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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.)

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.)

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.)

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.)

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.

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.

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.

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 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.