Simultaneous Quantification of Haloperidol and Other Antipsychotics in Human Serum by LC-MS/MS: Method Development and Clinical Application

Objective: To develop a high-throughput method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of serum concentrations of five commonly used antipsychotics (haloperidol, amisulpride, olanzapine, clozapine, and perphenazine) in order to address the issues of incomplete coverage and low efficiency in clinical monitoring during polypharmacy, thereby providing experimental evidence for personalized and precise medication in psychiatry. Methods: Haloperidol, amisulpride, olanzapine, clozapine, perphenazine and their corresponding deuterated internal standards (IS) were selected as analytes. Sample pretreatment procedures, chromatographic separation conditions, and mass spectrometric detection parameters were systematically optimized. Protein precipitation using acetonitrile as the precipitant was employed for serum sample preparation. Chromatographic separation was performed on an Agilent ZORBAX Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) using a gradient elution program with mobile phases consisting of ultrapure water containing 0.1% formic acid and acetonitrile. The mass spectrometer operated in electrospray positive ionization mode with multiple reaction monitoring (MRM) for quantification. Comprehensive method validation, including specificity, linearity, lower limit of quantification (LLOQ), precision, accuracy, matrix effect, extraction recovery, and stability, was conducted according to the guidelines of “9012 Guidance for Validation of Quantitative Analytical Methods for Biological Samples” from the Chinese Pharmacopoeia (2020 Edition, Volume IV). The established method was applied to therapeutic drug monitoring (TDM) in 150 patients with psychiatric disorders admitted to Baise Second People’s Hospital from January 2025 to December 2025. Results: An LC-MS/MS method capable of simultaneous analysis within 8 minutes was successfully established. All analytes showed good linearity in the range of 10 - 250 ng/mL with correlation coefficients (r²) greater than 0.998. The LLOQ was 10 ng/mL, with signal-to-noise ratios (S/N) exceeding 15. For all concentration levels, intra-day and inter-day precision were ≤8.1% and ≤9.7%, respectively; accuracy ranged from 94.2% to 106.5%. The matrix effect values were between 85.6% and 113.2%, and extraction recoveries ranged from 86.3% to 102.4%. Samples demonstrated good stability under various storage conditions. Clinical application to 150 samples revealed that in 97 samples (64.7%), the