Tran Mai Thi Chi, Baglin James, Tran Trang Thi Thu, Hoang Kien Trung, Phung Lan Tuyet, Read Ann, Greaves Ronda F
Clinical Biochemistry Laboratory, National Hospital of Pediatrics, Ha Noi, Viet Nam; Hanoi Medical University, Ha Noi, Viet Nam.
School of Mathematical and Geospatial Sciences, RMIT University, Victoria, Australia.
Clin Biochem. 2014 Feb;47(3):206-15. doi: 10.1016/j.clinbiochem.2013.11.016. Epub 2013 Dec 1.
The aim of this study was to develop an accurate robust testing method to simultaneously measure urine levels of HVA and VMA using gas chromatography mass spectrometry (GCMS) and to establish age-specific reference intervals of HVA and VMA in random urines for Vietnamese children.
The assay for urinary HVA and VMA was developed based on a classical urinary organic acid profiling method. Briefly, this incorporated 3-phenyl butyric acid as the internal standard and liquid-liquid extraction with ethyl acetate followed by derivatization with BSTFA. The Agilent 7890A GC and 5975C Mass Selective Detector in single ion monitoring mode was used for analysis. Reference intervals were developed from random urine samples collected from 634 disease free Vietnamese children and compared to 50 known neuroblastoma patient samples. Results were reported relative to creatinine concentration. Age related 95% reference intervals for urinary HVA and VMA were estimated from sample quantiles. The analytes (expressed as analyte/creatinine ratios) diagnostic values were determined by calculating the related sensitivity, specificity and likelihood ratios.
HVA and VMA were linear to at least 193 and 221μmol/L, respectively. The limit of quantitation for both analytes was 0.9μmol/L. Using the bi-level control (n=15), the within-batch coefficients of variations (CVs) were less than 3% for both analytes across the assay range. The between-batch CVs (n=20 over three months), were 3.6% at 11μmol/L and 2.1% at 88μmol/L for HVA, 6.6% at 18.2μmol/L and 2.6% at 90.6μmol/L for VMA. Vietnamese age related reference intervals were established for urinary HVA and VMA per creatinine. HVA for children <6months (n=91) was 5.3-37.0μmol/mmol; 6months to <1year (n=141) was 2.7-27.7μmol/mmol; 1 to 5years (n=139) was 3.4-17.9μmol/mmol; 6 to 10years (n=136) was 2.7-8.8μmol/mmol; and 11 to 15years (n=127) was 1.1-9.4μmol/mmol. VMA for children <6months was 1.8-12.2μmol/mmol; 6months to <1year was 1.5-9.3μmol/mmol; 1 to 5years was 1.9-7.8μmol/mmol; 6 to 10years was 1.6-5.1μmol/mmol; and 11 to 15years was <0.9-6.3μmol/mmol.
A robust testing method for simultaneous quantitation of urinary HVA and VMA by GCMS was developed. This method is accurate, precise and fit for its clinical purpose and suitable for developing countries. Age-related reference intervals of urinary HVA and VMA were established for Vietnamese children and the intervals declined progressively with increasing age for each analyte.
本研究的目的是开发一种准确可靠的检测方法，使用气相色谱 - 质谱联用仪（GCMS）同时测量尿中高香草酸（HVA）和香草扁桃酸（VMA）的水平，并建立越南儿童随机尿中HVA和VMA的年龄特异性参考区间。
基于经典的尿有机酸分析方法开发了尿HVA和VMA的检测方法。简而言之，该方法采用3 - 苯基丁酸作为内标，用乙酸乙酯进行液 - 液萃取，然后用BSTFA进行衍生化。使用安捷伦7890A气相色谱仪和5975C质量选择性检测器在单离子监测模式下进行分析。参考区间由从634名无疾病的越南儿童收集的随机尿样得出，并与50份已知神经母细胞瘤患者样本进行比较。结果以相对于肌酐浓度的形式报告。尿HVA和VMA的年龄相关95%参考区间通过样本分位数估计。分析物（以分析物/肌酐比值表示）的诊断价值通过计算相关的敏感性、特异性和似然比来确定。
HVA和VMA分别在至少193和221μmol/L范围内呈线性。两种分析物的定量限均为0.9μmol/L。使用双水平对照（n = 15），在整个检测范围内，两种分析物的批内变异系数（CVs）均小于3%。批间CVs（三个月内n = 20），HVA在11μmol/L时为3.6%，在88μmol/L时为2.1%；VMA在18.2μmol/L时为6.6%，在90.6μmol/L时为2.6%。建立了越南儿童尿HVA和VMA相对于肌酐的年龄相关参考区间。6个月以下儿童（n = 91）的HVA为5.3 - 37.0μmol/mmol；6个月至1岁（n = 141）为2.7 - 27.7μmol/mmol；1至5岁（n = 139）为3.4 - 17.9μmol/mmol；6至10岁（n = 136）为2.7 - 8.8μmol/mmol；11至15岁（n = 127）为1.1 - 9.4μmol/mmol。6个月以下儿童的VMA为1.8 - 12.2μmol/mmol；6个月至1岁为1.5 - 9.3μmol/mmol；1至5岁为1.9 - 7.8μmol/mmol；6至10岁为1.6 - 5.1μmol/mmol；11至15岁为<0.9 - 6.3μmol/mmol。
开发了一种通过GCMS同时定量尿中HVA和VMA的可靠检测方法。该方法准确、精密，适用于临床目的，且适合发展中国家。建立了越南儿童尿HVA和VMA的年龄相关参考区间，每种分析物的参考区间随年龄增长而逐渐下降。
