 2
Insert the Plate into the Reader
Place the plate into the reader for measurement.
On the device, press the "Drawer" button, then insert the plate into the reader.
Close the reader by pressing the "Drawer" button again.
Start the measurement by clicking the "Read" button in the SoftMax Pro software.
After the measurement, remove the plates and dispose of them properly.
Evaluation of Results
The concentrations of compounds in PBS filtrate are calculated using a dedicated Microsoft Excel calculation script.
Proper absorbance wavelengths for calculations are selected for each compound manually based on absorbance maximums (absolute absorbance unit values for the minimum and maximum concentration points within the 0 – 3 OD range).
Each final dataset is visually evaluated by the operator, and goodness of fit (R2) is calculated for each calibration curve.
The effective range of this assay is approximately 2 μM -400 μM and the compounds returning values close to the upper limit of the range may have higher actual solubility (e.g.5'-deoxy-5-fluorouridine).
Protocol references
1. Thomas O., G. Kazan Quantitative method to determine drug aqueous solubility: optimization and correlation to standard methods / Millipore Corporation Technical Note AN1730EN00
2. Thomas Onofrey, Ph.D. and Greg Kazan MultiScreen® Solubility Filter Plate Performance and correlation of a 96-well high throughput screening method to determine aqueous drug solubility/Millipore Corporation Technical Note AN1731EN00
3. MultiScreen® Solubility Filter Plate Determination of aqueous compound solubility using a 96-well filter plate to remove precipitated solids prior to UV/Vis spectroscopic analysis/Millipore Corporation Technical Note PC2445EN00
4. The MultiScreen Solubility Quantitative Method Protocol: With Data Acquisition and Analysis using Molecular Devices SpectraMax®Plus with SoftMax® Pro Software/Millipore Corporation Technical Note TN1176EN00