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Evidence is accumulating which may result in plasma free metadrenalines (PMets) becoming the preferred test for diagnosing phaeochromocytoma and paraganglioma. Moreover, increased availability and benefits over other analytical methods like liquid chromatography with electrochemical detection and immunoassay are causing liquid chromatography tandem mass spectrometry (LC-MS/MS) to become the method of choice for PMet measurement. This review explores the evidence-base supporting this, and summarises published LC-MS/MS analytical methods for PMet analysis. Key aspects of methods (including SPE extraction, HILIC chromatography, MRM MS-detection and standardisation) are discussed. Common causes of analytical interference (e.g. ion suppression/enhancement, ionic cross talk, in source transformation and isobaric interferences) are outlined to illustrate the importance of sample purification and chromatographic resolution. The importance of supine, fasting sampling and Bayesian interpretation against supine, fasting reference intervals are explained, as well as the importance of age-specific reference intervals for normetadrenaline. Confounding factors like diet, drugs, renal function and acute illness are explored, along with potential strategies to address these (e.g. CKD-specific reference intervals)., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Background: Availability of appropriately established reference intervals for biochemical tests can be troublesome in pediatrics. Here we establish age-specific continuous reference intervals for catecholamine O-methylated metabolites in children evaluated for catecholamine producing tumors, particularly younger children with suspected neuroblastoma., Methods: Plasma concentrations of 3-methoxytyramine, normetanephrine, metanephrine, and 3-O-methyldopa were analyzed by liquid chromatography tandem mass spectrometry in 533 children aged 2 days to 18 years., Results: Concentrations of plasma free normetanephrine, 3-methoxytyramine and 3-O-methyldopa were higher in neonates up until six months of age, but thereafter declined steeply to levels after one year that were <38% those of neonatal concentrations and to further lower concentrations in teenagers that were <23% those in neonates. In contrast, concentrations of plasma free metanephrine showed a reciprocal pattern with 50% lower concentrations in infants below one year compared to later in childhood., Conclusion: The dynamic reciprocal changes in plasma concentrations of normetanephrine, 3-methoxytyramine and 3-O-methyldopa compared to metanephrine during early childhood suggest underlying developmental changes in extra-adrenal and adrenal chromaffin tissue that must be considered for pediatric reference intervals, particularly in infants. With such reference intervals at hand, biochemical testing for catecholamine producing tumors in young children is substantially improved., (Copyright © 2019 Elsevier B.V. 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.)

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.)

Background: Total (i.e. free+sulfated) metanephrines in plasma is a biomarker for the diagnosis of pheochromocytoma/paraganglioma. Sulfated metanephrines must be completely deconjugated by perchloric acid hydrolysis or sulfatase treatment prior to analytical measurement to enable quantification by current techniques. In this report, we compare the yield and efficiency of both methods., Methods: The deconjugation rate of synthetic sulfated metanephrines (normetanephrine (S-NMN), metanephrine (S-MN) and methoxytyramine (S-MT)) spiked in charcoal-stripped plasma was determined by boiling perchloric acid and compared to sulfatase treatment. Total plasma metanephrines (MN, NMN and MT) were also determined in patient samples by both methods., Results: The complete deconjugation of sulfated metanephrines is achieved after 30 min incubation with 0.1M boiling perchloric acid or upon sulfatase treatment. Ten minutes of acid hydrolysis (gold-standard) leads to a 30% underestimation of metanephrine concentrations. The enzyme hydrolysis is time and amount of sulfatase dependent. The rate of hydrolysis is analyte-dependent (MT>>NMN>MN), although it must contain at least 0.8 U/ml of sample. The Deming regression curves comparing acid versus enzyme hydrolysis on patient samples assessed that both methods gave similar unbiased concentrations., Conclusion: Enzyme and acid treatments are equivalent and efficient for removing sulfate from metanephrines as long as the optimal protocol is used for each method. However, the gold standard method for acid hydrolysis at 10 min established more than 20 years ago was not satisfactory regarding the hydrolysis of metanephrines in plasma., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Catecholamines and their metabolites, metanephrines, are produced excessively in pheochromocytoma tumors of the chromaffin cells. Increased concentrations of these compounds produce symptoms and allow for clinical evaluation of disease. Historically, screening for such tumors by determination of catecholamines and metabolites in urine yielded false negative results in individuals with a genetic predisposition for the disease and those with paroxysmal hypertension. Analysis of metanephrines in plasma, however, is of decisive diagnostic importance. This test exhibits high sensitivity and specificity for the analytes produced by tumors., Methods: Plasma proteins are removed by solid phase extraction. Chromatographic isolation of the analytes and stable isotope internal standards is achieved by elution on a HILIC column connected to a UPLC MS/MS system. Metanephrines are measured using multiple reaction monitoring with an electrospray source operating in positive ion mode., Results: The method was validated for linearity, limit of quantification, accuracy, and precision. The method was accurate and correlated well to a comparison HPLC method. Potential interferences were evaluated., Conclusions: Results from this LC-MS/MS assay enable clinical diagnosis of pheochromocytoma and aid in monitoring treatment outcomes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Plasma free metanephrines have proved a highly sensitive biochemical test for the diagnosis of pheochromocytoma. We have developed and validated a simple, LC-MS/MS method to determine plasma metanephrines and compared the diagnostic efficacy of the method with an enzyme immunoassay procedure in 151 patients, 38 with histologically confirmed pheochromocytoma., Methods: Off-line solid phase extraction in a 96-well plate format was used to isolate metanephrines from 100-microL of plasma, followed by rapid separation with hydrophilic interaction chromatography. Mass spectrometry detection was performed in multiple-reaction monitoring mode using a tandem quadrupole mass spectrometer with positive electrospray ionization., Results: Detection limits were <0.1nmol/l with method linearity up to 23.0nmol/L for normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT). Method comparison with an automated LC-MS/MS yielded Deming regression slopes of r=0.94 for NMN, r=0.98 for MN and r=0.94 for 3-MT. Method comparison with enzyme immunoassay revealed regression slope of r=1.28 (NMN) and 1.25 (MN) with values approximately 25% lower than LC-MS/MS. Plasma metanephrines by LC-MS/MS identified all 38 patients with phaeochromocytoma compared with 36 cases by immunoassay., Conclusions: Plasma metanephrines measured by LC-MS/MS are a reliable and sensitive test for the biochemical detection of pheochromocytoma., (2010 Elsevier B.V. All rights reserved.)

Background: Intravenous epinephrine (EPI) is used as a pharmacologic agent to acutely treat patients in cardiac arrest. Unfortunately, there have been several homicide cases where hospitalized patients died due to a purposeful overdose of epinephrine. We measured plasma epinephrine metabolites (metanephrine, MET, and normetanephrine, NMET) to determine if exogenous epinephrine can be distinguished from endogenous epinephrine concentrations in a controlled animal study., Methods: Rabbits were subjected to three different protocols. In the physiologic stress group (n=8), rabbits were immobilized for 30 min in a restraining tube. In the sub-lethal dose (n=9), 0.01 mg/kg of epinephrine was injected into anesthetized rabbits. In the lethal dose group (n=8), 1.0 mg/kg of epinephrine was administered into anesthetized rabbits. Blood was collected at regular intervals for up to 480 min. The plasma metanephrine and normetanephrine concentrations were measured by liquid chromatography/mass spectrometry and the serum cortisol concentrations by immunoassay., Results: Serum cortisol and plasma metanephrine and normetanephrine concentrations increased in the stressed animals during immobilization demonstrating the endogenous stress model. Following a sub-therapeutic epinephrine dose, plasma metanephrine increased while plasma normetanephrine decreased. The peak plasma metanephrine concentrations were similar to the concentrations observed in the stressed animals; however, the ratio of plasma metanephrine to normetanephrine was significantly different. In the lethal epinephrine dose, both the plasma metanephrine concentrations and ratio of metabolites were significantly greater than those observed in the endogenously stressed animals., Conclusions: The ratio of plasma metanephrine to normetanephrine is the best marker to determine the presence of exogenous therapeutic and lethal epinephrine administration. However, there were limitations to the study design that could alter these conclusions.

Plasma 3-O-methylated catecholamines, i.e. 3-methoxytyramine, normetanephrine and metanephrine, were separated from catecholamines by passing through alumina and further purified by adsorbing on weakly acidic resin and Amberlite XAD-4. The amines were trifluoroacetylated and determined by gas chromatography or mass fragmentography. Tracer quantities of tritiated 3-MT, NMN or MN were used as internal standards for total recovery estimations. The contents of 3-O-methylated catecholamines in the plasma of normal persons and patients with hyperthyroidism, hypertension, neuroblastoma and pheochromocytoma were measured.