Analytical method for urinary homovanillic acid and 5-hydroxyindoleacetic acid levels using HPLC with electrochemical detection applied to evaluate children environmentally exposed to manganese.

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) are the urinary metabolites of dopamine (DA) and serotonin (5-HA), respectively. We aimed to develop an extraction method for the determination of HVA and 5-HIAA, using strong anionic exchange cartridges combined with HPLC with electrochemical detection, and apply it to measure the levels of HVA and 5-HIAA in children living near a ferro-manganese alloy plant in Simões Filho, Brazil. The validated method showed good selectivity, sensitivity, precision, and accuracy. The limits of detection (LOD) were 4 and 8 µmol/L for 5-HIAA and HVA, respectively, in urine. Recoveries ranged from 85.8 to 94%. The coefficients of determination (R2 ) of the calibration curves were greater than 0.99. Spot urine samples of 30 exposed children and 20 nonexposed ones were processed accordingly. The metabolite levels in exposed and reference children were within the physiological ranges. The medians (range) for 5-HIAA and HVA of the exposed ones were 36.4 µmol/L (18.4-58.0) and 32.9 µmol/L (

Optimising urinary catecholamine metabolite diagnostics for neuroblastoma.

INTRODUCTION: The analysis of urinary catecholamine metabolites is a cornerstone of neuroblastoma diagnostics. Currently, there is no consensus regarding the sampling method, and variable combinations of catecholamine metabolites are being used. We investigated if spot urine samples can be reliably used for analysis of a panel of catecholamine metabolites for the diagnosis of neuroblastoma. METHODS: Twenty-four-hour urine or spot urine samples were collected from patients with and without neuroblastoma at diagnosis. Homovanillic acid (HVA), vanillylmandelic acid (VMA), dopamine, 3-methoxytyramine, norepinephrine, normetanephrine, epinephrine and metanephrine were measured by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) and/or ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-MS/MS). RESULTS: Catecholamine metabolite levels were measured in urine samples of 400 neuroblastoma patients (24-hour urine, n = 234; spot urine, n = 166) and 571 controls (all spot urine). Excretion levels of catecholamine metabolites and the diagnostic sensitivity for each metabolite were similar in 24-hour urine and spot urine samples (p > .08 and >.27 for all metabolites). The area under the receiver-operating-characteristic curve (AUC) of the panel containing all eight catecholamine metabolites was significantly higher compared to that of only HVA and VMA (AUC = 0.952 vs. 0.920, p = .02). No differences were observed in metabolite levels between the two analysis methods. CONCLUSION: Catecholamine metabolites in spot urine and 24-hour urine resulted in similar diagnostic sensitivities. The Catecholamine Working Group recommends the implementation of spot urine as standard of care. The panel of eight catecholamine metabolites has superior diagnostic accuracy over VMA and HVA.

A Simple, Fast, and Reliable LC-MS/MS Method for the Measurement of Homovanillic Acid and Vanillylmandelic Acid in Urine Specimens.

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are catecholamine metabolites used in the diagnostic workup of neuroendocrine tumors. Here we describe a simple dilute-and-shoot method for simultaneously quantitating HVA and VMA in human urine specimens. The method employs analyte separation on a reverse-phase liquid chromatography column followed by detection using electrospray ionization triple quadrupole mass spectrometry (ESI-MS/MS), wherein qualifier and quantifier ion transitions are monitored. This is a simple and fast analytical method with an injection-to-injection time of 4 min.

Quantitation of Neuroblastoma Markers Homovanillic Acid (HVA) and Vanillylmandelic Acid (VMA) in Urine by Gas Chromatography-Mass Spectrometry (GC/MS).

Neuroblastoma and other neural crest tumors can be characterized by the increased production and excretion of catecholamines and their metabolites. Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are important catecholamine metabolites that can be measured to provide relatively rapid laboratory diagnosis and clinical follow-up of neuroblastoma. We present a procedure to quantify HVA and VMA in urine samples which have been diluted to a creatinine concentration of 2 mg/dL. Diluted samples are spiked with deuterated internal standards, acidified, and extracted with an organic solvent. A bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and pyridine mixture is added to the dried extract to create trimethylsilyl derivatives of HVA and VMA. The derivatized compounds are measured using gas chromatography-mass spectrometry (GC/MS).

Adverse health effects of PCBs on fine motor performance - Analysis of a neurophysiological pathway in the HELPcB surveillance program.

Since research literature indicates neurotoxic health effects of polychlorinated biphenyls (PCBs), it is necessary to identify by which mechanism PCBs might affect the human central nervous system and human behavior. In the present study, a neurophysiological pathway is assumed to explain the negative association of PCB exposure and performance in fine motor tasks mediated by the level of the dopamine (DA) metabolite homovanillic acid (HVA). A total of 113 occupationally PCB exposed workers and their relatives from an occupational health monitoring program were examined (89.4 % men). PCBs were analyzed in plasma via human biomonitoring and HVA was assessed in urine. The motor performance series was used to measure two dimensions of fine motor skills with 5 subgroups (accuracy: steadiness, line tracking accuracy; speed: line tracking speed, aiming, tapping). The direct effects of PCBs on fine motor performance and the indirect effects of PCBs on fine motor performance via DA metabolite HVA were tested with multiple regressions. We found significant effects for the accuracy dimension, namely a negative direct effect of PCBs on line tracking accuracy mediated by HVA. Further, an indirect effect could be found for PCBs with steadiness accuracy through HVA. There were no significant effects related to fine motor performances in the speed dimension. These results provide first indications for an underlying neurochemical pathomechanism involving the dopamine system of PCB-related deterioration of fine motor performance regarding accuracy.

Towards using high-performance liquid chromatography at home.

In order to make high-performance liquid chromatography (HPLC) more widely available at home and in small-scale settings, we have simplified two of its most costly modules, namely the pump and the detector. This should make the setup affordable for home or small laboratory use. A manual HPLC pump was constructed so as to fit into a caulk gun from a local hardware store enabling the generation of 100-150 bar of pressure. In order to limit the pressure drop during the running of a chromatogram, a pulse dampener was developed. We further modified the electrochemical detection (ECD) system so as to use a cheap boron-doped diamond electrode with an overlay of thin filter paper, causing an eluent flow over the electrode by wicking and gravity. Both the pump and the detector are at least ten times cheaper than conventional HPLC modules. Using a home-packed JupiterⓇ Proteo reversed phase capillary column we show how this low-cost HPLC system generates well resolving chromatograms after direct injection of fresh urine. The ECD did not lose its sensitivity during regular use over more than half a year. For homovanillic acid (HVA), which is of medical interest, we measured a linear dynamic range of two orders of magnitude, a detection limit of HVA in the injected sample of 3 µM and a coefficient of variation <10%. The contribution to peak broadening by the detector was much smaller than the contributions by the injector and by the column. After consumption of table olives containing hydroxytyrosol (HT), its metabolite HVA in the corresponding urine could be measured quantitatively. An approach to quantify HT in table olives is presented, as well. This method provides a new tool for investigating physiology of oneself or of dear ones at home.

Mass screening for neuroblastoma in infants.

Neuroblastoma (NB) is the only pediatric tumor that is screened for nationwide by detecting the urinary levels of homovanillic acid and/or vanillylmandelic acid; however, whether NB screening reduces the mortality rate has not been established. This review compared the incidence and mortality rates among data from international mass screening for NB, as well as an analysis of differences in age of screening, detection methods, and diagnostic biomarkers. A well-designed trial exploring possible benefits and hazards is warranted prior to resuming mass screening for NB.

Semi-quantitative detection of a vanillactic acid/vanillylmandelic acid ratio in urine is a reliable diagnostic marker for aromatic L-amino acid decarboxylase deficiency.

BACKGROUND: Aromatic L-amino acid decarboxylase (AADC) deficiency is a primary neurotransmitter defect of the biosynthesis of catecholamines and serotonin. The phenotype consists of varying degrees of neurological impairment, including motor and non-motor symptoms. Treatment outcomes correlate with the time point of diagnosis and treatment initiation; therefore, reliable diagnostic markers are necessary. Increased vanillactic acid (VLA) concentrations in the analysis of organic acids in urine have been reported in AADC deficiency. However, this elevation is often subtle and easily missed. In this study, we evaluate the semi-quantitative determination of VLA and vanillylmandelic acid (VMA) concentrations and establish the ratio of a VLA/VMA as a novel diagnostic marker for AADC deficiency. METHODS: Urine samples obtained from 10,095 non-AADC deficient controls and 14 confirmed AADC deficient patients were used for organic acid analysis by liquid-liquid extraction of the acidified samples and gas chromatographic-mass spectrometric separation after trimethylsilylation. The semi-quantitative determination of VLA and VMA concentrations and the calculation of a VLA/VMA ratio were evaluated as a diagnostic marker for AADC deficiency. RESULTS: The mean VLA and VMA concentrations in 10,095 non-AADCD samples was 0.3 mmol/mol creatinine (SD = 1.18, range 0-57.79) and 5.59 mmol/mol creatinine (SD = 3.87, range 0.04-60.62), respectively. The mean concentration of VLA in 14 patient-derived samples was 10.24 mmol/mol creatinine, (SD = 11.58, range = 0.37-33.06) and 0.45 mmol/mol creatinine for VMA (SD = 0.29, range 0.11-1.27). The mean VLA/VMA ratio in non-AADC controls was 0.07 (SD = 0.37, range 0.0-23.24), whereas AADC deficient patients revealed a mean VLA/VMA ratio of 23.16 (SD = 22.83, range 0.97-74.1). The VLA/VMA ratio thus allows a reliable identification of patients with AADC deficiency, especially in the young age cohort as it decreases with age. To take this into account, age-adjusted thresholds have been developed. CONCLUSION: Determination of individual concentrations of VLA and VMA in urine does not allow a reliable diagnosis of AADC deficiency. In this study, we could demonstrate that a semi-quantitative analysis of organic acids in urine allows the formation of metabolite ratios and that the VLA/VMA ratio is a reliable, easily accessible, new parameter for the diagnosis of AADC deficiency.

Altered dopamine signalling in chronic epigastric pain syndrome.

Chronic epigastric pain syndrome (CEPS) is an important diagnostic problem, especially in patients without macroscopic and microscopic changes in gastric mucosa. The cause of this ailment is unclear. The aim of this study was the assessment of coexistence between symptoms of this syndrome and secretion level of dopamine (DA), as well as the efficacy of peripheral and central D2 receptors antagonist. Sixty depressive patients with CEPS occurring independently of the diet and with no Helicobacter pylori infection and 30 healthy subjects were enrolled in this study. Plasma DA and urinary homovanilic acid (HVA) concentration were measured by ELISA, and the mRNA expression of dopa decarboxylase (DDC) in gastric mucosa was evaluated by RT-PCR in 30 patients with CEPS and 30 controls. Severity of epigastric pain before and after 12 weeks 2 x 50 mg itopride or sulpiride treatment was evaluated using the modified 10-point Visual Analogue Scale. Higher average levels of plasma DA and urinary HVA levels in CEPS patients than controls 129.5 ± 22.0 versus 109.1 ± 18.4 pg/ml (p < 0.001) and 6.82 ± 1.55 versus 5.39 ± 1.04 mg/24 h, respectively were obtained. Moreover, the expression of DDC in gastric mucosa of CEPS patients was higher than in healthy subjects (p < 0.01). Sulpiride subsided epigastric pain in 73.3%, but itopride reduced it only in 6.6% of CEPS patients. We concluded that altered dopamine signalling may affect locally-and-centrally mediated chronic epigastric pain.

Target-based metabolomics for fast and sensitive quantification of eight small molecules in human urine using HPLC-DAD and chemometrics tools resolving of highly overlapping peaks.

Chemometrics multivariate calibration coupled with high performance liquid chromatography-diode array detection (HPLC-DAD) analytical strategy was applied for fast and sensitive quantification of the eight small molecules (uric acid, creatinine, tyrosine, homovanillic acid, hippuric acid, indole-3-acetic acid, tryptophan and 2-methylhippuric acid) in human urine. The objective of this work was to get the successful resolution of the complex matrix with minimum experimental time in the presence of highly overlapping peaks, of distortions in the time and baseline aspects among chromatograms, and of the presence of unknown and background interferences. All the analysis were based on a short C18 column with the chromatographic system operating in isocratic mode and all analytes can be successfully quantified within 6 min. The second-order HPLC-DAD data acquired were handled intelligently by two typical chemometrics tools including alternating trilinear decomposition (ATLD) and multivariate curve resolution-alternating least squares (MCR-ALS). Reasonable resolution and satisfactory quantification results were obtained regardless of the complex matrix interferences from the urine samples and the second-order advantage was fully exploited. With the validation by classic HPLC method, the proposed strategy could take extra advantages such as increased selectivity and sensitivity, shorter analysis time, undemanding elution conditions and sufficiency of lower limit of quantification benefit from multivariate calibration. The method was shown as a promising means for fast and sensitive determination of small molecules in human urine and also for fast diagnosis or surveillance in related diseases.

Specific Urinary Metabolites in Malignant Melanoma.

Background and objectives: Melanin, which has a confirmed role in melanoma cell behaviour, is formed in the process of melanogenesis and is synthesized from tryptophan, L-tyrosine and their metabolites. All these metabolites are easily detectable by chromatography in urine. Materials and Methods: Urine samples of 133 individuals (82 malignant melanoma patients and 51 healthy controls) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). The diagnosis of malignant melanoma was confirmed histologically. Results: Chromatograms of melanoma patients showed increased levels of 5,6-dihydroxyindole-2-carboxylic acid, vanilmandelic acid, homovanilic acid, tryptophan, 5-hydroxyindole-3-acetic acid, and indoxyl sulphate compared to healthy controls. Concentration of indoxyl sulphate, homovanilic acid and tryptophan were significantly increased even in the low clinical stage 0 of the disease (indoxyl sulphate, homovanilic acid and tryptophan in patients with clinical stage 0 vs. controls expressed as medium/ interquartile range in µmol/mmol creatinine: 28.37/15.30 vs. 5.00/6.91; 47.97/33.08 vs. 7.33/21.25; and 16.38/15.98 vs. 3.46/6.22, respectively). Conclusions: HPLC detection of metabolites of L-tyrosine and tryptophan in the urine of melanoma patients may play a significant role in diagnostics as well as a therapeutic strategy of melanoma cancer.

Analytical validation and clinical application of urinary vanillylmandelic acid and homovanillic acid by LC-MS/MS for diagnosis of neuroblastoma.

Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are clinical biomarkers for diagnosis of neuroblastoma (NB), which commonly occurs in the childhood. Development and application of a robust LC-MS/MS method for fast determination of these biomarkers for optimal laboratory testing of NB is essential in clinical laboratories. In present study, we developed and validated a simple liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quick clinical testing of VMA and HVA for diagnosis of NB. The method was validated according to the current CLSI C62-A and FDA guidelines. The age-adjusted pediatric reference intervals and diagnostic performance were evaluated in both 24 h urine and random urine. Injection-to-injection time was 3.5 min. Inter- and intra-assay coefficients of variation (CVs) were ≤3.88%. The lower limit of quantification and the limit of detection were 0.50 and 0.25 µmol/L for both VMA and HVA. Recoveries of VMA and HVA were in the ranges of 85-109% and 86-100% with CVs ≤5.76%. This method was free from significant matrix effect, carryover and interference. The establishment of age-adjusted pediatric reference intervals by this LC-MS/MS method was favorable for the improvement in diagnostic performance, which was crucial for correct interpretation of test results from children in both 24 h and random urine.

Olive ingestion causing a false suspicion of relapsed neuroblastoma: A case of "oliveblastoma?"

Measurement of the urine catecholamine metabolites homovanillic acid (HVA) and vanillylmandelic acid (VMA) are the standard method for detecting disease recurrence in neuroblastoma. We present a case of abnormal concentrations of catecholamine metabolites that prompted investigations for relapsed neuroblastoma. However, further study revealed that the abnormal biochemistry was likely due to ingestion of olives. Olive ingestion should be considered when interpreting urine HVA and VMA results, and excluded if concentrations are unexpectedly abnormal.

Quantification of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in urine using a dilute-and-shoot and ultra-high pressure liquid chromatography tandem mass spectrometry method.

BACKGROUND: Urinary vanillylmandelic acid (VMA) and homovanillic acid (HVA) are biomarkers for the diagnosis and follow-up of neuroblastoma, whereas urinary 5-hydroxyindoleacetic acid (5-HIAA) is used to assess a carcinoid tumor. These analytes are conventionally analyzed in a single run by chromatography (LC) coupled with electrochemical detection (LC-ECD) using commercial kits. A rapid dilute-and-shoot LC tandem mass spectrometry (LC-MS/MS) assay was validated in order to replace the LC-ECD method and therefore improve analytical specificity and throughput. METHODS: Sample preparation was carried out by dilution of the urine sample with a solution containing the deuterated internal standards. The separation was achieved on an ultra-high pressure LC system with MS detection using a triple quadrupole mass spectrometer. The method was validated according to the current EMA and FDA guidelines. RESULTS: The full chromatographic run was achieved in 8 min. The method validation showed excellent linearity (r2>0.999 for all three analytes), precision (CV <15%), negligible matrix effect (recoveries >90%), low carryover (<1%) and LLOQ of 0.25, 0.4 and 0.4 µM for VMA, HVA and 5-HIAA, respectively. Deming fits and Bland-Altman analyses showed no significant differences between the values obtained between the two assays. CONCLUSIONS: The LC-MS/MS method proposed in this study is fast and robust, and the simple sample preparation saves time and avoids the additional costs of dedicated kits used for the LC-ECD assays by switching to LC-MS/MS. Additionally, the near-perfect correlation observed herein between both assays allows the previously established reference ranges to be maintained.

Excellent prognosis of patients with intermediate-risk neuroblastoma and residual tumor postchemotherapy.

BACKGROUND/PURPOSE: The prognosis of patients with intermediate-risk neuroblastoma is favorable; therefore, a reduction therapy is desired. However, the long-term prognosis of those with residual tumor is unclear. The aim of this study was to clarify the necessity of residual tumor resection. METHODS: We retrospectively reviewed the records of patients diagnosed with intermediate-risk neuroblastoma who either were treated by chemotherapy only (nonresection group; n=16), or received postchemotherapy tumor resection (resection group; n=9). RESULTS: In the nonresection group, tumor size decreased in 14 patients; 5 had no detectable local tumor at the end of the follow-up period. Tumor size increased in 2 patients 1.5-2.5years postchemotherapy. Both patients received additional treatment and survived. All patients survived during the median follow-up time of 127months. In the resection group, 5 patients received complete resections and 4 patients received nearly complete resections. All patients survived during the median follow-up time of 84months. In 8 out of 9 resected tumors, regression or maturation was pathologically induced by chemotherapy-only treatment. CONCLUSION: Patients with intermediate-risk neuroblastoma with or without postchemotherapy residual tumor resection had an excellent long-term outcome. The tumor pathology with intermediate-risk neuroblastoma might be susceptible to change to regression or maturation by chemotherapy. LEVEL OF EVIDENCE: IV.

Polychlorinated biphenyls and depression: cross-sectional and longitudinal investigation of a dopamine-related Neurochemical path in the German HELPcB surveillance program.

BACKGROUND: Exposure to polychlorinated biphenyls (PCBs) is associated with depressive symptomatology. A cause of depressive symptoms is a disturbance in the neurotransmitter system of dopamine (DA). Animal as well as human studies report that PCBs can influence the DA system. This study examined whether PCB-related depressive symptoms are affected by DA metabolites in humans with high PCB body burden. METHODS: This study is part of the German HELPcB surveillance program (Health Effects in high Level exposure to PCB) for occupationally exposed workers and their relatives. Data was collected from 178 participants on two measurement time points (t1 and t2) with a one-year time lag in between the two time points. PCBs were analyzed in plasma via human biomonitoring and a validated questionnaire was used to identify existence and severity of depressive symptoms. As a surrogate for DA, we measured its metabolites homovanillic acid (HVA) and vanillylmandelic acid (VMA) in urine. Mediation analyses were performed to test whether the association between PCB exposure and severity of depressive symptoms is mediated by urinary concentration of DA metabolites HVA and VMA. The mediation was tested with the SPSS macro MEDIATE. RESULTS: We found a significant mediation over time for lower-chlorinated, higher-chlorinated and dioxin-like PCBs. The positive association between PCB exposure with severity of depressive symptoms was mediated by the main DA metabolite HVA. At t1 a higher exposure with PCBs was associated with lower concentration in urinary HVA. A reduced HVA concentration at t1 was correlated with increased depressive symptoms severity at t2. No meditations were found for VMA. CONCLUSIONS: This work indicates that the association of PCB exposure and an increase of depressive symptoms after one year is mediated by the DA metabolite HVA as a surrogate for DA. These are first steps towards finding an explanation for an underlying neurochemical pathomechanism of PCB-related depressive symptomatology.

Simple dilute-and-shoot method for urinary vanillylmandelic acid and homovanillic acid by liquid chromatography tandem mass spectrometry.

BACKGROUND: Neuroblastomas are pediatric tumors characterized by overproduction of catecholamines. The catecholamine metabolites, vanillylmandelic acid (VMA) and homovanillic acid (HVA), are used in clinical evaluation of neuroblastoma. Tandem mass spectrometry (LC-MS/MS) is an effective analytical method for measurement of VMA and HVA in urine. METHODS: Dilute-and-shoot sample preparation was performed in a 96-well format using a liquid handler. Chromatographic separation was achieved using a reverse phase column; detection was accomplished by triple quadrupole mass spectrometry with electrospray ionization in positive mode. Data were acquired by multiple reaction monitoring. Two transitions, quantifier and qualifier, were monitored for each analyte and its stable isotope-labeled internal standard. Analytical specificity studies were performed. RESULTS: Injection-to-injection time was 4min. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Linearity was 0.5-100mg/l for both analytes. Within-run, between-day, and total imprecision were 1.0-4.1% for VMA and 0.8-3.8% for HVA. The method correlated well with our established HPLC method. Interferences precluding quantitation of VMA in 3% of specimens were reduced significantly (to 0.1% of specimens) using a modified LC gradient to reanalyze affected samples. CONCLUSIONS: A simple, robust, economical, fast LC-MS/MS method was developed and validated for measurement of urinary VMA and HVA.

A novel l-leucine modified Sol-Gel-Carbon electrode for simultaneous electrochemical detection of homovanillic acid, dopamine and uric acid in neuroblastoma diagnosis.

Neuroblastoma is a pediatric neuroblastic tumor arising in the sympathetic nervous crest cells. A high grade of Neuroblastoma is characterized by a high urinary excretion of homovanillic acid and dopamine. In this work l-leucine modified Sol-Gel-Carbon electrode was used for a sensitive voltammetric determination of homovanillic acid and dopamine in urine. The electrochemical response characteristics were investigated by cyclic and differential pulse voltammetry; the modified electrode has shown an increase in the effective area of up to 40%, a well-separated oxidation peaks and an excellent electrocatalytic activity. High sensitivity and selectivity in the linear range of 0,4-100µML-1 of homovanillic acid and 10-120µML-1 of dopamine were also obtained. Moreover, a sub-micromolar limit of detection of 0.1µM for homovanillic acid and 1.0µM for the dopamine was achieved. Indeed, high reproducibility with simple preparation and regeneration of the electrode surface made this electrode very suitable for the determination of homovanillic acid and dopamine in pharmaceutical and clinical preparations. The mechanism of homovanillic acid and the electrochemical oxidation at l-leucine modified Sol-Gel-Carbon electrode is described out the B3P86/6-31+G(d,p) level of theory as implemented in Gaussian software.

Urinary metabolomics revealed arsenic exposure related to metabolic alterations in general Chinese pregnant women.

Arsenic exposure is considered a major environmental threat to human health. It is already known that high-level arsenic exposure has adverse effects on human health. Since the pregnant women are known to be more susceptible to some chemical exposures than ordinary people, the understanding regarding the health effects of low-level arsenic exposure on pregnant women is critical and remains unclear. The aim of this study is to investigate the urinary metabolic changes of pregnant women exposed to low-dose arsenic, and to identify biomarkers from metabolomics analysis. Urine samples of 246 pregnant women were collected in the first trimester of pregnancy and were divided into three groups based on the tertile distribution of urinary arsenic concentrations which were determined using inductively coupled plasma mass spectrometry (ICP-MS). Changes in the metabolite profile were measured using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF MS). Arsenic- related metabolic biomarkers were investigated by comparing the samples of the first and third tertiles of arsenic exposure classifications using a partial least-squares discriminant model (PLS-DA). Nine urine potential biomarkers were putatively identified, including LysoPC (14:0), glutathione, 18-carboxy-dinor-LTE4, 20-COOH-LTE4, cystathionine ketimin, 1-(beta-d-ribofuranosyl)-1,4-dihydronicotinamide, thiocysteine, p-cresol glucuronide and vanillactic acid. The obtained results showed that environmental arsenic exposure, even at low levels, could cause metabolite alterations in pregnant women which might be associated with adverse health outcomes. This is the first report on metabolic changes in pregnant women for arsenic exposure. The findings may be valuable for the arsenic risk assessment for pregnant women.