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高效液相色谱-荧光法同时测定儿童神经母细胞瘤患者尿液中香草扁桃酸和高香草酸

常雅举1, 贾媛媛1, 何一帆2, 张晓清1, 刘曼3,*, 丁敏1,*   

  1. 1.重庆医科大学检验医学院,临床检验诊断学教育部重点实验室,重庆 400016;
    2.重庆医科大学附属第一医院内分泌科,重庆 400016;
    3.重庆医科大学附属儿童医院检验医学科,国家儿童健康与疾病临床医学研究中心,儿童发育疾病研究教育部重点实验室,儿童代谢与炎症性疾病重庆市重点实验室,重庆 400016

Simultaneous determination of vanillylmandelic acid and homovanillic acid in urine of children with neuroblastoma by high performance liquid chromatography-fluorescence detection

CHANG Yaju1, JIA Yuanyuan1, HE Yifan2, ZHANG Xiaoqing1, LIU Man3,*, DING Min1,*   

  1. 1. Key Laboratory of Clinical Laboratory Dignostics (Ministry of Education of China), School of Laboratory Medicine, Chongqing Medical University, Chongqing 40016, China;
    2. Endocrinology Department, the First Affiliated Hospital of Chongqing Medical University, Chongqing 40016, China;
    3. Department of Laboratory Medicine Children' s Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatric Metabolism and Inflammatory Diseases, Chongqing 40016, China

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摘要： 建立一种高效液相色谱-荧光检测（HPLC-FLD）方法同时检测儿童24 h尿液中的香草扁桃酸（vanillylmandelic acid,VMA）和高香草酸（homovanillic acid,HVA）。以液液萃取法对样本进行预处理，采用Shim-Pack VP-ODS柱（150 mm×4.6 mm,5 μm）分离，以70 mmol/L磷酸盐缓冲液（pH 5.54）和甲醇作为流动相进行梯度洗脱，柱温为30 ℃，流速为0.8 mL/min，荧光激发波长与发射波长分别为278 nm和320 nm,基质匹配标准曲线法定量。VMA在1.50~300.0 mg/L、HVA在3.00~600.0 mg/L线性范围内线性关系良好，相关系数（R2）均大于0.997。VMA和HVA的检出限（LOD）分别为0.08 mg/L和0.13 mg/L，定量限（LOQ）分别为0.31 mg/L和0.53 mg/L;在低、中、高3个水平下的加标回收率为96.60%~108.98%，日内精密度为1.51%~3.18%，日间精密度为3.29%~4.49%。用该方法测定21例初诊神经母细胞瘤（neuroblastoma,NB）患者和38例对照组患者24 h尿液中VMA和HVA含量，同时与临床采用微柱比色（MC）测定VMA的结果进行对比。采用受试者工作特征（ROC）曲线评估诊断NB的效能。MC检测VMA的曲线下面积（AUC）为0.94;HPLC检测VMA和HVA的AUC分别为0.86和0.96，而二者联合的AUC提升至0.98，具有良好的诊断效能。应用该方法还对NB术后化疗组（n=22）和NB结疗随访组（n=22）的患者24 h尿进行检测，有两例患者VMA和HVA含量均高于正常值，经电子计算机断层扫描（computed tomography,CT）影像学检查显示这两例患者肿瘤复发并发生转移，表明VMA和HVA含量对NB患者具有较好的预后评估能力。建立一种可靠的同时分析24 h尿液中VMA和HVA的方法，有助于改善儿科NB患者的临床管理。

关键词: 高效液相色谱, 荧光检测, 香草扁桃酸, 高香草酸, 神经母细胞瘤

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)

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