B-261 Comparing Selectivity of Core-Shell and Modified Fully Porous HPLC Columns to Optimize Separation of 3 Classes of 47 TDM Analytes
Authors: Huq S , Marin S , Orquera-Aguirre G 
Published: October 2024

Abstract

Background
Therapeutic drug monitoring (TDM) research requires quantitation within narrow concentration ranges to distinguish between acceptable and toxic drug concentrations that could trigger potential adverse effects. In this communication a robust analytical LC-MS/MS method was developed for proper identification and measurement of a large TDM panel. This panel was comprised of three categories: 22 antidepressants, 14 antipsychotics and 11 anticonvulsants for a total of 47 analytes. A parallel comparison of two LC columns packed with modified fully porous and core-shell particles was conducted to determine the best columns and conditions for fast LC separation while achieving baseline resolution of critical isobaric compounds in each drug class.

Methods
In this study, a fully porous, thermally modified Luna Omega 3.0 μm Polar C18, 50x3 mm was compared against a core-shell Kinetex 2.6 μm Biphenyl 50x3.0 mm column. The Biphenyl column used 0.1 % formic acid in water and methanol as mobile phase A and B respectively for gradient elution. The Luna Omega column showed affinity for a buffered mobile phase. Therefore, 2 mM ammonium acetate in water and methanol were used as mobile phase A and B, respectively. Additionally, an Agilent 1290 Infinity series LC system was employed with a SCIEX 6500 QTRAP mass spectrometer and ESI for MS detection.

Results
The anticonvulsant panel represents analytes with a vast range of polarity and chemical properties featuring zwitterionic, weakly acidic, and neutral compounds. The preferential affinity of these compounds was highly noticeable when using the Luna Omega column and revealed the superiority of this fully porous column for baseline separation of the critical isobaric anticonvulsant analyte pair, Oxcarbazepine and Carbamazepine epoxide. In contrast, the unique selectivity of the core-shell particles renders longer retention and better separation of the isobaric compounds, Venlafaxine and amitriptyline, and Mirtazapine and N-desmethyldoxepin in the antidepressant panel. The Biphenyl column was also better for the separation of antipsychotics, where most of the analytes are basic and relatively more hydrophobic in nature. Furthermore, the fully porous column failed to differentiate between the isobaric antipsychotic analogs, Norclozapine and Olanzapine. The chromatographic run time for the above scenarios was 5 minutes, which includes a 1.5-minute column equilibration. Lastly, for the antidepressant panel, both columns showed successful separation of the two isobaric pairs, however, higher MS signal response and wider separation of the isobaric pairs was observed with the Biphenyl column.

Conclusions
The modified fully porous Luna Omega particle enhanced the selectivity and retention of polar and neutral analytes, making it suitable for analysis of the more challenging anticonvulsant panel. The high efficiency Kinetex Biphenyl column demonstrates better separation of antipsychotics and antidepressant panels with good resolution for the critical isobaric pairs.