Purpose: Chiral separation is important but also challenging in bioanalytical matrix analysis. The investigation of antifungal drug luliconazole efficacy requires the measurement of its two geometric isomers (E/Z or trans/cis forms), as well as their chiral (R and S) forms. Typical reverse phase chromatographic conditions on non-chiral columns (like BDS Hypersil), baseline separation was only achieved between E and Z forms. With chiral columns under typical reverse phase conditions, RE, SE, RZ and SZ still cannot be baseline separated and achieve the required sensitivity in pg/mL.
Since typical reverse phase chromatography conditions used in electrospray or APCI- MS/MS could not achieve the required separation of the 4 enantiomers, we employed APPI (Atmospheric Pressure Photo Ionization) conditions instead.
Methods: In this work, APPI (Atmospheric Pressure Photo Ionization) ion source was used to replace the more common ion sources like ESI (Electrospray Ionization) or APCI (Atmospheric Pressure Chemical Ionization). APPI is well suited to the organic solvents (e.g., hexane), which eliminates the requirement of post column aqueous addition for ESI or APCI. In practice, it was very difficult to make the aqueous addition homogenous therefore, cause signal fluctuation and seriously deteriorated the quantitative measurement.
The organic mobile phase solvents are good dopants for APPI and lead to significantly enhanced sensitivity of LC-APPI-MS/MS.
Results: Under the optimized conditions, 4 chiral compounds were baseline separated and eluted at 5.4, 7.5, 8.3 and 11.4 min, respectively. The internal standard was also separated as two chiral forms, with RT at 9.0 and 11.1 min. It was demonstrated that better accuracy was obtained by applying a second IS for correcting the latest eluting chiral compound, while using the IS at 8.9 min for SZ, RZ, RE and 11.1 min for SE. LLOQ was 50 pg/mL for all four chiral compounds.
Conclusion: LC-APPI-MS/MS is a viable alternative to typical electrospray or chemical ionization techniques for bioanalysis of chiral analytes. The APPI source allowed for use of mobile phase which provided both the needed chromatographic separation, as well as enhanced sensitivity needed, in order to quantitate all 4 chiral analytes with a 15 minute run time.
Yafei Xu– Frontage Laboratories, Inc.
Harry Zhao– Frontage Laboratories, Inc.
Zhongping(John) Lin– Frontage Laboratories, Inc
Wei Zhang– Bioanalytical Scientist, Frontage Laboratories, Inc.