Determination of Solubility by Hyphenated HPLC Methods

As described in the general introduction for solubility assays, there is a difference between kinetic and thermodynamic solubility. Thermodynamic solubility needs solid material of good quality whereas kinetic solubility relies on organic stock solutions. The sample preparation is therefore different (Kibbey). 

pg/ml up to 2000 pg/ml. Gradient time is set either to have higher throughput (fast gradient) or better chromatographic resolution. 

The quantification is done via a calibration curve where the compound is weighed as standard and dissolved in an appropriate organic solvent (in most cases acetonitrile/water). The throughput of this method is 20 compounds a day. 

If the standard stock solution is delivered from a centralized stock solution the throughput can be increased significantly. Solubility obtained from organic stock solutions leads to kinetic solubility data (see also chapter 1.2). 

An aliquot of 30 pL is transferred in 250 pi buffer. The solution is shaken for 24 hours at 25 °C. If precipitation occurs the sample is centrifuged and filtrated. The following procedure is the same as for the solubility from solids described above. The achieved throughput is mainly limited by the gradient time. Pan et al. investigated the effect of filtration on the quantification of solubility. They recommend poly(tetrafluoroethylene) (PTFE) as filter material of choice. In their compound set they found 98% recovery after filtration. 

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