Abstract: Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are clinically significant biomarkers used in diagnosing and monitoring pediatric neuroblastoma (NB). Accurate quantification of their concentrations in 24-h urine samples provides critical information for clinical decision-making. This study aimed to develop a robust and reliable analytical method for the simultaneous determination of VMA and HVA. A high performance liquid chromatography with fluorescence detection (HPLC-FLD) method was established and validated. Sample preparation involved liquid-liquid extraction (LLE) using ethyl acetate as the extraction solvent which effectively isolated VMA and HVA from urinary matrices. Chromatographic separation was performed on a Shim-Pack VP-ODS column (150 mm×4.6 mm, 5 μm) under gradient elution conditions. The mobile phase consisted of 70 mmol/L phosphate-buffered saline (pH 5.54) and methanol. The column temperature was maintained at 30 °C throughout the analysis and the flow rate was set at 0.8 mL/min. Fluorescence detection was carried out at an excitation wavelength of 278 nm and an emission wavelength of 320 nm. The injection volume for each sample was 20 μL and the total run time was 11 min. Calibration curves were constructed for both analytes in two segments to enhance accuracy across the measurement range. VMA exhibited linearity over 1.50-300.0 mg/L, while HVA showed linearity from 3.00 to 600.0 mg/L, with correlation coefficients (R²) ≥0.997 for both. The limit of detection (LOD) was 0.08 mg/L for VMA and 0.13 mg/L for HVA; the corresponding limits of quantification (LOQs) were 0.53 mg/L and 0.31 mg/L, respectively. Accuracy was evaluated through recovery experiments using spiked urine samples at three levels. Recoveries ranged from 96.60% to 106.78% for VMA and from 99.32% to 108.98% for HVA, indicating high analytical accuracy. Precision was assessed by relative standard deviation (RSD). Intra-day RSDs were 1.89%-3.18% for VMA and 1.51%-2.22% for HVA, while inter-day RSDs were 3.92%-4.49% and 3.29%-3.85%, respectively, demonstrating good repeatability and intermediate precision. The validated method was applied to measure 24-h urinary VMA and HVA levels of 21 patients with newly diagnosed NB and 38 patients in the control group. Concurrently, VMA measurements obtained using the clinical microcolumn colorimetric (MC) method were collected for comparison. Median urinary VMA levels in NB patients were 129.07 mg/L by HPLC, compared to 42.04 mg/L by MC. Statistical analysis revealed a strong correlation (R=0.85, P<0.001) between the two methods for VMA measurements, although HPLC generally yielded higher concentrations, particularly at elevated levels. Diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.941 for MC-measured VMA and 0.863 for HPLC-measured VMA. HPLC-measured HVA achieved an AUC of 0.964, while the combined HPLC assay for VMA and HVA yielded the highest AUC of 0.979, indicating superior diagnostic accuracy. False-positive rates were 15.79% for MC (VMA only) versus 2.63% for the combined HPLC assay; false-negative rates were reduced from 9.52% to 4.76%. The utility of the method for disease monitoring was further evaluated in two additional cohorts: 22 patients undergoing chemotherapy and 22 subjects in post-treatment follow-up. Elevated levels of 24-h urinary VMA and HVA were observed in two individuals, exceeding normal reference ranges. Subsequent computed tomography (CT) imaging confirmed tumor recurrence and metastasis in these cases, underscoring the prognostic value of the method. A reliable HPLC-FLD method has been developed for the simultaneous quantification of 24-h urinary VMA and HVA. The combined determination of VMA and HVA is recommended to enable a more comprehensive biochemical assessment, thereby facilitating improved clinical management of pediatric NB patients. This study presents a valuable analytical tool for pediatric oncology practice, contributing to earlier and more accurate diagnosis and monitoring of NB.

Key words: high performance liquid chromatography, fluorescence detection (HPLC-FLD), vanillylmandelic acid (VMA), homovanillic acid (HVA), neuroblastoma (NB)