Surfactant-enhanced emulsification liquid-liquid microextraction combined with sweeping micellar electrokinetic chromatography-tandem mass spectrometry for therapeutic drug monitoring of alpelisib and fulvestrant in human plasma

A novel bioanalytical method combining surfactant-enhanced emulsification liquid-liquid microextraction (SE-LLME) with sweeping micellar electrokinetic chromatography-tandem mass spectrometry (MEKC-MS/MS) was developed and validated for therapeutic drug monitoring (TDM) of alpelisib (ALP) and fulvestrant (FUL) in human plasma. This method addresses the need for sensitive, selective quantification in patients with PIK3CA-mutated, HR+/HER2- breast Cancer. Sample preparation involved protein precipitation followed by SE-LLME using pentadecafluorooctanoic acid (PFOA) and chloroform, yielding recoveries greater than 88.4% for ALP and 78.7% for FUL. Optimised MEKC conditions were 50 mM ammonium pentadecafluorooctanoate at pH 9.75 with 25% methanol, 30 kV separation voltage, 30 °C capillary temperature, and 100 mbar additional pressure during the analysis. Sweeping preconcentration significantly enhanced sensitivity-109-fold for ALP and 11.2-fold for FUL. The method was validated per ICH guidelines, demonstrating excellent linearity ( $r\ge$ 0.9963) across the calibration ranges (200-2000 ng/mL for ALP, 10-100 ng/mL for FUL), accuracy (mean biases -10.3% to 7.5%), and precision (RSD < 12.6%). Despite notable matrix effects for ALP, consistency across six different plasma sources (RSD ≤ 12.7%) ensured reliability. Analytes were stable under benchtop, autosampler, freeze-thaw, and long-term conditions (bias ≤ 11.1%). No carry-over was detected, and dilution integrity was confirmed (bias ≤ 2.7%). Application to patient samples validated the method's clinical relevance, with measured concentrations aligning with the expected ones. This is the first capillary electrophoresis method for ALP in biological matrices and the only method for simultaneous TDM of ALP and FUL, offering a robust, cost-effective, and eco-friendly alternative to traditional chromatographic approaches.

Bioanalytical method; Breast cancer; Capillary electrophoresis; Mass spectrometry; Personalised treatment; Surfactant-enhanced emulsification liquid-liquid microextraction.