Which Detection Method Should I Use for my Solubility Project?

Solubility is a critical physicochemical property to measure in drug discovery. The data can be used to guide lead optimization, troubleshoot erratic bioassay results, and identify potential downstream liabilities such as insufficient solubility for bioassays or oral bioavailability. Analiza uses the miniaturized, gold standard shake-flask method to measure solubility, and offers detection using chemiluminescent nitrogen detection (CLND), HPLC-UV, or HPLC-MS in order to accommodate clients’ varying budgets and uses for solubility data.

Chemiluminescent nitrogen detection (CLND)

Chemiluminescent nitrogen detection (CLND) assesses solubility by measuring the nitrogen content in the sample of interest. The concentration of the analyte is determined from the CLND signal and the number of nitrogen atoms in the analyte using a generic nitrogen calibration curve. The limit of detection (LOD) will vary between compounds because it is based on the compound’s molecular weight, the number of nitrogens in the compound, and the concentration of the compound in the filtrate. Because the measurement is based on an equimolar nitrogen response, the following considerations must be made:

1. The compound(s) being analyzed must contain nitrogen. 
2. The compound(s) being analyzed must be free of impurities.
3. The assay buffer cannot contain nitrogen.

Unlike HPLC-UV and HPLC-MS, CLND detection does not require compound-specific calibration. This reduces compound consumption and increases throughput, making CLND detection the least expensive detection option and a very useful screening tool for rank-ordering chemical compounds within a chemical series and identifying high-liability compounds.

HPLC-UV detection

HPLC-UV detection is appropriate when seeking more robust solubility data. It is also recommended as the detection method to use if your compounds do not contain nitrogen. Analiza’s standard method for HPLC-UV detection collects data at 215, 254, and 280nm (other wavelengths available). To successfully analyze a compound using HPLC-UV detection, the compound must have UV absorbance. Because a compound specific calibration curve is created for each compound, using HPLC-UV detection requires clients to submit a larger volume of compound and costs more compared to CLND analysis.

HPLC-MS detection

HPLC-MS detection is recommended to clients seeking data with high sensitivity or absolute integrity. With sub 2ppm mass accuracy, our QTOF-MS provides absolute identification of the test article. Like HPLC-UV detection, a compound specific calibration curve is created for each compound, thus requiring a larger volume of compound. Unlike HPLC-UV detection, the compound being analyzed does not have to contain a chromophore. The compound must, however, have adequate ionization in positive (+) mode electrospray, and the compound’s molecular formula must be provided to calculate exact mass.

Another factor that must be taken into consideration when using HPLC-MS to measure solubility is the instrument’s limit of detection (LOD). Because HPLC-MS generates data with higher sensitivity compared to CLND and HPLC-UV, highly soluble compounds may require additional dilution.