Urinary Free Metanephrines Research Articles

Development and validation of a simple, fast and sensitive liquid chromatography-tandem mass spectrometry method to establish reference intervals for 24-h urinary free normetanephrine, metanephrine and methoxytyramine

ObjectiveTo develop and validate a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect urinary free metanephrines and methoxytyramine, establishing reference intervals. MethodsUrine samples were diluted with isotope internal standard solution, then analyzed directly using tandem mass spectrometry with multiple reaction monitoring measurement and electrospray ionization source in positive ion mode. Analytical parameters including linearity, lower limit of quantitation, imprecision and accuracy of the method were evaluated. The reference intervals for urinary catecholamine metabolites were established by analyzing 24-h urine samples collected from 81 apparently healthy volunteers. ResultsThe analytical times for MN, NMN, and 3-MT were at 2.79, 2.80, and 2.74 min, respectively. The method displayed excellent linearity (r > 0.99) in the range of 1-1000 ng/mL, with lower limits of quantification (LLOQ) at 0.50 ng/mL for MN and NMN, and 0.25 ng/mL for 3-MT. The method's intra-day and inter-day imprecisions were less than 8 %. The method recovery ranged from 96.8% to 105.8 % for MN, 89.7%–106.4 % for NMN, and 93.5%–106.2 % for 3-MT. No carry-over was observed during the analysis of all analytes. The LC-MS/MS method was used to establish reference intervals in 24-h urine samples from 81 apparently healthy volunteers. There was no association of sex with urinary free metabolites. ConclusionThis study established a novel, fast and sensitive LC-MS/MS method for determining urinary free catecholamine metabolites, which could facilitate screening and diagnosis for catecholamine-related tumors more conveniently and quickly.

FRI188 Exploratory Genomic Analysis From A Brazilian PMAH-Family With ARMC5 Alteration With Insidious Pattern Progression Of The Disease

Abstract Disclosure: H.L. Charchar: None. A.W. Kuhn: None. B.M. Mariani: None. M.Y. Nishi: None. F.L. Ledesma: None. M.C. Zerbini: None. F.Y. Tanno: None. V. Srougi: None. J.L. Chambo: None. M.Q. Almeida: None. B.B. Mendonca: None. M.C. Fragoso: None. Background: The clinical evolution of primary macronodular adrenal hyperplasia (PMAH) is usually slow over many years and is related to the insidious progression of hypercortisolism. The disease presents an autosomal dominant inheritance pattern with incomplete penetrance. Germline heterozygous mutations in the ARMC5 are present in 10% to 55% of the patients. The long-term follow-up of patients harboring germline ARMC5 pathogenic variants with normal evaluation will provide us with important insights regarding the outcomes. Clinical case: A 53-year-old man (index-case) was referred by the cardiologist to our service in October 2012, due to resistant hypertension (HTA), type 2 diabetes mellitus, dyslipidemia, and BMI of 32.4 kg/m2. Hormonal data: low ACTH (<5 pg/mL) and no suppression of cortisol after the overnight DST-1mg (22.7 µg/dL); late-night salivary cortisol, urinary free cortisol, aldosterone/renin ratio and plasma metanephrines were in the normal range. A CT scan showed bilateral enlargement of the adrenals associated with multiple macronodules. He underwent an adrenal-sparing surgery (total adrenalectomy of the left adrenal and partial adrenalectomy of the right adrenal). The macroscopic and histological appearance was consistent with PMAH. Leucocyte DNA genotyping identified the germline heterozygous ARMC5 pathogenic variant in exon 1 c.280del, p.(Ser94Argfs*43). This variant was identified in 3 of the 10 relatives investigated (son#1, daughter#2 and grandson#3). In 2021, the 37-year-old son#1 presented with a BMI of 32.6 kg/m2. Autonomous cortisol secretion was confirmed (cortisol after DST-1mg = 6.7 µg/dL). CT scan showed bilateral enlargement of the adrenals, similar to his father (index-case), with a larger left adrenal. Unilateral left adrenalectomy was performed, and PMAH was histologically confirmed. In 2018, the 21-year-old daugher#2 had normal adrenal function. More recently, in December 2022, she presented with an important weight gain (44kg in 4 years), without any specific signs and symptoms of hypercortisolism. Hormonal investigation showed a possible autonomous cortisol secretion (cortisol after DST-1mg = 2.2 µg/dL); the CT scan demonstrated an adrenal diffusely thickened with several nodules, especially on the left side, as the other relatives. Up to now, she has been using ketoconazole. All of them did not present meningioma of CNS. The grandson#3 with 11-year-old, is asymptomatic so far. Clinical Lesson: PMAH is a heterogenous and underdiagnosed disorder and is more genetically associated than previously thought. Therefore, a careful hormonal-genetic-clinical-radiological study of the relatives of index cases permits the diagnosis of many unsuspected familial-cases, including asymptomatic patients. Surgical or medical treatment should be considered for these patients to avoid progression to a possible overt Cushing syndrome. Presentation: Friday, June 16, 2023

Urinary Free Metanephrines for Diagnosis of Pheochromocytoma and Paraganglioma.

BackgroundPheochromocytoma and paraganglioma (PPGL) is diagnosed through biochemical confirmation of excessive catecholamines in urine and plasma. Recent technological developments have allowed us to measure urinary free metanephrines; however, the diagnostic accuracy of these new methods and the diagnostic cutoff values have not been evaluated.MethodsThis is a retrospective study of 595 subjects, including 71 PPGL cases and 524 controls. PPGL was based on pathological confirmation. Subjects with no evidence of PPGL over 2 years were included in the control group.ResultsUrinary free metanephrines yielded similar area under the curve (AUC) to urinary fractionated metanephrines and plasma free metanephrines. However, urinary free normetanephrine yielded a better AUC than did urinary fractionated normetanephrine. The optimal cutoff for urinary free metanephrine and normetanephrine corrected for urinary creatinine yielded 97.2% sensitivity and 98.1% specificity.ConclusionUrinary free metanephrines are a reliable method for diagnosing PPGL in Asian populations compared with existing biochemical methods.

Quantitative Measurement of Plasma Free Metanephrines by a Simple and Cost-Effective Microextraction Packed Sorbent with Porous Graphitic Carbon and Liquid Chromatography-Tandem Mass Spectrometry.

Plasma free metanephrines are widely used for the diagnosis of pheochromocytoma and paraganglioma (PPGL), yet quantifying metanephrines using a simple and cost-effective approach may be challenging due to preanalytical and analytical constraints. In this study, we established and validated a new method for quantitative measurement of plasma free metanephrines based on microextraction by packed sorbent (MEPS) with porous graphitic carbon (PGC) and liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The elution step was fully compatible with HILIC mode without evaporation and reconstitution. The analytes were well resolved, and potential interferences (54 substances) were investigated. This method was linear from 24.7–2717 pg/mL for metanephrine (MN) and 24.5–4010 pg/mL for normetanephrine (NMN) with a coefficient of determination (R2) higher than 0.994. The limit of MN and NMN detection were 12.4 pg/mL and 12.3 pg/mL, respectively. The intra- and interassay impressions were ≤12.8% for spiked quality controls and ≤13.6% for commercial quality controls; the method recoveries ranged within 88.0–109.0%, respectively. The area under the receiver operating characteristic (ROC) curve was 0.848 ± 0.047 for MN and 0.979 ± 0.021 for NMN. Validation that was performed by comparing clinical specimens with various biochemical results showed that plasma free metanephrines in a seated position had comparable sensitivity and lower specificity to urinary free metanephrines, which could be compensated by combining other biochemical tests. The newly developed MEPS method resulted as a time-saving, reliable, and cost-effective microextraction technique that can be applied for a successful screening of PPGL.

Urinary free metanephrines measurement in dogs with adrenal gland diseases using a new simple liquid chromatography tandem mass spectrometry method.

Measurement of urinary metanephrines in spot samples is used for the diagnosis of canine pheochromocytoma (PC). We describe a simple analytical method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for measuring free metanephrine (MN) and normetanephrine (NMN) in spot urine samples. Using the developed method, we evaluated the stability of urinary free-MN and free-NMN at various storing conditions. In addition, we assessed the feasibility of urinary free-MN and -NMN measurement for diagnosing PC. Urine samples were mixed with stable isotope internal standards and thereafter purified by ultrafiltration. The purified samples were analyzed by LC-MS/MS in the multiple reaction monitoring mode after separation on a multimode octa decyl silyl column. The coefficient of variation of free-MN and -NMN measurement was 7.6% and 5.5%, respectively. The linearity range was 0.5–10 µg/l for both analytes. Degradation was less than 10% for both analytes under any of the storage conditions. The median free-NMN ratio to creatinine of 9 PC dogs (595, range 144–47,961) was significantly higher (P<0.05) than that of 13 dogs with hypercortisolism (125, range 52–224) or 15 healthy dogs (85, range 50–117). The developed method is simple and may not require acidification of spot urine. The results of this preliminary retrospective study suggest that the measurement of urinary free metanephrines is a promising tool for diagnosing canine PC.

Detection of spot urinary free metanephrines and 3-methoxytyramine with internal reference correction for the diagnosis of pheochromocytomas and paragangliomas

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.

Simple, rapid, and cost-effective microextraction by the packed sorbent method for quantifying of urinary free catecholamines and metanephrines using liquid chromatography-tandem mass spectrometry and its application in clinical analysis.

Pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumors arising from adrenal and extra-adrenal chromaffin cells. They produce excessive amounts of catecholamines and their metabolites. A newly analytical procedure based on the semi-automated microextraction by packed sorbent (MEPS) technique, using a digitally controlled syringe (eVol) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed to quantify free urinary catecholamines and metanephrines. The important parameters affecting MEPS performance, namely the type of sorbent material (porous graphitized carbon (PGC), polar enhanced polymer (PEP), cation-exchange (CX) and C18), number of extraction cycles, and elution solvent system, were evaluated. The optimal experimental conditions involved the loading of sample mixture in seven extraction cycles through a C18 sorbent in a MEPS syringe, followed by using elution solutions (water/acetonitrile/formic acid, 95/4.75/0.25). The entire sample preparation took about 4min. Chromatographic separation was well achieved with an HSS PFP column using the gradient elution. The linearity range of the method was 0.167-33.4ng/mL for epinephrine, 0.650-130ng/mL for norepinephrine, 1.53-306ng/mL for dopamine, 1.34-268ng/mL for metanephrine, 3.43-686ng/mL for normetanephrine, and 1.33-265ng/mL for 3-methoxytyramine. The intra- and interassay precisions were ≤ 12.8%, and the respective accuracies were 88.4-112.0% and 89.0-109.5%. The carryover and sample stability without acidification were also investigated. Validation using clinical urine specimens showed that the proposed method had higher sensitivity compared with other urinary biochemical tests. The developed MEPS-LC-MS/MS method was simple, fast, and cost-effective; it helped to obtain information about multiple metabolites. It is applicable in routine clinical laboratories for the screening of PPGL. Graphical abstract.

Biochemical Diagnosis of Chromaffin Cell Tumors in Patients at High and Low Risk of Disease: Plasma versus Urinary Free or Deconjugated O-Methylated Catecholamine Metabolites

Measurements of plasma or urinary metanephrines are recommended for diagnosis of pheochromocytoma and paraganglioma (PPGL). What test offers optimal diagnostic accuracy for patients at high and low risk of disease, whether urinary free metanephrines offer advantages over deconjugated metanephrines, and what advantages are offered by including methoxytyramine in panels all remain unclear. A population of 2056 patients with suspected PPGLs underwent prospective screening for disease using mass spectrometric-based measurements of plasma free, urinary deconjugated, and urinary free metanephrines and methoxytyramine. PPGLs were confirmed in 236 patients and were excluded in others on follow-up evaluation. Measurements of plasma free metabolites offered higher (P < 0.01) diagnostic sensitivity (97.9%) than urinary free (93.4%) and deconjugated (92.9%) metabolites at identical specificities for plasma and urinary free metabolites (94.2%) but at a lower (P < 0.005) specificity for deconjugated metabolites (92.1%). The addition of methoxytyramine offered little value for urinary panels but provided higher (P < 0.005) diagnostic performance for plasma measurements than either urinary panel according to areas under ROC curves (0.991 vs 0.972 and 0.964). Diagnostic performance of urinary and plasma tests was similar for patients at low risk of disease, whereas plasma measurements were superior to both urinary panels for high-risk patients. Diagnosis of PPGLs using plasma or urinary free metabolites provides advantages of fewer false-positive results compared with commonly measured deconjugated metabolites. The plasma panel offers better diagnostic performance than either urinary panel for patients at high risk of disease and, with appropriate preanalytics, provides the test of choice. Measurements of methoxytyramine in urine show limited diagnostic utility compared with plasma.

High specificity of spot urinary free metanephrines in diagnosis and prognosis of pheochromocytomas and paragangliomas by HPLC with electrochemical detection

BackgroundThe 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. MethodsWe 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. ResultsThe 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. ConclusionsSpot urinary free MNs/Cr, superior to plasma free MNs, presented a promising biomarker for the diagnosis and prognosis of PPGLs.

Dilute, derivatise and shoot: Measurement of urinary free metanephrines and catecholamines as ethyl derivatives by LC-MSMS

BackgroundThe measurement of catecholamines and their metabolites in either urine or plasma is an important diagnostic test used to exclude the presence of neuroendocrine tumours. Because of weak chromatographic retention and potential ion-suppression, reverse-phase LC-MSMS is not ideal for analysis of these polar molecules. Here, we investigate derivatisation by ethylation as an alternative approach. MethodsA simple and rapid method involving acetaldehyde and a reducing agent was used to convert urine free metanephrines and catecholamines, and their deuterated analogues as internal standards, to mono-ethyl or diethyl- derivatives. Using an Agilent 6460 triple-quadrupole mass spectrometer, precursor and product ion mass spectra were recorded to allow comparison of multiple reaction monitoring methods for both derivatised and non-derivatised analytes under reverse-phase LC-MSMS conditions with positive electrospray ionization. ResultsConversion of biogenic amines to less polar ethyl derivatives increased their mass and enhanced the intensity of their molecular ions and fragments. Ethylation also improved the chromatographic properties of the amines, with greater retention and elution from reverse-phase HPLC columns with a methanol or acetonitrile gradient. The signal response of tandem mass spectrometric detection was increased up to 50-fold for ethyl metanephrines compared to non-derivatised compounds. This increase allowed for the omission of solid-phase extraction of urine as a clean-up step prior to analysis. The ‘dilute-derivatise-shoot’ method maintained analytical performance with respect to between-run imprecision (CV<6%) and accuracy in an external quality assurance program. Gender-related ranges for free metanephrines in early-morning spot urines, collected from adult patients, were similar using either derivatised or non-derivatised samples. ConclusionsThe LC-MSMS detection of free urine biogenic amines can be greatly enhanced by ethyl derivatisation, which is easy and rapid to perform. Advantages include improved chromatography and lower limits of quantitation, that negate the requirement for solid-phase clean-up of urine prior to analysis. A disadvantage is the potential toxicity of the derivatising agents used if they are not handled appropriately.

A simple and rapid analytical method based on solid-phase extraction and liquid chromatography–tandem mass spectrometry for the simultaneous determination of free catecholamines and metanephrines in urine and its application to routine clinical analysis

ObjectivesUrinary catecholamines and metanephrines are biochemical indicators of pheochromocytoma. We developed and validated a rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to tandem mass spectrometry (LC–MS/MS) for measuring urinary free catecholamines and metanephrines in a clinical setting. MethodsFollowing SPE purification of catecholamines and metanephrines from urine specimens, chromatographic separation and quantitative detection were performed using LC–MS/MS. The developed method for simultaneous measurement of urinary free catecholamines and metanephrines was validated with clinical urine specimens and was compared with other clinical and biochemical results, including urinary total metanephrines, vanillylmandelic acid (VMA), and plasma free metanephrines. ResultsThe performance of our newly developed method for measuring urinary free epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), and normetanephrine (NMN), was acceptable. The recoveries and matrix effects of analytes were 61–107% and 84.5–130.7%. The linear ranges of each analyte were 3.8–2163μg/L, 7.4–2,359μg/L, 5.4–2,825μg/L, 3.5–2,466μg/L, and 3.7–2,569μg/L, and the coefficients of variation (CV) were less than 10% with respect to imprecision. Carryover and sample stability were also validated. Validation using clinical urine specimens by comparison with various biochemical results showed that urinary free metanephrines had comparable sensitivity (100%) and superior specificity (97.1%) to urinary total and plasma free metanephrines. ConclusionsThe facile and reliable simultaneous measurement method for urinary free catecholamines and metanephrines using LC–MS/MS developed in this study is helpful in obtaining information about multiple metabolites and is applicable to routine clinical settings for the screening of pheochromocytoma.

A comprehensive LC/MS assay for biogenic amines in urine

Urinary catecholamines and metanephrines are biochemical indicators of pheochromocytoma. We developed and validated a rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to tandem mass spectrometry (LC–MS/MS) for measuring urinary free catecholamines and metanephrines in a clinical setting.Following SPE purification of catecholamines and metanephrines from urine specimens, chromatographic separation and quantitative detection were performed using LC–MS/MS. The developed method for simultaneous measurement of urinary free catecholamines and metanephrines was validated with clinical urine specimens and was compared with other clinical and biochemical results, including urinary total metanephrines, vanillylmandelic acid (VMA), and plasma free metanephrines.The performance of our newly developed method for measuring urinary free epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), and normetanephrine (NMN), was acceptable. The recoveries and matrix effects of analytes were 61–107% and 84.5–130.7%. The linear ranges of each analyte were 3.8–2163 μg/L, 7.4–2,359 μg/L, 5.4–2,825 μg/L, 3.5–2,466 μg/L, and 3.7–2,569 μg/L, and the coefficients of variation (CV) were less than 10% with respect to imprecision. Carryover and sample stability were also validated. Validation using clinical urine specimens by comparison with various biochemical results showed that urinary free metanephrines had comparable sensitivity (100%) and superior specificity (97.1%) to urinary total and plasma free metanephrines.The facile and reliable simultaneous measurement method for urinary free catecholamines and metanephrines using LC–MS/MS developed in this study is helpful in obtaining information about multiple metabolites and is applicable to routine clinical settings for the screening of pheochromocytoma.

Simultaneous liquid chromatography tandem mass spectrometric determination of urinary free metanephrines and catecholamines, with comparisons of free and deconjugated metabolites

ObjectiveWe 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. MethodsMethod 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. ResultsAnalyte 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 1.2nmol/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). ConclusionMeasurements of urinary catecholamines and their free O-methylated metabolites by our method provide potential advantages over urinary deconjugated metanephrines for diagnosis of pheochromocytoma.

Comparison of Diagnostic Accuracy of Urinary Free Metanephrines, Vanillyl Mandelic Acid, and Catecholamines and Plasma Catecholamines for Diagnosis of Pheochromocytoma

Recent evidence suggests that plasma-free metanephrines provide a highly sensitive test in patients requiring exclusion of pheochromocytoma. The diagnostic efficacy of urinary free metanephrines, however, has not been evaluated. OBJECTIVE, DESIGN, SETTING, PATIENTS, AND OUTCOME MEASURES: We compared retrospectively the diagnostic efficacy of 24-h urinary free metanephrines with our currently available measurements of 24-h urinary vanillyl mandelic acid (VMA), urinary catecholamines, and plasma catecholamines in 159 outpatients tested in a tertiary referral center for pheochromocytoma over a 4-yr period. The sensitivity of urinary free metanephrines was 100% [25 of 25 patients; 95% confidence interval (CI) 86-100%)] compared with the sensitivity of 84% (21 of 25; 95% CI 64-95%) for urinary catecholamines; 72% (18 of 25; 95% CI 51-88%) for urinary VMA; and 76% (16 of 21; 95% CI 53-92%) for plasma catecholamines. The specificity of urinary free metanephrines was 94% (116 of 123; 95% CI 89-98%), compared with the specificity of 99% (127 of 129; 95% CI 96-100%) for urinary catecholamines; 96% (130 of 134; 95% CI 91-98%) for urinary VMA; and 88% (66 of 75; 95% CI 78-94%) for plasma catecholamines. Receiver operating characteristic curves for all test groups were generated. Pairwise comparisons of the area under the receiver operating characteristic curve for urinary free metanephrines with that of each of the other three test groups individually were: 0.993 (95% CI 0.962-0.999) vs. 0.919 (95% CI 0.862-0.957, P = 0.032) for urine catecholamines; 0.993 (95% CI 0.962-0.999) vs. 0.846 (95% CI 0.778-0.900, P = 0.002) for urine VMA; and 0.992 (95% CI 0.945-0.998) vs. 0.852 (95% CI 0.762-0.918, P = 0.009) for plasma catecholamines. Testing with urinary free metanephrines failed to misidentify a single case of pheochromocytoma, compared with four missed cases for urinary catecholamines, seven missed cases for urinary VMA, and five missed cases for plasma catecholamines. Urinary free metanephrines were superior to urinary VMA, urinary catecholamines, and plasma catecholamines and can provide a valuable test for diagnosis of pheochromocytoma in adults.

Attomole Electrophoretic Analysis of Catecholamines Using Copper-Catalyzed Intramolecular Cyclization

Urinary catecholamines and metanephrines are biochemical indicators of pheochromocytoma. We developed and validated a rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to tandem mass spectrometry (LC–MS/MS) for measuring urinary free catecholamines and metanephrines in a clinical setting.Following SPE purification of catecholamines and metanephrines from urine specimens, chromatographic separation and quantitative detection were performed using LC–MS/MS. The developed method for simultaneous measurement of urinary free catecholamines and metanephrines was validated with clinical urine specimens and was compared with other clinical and biochemical results, including urinary total metanephrines, vanillylmandelic acid (VMA), and plasma free metanephrines.The performance of our newly developed method for measuring urinary free epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), and normetanephrine (NMN), was acceptable. The recoveries and matrix effects of analytes were 61–107% and 84.5–130.7%. The linear ranges of each analyte were 3.8–2163 μg/L, 7.4–2,359 μg/L, 5.4–2,825 μg/L, 3.5–2,466 μg/L, and 3.7–2,569 μg/L, and the coefficients of variation (CV) were less than 10% with respect to imprecision. Carryover and sample stability were also validated. Validation using clinical urine specimens by comparison with various biochemical results showed that urinary free metanephrines had comparable sensitivity (100%) and superior specificity (97.1%) to urinary total and plasma free metanephrines.The facile and reliable simultaneous measurement method for urinary free catecholamines and metanephrines using LC–MS/MS developed in this study is helpful in obtaining information about multiple metabolites and is applicable to routine clinical settings for the screening of pheochromocytoma.

Induction of the acute phase reaction increases heparin-binding proteins in rat plasma

Urinary catecholamines and metanephrines are biochemical indicators of pheochromocytoma. We developed and validated a rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to tandem mass spectrometry (LC–MS/MS) for measuring urinary free catecholamines and metanephrines in a clinical setting.Following SPE purification of catecholamines and metanephrines from urine specimens, chromatographic separation and quantitative detection were performed using LC–MS/MS. The developed method for simultaneous measurement of urinary free catecholamines and metanephrines was validated with clinical urine specimens and was compared with other clinical and biochemical results, including urinary total metanephrines, vanillylmandelic acid (VMA), and plasma free metanephrines.The performance of our newly developed method for measuring urinary free epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), and normetanephrine (NMN), was acceptable. The recoveries and matrix effects of analytes were 61–107% and 84.5–130.7%. The linear ranges of each analyte were 3.8–2163 μg/L, 7.4–2,359 μg/L, 5.4–2,825 μg/L, 3.5–2,466 μg/L, and 3.7–2,569 μg/L, and the coefficients of variation (CV) were less than 10% with respect to imprecision. Carryover and sample stability were also validated. Validation using clinical urine specimens by comparison with various biochemical results showed that urinary free metanephrines had comparable sensitivity (100%) and superior specificity (97.1%) to urinary total and plasma free metanephrines.The facile and reliable simultaneous measurement method for urinary free catecholamines and metanephrines using LC–MS/MS developed in this study is helpful in obtaining information about multiple metabolites and is applicable to routine clinical settings for the screening of pheochromocytoma.