Development and validation of dispersive liquid-liquid microextraction coupled with LC-MS/MS for plasma 5-hydroxyindoleacetic acid quantification with application as a biomarker in major depressive disorder

A sensitive dispersive liquid-liquid microextraction (DLLME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for plasma 5-hydroxyindoleacetic acid (5-HIAA) quantification as a depression biomarker. Box-Behnken design evaluated four DLLME factors (disperser volume, extraction volume, pH, centrifugation time) where second-order polynomial model identified optimal conditions with R² = 0.9444, achieving 95.1% extraction recovery. Chromatographic separation employed Poroshell 120 EC-C18 column (2.7 μm, 50 × 3.0 mm) with isocratic elution (20% acetonitrile with 0.1% formic acid) and 5-minute run time. Positive electrospray ionization with multiple reaction monitoring transitions (m/z 192.1 → 146.1 for 5-HIAA; 197.1 → 151.1 for 5-HIAA-d 5 ) enabled selective detection. Validation following ICH M10 guidelines with surrogate matrix calibration demonstrated excellent linearity (r² = 0.9997, 5-1000 ng/mL), adequate sensitivity (LLOQ 5 ng/mL), satisfactory accuracy (-10.3% to + 0.2%) and precision (6.7–14.6% CV), minimal matrix effects (96.8-102.5%), and successful parallelism assessment confirming surrogate-authentic matrix equivalence. Clinical application revealed significantly elevated plasma 5-HIAA in depression (25.73 vs. 17.35 ng/mL, 48% elevation, p < 0.001, Cohen’s d = 3.059), strong correlation with Hamilton Depression Rating Scale scores ( r = 0.791, r² = 0.625), and excellent diagnostic performance (AUC = 0.985, sensitivity 88.2%, specificity 100%). This validated method demonstrates clinical utility for objective depression assessment combining microextraction sample preparation, sensitive detection, and comprehensive biomarker evaluation.