An Electrochemical Sensor for Detection of Neuroblastoma Markers: Complexation Studies as a Tool for the Selection of a Suitable Receptor for Electrode Coating.

Homovanillate (HVA) and vanilmandelate (VMA) are recognized markers of diseases, including neuroblastoma. However, their detection in urine represents a challenging task due to the complexity of the matrix. Here, a design, synthesis and thorough investigation of polymerizable urea-based receptors interacting with HVA and VMA are reported. The selection of receptor with the best anion recognition properties for electrode coating is based on 1 H-NMR and UV-Vis complexation studies. The sensor is prepared by electropolymerization with progress monitoring by cyclic voltammetry. The deposited layer is characterized by IR and scanning electron microscopy. The obtained sensor shows an electrochemical impedance spectroscopy response to VMA with linear range 9.9×10-6 to 1.2×10-3  M and LOD of 3.4×10-6  M. The sensor selectivity was demonstrated by the determination of VMA level in the presence of 16 µM HVA and in artificial urine with and without phosphates, with standard deviations of 0.11, 0.17 and 0.09, respectively.

Urinary 3-Methoxytyramine Is a Biomarker for MYC Activity in Patients With Neuroblastoma.

PURPOSE: Elevated urinary 3-methoxytyramine (3MT) level at diagnosis was recently put forward as independent risk factor for poor prognosis in neuroblastoma. Here, we investigated the biologic basis underlying the putative association between elevated 3MT levels and poor prognosis. METHODS: Urinary 3MT levels and prognosis were investigated in both retrospective Italian (N = 90) and prospective Dutch (N = 95) cohorts. From the Dutch Cancer Oncology Group cohort (N = 122), patients with available urinary 3MT and gene expression data (n = 90) were used to generate a 3MT gene signature. The 3MT gene signature score was then used to predict survival outcome in the Children's Oncology Group (N = 247) and German Pediatric Oncology Group (N = 498) cohorts and compared with other known gene signatures. Immunohistochemistry of MYCN and dopamine ß-hydroxylase proteins was performed on primary tumors. RESULTS: Elevated urinary 3MT levels were associated with poor prognosis in a retrospective cohort and a prospective cohort. Moreover, elevated urinary 3MT levels were associated with eight differentially expressed genes, providing a 3MT gene signature that successfully predicted poor clinical outcome. Even among low-risk patients, high 3MT signature score was associated with poor 5-year overall survival (72% v 99% among low-risk patients with a low 3MT signature score), and the 3MT signature score was correlated with MYC activity in the tumor (R = 82%, P < .0001). Finally, a strong MYCN and weak dopamine ß-hydroxylase staining of tumors derived from patients with elevated urinary 3MT levels was observed, linking MYC activity in the tumor to both catecholamine biosynthesis and elevated urinary 3MT levels. CONCLUSION: Elevated urinary 3MT is a promising biomarker for poor prognosis and reflects increased MYC activity in the tumor. Therefore, urinary 3MT levels should be measured at diagnosis and may assist in assessing risk.

Catecholamines in neuroblastoma: Driver of hypertension, or solely a marker of disease?

BACKGROUND: Neuroblastoma is a common solid tumor of childhood and is often associated with hypertension. Potential etiologies contributing to hypertension include renal compression, pain, volume overload, and catecholamine secretion. CASES: We completed a single center retrospective review of children with neuroblastoma and ≥stage II hypertension (per Hypertension Canada guidelines) over a 2-year period. All patients (n = 10) had elevated urine normetanephrine levels and eight had intra-abdominal tumors. Four patients had refractory hypertension requiring > three agents, of which three required alpha/beta blockade. CONCLUSION: Although multifactorial, hypertension in neuroblastoma often has a neuroendocrine component. Excess normetanephrine production in neuroblastoma may be a more common hypertensive mechanism than previously appreciated. Urinary normetanephrine elevation could suggest potential neuroendocrine-mediated hypertension.

Identification of novel neuroblastoma biomarkers in urine samples.

Urine is a complex liquid containing numerous small molecular metabolites. The ability to non-invasively test for cancer biomarkers in urine is especially beneficial for screening child patients. This study attempted to identify neuroblastoma biomarkers by comprehensively analysing urinary metabolite samples from children. A total of 87 urine samples were collected from 54 participants (15 children with neuroblastoma and 39 without cancer) and used to perform a comprehensive analysis. Urine metabolites were extracted using liquid chromatography/mass spectrometry and analysed by Metabolon, Inc. Biomarker candidates were extracted using the Wilcoxon rank sum test, random forest method (RF), and orthogonal partial least squares discriminant analysis (OPLS-DA). RF identified three important metabolic pathways in 15 samples from children with neuroblastoma. One metabolite was selected from each of the three identified pathways and combined to create a biomarker candidate (3-MTS, CTN, and COR) that represented each of the three pathways; using this candidate, all 15 cases were accurately distinguishable from the control group. Two cases in which known biomarkers were negative tested positive using this new biomarker. Furthermore, the predictive value did not decrease in cases with a low therapeutic effect. This approach could be effectively applied to identify biomarkers for other cancer types.

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.

Crisis adrenérgica como forma de debut de un neuroblastoma / Adrenergic crisis as a debut form of a neuroblastoma

INTRODUCCIÓN: La forma clínica de presentación más común del neuroblastoma es el de una masa abdominal, pero puede presentarse con sintomatología menos habitual, como es la crisis adrenérgica por liberación de catecolaminas. OBJETIVO: Describir una forma de presentación inusual de neuroblastoma y el amplio diagnóstico diferencial que existe en un lactante con síntomas adrenérgicos. CASO CLÍNICO: Lactante femenina de 7 semanas de vida, consultó por historia de tres semanas de sudoración e irritabilidad a lo que se asoció fiebre de 24 h de evolución y dificultad respiratoria. Al ingreso presentaba mal esta do general, irritabilidad, sudoración, enrojecimiento facial, taquipnea y palidez cutánea, taquicardia sinusal extrema e hipertensión arterial (HTA), interpretadas como sintomatología adrenérgica. Se completó el estudio con una ecografía abdominal y resonancia magnética que mostraron una gran masa retroperitoneal compatible con neuroblastoma. Las catecolaminas en sangre y en orina mostraron altos niveles de dopamina, adrenalina y noradrenalina, probablemente de origen tumoral. Se inició tratamiento antihipertensivo con fármacos alfa bloqueantes con buen control de la tensión arterial. Se resecó quirúrgicamente el tumor sin incidencias y con una adecuada recuperación posterior. La paciente presentó evolución favorable a tres años de seguimiento. CONCLUSIONES: en un lactante con sintomatología adrenérgica como irritabilidad, enrojecimiento, sudoración asociada a HTA, se debe descartar patología cardiaca, metabólica (hipoglucemia), intoxicaciones y/o patología suprarrenal. Dentro de esta última, el neuroblastoma es la primera posibilidad diagnóstica, por ser uno de los principales tumores en la infancia y aunque esta presentación no es habitual puede producir estos síntomas.

INTRODUCTION: The most common clinical presentation of neuroblastoma is an abdominal mass, but it can present with uncommon symptoms, such as adrenergic storm due to catecholamine release. OBJECTIVE: To describe an unusual presentation of neuroblastoma and the wide differential diagnosis that exists in an infant with adrenergic symptoms. CLINICAL CASE: A 7-week old female infant was evaluated due to a 3-week history of sweating and irritability associated with a 24-hour fever and respiratory distress. At admission, she presented poor general condition, irritability, sweating, facial redness, tachypnea and skin paleness, extreme sinus tachycardia, and high blood pressure (HBP), interpreted as adrenergic symptoms. The study was completed with abdominal ultrasound and magnetic reso nance imaging that showed a large retroperitoneal mass compatible with neuroblastoma. Plasma and urinary catecholamines tests showed high levels of dopamine, adrenaline, and noradrenaline, probably of tumor origin. We started antihypertensive treatment with alpha-blocker drugs, showing a good blood pressure control. The tumor was surgically resected without incidents and adequate subsequent recovery. The patient presented a favorable evolution after three years of follow-up. CONCLUSIONS: In an infant with adrenergic symptoms such as irritability, redness, sweating associated with HBP, it should be ruled out pathology heart or metabolic (hypoglycemia) pathology, intoxications, and/or adrenal pathology. Within this last one, neuroblastoma is the first diagnostic possibility, since it is one of the main tumors in childhood and, although this presentation is not usual, it can produce these symptoms.

Biochemical testing for neuroblastoma using plasma free 3-O-methyldopa, 3-methoxytyramine, and normetanephrine.

BACKGROUND: Neuroblastoma, the most common extracranial solid tumor of childhood, produces catecholamines that are metabolized within tumor cells. Homovanillic acid (HVA) and vanillylmandelic acid (VMA), the end products of catecholamine metabolism, have limited accuracy for testing of the tumors. This study assessed whether metabolites produced in earlier steps of catecholamine metabolism might offer improved diagnostic accuracy over urinary HVA and VMA. PROCEDURE: Plasma concentrations of 3-methoxytyramine, normetanephrine, and metanephrine were measured in two pediatric cohorts: (i) 96 children with confirmed neuroblastoma and (ii) 41 children with signs and symptoms of a catecholamine-producing tumor or other neoplasms and in whom neuroblastoma was excluded. Additional measurements of plasma 3-O-methyldopa and relationships of metabolites to MYCN amplification were examined in patient subgroups. RESULTS: Overall, 94 of the 96 patients with neuroblastoma had concentrations of 3-methoxytyramine or normetanephrine above age-specific upper limits of reference intervals, providing a diagnostic sensitivity of 97.9% that was higher (P < 0.0001) than that of 82.2% for HVA and VMA. One of the two patients with normal plasma results showed an elevation of plasma 3-O-methyldopa. Diagnostic specificities were, respectively, 95.1% and 84.8%. Areas under receiver-operating characteristic curves confirmed the superior diagnostic power of the plasma than the urinary test (0.994 vs 0.945; P = 0.0095). Ratios of plasma 3-methoxytyramine to normetanephrine were 7.2-fold higher (P < 0.0001) for patients who had neuroblastomas with MYCN amplification than without MYCN amplification. CONCLUSIONS: Measurements of plasma 3-methoxytyramine and normetanephrine provide a highly accurate diagnostic test for neuroblastoma and also offer potential for prognostic risk stratification.

Adrenergic crisis as a debut form of a neuroblastoma. / Crisis adrenérgica como forma de debut de un neuroblastoma.

INTRODUCTION: The most common clinical presentation of neuroblastoma is an abdominal mass, but it can present with uncommon symptoms, such as adrenergic storm due to catecholamine release. OBJECTIVE: To describe an unusual presentation of neuroblastoma and the wide differential diagnosis that exists in an infant with adrenergic symptoms. CLINICAL CASE: A 7-week old female infant was evaluated due to a 3-week history of sweating and irritability associated with a 24-hour fever and respiratory distress. At admission, she presented poor general condition, irritability, sweating, facial redness, tachypnea and skin paleness, extreme sinus tachycardia, and high blood pressure (HBP), interpreted as adrenergic symptoms. The study was completed with abdominal ultrasound and magnetic reso nance imaging that showed a large retroperitoneal mass compatible with neuroblastoma. Plasma and urinary catecholamines tests showed high levels of dopamine, adrenaline, and noradrenaline, probably of tumor origin. We started antihypertensive treatment with alpha-blocker drugs, showing a good blood pressure control. The tumor was surgically resected without incidents and adequate subsequent recovery. The patient presented a favorable evolution after three years of follow-up. Con clusions: In an infant with adrenergic symptoms such as irritability, redness, sweating associated with HBP, it should be ruled out pathology heart or metabolic (hypoglycemia) pathology, intoxications, and/or adrenal pathology. Within this last one, neuroblastoma is the first diagnostic possibility, since it is one of the main tumors in childhood and, although this presentation is not usual, it can produce these symptoms.

Likelihood of Diagnosing Neuroblastoma in Isolated Horner Syndrome.

BACKGROUND: The need for an extensive evaluation for neuroblastoma in children with Horner syndrome is controversial. METHODS: A retrospective study design was used. The cohort included 47 children with anisocoria who were diagnosed with Horner syndrome and 135 children with neuroblastoma evaluated at a pediatric medical center between 2007 and 2015. To detect neuroblastoma, patients with Horner syndrome underwent brain and cervical MRI, abdominal ultrasound, and/or measurement of urinary vanillylmandelic acid (VMA). The neuroblastoma group was evaluated for signs/symptoms of Horner syndrome at the time of diagnosis. RESULTS: Seven patients with Horner syndrome were lost to follow-up, and the findings of the remaining 40 were categorized according to the age of the patient. Horner syndrome most frequently was idiopathic (58%), and in only 1 patient did the discovery of neuroblastoma precede the appearance of Horner syndrome. In the 21 patients aged 1-18 years, Horner syndrome was acquired in 15 patients and congenital in 6. The most common etiology was trauma (62%). Imaging was performed in 14 patients and VMA testing in 13. Neuroblastoma was diagnosed in 5 patients; in none was it related to Horner syndrome. In the 135 patients with neuroblastoma, most of the tumors were diagnosed at Stage 4 (60%) or Stage 3 (30%) with 53% originating in the abdomen. In one patient (0.74%) with signs/symptoms of Horner syndrome at diagnosis of neuroblastoma, the tumor had been identified prenatally and the diagnosis confirmed by imaging postnatally. CONCLUSIONS: The absence of occult neuroblastoma in children with Horner syndrome and of signs/symptoms of Horner syndrome in the children diagnosed with neuroblastoma suggests that Horner syndrome might not be as frequent a cause of neuroblastoma as previously thought. We recommend that full investigation for neuroblastoma be reserved for suspicious cases associated with additional systemic signs or symptoms.

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.

Circulating tumor cells detection in neuroblastoma patients by EpCAM-independent enrichment and immunostaining-fluorescence in situ hybridization.

Detecting circulating tumor cells (CTCs) has proven valuable for evaluating the prognosis of cancer patients and for studying the mechanisms of treatment resistance. Owing to the lack of universal and specific tumor markers for neuroblastoma (NB), in this prospective study, we adopted an EpCAM-independent method to detect CTCs in the peripheral blood of NB patients. We used an EpCAM-independent assay to delete leukocytes and to enrich the CTCs. CTCs were identified by immunostaining of CD45, DAPI and DNA fluorescence in situ hybridization (FISH) of the centromere of chromosome 8 probe (CEP8). Cells that were DAPI+/CD45-/CEP8 ≥ 3 were considered CTCs. We collected peripheral blood from 28 NB patients as well as clinical and follow-up data. The number of CTCs among the different risk groups were significantly different (p = .0208, Kruskal-Wallis test). Patients with metastasis had more CTCs than those without metastasis (p < .0001, Mann-Whitney test). Patients with ≥3 CTCs per 4 ml of peripheral blood had an increased likelihood of having metastasis (sensitivity, 88.89%; specificity, 78.59%), and patients with ≥10 CTCs per 4 ml of peripheral blood had poorer overall survival. The EpCAM-independent assay along with immunostaining-FISH (i-FISH) described here can detect CTCs in patients with NB at a high sensitivity and may have clinical value for prognosis evaluation and diagnosing metastasis when imaging data are ambiguous.

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.

3-Methoxytyramine: An independent prognostic biomarker that associates with high-risk disease and poor clinical outcome in neuroblastoma patients.

INTRODUCTION: Prognosis of neuroblastoma patients is very diverse, indicating the need for more accurate prognostic parameters. The excretion of catecholamine metabolites by most neuroblastomas is used for diagnostic purposes, but their correlation with prognosis has hardly been investigated. Therefore, we performed an in-depth analysis of a panel of elevated urinary catecholamine metabolites at diagnosis and their correlation with prognosis. PATIENTS AND METHODS: Retrospective study of eight urinary catecholamine metabolites in a test (n = 96) and validation (n = 205) cohort of patients with neuroblastoma (all stages) at diagnosis. RESULTS: Multivariate analyses, including risk factors such as stage and MYCN amplification, revealed that 3-methoxytyramine (3MT) was an independent risk factor for event-free survival (EFS) and overall survival (OS). Furthermore, only 3MT appeared to be an independent risk factor for both EFS and OS in high-risk patients, which was independent of modern high-risk therapy and immunotherapy. Among high-risk patients, those with elevated 3MT and older than 18 months had an extremely poor prognosis compared to patients with non-elevated 3MT and younger than 18 months (5-year EFS of 14.3% ± 4% and 66.7% ± 18%, respectively, p = 0.001; 5-year OS of 21.8% ± 5% and 87.5% ± 12%, respectively, p < 0.001). CONCLUSIONS: Elevated 3MT at diagnosis was associated with high-risk disease and poor prognosis. For high-risk patients, elevated 3MT at diagnosis was the only significant risk factor for EFS and OS. 3MT was also able to identify subgroups of high-risk patients with favourable and extremely poor prognosis.

Catecholamines profiles at diagnosis: Increased diagnostic sensitivity and correlation with biological and clinical features in neuroblastoma patients.

INTRODUCTION: Neuroblastoma (NBL) accounts for 10% of the paediatric malignancies and is responsible for 15% of the paediatric cancer-related deaths. Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are most commonly analysed in urine of NBL patients. However, their diagnostic sensitivity is suboptimal (82%). Therefore, we performed in-depth analysis of the diagnostic sensitivity of a panel of urinary catecholamine metabolites. PATIENTS AND METHODS: Retrospective study of a panel of 8 urinary catecholamine metabolites (VMA, HVA, 3-methoxytyramine [3MT], dopamine, epinephrine, metanephrine, norepinephrine and normetanephrine [NMN]) from 301 NBL patients at diagnosis. Special attention was given to subgroups, metaiodobenzylguanidine (MIBG) non-avid tumours and VMA/HVA negative patients. RESULTS: Elevated catecholamine metabolites, especially 3MT, correlated with nine out of 12 NBL characteristics such as stage, age, MYCN amplification, loss of heterozygosity for 1p and bone-marrow invasion. The combination of the classical markers VMA and HVA had a diagnostic sensitivity of 84%. NMN was the most sensitive single diagnostic metabolite with overall sensitivity of 89%. When all 8 metabolites were combined, a diagnostic sensitivity of 95% was achieved. Among the VMA and HVA negative patients, were also 29% with stage 4 disease, which usually had elevation of other catecholamine metabolites (93%). Diagnostic sensitivity for patients with MIBG non-avid tumour was improved from 33% (VMA and/or HVA) to 89% by measuring the panel. CONCLUSIONS: Our study demonstrates that analysis of a urinary catecholamine metabolite panel, comprising 8 metabolites, ensures the highest sensitivity to diagnose NBL patients.

L-DOPA therapy interferes with urine catecholamine analysis in children with suspected neuroblastoma: a case series.

Neuroblastoma is the most common solid extracranial malignancy diagnosed in childhood. Clinical presentation is variable, and metastatic disease is common at diagnosis. Analyses of urinary catecholamines and their metabolites are commonly requested as a first-line investigation when clinical suspicion exists. Levodopa (L-Dopa) therapy is utilized as a treatment for a number of disorders in childhood, including Dopa-responsive dystonia. Neuroblastoma may mimic some of the clinical features of this disorder. L-Dopa can interfere with analysis of urinary catecholamines and their metabolites and complicate the interpretation of results. We present the cases of three children who were prescribed L-dopa at the time of analysis of urinary catecholamines and metabolites as a screen for neuroblastoma, but who did not have the disease. Comparison of their results with those from cases with true neuroblastoma reveal that it is impossible to reliably distinguish true neuroblastoma from L-Dopa therapy using these tests. We recommend that patients should be off L-dopa therapy, if possible when these tests are performed. These cases illustrate the importance of providing clinical details and drug history to the laboratory in order to avoid diagnostic confusion.

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.

Clinical validation of urine 3-methoxytyramine as a biomarker of neuroblastoma and comparison with other catecholamine-related biomarkers.

Background Urinary dopamine, homovanillic acid and 4-hydroxy-3-methoxymandelic acid are established tests for diagnosis and monitoring of neuroblastic disease. We compared the diagnostic performance of total urinary 3-methoxytyramine, the O-methylated product of dopamine, to these three established tumour markers. Methods Urinary 3-methoxytyramine, dopamine, homovanillic acid and 4-hydroxy-3-methoxymandelic acid were measured by high-performance liquid chromatography with electrochemical detection on consecutive urine samples from histologically proven neuroblastic patients and controls. Patients with neuroblastic disease were further classified as untreated, advancing, residual or absent disease based on clinical and radiological criteria. Receiver operating characteristic curve analysis was used to compare the diagnostic performance of the four tumour markers. Results Urinary 3-methoxytyramine was well correlated with established tumour markers and its concentration correlated with disease activity. It was the most commonly elevated tumour marker in neuroblastic disease and showed similar sensitivity to dopamine and homovanillic acid. The diagnostic utility of urinary 3-methoxytyramine as measured by area under the receiver operating characteristic curve was similar to dopamine and homovanillic acid. Conclusion Our results support the use of urinary 3-methoxytyramine as a tumour marker in the diagnosis and the monitoring of neuroblastoma disease.

Determination of urinary biogenic amines' biomarker profile in neuroblastoma and pheochromocytoma patients by MEKC method with preceding dispersive liquid-liquid microextraction.

The unbalanced secretion of biogenic amines (BAs) is considered to be a relevant biochemical biomarker in the screening for neuroendocrine tumors, such as: neuroblastoma and pheochromocytoma. However, there is still a need to improve the bioanalytical procedures for BA determination in biological samples due to their instability (photo- and thermosensitivity, easy oxidation) and low concentration in the body fluids. In this study, the primary analytical challenge was to optimize the method of extraction of seven compounds from among BAs and their precursors from urine samples. Several methods based on liquid-liquid extraction (LLE) or solid phase extraction (SPE) techniques were tested. By optimization of the extraction and data analysis using chemometric tool, the dispersive liquid-liquid microextraction (DLLME) has been chosen due to its low solvents consumption, high efficiency of isolation, preconcentration and suitable clean-up of biological matrix. Further, α-cyclodextrin-modified micellar electrokinetic chromatography (MEKC) with ultraviolet detection (UV) has been applied for quantification of the analyzed biologically active compounds with limits of detection (LOD) and limits of quantification (LOQ) at 0.15 and 0.5µgmL-1, respectively. Finally, the optimized and validated DLLME-MEKC-UV method has been employed for the analysis of real urine samples, obtained from 6 children with neuroendocrine tumors and 6 healthy children. It was stated that concentrations of BA could serve to differentiate between the patients and healthy children. This pilot study indicates that the elaborated fast and sensitive DLLME-MEKC-UV method for determination of panel of biomarkers could be successfully applied in everyday clinical practice to help to confirm the clinical diagnosis of neuroendocrine tumors in children.