Establishment and clinical application of a candidate reference measurement procedure for quantification of urinary vanillylmandelic acid and homovanillic acid using ID-LC-MS/MS method.

Neuroblastoma (NB), an embryonic tumor of the autonomous nervous system, poses a significant threat to the health and lives of children. Accurate measurement of vanillylmandelic acid (VMA) and homovanillic acid (HVA) in human urine is crucial for screening and diagnosis of NB. Although various laboratories have developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect VMA and HVA, the comparability between the results obtained from different laboratories and methods was poor. The absence of reference method for VMA and HVA hinders the standardization of their measurements. Therefore, a candidate reference measurement procedure (cRMP) based on isotope dilution LC-MS/MS (ID-LC-MS/MS) for the detection of VMA and HVA in human urine was established. Urine samples were spiked with VMA-d3 and HVA-d5 as internal standards and extracted using a protein precipitation method. The cRMP exhibited desirable precision with the total imprecision below 5 %. The accuracy of this cRMP was demonstrated by the high analytical recovery (98.64 % - 102.22 % and 98.41 % - 100.97 % for VMA and HVA, respectively), and comparability between different reference systems. The limit of detection for HVA and VMA were 15.625 ng/mL and 3.906 ng/mL, respectively; the quantification limits were 62.5 ng/mL and 7.813 ng/mL, respectively, which can meet the clinical detection requirements. The linear range was from 78.125 ng/mL to 20 µg/mL. Specificity evaluations showed no corresponding interference from structurally similar analogs. In conclusion, we have established a cRMP based on ID-LC-MS/MS for the measurement of VMA and HVA in urine samples, demonstrating well-defined method performance including accuracy, precision, and specificity. This newly established cRMP is suitable for routine assay standardization and evaluation of clinical samples. Furthermore, this method has the potential to significantly enhance the diagnostic accuracy for neuroblastoma.

Analysis of the relationship between MYCN gene and serum NSE and urinary VMA levels and neuroblastoma pathological features and prognosis.

The purpose of this study was to explore the relationship between the MYCN gene, serum neuron-specific enolase (NSE), urinary vanillylmandelic acid (VMA) levels, and neuroblastoma pathological features and prognosis. Ninety-four children with neuroblastoma treated in the hospital were selected to compare the differences in MYCN gene amplification, serum NSE, and urinary VMA levels in children with different clinicopathological features and prognoses. The proportion of children with MYCN gene copy number ≥10 in INSS stage 3-4 was higher than that of children with INSS stage 1-2 (P < 0.05); the proportion of children with MYCN gene copy number ≥10 in high-risk children in the COG risk stratification was higher than that of children with intermediate and low risk (P < 0.05); the serum NSE of children aged >12 months higher than that of children aged ≤12 months (P < 0.05); serum NSE of children with tumors >500 cm3 higher than that of children with tumors ≤500 cm3 (P < 0.05); serum NSE and urinary VMA of children with INSS staging of stages 3-4 were higher than that of children with stages 1 to 2 (P < 0.05); serum NSE and urinary VMA in children with lymph node metastasis were higher than that of children without lymph node metastasis (P < 0.05); serum NSE of children with MYCN gene copy number ≥10 was higher than that of children without lymph node metastasis (P < 0.05); the proportion of children with MYCN gene copy number ≥10 who died, and the percentages of serum NSE and urinary VMA were higher than those of the surviving children (P < 0.05). MYCN gene amplification and serum NSE and urinary VMA levels were related to the age, tumor size, INSS stage, COG stage, lymph node metastasis, and prognosis of the children with neuroblastoma.

Scoring system for diagnosis and pretreatment risk assessment of neuroblastoma using urinary biomarker combinations.

The urinary catecholamine metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA), are used for the adjunctive diagnosis of neuroblastomas. We aimed to develop a scoring system for the diagnosis and pretreatment risk assessment of neuroblastoma, incorporating age and other urinary catecholamine metabolite combinations. Urine samples from 227 controls (227 samples) and 68 patients with neuroblastoma (228 samples) were evaluated. First, the catecholamine metabolites vanillactic acid (VLA) and 3-methoxytyramine sulfate (MTS) were identified as urinary marker candidates through comprehensive analysis using liquid chromatography-mass spectrometry. The concentrations of these marker candidates and conventional markers were then compared among controls, patients, and numerous risk groups to develop a scoring system. Participants were classified into four groups: control, low risk, intermediate risk, and high risk, and the proportional odds model was fitted using the L2-penalized maximum likelihood method, incorporating age on a monthly scale for adjustment. This scoring model using the novel urine catecholamine metabolite combinations, VLA and MTS, had greater area under the curve values than the model using HVA and VMA for diagnosis (0.978 vs. 0.964), pretreatment risk assessment (low and intermediate risk vs. high risk: 0.866 vs. 0.724; low risk vs. intermediate and high risk: 0.871 vs. 0.680), and prognostic factors (MYCN status: 0.741 vs. 0.369, histology: 0.932 vs. 0.747). The new system also had greater accuracy in detecting missing high-risk neuroblastomas, and in predicting the pretreatment risk at the time of screening. The new scoring system employing VLA and MTS has the potential to replace the conventional adjunctive diagnostic method using HVA and VMA.

Abnormalities in Clostridioides and related metabolites before ACTH treatment may be associated with its efficacy in patients with infantile epileptic spasm syndrome.

OBJECTIVE: Adrenocorticotropic hormone (ACTH) is the first-line treatment of infantile epileptic spasm syndrome (IESS). Its reported effectiveness varies, and our current understanding regarding the role of gut microbiota composition in IESS treatment response is limited. This study assessed the microbiome-metabolome association to understand the role and mechanism of gut microbiota composition in IESS treatment outcomes. METHODS: Children with IESS undergoing ACTH treatment were enrolled. Pre-treatment stool and serum samples were collected for 16S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry, respectively. The children were divided into "responsive" and "non-responsive" groups, and gut microbiota and serum metabolome differences were analyzed. RESULTS: Of the 30 patients with IESS, 14 responded to ACTH and 16 did not. The "non-responsive" group had larger maleficent Clostridioides and Peptoclostridium_phage_p630P populations (linear discriminant analysis >2; false discovery rate q < 0.05). Ten metabolites were upregulated (e.g., xanthurenic acid) and 15 were downregulated (e.g., vanillylmandelic acid) (p < 0.05). Association analysis of the gut microbiome and serum metabolome revealed that Clostridioides and Peptoclostridium_phage_p630P2 were positively correlated with linoleic and xanthurenic acids, while Clostridioides was negatively correlated with vanillylmandelic acid (p < 0.05). A classifier using differential gut bacteria and metabolites achieved an area under the receiver operating characteristic curve of 0.906 to distinguish responders from non-responders. CONCLUSION: This study found significant differences in pre-treatment gut microbiota and serum metabolome between children with IESS who responded to ACTH and those who did not. Additional exploration may provide valuable information for treatment selection and potential interventions. Our results suggest that varying ACTH responses in patients with IESS may be associated with increased gut Clostridioides bacteria and kynurenine pathway alteration, but additional experiments are needed to verify this association.

Smart dual imprinted Origami 3D-ePAD for selective and simultaneous analysis of vanillylmandelic acid and 5-hydroxyindole-3-acetic acid carcinoid cancer biomarkers using graphene quantum dots coated with dual molecularly imprinted polymers.

Measuring the levels of the biomarkers vanillylmandelic acid (VMA) and 5-Hydroxyindole-3-acetic acid (5-HIAA) is a valuable tool for clinical diagnosis not only of neuroblastoma or carcinoid syndrome, but also of essential hypertension, depression, migraine, and Tourette's syndrome. Herein, we explore using graphene quantum dots (GQDs) coated with molecularly imprinted polymer (MIP) as novel dual-imprinted sensors for selective and simultaneous determination of VMA and 5-HIAA in urine and plasma samples. The dual-MIP was successfully coated on the GQDs core via co-polymerization of (3-aminopropyl) triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS), acting as functional and cross-linking monomers, respectively. In addition, we successfully created the dual imprinted VMA and 5-HIAA shell on the GQDs' core via a one-pot synthesis. We fabricated a facile and ready-to-use Origami three-dimensional electrochemical paper-based analytical device (Origami 3D-ePAD) for simultaneous determination of VMA and 5-HIAA using a GQDs@dual-MIP modified graphene electrode (GQDs@dual-MIP/SPGE). The Origami 3D-ePAD was designed to form a voltammetric cell on a three-layer foldable sheet with several advantages. For example, they were quickly assembled and enhanced the device's physical durability with the hydrophobic backup sheet. The developed dual imprinted Origami 3D-ePAD leads to substantially enhanced sensitivity and selectivity to electrochemical signal amplification generated from increasing the electrode-specific surface area, electrocatalytic activity, and the large numbers of dual imprinted sites for VMA and 5-HIAA detection. The synthetic recognition sites are highly selective for 5-HIAA and VMA molecules with an imprinting factor of 8.46 and 7.10, respectively. Quantitative analysis relying on square wave voltammetry reveals excellent linear dynamic ranges of around 0.001-25 µM, with detection limits of 0.023 nM for 5-HIAA and 0.047 nM for VMA (3Sb, n = 3). The Origami 3D-ePAD provides high accuracy and precision (i.e., recovery values of 5-HIAA ranged from 82.98 to 98.40 %, and VMA ranged from 83.28 to 104.39 %), and RSD less than 4.37 %) in urine and plasma samples without any evidence of interference. Hence, it is well suited as a facile and ready-to-use disposable device for point-of-care testing. It is straightforward, cost-effective, reproducible, and stable. Furthermore, it allows for rapid analysis (analysis time ∼20s) useful in medical diagnosis and other relevant fields.

Clinical value of combined serum CA125, NSE and 24-hour urine VMA for the prediction of recurrence in children with neuroblastoma.

BACKGROUND: In this study, we intend to retrospectively analyze the clinical data of postoperative neuroblastoma children, including the results of follow-up examinations and laboratory tests, to explore the clinical value of combined serum Carbohydrate antigen 125 (CA125), neuron-specific enolase (NSE) and 24-hour urine vanillylmandelic acid (VMA) levels at baseline for the prediction of recurrence in children with neuroblastoma. METHODS: 265 children with neuroblastoma were successfully followed up, including 163 cases without recurrence (non-recurrence group) and 102 cases with recurrence (recurrence group). The levels of 24-hour urine VMA were determined using spectrophotometric methods. Additionally, the serum levels of CA125 and NSE were measured using electrochemiluminescence immunoassay. RESULTS: The serum CA125, NSE and 24-hour urine VMA levels were significantly higher in the recurrence group than in the non-recurrence group. It demonstrated a significant positive correlation between the levels of serum CA125, NSE, and 24-hour urine VMA in all children with neuroblastoma. All children in stage IV of neuroblastoma had the highest level of serum CA125, NSE and 24-hour urine VMA and vice versa. The combined CA125, NSE and VMA had significantly better sensitivity and specificity than an individual marker. CONCLUSIONS: Combined serum CA125, NSE and 24-hour urine VMA had the potential to predict neuroblastoma recurrence more effectively.

Profiling the metabolome of uterine fluid for early detection of ovarian cancer.

Ovarian cancer (OC) causes high mortality in women because of ineffective biomarkers for early diagnosis. Here, we perform metabolomics analysis on an initial training set of uterine fluid from 96 gynecological patients. A seven-metabolite-marker panel consisting of vanillylmandelic acid, norepinephrine, phenylalanine, beta-alanine, tyrosine, 12-S-hydroxy-5,8,10-heptadecatrienoic acid, and crithmumdiol is established for detecting early-stage OC. The panel is further validated in an independent sample set from 123 patients, discriminating early OC from controls with an area under the curve (AUC) of 0.957 (95% confidence interval [CI], 0.894-1). Interestingly, we find elevated norepinephrine and decreased vanillylmandelic acid in most OC cells, resulting from excess 4-hydroxyestradiol that antagonizes the catabolism of norepinephrine by catechol-O-methyltransferase. Moreover, exposure to 4-hydroxyestradiol induces cellular DNA damage and genomic instability that could lead to tumorigenesis. Thus, this study not only reveals metabolic features in uterine fluid of gynecological patients but also establishes a noninvasive approach for the early diagnosis of OC.

Voltammetric Detection of Vanillylmandelic Acid and Homovanillic Acid Using Urea-Derivative-Modified Graphite Electrode.

Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are diagnostic markers of neuroblastoma. The purpose of this study was to understand the reason for the discrimination of structural analogues (VMA and HVA) onto a graphite electrode coated with an electrochemically oxidized urea derivative. Density functional theory calculations (DFT), FTIR spectroscopic measurements, and electrochemical impedance spectroscopic measurements were used in this work. Density functional theory calculations (DFT) were used to identify the most suitable binding sites of the urea derivative and to describe possible differences in its interaction with the studied analytes. The FTIR measurement indicated the enhancement and disappearance of NH vibrations on graphite and platinum surfaces, respectively, that could be connected to a different orientation and thus provide accessibility of the urea moiety for the discrimination of carboxylates. Additionally, the higher the basicity of the anion, the stronger the hydrogen-bonding interaction with -NH-groups of the urea moiety: VMA (pKb = 10.6, KAds = (5.18 ± 1.95) × 105) and HVA (pKb = 9.6, KAds = (4.78 ± 1.58) × 104). The differential pulse voltammetric method was applied to detect VMA and HVA as individual species and interferents. As individual analytes, both HVA and VMA can be detected at a concentration of 1.99 × 10-5 M (RSD ≤ 0.28, recovery 110-115%).

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 nomogram for predicting recurrence-free survival of intermediate and high-risk neuroblastoma.

This study aimed to confirm the independent risk factors for recurrence-free survival (RFS) in intermediate and high-risk neuroblastoma (NB) patients and set up an effective nomogram model for predicting the recurrence of NB. A total of 212 children with intermediate- and high-risk neuroblastoma, who had ever achieved complete remission (CR) or very good partial remission (VGPR) after standardized treatment in this hospital, were chosen as study objects. After retrospective analysis of the clinical data, Cox regression model was used to explore the factors related to the recurrence of neuroblastoma, to determine the variables to construct the Nomogram. The consistency index would predict the accuracy of this nomogram. RFS rate in 1-year, 3-year, 5-year, and 10-year was 0.811, 0.662, 0.639, and 0.604, respectively. Children with MYCN amplification had a higher neuron-specific enolase (NSE) value (P = 0.031) at the initial diagnosis than MYCN non-amplification. The univariate analysis predicted that increased vanillylmandelic acid (VMA) and NSE value and dehydrogenase (LDH) > 1000 U/L were important adverse factors for the recurrence of NB. Multivariate analysis demonstrated that age at diagnosis, tumor localization, MYCN state, histologic subtype, and tumor capsule were significantly associated with RFS (all P values < 0.05). Nomograms were established for predicting the recurrence of NB according to the Cox regression analysis. Internal verification by the Bootstrap method showed that the prediction of the nomogram's consistency index (C-index) was 0.824 (P = 0.023).   Conclusion: Age at diagnosis, tumor localization, MYCN state, histologic category, and tumor capsule were independent risk factors for the recurrence of NB. The nomogram model could accurately predict the recurrence of children with neuroblastoma. What is Known: • The prognoses of neuroblastoma (NB) could vary greatly due to the high heterogeneity, the 5-year survival rate of low-risk NB exceeded 90%, while the 5-year survival rate of children in the intermediate and high-risk groups was not satisfactory.. What is New: • Increased vanillylmandelic acid (VMA) and neuron-specific enolase (NSE) value, and lactate dehydrogenase (LDH)>1000U/L were important adverse factors for the recurrence of NB. • NSE value was more valuable for predicting NB recurrence.

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).

Disturbance of neurotransmitter metabolism in drug-naïve, first-episode major depressive disorder: a comparative study on adult and adolescent cohorts.

Neurotransmitter metabolism plays a critical role in the pathophysiology of major depressive disorder (MDD). However, whether the neurotransmitter metabolism in adolescent MDD is differentiated from adult MDD is still elusive. In the current study, plasma concentrations of monoamine and amino acid neurotransmitters as well as their metabolites, including tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE), vanillylmandelic acid (VMA), 3-methoxy-4-hydroxyphenylglycol (MHPG), glutamine (GLN), glutamate (GLU) and gamma-aminobutyric acid (GABA), were measured and compared in two cohorts of subjects (adult cohort: 31 first-episode MDD vs. 35 healthy controls; adolescent cohort: 33 first-episode MDD vs. 30 healthy controls). To assess the effects of antidepressant treatment, we also analyzed the concentrations of these indexes pre- and post-treatment in adult and adolescent cohorts. At baseline, the deficits of neurotransmitter metabolism in adult MDD were manifested in all the neurotransmitter systems. In contrast, for adolescent MDD, the dysregulation of neurotransmission was mainly indicated in the catecholaminergic systems. After antidepressant treatment, adult MDD showed increased TRP, KYN, KYNA and GLU levels, together with decreased levels of 5-HIAA and DOPAC. Adolescent MDD illustrated an increased level of 5-HT and decreased levels of TRP and GABA. The improvements of Hamilton total scores correlated with the changes in plasma TRP and the turnover of KYN/TRP after treatment in all MDD patients. However, these correlations were only manifested in the adult MDD rather than in adolescent MDD patients. The findings highlight the shared and distinguished neurotransmitter pathways in MDD and emphasize the different antidepressant responses between adults and adolescents. Potentially, the neurotransmitters above could serve as diagnostic biomarkers and provide a novel pharmacological treatment strategy for MDD.

Values of serum CA125, NSE and 24-hour urine VMA in diagnosis and prediction of treatment of paediatric neuroblastoma.

BACKGROUND: Paediatric neuroblastoma is a relatively common type of malignant tumour originating from neural crest tissues. Early diagnosis and the performance of specific therapeutic strategies can increase the survival rate and improve the prognosis of children with neuroblastoma. METHODS: A total of 86 children with neuroblastoma were recruited in this research, and 50 healthy children aged 1-12 years were also selected as controls. Twenty-four-hour urine vanillylmandelic acid (VMA) was evaluated by high-performance liquid chromatography. Serum carbohydrate antigen 125 CA125 and neuron-specific enolase (NSE) levels were evaluated by electrochemiluminescence in Cobas E411 autoanalyser. RESULTS: The serum CA125, NSE and 24-hour urine VMA levels of children with stage III-IVs neuroblastoma were significantly higher than those of children with clinical stages I-II; the serum CA125, NSE and 24-hour urine VMA levels of children in the effective treatment group were significantly lower than those in the treatment-ineffective group. The serum CA125 generated sensitivity and specificity of 71.88% and 59.26%, combined with an AUC (area under the curve) of 0.7049. The serum NSE generated sensitivity and specificity of 68.75% and 81.48%, combined with an AUC of 0.7407. The 24-hour urine VMA generated sensitivity and specificity of 90.63% and 59.26%, combined with an AUC of 0.7986. CONCLUSION: In conclusion, serum CA125, NSE and 24-hour urine VMA levels before treatment could assess the condition of children with neuroblastoma and predict the effect of treatment.

Results of mass screening for neuroblastoma in 18-month-old infants in Osaka area, Japan.

PURPOSE: In 2004, the Japanese government halted nationwide mass screening for neuroblastoma in 6-month-old infants as it led to overdiagnosis of localized tumors with favorable prognoses and failed to reduce neuroblastoma-related mortality. However, a new mass screening program for neuroblastoma in 18-month-old infants (18MS) was conducted in the Osaka prefecture. We assessed the efficacy of the 18MS in screening unfavorable cases. METHODS: Public health centers in Osaka prefecture, excluding the Osaka city area, provided test kits to the guardians of infants who received a check-up at 18 months of age between 2004 and 2017. For patients whose standardized urinary levels of vanillylmandelic acid or homovanillic acid exceeded the threshold, they were further examined and treated in two specific hospitals Osaka University Hospital and Osaka Women's and Children's Hospital. Screening-positive patients with and without neuroblastoma were retrospectively reviewed. RESULTS: Among 142,423 children screened during the 18MS, 85 tested positive, and 14 were diagnosed with neuroblastoma. Twelve patients were classified as very low risk, while 2 were classified as high risk, based on the International Neuroblastoma Risk Group risk classification. CONCLUSION: The 18MS did not screen unfavorable cases with neuroblastoma efficiently, although few participants benefited from it.

Application of an LC-MS/MS Method for the Simultaneous Quantification of Homovanillic Acid and Vanillylmandelic Acid for the Diagnosis and Follow-Up of Neuroblastoma in 357 Patients.

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC-MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa's k = 0.6) and a strong correlation (Pearson's r = 0.73). To our knowledge, this is the first study to utilize LC-MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.

Urinary vanillylmandelic acid:creatinine ratio in dogs with pheochromocytoma.

Pheochromocytoma diagnosis in dogs is challenging because biochemical tests are not always available. In humans, urinary vanillylmandelic acid (VMA) is part of a pheochromocytoma biochemical diagnostic profile, whereas its diagnostic accuracy is currently unknown in dogs with pheochromocytoma. Prospectively, VMA was determined by HPLC and expressed as the ratio with respect to urinary creatinine (VMA:C). The diagnostic accuracy of the VMA:C ratio was evaluated by analyzing the receiver operating characteristic (ROC) curve in 10 healthy dogs, 8 dogs with pituitary-dependent hypercortisolism, 8 dogs with adrenal-dependent hypercortisolism, and 7 dogs with pheochromocytoma. The pheochromocytoma diagnosis was confirmed by histology and immunohistochemistry in all tumors. The VMA:C ratio was significantly higher in dogs with pheochromocytoma (158 [53.4 to 230.8] × 10-3) than in dogs with adrenal-dependent hypercortisolism (48.1 [24.3 to 144.9] × 10-3; P < 0.05), dogs with pituitary-dependent hypercortisolism (37.5 [32 to 47.1] × 10-3; P < 0.001), and healthy dogs (33.8 [13.3 to 87.9] × 10-3; P < 0.001). When using a VMA:C ratio >58.2 × 10-3 for pheochromocytoma diagnosis, a sensitivity of 85.7% and a specificity of 88.4% were obtained. Nevertheless, when using a cut-off ratio of 4 times the median VMA:C ratio determined in healthy dogs, there was no overlap (100% specificity). The area under the ROC curve indicated that the VMA:C ratio test could be used to discriminate between dogs with and without pheochromocytoma, what leads to the conclusion that it is useful for pheochromocytoma diagnosis in dogs.

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.

Label-free silver triangular nanoplates for spectrophotometric determination of catecholamines and their metabolites.

A novel method towards spectrophotometric determination of catecholamines and their metabolites differing in their functional groups has been developed. This method is based on a change in morphology of silver triangular nanoplates upon the action of cateсholamines and their metabolites, which is manifested by the decrease of the nanoparticle local surface plasmon resonance (LSPR) band intensity or its shift to the short-wavelength region of the spectrum. The shift value of the LSPR band or the change of its intensity increases with increasing concentration of catecholamines or their metabolites, which is proposed for their spectrophotometric determination. The limits of detection of catecholamines and their metabolites under selected conditions increase in the series homovanillic acid < vanillylmandelic acid < L-epinephrine < L-norepinephrine < dopamine and are 0.25, 1.2, 3.0, 64, and 130 µmol L-1, respectively. The selectivity of the proposed method was assessed using vanillylmandelic acid as example. It was found that the determination of vanillylmandelic acid does is not interfered in the presence of 4000-fold excess of Na+, K+, CH3COO-, and 1000-fold excess of Mg2+, Ca2+, Al3+, NO3-. The method also allows for the selective determination of vanillylmandelic acid in the presence of a 1000-fold excess of structurally related substances that do not contain either a catechol fragment or an electron donor substituent. The proposed approach was successfully applied to the determination of catecholamines in pharmaceuticals and artificial urine. Graphical abstract.