High-throughput quantification of tacrolimus in whole blood by acoustic ejection mass spectrometry: analytical validation and comparative assessment against conventional LC-MS/MS for therapeutic drug monitoring

Background Therapeutic drug monitoring (TDM) of tacrolimus requires accurate, high-throughput methods, but conventional liquid chromatography–tandem mass spectrometry (LC-MS/MS) is limited by lengthy run times and complex sample preparation. Methods We developed a chromatography-free assay for quantification of tacrolimus in whole blood by integrating cold-induced phase separation with acoustic ejection mass spectrometry (AEMS). This workflow only requires less than 10 μL of whole blood, eliminates centrifugation, and enables direct nanoliter-scale injection into the mass spectrometer. The AEMS assay performance was validated in accordance with CLSI C62-A and ICH M10 guidelines using 62 patient samples and matrix-matched calibrators, demonstrating its suitability for high-throughput therapeutic drug monitoring. Results The AEMS assay exhibited excellent linearity (0.5–50 μg/L; R 2  = 0.9987), with a lower limit of quantification of 0.5 μg/L, which was well below clinically relevant trough concentrations. Accuracy (93.3%–100.1%) and precision (intra-day RSD: 3.7%–11.3%; inter-day RSD: 2.3%–12.4%) met regulatory acceptance criteria. Results showed strong concordance with routine LC-MS/MS (Spearman ρ = 0.972; Passing–Bablok slope = 1.100 [95% CI: 1.026–1.177]). The total analysis cycle time was less than 3 s per sample, enabling a throughput exceeding 1000 samples per hour theoretically. Conclusion This study establishes the first chromatography-free mass spectrometry platform validated for clinical TDM of TAC. By integrating ultra-high throughput capability, minimal sample consumption, and robust analytical performance, the AEMS workflow offers a transformative analytical solution for high-demand clinical environments, including transplant centers and pediatric monitoring settings.