Purpose: High performance liquid chromatography (HPLC) coupled with mass spectrometry is one of the most common tools used to determine levels of drug candidates from both in vivo and in vitro experiments. However, HPLC has some limitations when trying to separate isomeric drug analytes. In recent years, supercritical fluid chromatography (SFC) has gained popularity. SFC uses carbon dioxide as the major component of the mobile phase. As a result of using supercritical conditions, the mobile phases have higher diffusivity, less viscosity, and can achieve better separations under shorter run times compared to liquid mobile phases. SFC in drug discovery screening could result in faster, green and more cost effective approach compared to HPLC. However, the robustness, as well as the accuracy of the instrument needs to be investigated. Here, triazolam and its two isomeric metabolites, 1-OH triazolam and 4-OH triazolam, were used to demonstrate how the method can be easily transferred from HPLC-MS to SFC-MS.
Methods: Methods will be modified to demonstrate the SFC’s capability to separate the isomeric metabolites under a shorter run time, and experimental data will be compared side-by-side to demonstrate the system’s reproducibility and accuracy.
Results: Using HPLC-MS, a 4 minute long run was needed to achieve a separation of 0.09 minute. Using SFC-MS, however, an improved separation of 0.26 minute was achieved in a 1.5 minute long run. Raw data from both HPLC-MS and SFC-MS was comparable. Sample preparation steps were also shorter.
Conclusion: In conclusion, based on the results for triazolam, 1-OH Triazolam and 4-OH triazolam, SFC-MS can be used to support in drug discovery screening. Due to the unique supercritical conditions, using SFC would lead to a faster, more green and more cost effective approach compared to HPLC.