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OBJECTIVE	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 .
METHODS	The assay for urinary HVA and VMA was developed based on a classical urinary organic acid profiling method .
METHODS	Briefly , this incorporated 3-phenyl butyric acid as the internal standard and liquid-liquid extraction with ethyl acetate followed by derivatization with BSTFA .
METHODS	The Agilent 7890A GC and 5975C Mass Selective Detector in single ion monitoring mode was used for analysis .
METHODS	Reference intervals were developed from random urine samples collected from 634 disease free Vietnamese children and compared to 50 known neuroblastoma patient samples .
METHODS	Results were reported relative to creatinine concentration .
METHODS	Age related 95 % reference intervals for urinary HVA and VMA were estimated from sample quantiles .
METHODS	The analytes ( expressed as analyte/creatinine ratios ) diagnostic values were determined by calculating the related sensitivity , specificity and likelihood ratios .
RESULTS	HVA and VMA were linear to at least 193 and 221mol/L , respectively .
RESULTS	The limit of quantitation for both analytes was 0.9 mol/L .
RESULTS	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 .
RESULTS	The between-batch CVs ( n = 20 over three months ) , were 3.6 % at 11mol/L and 2.1 % at 88mol/L for HVA , 6.6 % at 18.2 mol/L and 2.6 % at 90.6 mol/L for VMA .
RESULTS	Vietnamese age related reference intervals were established for urinary HVA and VMA per creatinine .
RESULTS	HVA for children < 6months ( n = 91 ) was 5.3-37 .0 mol/mmol ; 6months to < 1year ( n = 141 ) was 2.7-27 .7 mol/mmol ; 1 to 5years ( n = 139 ) was 3.4-17 .9 mol/mmol ; 6 to 10years ( n = 136 ) was 2.7-8 .8 mol/mmol ; and 11 to 15years ( n = 127 ) was 1.1-9 .4 mol/mmol .
RESULTS	VMA for children < 6months was 1.8-12 .2 mol/mmol ; 6months to < 1year was 1.5-9 .3 mol/mmol ; 1 to 5years was 1.9-7 .8 mol/mmol ; 6 to 10years was 1.6-5 .1 mol/mmol ; and 11 to 15years was < 0.9-6 .3 mol/mmol .
CONCLUSIONS	A robust testing method for simultaneous quantitation of urinary HVA and VMA by GCMS was developed .
CONCLUSIONS	This method is accurate , precise and fit for its clinical purpose and suitable for developing countries .
CONCLUSIONS	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 .

