A pH-mediated stacking CE-MS method for the sensitive determination of clozapine and amisulpride drugs in high ionic strength biomatrices.

Online preconcentration in capillary electrophoresis (CE) is severely limited by the high ionic strength of biomatrices, which eliminates the conductivity gradient required for field-amplified stacking. pH-Mediated stacking overcomes this problem by injecting a strong acid plug to neutralize matrix anions, thereby restoring field amplification for efficient on-column enrichment. In this work, a pH-mediated stacking CE method was developed for the simultaneous determination of the atypical antipsychotics clozapine and amisulpride in plasma, achieving sensitivity enhancement factors of approximately 114-fold for clozapine and 220-fold for amisulpride over conventional capillary zone electrophoresis. Upon coupling with MS detection, the method was fully validated. Both analytes exhibited good linearity over 5 to 400 ng/mL (R² ≥ 0.9926), with LODs of 1 ng/mL and LOQs of 5 ng/mL. Intra- and inter-day precision (RSD) were below 10% for peak areas and below 1.34% for migration times, and accuracy (RE) was within ±10%. Analysis of patient plasma samples yielded relative differences of 0.9% (clozapine) and 2.8% (amisulpride) compared with UHPLC-MS/MS, confirming good agreement between the two methods. These results indicate that pH-mediated stacking CE-MS provides a practical alternative to UHPLC-MS/MS for therapeutic drug monitoring, with comparable sensitivity, minimal sample consumption, and requiring only liquid-liquid extraction without additional offline enrichment.