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Urinary catecholamines and their methylated metabolites are biochemical indicators of pheochromocytoma, paraganglioma and neuroblastoma. A rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to high-resolution mass spectrometry (LC-HRMS) was developed and validated to measure urinary catecholamines (epinephrine (E), norepinephrine (NorE), dopamine (D)) and total methylated metabolites (normetanephrine (NorMN), metanephrine(MN) and 3-methoxytyramine (3-MT)) in a clinical setting. Results of 51 urine specimens measured using this LC-HRMS method were compared with a liquid chromatography assay with electrochemical detection (LC-EC). Urine samples (200 μL) were spiked with an internal standard solution followed by SPE purification. In the case of total methylated metabolites, urine was hydrolyzed before SPE purification. Separation was achieved on an Acclaim Mixed Mode WCX column, with an 8.5 min runtime. All compounds were detected in electrospray positive ionization mode with a parallel reaction monitoring acquisition and quantified with a linear regression (r2 > 0.998) between 2 and 200 µg/L (10.9-1090; 11.8-1182 nmol/L) for E and NorE respectively and between 10 and 1000 µg/L for others (65.2-6520; 50.7-5070; 54.5-5450 ; 59.8-5980 nmol/L for D, M, NorMN and 3-MT, respectively). Overall imprecision and bias did not exceed 15%. No significant matrix effect was observed. Correlation between the two assays was good except for epinephrine. Epinephrine concentrations measured by LC-EC method were slightly higher than values obtained with LC-HRMS method but without impact on clinical decision. This LC-HRMS assay provides a new tool for simultaneous quantitative catecholamine determination and was successfully applied in routine for the screening or follow up of pheochromocytoma, paraganglioma and neuroblastoma. LC-HRMS method offers significant advantages compared to LC-EC with good sensitivity, an unambiguous analyte determination and high sample throughput., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Detection of normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT) could be used to diagnose pheochromocytomas and paragangliomas (PPGLs). The accuracy for the diagnosis of PPGLs is only 6% by virtue of the classic symptom triad. In addition, false-positive results were found using plasma free MNs as biomarkers. Spot urinary free metanephrines (MNs) presented high specificity for PPGLs diagnosis in our previous work by HPLC with the electrochemical detection. Whereas, MNs and creatinine (Cr) need to be detected separately. A simple and specific method was urgently needed for the diagnosis of PPGLs. Here, we established a new HPLC method for spot urinary free MNs and 3-MT by the fluorescence detection and Cr by the ultraviolet detection simultaneously. It was worth mentioning that Cr for the virtue of being fairly constant in a given subject was used as an internal reference correction to eliminate the effect of spot urine volume for the diagnosis of PPGLs. Thirty-seven patients with PPGLs and 164 control subjects were detected by the established method and the peak area ratios of MNs and 3-MT to Cr were used innovatively for the diagnosis of PPGLs. The results showed acceptable precisions and recoveries. The sensitivities of the method were 94.6%, 91.9% and 86.5% and the specificities were 96.3%, 93.9% and 82.3%, respectively by the peak area of NMN/Cr, MN/Cr and 3-MT/Cr for the diagnosis. The established method provides a promising way for simple, rapid and accurate diagnosis of PPGLs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pheochromocytomas are rare neuroendocrine chromaffin-derived tumors that arise within the adrenal medulla. They are usually benign, but if not diagnosed or if left untreated, they can have devastating consequences. Clinical consideration of the diagnosis is paramount, as they may have protean manifestations, and a high index of suspicion is essential if serious consequences are to be avoided. An accurate biochemical diagnosis is crucial for the management of these patients: either plasma or urinary metanephrines are both highly sensitive and specific if correctly employed, but knowledge of pre- and post-analytic interference is essential. Diagnostic imaging with cross-sectional CT and/or MRI offers high sensitivity in their detection, but lack specificity. The introduction of PET/CT/MR has led to a dramatic improvement in the localization of both pheochromocytomas and paragangliomas, together with the increasing availability of new functional imaging radionuclides. Optimal investigation and accurate diagnosis is best achieved at 'centers of excellence' with expert multidisciplinary teams., (Copyright © 2019. Published by Elsevier Ltd.)

Pheochromocytoma during pregnancy, although rare, is a perilous condition. The wellbeing of mother and fetus are at stake if not diagnosed and treated antenatally and timely. The diagnosis is frequently overlooked because of the aspecific nature of signs and symptoms and confusion with pregnancy-related hypertension. Measurements of plasma or urinary free metanephrines have the highest diagnostic accuracy. MRI is preferred over ultrasonography. The optimal time for surgical removal is before 24 weeks of gestation or at/after delivery. Laparoscopic adrenalectomy should be preceded by medical pretreatment. Cesarean delivery is preferred in these patients; vaginal delivery might be considered in selected pretreated patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Plasma or urinary metanephrines are recommended for screening of pheochromocytomas and paragangliomas (PPGLs). Measurements of urinary free rather than deconjugated metanephrines and additional measurements of methoxytyramine represent other developments. For all measurements there is need for reference intervals., Methods: Plasma free, urinary free and urinary deconjugated O-methylated catecholamine metabolites were measured by LC-MS/MS in specimens from 590 hypertensives and normotensives. Reference intervals were optimized using data from 2,056 patients tested for PPGLs., Results: Multivariate analyses, correcting for age and body surface area, indicated higher plasma and urinary metanephrine in males than females and sex differences in urinary normetanephrine and free methoxytyramine that largely reflected body size variation. There were positive associations of age with plasma metabolites, but negative relationships with urinary free metanephrine and methoxytyramine. Plasma and urinary normetanephrine were higher in hypertensives than normotensives, but differences were small. Optimization of reference intervals using the data from patients tested for PPGLs indicated that age was the most important consideration for plasma normetanephrine and sex most practical for urinary metabolites., Conclusion: This study clarifies impacts of demographic and anthropometric variables on catecholamine metabolites, verifies use of age-specific reference intervals for plasma normetanephrine and establishes sex-specific reference intervals for urinary metabolites., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The metanephrines (MNs) in plasma and urine were proposed as biomarkers for the diagnosis of pheochromocytomas and paragangliomas (PPGLs). However, plasma free MNs and 24h urinary fractionated MNs were not satisfactory enough in specificity for the diagnosis of PPGLs. Moreover, the collection of 24h urine was inconvenient. This work examined the diagnostic and prognostic efficiency of free MNs in spot urine for PPGLs., Methods: We measured free MNs concentration in spot urine and plasma of 28 PPGLs patients and 155 control subjects by HPLC with electrochemical detection. Postoperative free MNs levels in spot urine and plasma of 14 PPGLs patients were also determined. Creatinine (Cr) concentration was used for the correction of urine volume., Results: The specificity of spot urinary free MNs/Cr in the diagnosis of PPGLs was significantly higher than that of plasma free MNs [normetanephrine (NMN), 98.7% (95.4%-99.8%) vs 93.0% (87.4%-96.6%); metanephrine (MN), 93.6% (88.5%-96.9%) vs 84.5% (77.5%-90.0%)]. Meanwhile, the positive likelihood ratios for spot urinary free NMN/Cr and MN/Cr were 69.21 and 13.29, compared with 12.68 and 5.30 for plasma free NMN and MN, respectively. For the PPGLs patients underwent surgery, the plasma free MNs level appeared an abnormal elevation and yielded false-positive results for some patients. Our findings were validated in an independent cohort, resulting in the specificity of 100% for both urinary free NMN/Cr and MN/Cr, and 97.3% and 83.8% for plasma free NMN and MN, respectively., Conclusions: Spot urinary free MNs/Cr, superior to plasma free MNs, presented a promising biomarker for the diagnosis and prognosis of PPGLs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

It remains a challenge to simultaneously quantify catecholamines and metanephrines in a simple, sensitive and cost-effective manner due to pre-analytical and analytical constraints. Herein, we describe such a method consisting of a miniaturized sample preparation and selective LC-MS/MS detection by the use of second morning spot urine samples. Ten microliters of second morning urine sample were subjected to solid phase extraction on an Oasis HLB microplate upon complexation with phenylboronic acid. The analytes were well-resolved on a Luna PFP column followed by tandem mass spectrometric detection. Full validation and suitability of spot urine sampling and biological variation were investigated. The extraction recovery and matrix effect are 74.1-97.3% and 84.1-119.0%, respectively. The linearity range is 2.5-500, 0.5-500, 2.5-1250, 2.5-1250 and 0.5-1250ng/mL for norepinephrine, epinephrine, dopamine, normetanephrine and metanephrine, respectively. The intra- and inter-assay imprecisions are ≤9.4% for spiked quality control samples, and the respective recoveries are 97.2-112.5% and 95.9-104.0%. The Deming regression slope is 0.90-1.08, and the mean Bland-Altman percentage difference is from -3.29 to 11.85 between a published and proposed method (n=50). A correlation observed for the spot and 24h urine collections is significant (n=20, p<0.0001, r: 0.84-0.95, slope: 0.61-0.98). No statistical differences are found in day-to-day biological variability (n=20). Reference intervals are established for an apparently healthy population (n=88). The developed method, being practical, sensitive, reliable and cost-effective, is expected to set a new stage for routine testing, basic research and clinical applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Urinary vanillylmandelic acid (VMA) is used to diagnose and monitor catecholamine secreting neuroendocrine tumors (NETs). We developed and validated a new liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for determination of serum VMA., Methods: We used serum samples from healthy volunteers (n=314) and patients suspected for NET (n=36). Deuterated VMA as an internal standard was added to samples before solid phase extraction (SPE) and LC-MS/MS analysis. We studied the effects of sample storage, sampling device and a meal on serum VMA and metanephrine concentrations. Diurnal variation and age-dependent reference intervals were established. The diagnostic performance was compared with a urinary HPLC assay for VMA and metanephrines and a serum metanephrine LC-MS/MS assay., Results: Serum VMA is stable at least for one day at +4°C, seven days at room temperature and 98 days at -20°C. Type of sampling device was not critical, but elevated serum VMA occurs after a meal (p = 0.031). Serum VMA increased with age. Therefore, we suggest clinical cut-off values of 62 nmol/L, 80 nmol/L and 108 nmol/L for age groups 18-50 yrs, 51-70 yrs and > 70 yrs, respectively. Comparison between a urinary VMA HPLC assay and serum VMA LC-MS/MS assay showed good correlation., Conclusions: Our LC-MS/MS assay is fast and sensitive and suits well for use in a clinical laboratory. Compared to 24-h urine collection our serum assay enables well controlled sampling and convenient preanalytical steps., (Copyright © 2015 Elsevier B.V. All rights reserved.)

For more than 20 years, measurement of catecholamines in plasma and urine in clinical chemistry laboratories has been the cornerstone of the diagnosis of neuroendocrine tumors deriving from the neural crest such as pheochromocytoma (PHEO) and neuroblastoma (NB), and is still used to assess sympathetic stress function in man and animals. Although assay of catecholamines in urine are still considered the biochemical standard for the diagnosis of NB, they have been progressively abandoned for excluding/confirming PHEOs to the advantage of metanephrines (MNs). Nevertheless, catecholamine determinations are still of interest to improve the biochemical diagnosis of PHEO in difficult cases that usually require a clonidine-suppression test, or to establish whether a patient with PHEO secretes high concentrations of catecholamines in addition to metanephrines. The aim of this chapter is to provide an update about the catecholamine assays in plasma and urine and to show the most common pre-analytical and analytical pitfalls associated with their determination., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: We introduce a novel liquid chromatographic tandem-mass spectrometric method for simultaneous measurements of urinary catecholamines and their free O-methylated metabolites, which we compare to the deconjugated metabolites., Methods: Method performance was validated for recovery, linearity, precision and accuracy, analyte stability, ion suppression and carry over. Results from 53 patients with and 138 volunteers without pheochromocytoma were compared., Results: Analyte recoveries ranged from 60 to 96% and intra- and inter-assay coefficients of variation from 2.7 to 13.2%. The method showed excellent linearity over 3 orders of magnitude with analytical sensitivity sufficient to measure to 1.2 nmol/L. Free O-methylated metabolites were excreted at less than 20% the rates of the deconjugated metabolites, but were easily measureable. Increases in urinary normetanephrine in pheochromocytoma patients relative to volunteers were higher for free than deconjugated metabolites and higher for both than for norepinephrine (10 vs 5.5 vs 3.7 fold increases). In contrast, relative increases in urinary free versus deconjugated metanephrine (2.7 and 3.2) and methoxytyramine (2.1 and 1.9) did not differ, but for methoxytyramine were larger than for dopamine (1.2)., Conclusion: Measurements of urinary catecholamines and their free O-methylated metabolites by our method provide potential advantages over urinary deconjugated metanephrines for diagnosis of pheochromocytoma., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The silent pheochromocytoma, a hidden form of pheochromocytoma, exposes the patient to an increased risk of mortality if the diagnosis is not established on time. Biological diagnosis of pheochromocytoma can be difficult. Catecholamine secretion is dependent on tumor size and a large number of physiological, pharmacological, lifestyle modifications and sampling conditions influence the measurement of urinary and plasma metanephrines. The prevalence of pheochromocytoma is 2% among adrenal incidentaloma smaller than 3 cm (2/3 of tumors). Recent studies suggest the almost zero risk of pheochromocytoma among these tumors if they are hypodense (<10 housefield units) on adrenal tomography. Addison's disease is a pathology affecting about 1 in 8000. Immunopathology is still unknown, but some elements advocated the hypothesis of a predominant cell-mediated immunity in particular Interferon-gamma production by CD4 T lymphocytes in the presence of an epitope from the 21-hydroxylase, as well as IgG1 subtype produced by activated B lymphocytes, autoantibodies do appear to be a simple marker of the disease. Subclinical Addison's disease is defined by the presence of anti-21-hydroxylase autoantibodies, without clinical symptoms. It evolves faster to the clinical phase in young subjects, male, having high levels of autoantibodies and with an initially impaired adrenal function. Dosage of ACTH, plasma renin active, and basal cortisol and after Synacthen allow to discriminate the subjects with low or high risk of evolution and establish an appropriate monitoring., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Background: The quantification of total (free+sulfated) metanephrines in urine is recommended to diagnose pheochromocytoma. Urinary metanephrines include metanephrine itself, normetanephrine and methoxytyramine, mainly in the form of sulfate conjugates (60-80%). Their determination requires the hydrolysis of the sulfate ester moiety to allow electrochemical oxidation of the phenolic group. Commercially available urine calibrators and controls contain essentially free, unhydrolysable metanephrines which are not representative of native urines. The lack of appropriate calibrators may lead to uncertainty regarding the completion of the hydrolysis of sulfated metanephrines, resulting in incorrect quantification., Methods: We used chemically synthesized sulfated metanephrines to establish whether the procedure most frequently recommended for commercial kits (pH 1.0 for 30 min over a boiling water bath) ensures their complete hydrolysis., Results: We found that sulfated metanephrines differ in their optimum pH to obtain complete hydrolysis. Highest yields and minimal variance were established for incubation at pH 0.7-0.9 during 20 min., Conclusion: Urinary pH should be carefully controlled to ensure an efficient and reproducible hydrolysis of sulfated metanephrines. Synthetic sulfated metanephrines represent the optimal material for calibrators and proficiency testing to improve inter-laboratory accuracy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Determination of urinary metanephrines is requested frequently for the differential diagnosis and monitoring of pheochromocytoma. Although numerous methods have been developed, interferences are common and hinder most available assays. This study describes the development, validation and implementation of a reliable high-throughput LC-MS/MS method for the measurement of metanephrine and normetanephrine in urine. Metanephrine and normetanephrine were isolated from urine samples subjected to acid hydrolysis using solid phase extraction on a mixed mode cation exchange sorbent in 96-well format. The extracts were injected directly onto a Restek perfluorophenyl column and separated isocratically in 0.2% formic acid in 5% methanol with a gradient cleanout step to 50% methanol. Detection was accomplished using an API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Data were acquired in multiple reaction monitoring mode. Three transitions were monitored for metanephrine and its deuterated internal standard; two transitions were monitored for normetanephrine and its deuterated internal standard. Two quantification methods were used to address metanephrine interferences without reducing throughput. The method was linear to 15,000 nmol/L. The limits of detection and quantification were 2.5 and 10 nmol/L, respectively. Within run, between-day and total imprecision values were at or below 1.9%, 2.5% and 2.7% for both analytes. The method correlated well with our previously used GC-MS method. Injection-to-injection time was 6 min. The validated LC-MS/MS method for measurement of metanephrine and normetanephrine in urine specimens was placed into service in August 2010 and has performed exceptionally well., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Paragangliomas are rare tumors arising from extraadrenal chromaffin cells. We report a 43-year-old man who presented with abdominal pain. An abdominal computed tomography scan revealed a large retroperitoneal mass. During an endoscopic biopsy of this tumor, the patient experienced marked hemodynamic fluctuations with tachycardia and high blood pressure, and an extraadrenal pheochromocytoma was suspected. Measurements of plasma and urinary catecholamines and urinary total metanephrines ruled in the diagnosis. Echocardiography disclosed acute myocardial dysfunction that returned to normal after surgical resection of the paraganglioma. This report also underlines the importance of the anesthetic preparation and monitoring around the surgical procedure and the need of a long-term follow-up to detect malignant paraganglioma in the absence of histological criteria of benign tumor., (Copyright © 2010 Société nationale française de médecine interne (SNFMI). Published by Elsevier SAS. All rights reserved.)

The silica monolith with ionizable silanol groups and large surface area was found able to function as an offline cation exchange solid phase extraction (SPE) cartridge for extracting polar analytes. The prepared cartridge was housed in a 2-mL syringe fixed over a SPE vacuum manifold. The unique property of this silica monolithic cartridge was demonstrated by extracting epinephrine, normetanephrine and metanephrine from urine samples. These analytes were chosen as model compounds for testing because of their high hydrophilicity, and being candidates monitored for clinical diagnosis. The extracted analytes, after concentration and reconstitution were then quantitated by high-performance liquid chromatography coupled to mass spectrometer (HPLC/ESI/MS). Multiple reactions monitoring was carried out with transitions: 184-->107, 184-->134 and 198-->148 for analyzing epinephrine, normetanephrine and metanephrine, respectively. The limit of detection was 3 ng/mL for metanephrine and 5 ng/mL for normetanephrine and epinephrine. The relative standard deviations of measurements ranged from 2 to 10%. The sorbent offered good linearity with coefficient of determination (r(2))>0.99, over a concentration range of 20-200 ng/mL. The relative recoveries ranged from 60 to 67%, 55 to 59% and 99 to 105% for epinephrine, normetanephrine and metanephrine, respectively. The prepared cartridge had shown potential and was found robust in extracting the polar analytes repeatedly without any significant loss in efficiency., (Copyright 2010 Elsevier B.V. All rights reserved.)

Introduction: The paraganglioma is a rare tumour that can sometimes develop near to the pancreatic gland and can occasionally mimic an intra pancreatic mass. Invasive investigations with puncture or primary surgical resection must be avoided because of the risk of vital complications., Exegesis: We report on the case of a 41 years old woman presenting with a mass apparently affecting the pancreatic gland. She also presented with malaises. Paraganglioma was suspected because of the malaises and the blood pressure crises. The diagnosis was permitted by the findings of high urinary metanephrine levels. So the tumour was resected without previous fine needle aspiration and with specific anaesthetic measures. No complication was observed., Conclusion: When a patient presents with a pancreatic mass, physician must carefully take into account the possibility of a paraganglioma particularly when malaises are present.

The term 'metanephrines' is used to indicate the two catechol 3-O-methylated metabolites of epinephrine (E) and norepinephrine (NE): metanephrine and normetanephrine (NMN). The corresponding 3-O-methylated metabolite of dopamine is usually referred to as 3-methoxytyramine rather than 3-methoxydopamine and is not generally considered a "metanephrine". O-Methylation occurs outside the sympathetic neuron and neuroeffector junction. Metanephrines are products of the enzyme catechol-O-methyltransferase (COMT). Subsequent conjugation with sulfate or deamination by monoamine oxidase (MAO) followed by reduction to vanilmandelic acid (VMA) facilitates urinary excretion. For the clinician, measurement of normetanephrine provides an index of norepinephrine released during sympathetic nervous system activity, whereas metanephrine concentration provides an indication of adrenal medullary metabolism of epinephrine prior to its discharge into the circulation. Plasma epinephrine concentration is the preferable index of adrenal medullary epinephrine discharge. Pheochromocytomas, with their protean clinical manifestations, may be diagnostic challenges, but assay of metanephrines, especially plasma metanephrine, can be particularly helpful in diagnosis. These COMT metabolites may also help in elucidation of still undiscovered genetic and acquired disorders of catecholamine metabolism.

A method successfully coupling capillary electrophoretic separation to time-of-flight mass spectrometric (TOFMS) detection for the simultaneous analysis of catecholamines (dopamine, norepinephrine, and epinephrine) and their O-methoxylated metabolites (3-methoxytyramine, normetanephrine, and metanephrine) is described. The inner capillary wall was coated with polyvinyl alcohol in order to obtain baseline resolution of catecholamines and metanephrines and to ensure reproducibility without extensive restorative washing of the capillary. Using electrokinetic injection, detection limits of 0.3 microM for dopamine and norepinephrine, 0.2 microM for 3-methoxytyramine and normetanephrine, and 0.1 microM for epinephrine and metanephrine were achieved with standard solutions. The usefulness of this approach was demonstrated by applying the developed method to the analysis of a spot collection of human urine from a healthy volunteer. The catecholamines and metanephrines were removed from the urine samples and preconcentrated by simultaneous SPE on cation-exchange sorbents. The recoveries of all analytes, with the exception of epinephrine (75%), were over 80%. Catecholamines and metanephrines in the urine samples were quantitated using 3,4-dihydroxybenzylamine as an internal standard. Submicromolar concentrations, consistent with the catecholamine and metanephrine levels reported for normal human urine, were detected., (Copyright 2002 Elsevier Science B.V.)

Introduction: Pheochromocytoma is a catecholamine-secreting neoplasm of chromaffin tissue. It is a rare disease. Biochemical tests should be performed only in patients at high risk of pheochromocytoma, and an imaging procedure only in those with positive biochemical tests., Current Knowledge and Key Points: The most specific and sensitive diagnostic test for the disease is the determination of plasma or urinary metanephrines. The tumor can be located by computerized tomography, magnetic resonance imaging, and specific scintigraphy. Ten to 20% of pheochromocytomas result from hereditary diseases, including multiple endocrine neoplasia type 2, von Hippel Lindau disease, and neurofibromatosis 1. Familial cases are diagnosed earlier, and are more frequently bilateral and recurring than sporadic cases. About 10% of the cases are malignant either at first operation or during follow-up, malignancy being diagnosed by the presence of lymph node, visceral or bone metastases. The probability of a recurrence is positively correlated with the urinary excretion of metanephrines and tumor size. Recurrences are more frequent in cases with ectopic tumors and in those with a low plasma epinephrine to total catecholamine ratio. Patients, especially those with familial tumors or low epinephrine secretion, should be followed-up indefinitely., Future Prospects and Projects: Treatment for malignant recurrences includes surgery, therapeutic embolization, chemotherapy, and the application of therapeutic doses of metaiodobenzylguanidine. Metyrosine, phenoxybenzamine, or somatostatin analogs may help to control blood pressure and to alleviate symptoms in patients with malignant pheochromocytoma; however such a treatment has no antiproliferative effect.

A convenient HPLC-fluorescent assay of norepinephrine (NE), epinephrine (E), dopamine (DA) and their 3-O-methylated metabolites, normetanephrine (NM) and metanephrine (MN) was developed. These analytes were coupled to 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) before assays. Results showed that using a linear gradient elution, peaks of FMOC-NE, FMOC-E, FMOC-DA, FMOC-NM, FMOC-MN and FMOC-DHBA (3,4-dihydroxybenzylamine, internal standard) were simultaneously resolved within 40 min. Optimization of the chromatographic and derivatization conditions, and validation of the assay were further discussed in the paper. The structures of these derivatives were confirmed by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The molecular ions [M+H]- of FMOC-NE, FMOC-E, FMOC-DA, FMOC-NM and FMOC-MN were m/z 836, 850, 820, 628 and 642, respectively. Based on these findings, the FMOC-derivatives of metanephrines and catecholamines were confirmed to be bi-substituted and tri-substituted respectively at the amino and catechol functional groups. Finally, the assay was successfully applied to the measurement of urinary E, DA, NM and MN after direct derivatization and simple cleaning.

It is essential to validate an assay with regards to its precision, accuracy, reproducibility, selectivity and robustness. In pharmaceutical research, in addition to these requirements, an assay is often required to be proven beyond doubt to be stability-indicating. A stability-indicating assay is one that can accurately and selectively differentiate an intact drug from its potential decomposition products. An assay that fails to meet this requirement would produce stability data that are inaccurate and misleading. This concept could be applied to other fields of research, such as clinical laboratory chemistry where accurate and reliable clinical data are imperative in the diagnosis and following-up of diseases. In this study, we employed an amperometric HPLC assay developed for the measurements of urinary catecholamines and metanephrines to illustrate the validation process of this assay with regards to its stability-indicating capacity. The implications of this study are also discussed.

This study investigated the relationship between biological and psychological processes in the recovery phase of depression during treatment with cognitive therapy. In particular, we tested the hypothesis that biological and psychological processes are independent; the hypothesis that they are related in a linear or sequential process; and the hypothesis that there is a circular relationship between them. 35 depressed patients completed a 12 week program of cognitive therapy. Changes in measures of negative thinking and amine dysfunction over the course of therapy were compared for those who improved and those who did not. There was a significant relationship between improvement in symptoms and change for ATQ scores, DAS scores, and epinephrine levels. There was significant change in metanephrine levels during therapy which was not related to improvement. The changes were not consistent across indices. Although the results did not provide definite support for any of the four models of the relationship between biological and psychological variables during recovery from depression, the finding that there was some change in some of the indices of amine dysfunction during cognitive therapy indicates that the interface between biological and psychological processes in depression should continue to be studied.

A simplified liquid chromatographic method for the simultaneous determination of free or total catecholamines and methoxycatecholamines in rat and pig urine is presented. The extraction procedure involves a two-stage batch extraction, with successive adsorption on cation- (catecholamine elution) and anion-exchange columns (methoxycatecholamine elution). The column eluates are successively monitored by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection. The proportion of conjugates for each compound was assessed in both species, through the comparison of concentrations with or without hydrolysis pretreatment. Conjugates were found to account for a small fraction of total catecholamines and methoxycatecholamines excretion (0 to 35%). The free fraction of each compound was highly correlated with the total amount. Furthermore, the hydrolysis procedure leads to partial degradation of metanephrine (25%) and to the production of compounds giving artefactual peaks. Thus, we do not recommend hydrolysis of rat and pig urines for catecholamine and methoxycatecholamine determination.

A patient with preexisting multiple endocrine neoplasia type IIA had normal 24-hour urinary metanephrine and vanillylmandelic acid excretions before and during pregnancy. After a benign prenatal course, the patient had a term spontaneous vaginal delivery. Multiple endocrine neoplasia type IIA antedating pregnancy may be associated with a normal obstetric outcome in the absence of a phenochromocytoma.

The present paper compares a manual high-performance liquid chromatographic (HPLC) method with electrochemical detection (ED) with an on-line HPLC-ED method for the determination of free catecholamines (CAs) (norepinephrine, epinephrine, and dopamine) in urine. The manual method involves a two-stage extraction procedure with home-packed columns filled with weakly acidic cation-exchange resin followed by adsorption onto alumina while the on-line method uses a strong cation-exchange polymer as precolumn material which allows the simultaneous determination of free CAs and metanephrines (MNs) in urine. Reference control urine samples analysed with both methods gave levels of CAs within the acceptable reference ranges. Dihydroxybenzylamine was used as an internal standard in both methods and the separation was performed by ion-pairing reversed-phase HPLC.

Presently, the diagnosis of pheochromocytoma rests upon the determination of urinary catecholamines, and specially metanephrines which offer 100% sensitivity and 97% specificity. The 24 hours urine collection is problematic for practical reasons and patient compliance. Serum metanephrines determination by HPLC (High Performance Liquid Chromatography) avoids these difficulties making the investigation easier. We report the results of serum metanephrine (MN) and normetanephrine (NMN) in 10 patients presenting with a pheochromocytoma histologically confirmed and without renal insufficiency, compared to 18 control healthy subjects, 33 patients suffering from hypertension and 11 patients with terminal renal failure on haemodialysis. The mean value of total metanephrines (MN + NMN) for the patients presenting with a pheochromocytoma is 326 +/- 206 nmol/l (range 76 to 718). In the control group without hypertension this value is 6.8 +/- 2.9 nmol/l (range 0.5 to 10.7) and for the control group with hypertension 25 +/- 27 nmol/l (range 7 to 138). For the patients with renal failure, the mean value is 471 +/- 201 nmol/l (range 188 to 920). Thus total metanephrines serum level values were found higher in the patients with a pheochromocytoma than in those with or without hypertension, but they were not discriminatory when the cases of renal insufficiency were included. CONCLUSION In case of a normal renal function, serum metanephrines determination for the diagnosis of pheochromocytoma gives, above a threshold value of 50 nmol/l, a sensitivity of 100% and a specificity of 94%.

We developed a method for simultaneous quantification of the urinary 3-O-methylated catecholamine metabolites 3-methoxytyramine, normetanephrine and metanephrine by stable isotope-dilution ammonia chemical ionization mass fragmentography. Prepurification of lyophilized samples was done by simultaneous deconjugation and pentafluoropropionylation, followed by extraction and rederivatization. Compared with our previously described method, based on acid hydrolysis, alkaline extraction, derivatization and electron-impact mass fragmentography, the present method was found to be less laborious, more sensitive and presumably more accurate. New urinary excretion values were established for apparently healthy adults. The present prepurification method may prove applicable for profiling of a variety of naturally occurring mono-, di- and polyamines in biological samples.

Many clinical features of the neuroleptic malignant syndrome suggest that sympathetic nervous system hyperactivity is involved in the pathophysiology of this disorder. Only a few studies have examined levels of catecholamines or their metabolites in patients with NMS; results so far have been inconclusive. In the present study urinary catecholamine metabolites obtained during the course of NMS were studied with respect to frequently reported signs and symptoms of NMS. The principal findings are that (1) elevated urinary catecholamines and metabolites are a frequent but inconstant feature of NMS; (2) it is likely that sympathetic nervous system hyperactivity contributes to the picture of fulminant NMS; and (3) the role of the adrenal medulla in producing excess catecholamines during NMS is uncertain.

This report describes a high-performance liquid chromatographic method with electrochemical detection for the simultaneous quantitation of urinary metanephrine, normetanephrine and 3-methoxytyramine. This method, which involves manual dual-column purification steps for the routine determination of urinary metanephrines, is compared with the previously used spectrophotometric Pisano method and an on-line sample preparation procedure, where the automated sequential trace enrichment (ASTED) apparatus is used for the column-switching procedure. In order to automate the metanephrine assay, the enrichment technique was evaluated against the reference chromatographic method. Bio-Rad urine controls gave coefficients of variation of less than 9% at all levels for the reference method. Values of less than 19% were found in the reference range with the enrichment method, and the recovery of 3-methoxytyramine was also too poor to be measured in normal concentrations. The linearity of both methods is sufficient to determine pathological levels of these biogenic amines. Future developments should be focused on decreasing the variation of between-day assays in an on-line, automated procedure.

A simple routine method is described for simultaneous assay of total urinary norepinephrine, epinephrine, dopamine, normetanephrine and metanephrine. An internal standard of 3,4 dihydroxybenzylamine is added to the diluted urine and acidic hydrolysis of the conjugates is followed by reverse-phase HPLC separation and coulometric detection in the redox mode. The method is rapid and precise and it has a broad linear working range for all substances making it suitable for clinical analysis. Examples are shown of excretion patterns of catecholamines and metanephrines for healthy subjects and depressed patients.

A noninvasive method is described for assessing the disposition of NE produced by peripheral sympathetic neurons. Total NE production is estimated from integrated NE metabolite excretion. The rate at which NE enters the bloodstream (apparent NE secretion) is estimated by isotope dilution analysis. The rate at which NE is metabolized locally is calculated by subtracting apparent NE secretion from NE production. In 10 normal volunteers, basal NE production by the peripheral sympathetic nervous system was 11.1 +/- 1.0 nmol/m2/min. Apparent NE secretion, by contrast, was only 2.4 +/- 0.18 nmol/m2/min. Our data thus demonstrate that in normal individuals under basal conditions, at least 78% of the NE produced by the sympathetic nervous system undergoes local metabolism and that no more than 22% of NE diffuses into the circulation.

Results of 1414 measurements of urinary excretion of free and conjugated (total) metanephrine and normetanephrine (total metanephrines, tMN) and of 1630 measurements of vanilmandelic acid (VMA) excretion were analysed according to the month of sample collection. These collections had been carried out as a part of a study of hypertension at out institute over the 5-year period between 1970 and 1975. The means and scatters of tMN were significantly varied depending on the season of the year; the mean during 3 months of summer was 380 mug/day and the mean for 3 winter months 800 mug/day. The scatter of tMN in winter months was wider. A minor seasonal variation was observed for VMA: the means of summer and winter months were 3.8 mg/day and 4.5 mg/day, respectively. The results suggest a seasonal difference of catecholamine catabolism.

A sensitive and specific direct analysis of urinary normetanephrine (NMN) and metanephrine (MN) was achieved utilizing reversed-phase high performance liquid chromatography and electrochemical detection. Individual specimens from "control" subjects and those with pheochromocytoma were hydrolyzed and the metanephrines separated from other urinary constituents by elution with ammonia from a Dowex CG-50 resin. Chromatographic peaks were identified by retention behavior, co-chromatography with reference compounds, ratio of responses at various oxidation potentials and stopped-flow UV spectra of the collected fractions. The NMN and MN content for the control subjects was between 0.086 and 0.21 (mean - 0.14) microgram/mg creatinine and 0.012 and 0.092 (mean = 0.039) microgram/mg creatinine, respectively. The values for subjects with pheochromocytoma varied from 1.5 to 27.5 (mean = 9.9) microgram/mg creatinine for NMN and 0.10 to 1.60 (mean = 0.86) microgram/mg creatine for MN. The patient with ganglioneuroma had an NMN of 4.1 and an MN of 0.80 microgram/mg creatinine. While this method permits discrimination between those patients with pheochromocytoma and the overwhelming majority of hypertensive patients, it may ultimately be further extended to separate normal subjects from those with more subtle derangements in catecholamine metabolism.

A simple method for the simultaneous analysis of normetanephrine, metanephrine and 3-methoxytyramine (both free and conjugated) in human urine by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection has been developed. Existing methods have been optimized for extraction by study of analytical parameters. The hydrolysed urines are purified and concentrated by successive passages on two ion-exchange resins and ammoniacal elution to eliminate interference from pigments or related chemical compounds. The methoxyamines are separated by high-performance liquid chromatography on a reversed-phase column. Detection and quantitation are achieved with an electrochemical detector using a vitreous carbon electrode. Samples can be injected at 25-min intervals. Reference values of adults and children are given.

Urine after the addition of phenylephrine as internal standard is hydrolyzed and passed through a disposable BondElut SCX column. After washing the column, metanephrines and the internal standard are eluted with dilute ammonia. The eluate is treated with periodate and extracted with toluene. The toluene layer is collected and phenols are extracted into dilute tetramethylammonium hydroxide. An aliquot of the aqueous layer is chromatographed on a nonsilica resin base reversed phase column with an alkaline mobile phase. The peaks are detected by an absorbance detector at 350 nm. There is a baseline separation of vanillin formed by metanephrines and of m-hydroxybenzaldehyde formed by phenylephrine. The procedure is linear from 0.2 mg to 10 mg of metanephrine per liter of urine. The procedure has a high degree of specificity as the commonly prescribed antihypertensive drugs and their metabolites do not interfere.

A semi-automated fluorimetric method for the quantitation of urinary catecholamines (dopamine, noradrenaline and adrenaline) combined with manual methods for the quantitation of their metabolites (homovanillic acid, normetanephrine, metanephrine and vanillylmandelic acid) is described. It provides a sensitive and reproducible analytical technique for routine use. Values obtained in healthy adults are given.

A sensitive and specific assay for free and sulfoconjugated normetanephrine, metanephrine, and 3-methoxytyramine was achieved by tetraphenylboron complexing and by high-performance liquid chromatography with electrochemical detection. Sulfoconjugated metabolites were hydrolyzed by sulfatase. Complexing the 3-O-methylated catecholamines with tetraphenylboron followed by their extraction in either and reextraction in dilute hydrochloric acid succeeded in partially purifying the sample without the use of a prepacked column. Detection by electrochemical technique additionally eliminates unoxidable components in the urine. The relatively high sensitivity of this method permits measurement of metabolite concentrations as low as 10 ng/ml, allowing for the first time the determination of free 3-O-methylated catecholamines in human urine. Hydrolysis by sulfatase specifically identifies the form of the conjugated metabolite. The mean total (free plus sulfoconjugated) values of normetanephrine, metanephrine, and 3-methoxytyramine obtained from 25 normal volunteers were 214, 102, and 227 micrograms/day, respectively. Values were greatly increased in patients with pheochromocytoma. The degree of sulfoconjugation varied, and each individual metabolite was highest with normetanephrine (86%). This method is sensitive, rapid, simple, and can be easily standardized for clinical investigation of pheochromocytoma.

Urinary excretion of DA, DOPAC, 3-MT, HVA, NMA, MA, VMA and 5-HIAA were studied in 33 parkinsonian patients treated with 1.5-7.5 g of levodopa daily for up to six months and in 30 patients receiving levodopa (800-1,000 mg) combined with a dopa decarboxylase inhibitor, benserazide (200-250 mg). Basal urinary excretions were within normal limits except for that of 3-MT which was significantly lower in parkinsonian patients as compared to controls. Levodopa induced an increase of about 400 fold in urinary DA; DOPAC was increased about 300 fold, 3-MT only about 70 fold, but HVA about 300 fold. Urinary NMA and MA did not change but VMA was increased significantly. On the other hand, urinary 5-HIAA was significantly decreased. The amounts of excreted DA and its subsequent metabolities were increased with the continuation of treatment, suggesting inductive phenomena in enzyme systems. During combined treatment with levodopa and benserazide urinary DA was increased, but only to about one tenth the extent seen with levodopa alone. The excretion of DOPAC was about one 20th, of 3-MT about one fourth and of HVA one 25th that seen during levodopa treatment. No signs of enzyme induction were seen. NMA was lowered significantly but MA remained unchanged. VMA was increased and significantly more than during therapy with levodopa alone. 5-HIAA was again significantly decreased and the decrease was significantly greater than that seen with levodopa alone. Some statistically significant correlations were seen between the excretions of NMA, MA and VMA and cardiovascular side effects, indicating an affection on the NA-ergic system by levodopa treatment. Significant correlation between 5-HIAA excretion and clinical improvement of tremor during levodopa treatment may suggest that participation of 5-HT in the mechanism of tremor.

Patients with essential hypertension were treated for four weeks with the alpha- and beta-adreno-receptor blocking agent labetalol. Urinary excretion of total catecholamines, metanephrine plus normetanephrine and vanillylmandelic acid was measured with various methods before and during treatment. An unidentified substance interfering with the fluorimetric method for catecholamines and the photometric assay for metanephrines caused falsely high values of those substances. Using appropriate methodology no changes of total catecholamines, metanephrine plus normetanephrine and vanillylmandelic acid excretion were found after labetalol therapy. Our findings are important in preventing errors in the diagnosis of pheochromocytoma as well as in the evaluation of the effects of labetalol on the sympathetic nervous system in man.

A method is described which extends a high performance liquid chromatographic assay for urinary catecholamines to the assay of urinary metanephrines. The amines are separated from urine after acid hydrolysis of conjugates by ion-exchange chromatography, and then further purified by solvent extraction. The final extracts are suitable for direct HPLC assay using the endogenous fluorescence of the amines for detection. Twenty-four hour excretion of the amines in 35 hospital in-patients was found to be (mean and range: 169 micrograms/24 h normetanephrine (39-423), 102 micrograms/24 h metanephrine (19-290) and 138 micrograms/24 h 3-methoxytyramine (32-234)).

It is possible to separate metanephrine and normetanephrine from urine for the automated fluorimetric routine determination by means of a combination of liquid-liquid partition with ethylacetate and clean-up of the extract through Amberlite XAD-4. The differentiated fluorimetric determination is based on the oxidation with potassium hexacyanoferrate (III) in the presence of zinc ions and different pH values in an autoanalyzer. No quenching effects were observed. The recovery yields were in the range of 60%, the relative standard deviation being less than 10%.

A high-performance liquid chromatographic method has been developed for the determination of metapramine in human plasma and urine. After selective extraction and derivatization with dansyl chloride, metapramine and the internal standard (maprotiline) are chromatographed on a reversed-phase LiChrosorb RP-18 column using a mixture of water--acetonitrile (35:65) as mobile phase. The eluted compounds are measured using a fluorescence detector. The detection limit of the assay for plasma and urine samples is about 1 ng/ml. The method has been successfully applied in a pharmacokinetic study following intravenous administration of 35 mg of metapramine.

A method is described for quantitative analysis of urinary free and conjugated catecholamines and metanephrines. The compounds are isolated from the urine by cation exchange on Amberlite CG 50. Separation is performed by ion-pair reversed-phase high-performance liquid chromatography. For detection of the amines their native fluorescence emitted at 313 nm on excitation at 285 nm was monitored. There was good separation of the compounds of interest, while interference by exogenous compounds other than the catecholamines or metanephrines was minimal. The method is rapid and precise, and it has a broad linear working range for all six DOPA metabolites, making it suitable for clinical analysis. Reference values for children of 0-16 years were established. Examples are shown of excretion patterns of DOPA metabolites from patients with different types of neurogenic tumour.

A simple high-performance liquid chromatographic procedure was developed for the simultaneous determination of catecholamines and metanephrines in urine. One-step sample preparation was achieved with Bio-Rex 70 ion-exchange resin. The extract was assayed on a C18 reversed-phase column. Dihydroxybenzylamine was used as an internal standard. The eluent was monitored by an electrochemical detector with an oxidation potential of +0.85 V. The use of 1-heptanesulphonic acid in the mobile phase permitted the separation of norepinephrine, epinephrine, dopamine, normetanephrine and metanephrine in a single chromatogram. The corresponding detection limits were 5, 9, 14, 10 and 30 nmol/l, respectively. For the between-day precision, the coefficients of variation at physiological and pathological concentrations were less than 11%. Compounds with similar chemical structures and drugs commonly prescribed for the treatment of hypertension were assayed and found not to cause interferences in the chromatogram. The assay is reliable and is suitable for the analysis of clinical specimens. Reference values were established for normotensive Chinese patients with no neurological or endocrine disorders and also for patients suffering from essential hypertension.