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1.
Anal Chem ; 92(13): 9072-9078, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32484659

RESUMO

Plasma-free metanephrines and catecholamines are essential markers in the biochemical diagnosis and follow-up of neuroendocrine tumors and inborn errors of metabolism. However, their low circulating concentrations (in the nanomolar range) and poor fragmentation characteristics hinder facile simultaneous quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we present a sensitive and simple matrix derivatization procedure using propionic anhydride that enables simultaneous quantification of unconjugated l-DOPA, catecholamines, and metanephrines in plasma by LC-MS/MS. Dilution of propionic anhydride 1:4 (v/v) in acetonitrile in combination with 50 µL of plasma resulted in the highest mass spectrometric response. In plasma, derivatization resulted in stable derivatives and increased sensitivity by a factor of 4-30 compared with a previous LC-MS/MS method for measuring plasma metanephrines in our laboratory. Furthermore, propionylation increased specificity, especially for 3-methoxytyramine, by preventing interference from antihypertensive medication (ß-blockers). The method was validated according to international guidelines and correlated with a hydrophilic interaction LC-MS/MS method for measuring plasma metanephrines (R2 > 0.99) and high-performance liquid chromatography with an electrochemical detection method for measuring plasma catecholamines (R2 > 0.85). Reference intervals for l-DOPA, catecholamines, and metanephrines in n = 115 healthy individuals were established. Our work shows that analytes in the subnanomolar range in plasma can be derivatized in situ without any preceding sample extraction. The developed method shows improved sensitivity and selectivity over existing methods and enables simultaneous quantification of several classes of amines.


Assuntos
Catecolaminas/sangue , Metanefrina/sangue , Espectrometria de Massas em Tandem/métodos , Catecolaminas/isolamento & purificação , Catecolaminas/normas , Cromatografia Líquida de Alta Pressão/normas , Dopamina/análogos & derivados , Dopamina/sangue , Dopamina/isolamento & purificação , Dopamina/normas , Humanos , Levodopa/sangue , Levodopa/isolamento & purificação , Levodopa/normas , Limite de Detecção , Metanefrina/isolamento & purificação , Metanefrina/normas , Valores de Referência , Extração em Fase Sólida , Espectrometria de Massas em Tandem/normas
2.
Clin Chem Lab Med ; 58(5): 753-757, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-31348754

RESUMO

Background Analysis of plasma metanephrine (MN) and normetanephrine (NMN) with liquid chromatography tandem mass spectrometry (LC-MS/MS) is the gold standard for the screening of pheochromocytomas and paragangliomas (PPGLs). As scarce information is available on the stability of MNs in diagnostic samples, this study was aimed at analyzing the short-term stability of plasma free MNs in whole blood and plasma, using LC-MS/MS. Methods The stability of plasma MNs was evaluated after sample collection at 1, 2 and 3 h in whole blood, and at 2, 4 and 6 h in centrifuged samples. Both studies were performed while maintaining the samples at room temperature (RT) and at 4 °C. The ClinMass Complete Kit (Recipe, Munchen, Germany) was used for measuring MNs with LC-MS/MS (Nexera X2 UHPLC-4500MD Sciex). Differences from the baseline (T0) were assessed using repeated measures one-way ANOVA, Students' paired t-test and a comparison of the mean percentage changes with the total change limit (TCL). Results Statistically significant differences from T0 were found for both MNs (p < 0.001) in whole blood stored at RT, and for NMN (p = 0.028) but not MN (p = 0.220) at 4 °C. The mean difference exceeded the TCL after 1 h and 3 h at RT for MN, and after 1 h at RT for NMN. Statistically significant differences from T0 were only observed in the plasma samples for NMN at RT (p = 0.012), but the variation was within the TCL. Conclusions MN and NMN displayed different patterns of stability before and after centrifugation. Even short-time storage at RT in whole blood should hence be avoided.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metanefrina/sangue , Espectrometria de Massas em Tandem/métodos , Adulto , Feminino , Humanos , Masculino , Metanefrina/isolamento & purificação , Pessoa de Meia-Idade , Normetanefrina/sangue , Normetanefrina/isolamento & purificação , Fase Pré-Analítica , Temperatura , Fatores de Tempo
3.
Methods Mol Biol ; 1378: 149-57, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26602127

RESUMO

Measuring urinary metanephrines aides in the diagnosis of pheochromocytomas-catecholamine producing tumors. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows for greater sensitivity and simpler sample preparation as compared with other techniques. Here we describe a simple LC-MS/MS method for measuring metanephrines in urine. Each urine sample was treated with diphenylboronic acid to create boronate complexes, and then applied to a Bond-Elut Plexa cartridge. After solid phase extraction, samples were concentrated and analyzed on an Atlantis T3 column with chromatographic run time totaling 8.5 min. MS/MS was set in positive electrospray ionization mode with multiple reaction monitoring for data collection. The assay was linear from 0.2 to 27.4 µmol/L and 0.3 to 14.6 µmol/L for metanephrine and normetanephrine, respectively. Intra-assay and total precision at three concentration levels over 10 days were <5 % for metanephrine and <10 % for normetanephrine.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metanefrina/urina , Normetanefrina/urina , Espectrometria de Massas em Tandem/métodos , Urinálise/métodos , Métodos Analíticos de Preparação de Amostras , Ácidos Borônicos/química , Formiatos/química , Humanos , Metanefrina/química , Metanefrina/isolamento & purificação , Normetanefrina/química , Normetanefrina/isolamento & purificação , Extração em Fase Sólida
4.
Ann Lab Med ; 35(5): 519-22, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26206689

RESUMO

High-resolution imaging techniques have increased the detection rate of adrenal incidentaloma. We developed a method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection of plasma free metanephrine (MN) and normetanephrine (NMN) and evaluated its analytical performance and clinical efficacy in differential diagnosis of adrenal incidentaloma. After solid-phase extraction, chromatographic isolation of the analytes and internal standard was achieved by column elution in the LC-MS/MS system. The analytes were detected in multiple-reaction monitoring mode by using positive electrospray ionization: MN, transition m/z 180.1-->165.1; NMN, m/z 166.1-->134.1. This method was validated for linearity, precision, accuracy, lower limits of quantification and detection, extraction recovery, and the matrix effect. Plasma concentrations of MN and NMN of 14 patients with pheochromocytoma were compared with those of 17 healthy volunteers, 10 patients with essential hypertension, and 60 patients with adrenal adenoma. The assay's linear range was 0.04-50.0 and 0.08-100.0 nmol/L for MN and NMN, respectively. Assay imprecision was 1.86-7.50%. The accuracy ranged from -7.50% to 2.00%, and the mean recovery of MN and NMN was within the range 71.5-95.2%. Our LC-MS/MS method is rapid, accurate, and reliable and useful for differential diagnosis of adrenal incidentaloma.


Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Cromatografia Líquida de Alta Pressão , Metanefrina/sangue , Espectrometria de Massas em Tandem , Estudos de Casos e Controles , Diagnóstico Diferencial , Humanos , Metanefrina/isolamento & purificação , Normetanefrina/sangue , Feocromocitoma/diagnóstico , Extração em Fase Sólida
5.
Endocr J ; 59(9): 831-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22785075

RESUMO

The diagnosis of pheochromocytoma depends on the documentation of catecholamine overproduction. The use of urinary fractionated metanephrines has recently become common for the diagnosis of pheochromocytoma. In order to avoid false positive and false negative results, optimal cut-off levels are necessary; however, there have been few published reports on whether different cut-off levels are needed to diagnose pheochromocytoma according to sex. We reviewed the medical records of 815 subjects (including 103 pheochromocytoma patients) whose of 24-h urinary fractionated metanephrine was measured using high-performance liquid chromatography methods and adrenal imaging at Samsung Medical Center. Receiver operating characteristic (ROC) curves were used to determine cut-off values according to sex. The upper limit values of fractionated metanephrine in healthy volunteers and the control group were significantly higher in male subjects compared with females. When we applied cut-off values according to sex, the diagnostic efficacies (defining a positive test as either metanephrine or normetanephrine levels above the cut-off value) were a sensitivity of 96% in male subjects and 98.1% in female subjects and a specificity of 88.6% in male subjects and 94.1% in female subjects. However, when we applied cut-off values without considering sex, the specificity decreased from 88.6% to 77.8% in male subjects. In this study, urinary fractionated metanephrines had a high level of sensitivity and specificity for the diagnosis of pheochromocytoma. However, diagnostic cut-off values were higher in male subjects than in female subjects. Therefore, different cut-off values may be needed according to sex to diagnose pheochromocytoma in Koreans.


Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Metanefrina/urina , Normetanefrina/urina , Feocromocitoma/diagnóstico , Centros Médicos Acadêmicos , Neoplasias das Glândulas Suprarrenais/etnologia , Neoplasias das Glândulas Suprarrenais/patologia , Neoplasias das Glândulas Suprarrenais/urina , Glândulas Suprarrenais/patologia , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Feminino , Seguimentos , Humanos , Masculino , Prontuários Médicos , Metanefrina/isolamento & purificação , Pessoa de Meia-Idade , Normetanefrina/isolamento & purificação , Feocromocitoma/etnologia , Feocromocitoma/patologia , Feocromocitoma/urina , República da Coreia , Estudos Retrospectivos , Sensibilidade e Especificidade , Caracteres Sexuais , Carga Tumoral
6.
Anal Bioanal Chem ; 402(9): 3003-10, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22349327

RESUMO

Liquid chromatography-tandem mass spectrometry has become the preferred technology to measure unconjugated metanephrine and normetanephrine in plasma because of its high sensitivity and specificity over immunoassay and gas chromatography-mass spectrometry. In our earlier study, plasma metanephrines were extracted with offline ion-pairing solid-phase extraction and quantified by liquid chromatography-tandem mass spectrometry with porous graphitic carbon column based chromatography. In this study, we aim to automate the sample preparation with turbulent flow online extraction technology and maintain or improve the analytical performance previously achieved from the offline approach. The online extraction was done with a mixed-mode cation exchange turbulent flow chromatography column assisted with ion-pairing reagent and porous graphitic column was used for chromatographic separation. The total online extraction and analytical LC runtime was 12 min. This method was linear from 6.3 to 455.4 pg/mL for metanephrine; 12.6 to 954.5 pg/mL for normetanephrine with an accuracy of 80.6% to 93.5% and 80.9% to 101.7%, respectively. The lower limit of quantitation was 6.3 pg/mL for metanephrine and 12.6 pg/mL for normetanephrine. Inter-assay and intra-assay precision for metanephrine and normetanephrine at low and high concentration levels ranged from 2.0% to 10.5%. In conclusion, we have developed a fast and sensitive automated online turbulent flow extraction method for the quantitative analysis of plasma metanephrines. Ion-pairing reagent was necessary for the success of this method.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metanefrina/análise , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Metanefrina/sangue , Metanefrina/isolamento & purificação , Normetanefrina/análise , Normetanefrina/sangue , Normetanefrina/isolamento & purificação
7.
J Chromatogr B Analyt Technol Biomed Life Sci ; 879(23): 2355-9, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21723209

RESUMO

Plasma free metanephrine and normetanephrine are the best biomarkers for diagnosing pheochromocytoma. In the past few years, liquid chromatography-tandem mass spectrometry has become the preferred technology to measure plasma metanephrine and normetanephrine because of its high sensitivity and specificity, as well as fast and simple sample preparation. In this study, we report a liquid chromatography-tandem mass spectrometry method for measuring plasma metanephrine and normetanephrine. A solid phase extraction method using ion-pairing reagent and C18 stationary phase was used for sample preparation. We tested a porous graphitic carbon column and a HILIC column for chromatographic separation, and the former one showed better resolution with no interference from plasma matrix. This method was linear from 7.2-486.8 pg/mL for metanephrine and 18.0-989.1 pg/mL for normetanephrine with an accuracy of 92.2-111.8% and 92.1-115.0%, respectively. Inter-assay and intra-assay CV for metanephrine and normetanephrine at two different concentration levels ranged from 2.0% to 10.9%. In conclusion, this liquid chromatography-tandem mass spectrometry method using ion-pairing solid phase extraction and porous graphitic column was simple and efficient for measuring plasma metanephrines.


Assuntos
Cromatografia Líquida/métodos , Grafite/química , Metanefrina/sangue , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Neoplasias das Glândulas Suprarrenais/sangue , Neoplasias das Glândulas Suprarrenais/diagnóstico , Humanos , Metanefrina/isolamento & purificação , Feocromocitoma/sangue , Feocromocitoma/diagnóstico , Porosidade , Extração em Fase Sólida/instrumentação
8.
Clin Chem Lab Med ; 49(7): 1213-6, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21574886

RESUMO

BACKGROUND: Measuring urinary fractionated metanephrines is one of the initial tests in the diagnosis of pheochromocytoma. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) represents the most specific and accurate technology for this purpose. The goal of this work was to develop a simple LC-MS/MS method for measuring metanephrines in urine. METHODS: Each urine sample was complexed with diphenylboronic acid, and purified on a Bond-Elute Plexa cartridge. The extract was concentrated and analyzed on a short Atlantis T3 column in 8.5 min. Metanephrines and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring. RESULTS: Ion suppression was observed, but was compensated for by the respective internal standard. The analytical measurement range was 0.2-27.4 µmol/L and 0.3-14.6 µmol/L for metanephrine and normetanephrine, respectively. The intra-assay and total coefficient of variation throughout the linear ranges was 2.03%-2.11% and 2.20%-3.80% for metanephrine, and 4.50%-8.09% and 9.00%-10.00% for normetanephrine, respectively. Comparison with a commercial HPLC method using patient samples (n=65) by Passing-Bablok regression showed a slope of 1.000 and 1.014, y-intercept of -0.080 and -0.067, a correlation coefficient of 0.8830 and 0.9022, and a mean difference of 14.0% and -0.43% for metanephrine and normetanephrine, respectively. CONCLUSIONS: This simple method for urine metanephrines is suitable for clinical use.


Assuntos
Cromatografia Líquida/métodos , Metanefrina/urina , Normetanefrina/urina , Espectrometria de Massas em Tandem/métodos , Urinálise/métodos , Ácidos Borônicos/química , Humanos , Metanefrina/química , Metanefrina/isolamento & purificação , Normetanefrina/química , Normetanefrina/isolamento & purificação , Extração em Fase Sólida
9.
Anal Chim Acta ; 613(1): 108-15, 2008 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-18374708

RESUMO

This paper tackles a simple and efficient method for the simultaneous separation and stacking of neurotransmitters in capillary electrophoresis with UV detection. By using poly(diallyldimethylammonium chloride) (PDDAC) as a buffer additive, the high and reversed EOF are observed. Moreover, the mobility of indolamines and catecholamines decreases as the PDDAC concentration increases. Based on the difference in mobility in the presence and absence of PDDAC, the analytes were simply stacked between the boundary of the sample zone and the background electrolyte containing PDDAC. The separation of 14 analytes including indolamines, catecholamines, and metanephrines was accomplished within 33 min under optimal conditions (1.2% PDDAC and 5 mM formic acid at pH 4.0), and the values of relative standard deviation of their migration time were less than 3.1%. By applying stacking methods for fourteen analytes, we observed: (a) the sample injection volume of sample is up to 216 nL, (b) the limits of detection at signal-to-noise of 3 range from 15.4 to 122.1 nM, and (c) the sensitivity enhancements, compared to normal injection (12 nL), range from 110- to 220-fold. Under the optimal stacking conditions, the present method has been applied to analyze of vanillomandelic acid, 5-hydroxyindole-3-acetic acid, dopamine, tryptamine, and 3-indoxyl sulfate in urine samples.


Assuntos
Catecolaminas/isolamento & purificação , Indóis/isolamento & purificação , Metanefrina/isolamento & purificação , Polietilenos/química , Compostos de Amônio Quaternário/química , Adulto , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Humanos , Concentração de Íons de Hidrogênio , Masculino , Sensibilidade e Especificidade , Soluções , Raios Ultravioleta , Urina/química
10.
J Chromatogr ; 578(2): 165-74, 1992 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-1400794

RESUMO

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.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dopamina/análogos & derivados , Metanefrina/urina , Normetanefrina/urina , Adulto , Criança , Dopamina/isolamento & purificação , Dopamina/urina , Eletroquímica , Humanos , Metanefrina/isolamento & purificação , Normetanefrina/isolamento & purificação , Reprodutibilidade dos Testes
11.
J Chromatogr ; 563(1): 23-36, 1991 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-2061394

RESUMO

A simple method for the simultaneous assay of normetanephrine and metanephrine (both free and conjugated) in human plasma by high-performance liquid chromatography with electrochemical detection has been developed. The hydrolysed plasma is purified on a Dowex 50 H+ column, and the methoxylated amines are eluted with ammonia-methanol. The methoxyamines are assayed using a dual-series electrode arrangement with differential current measurement. This system greatly improves the assay specificity toward endogenous or exogenous metabolites and drugs. The high sensitivity of the method enables the determination in normal subjects of levels of normetanephrine and metanephrine.


Assuntos
Metanefrina/sangue , Normetanefrina/sangue , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica , Eletroquímica/métodos , Humanos , Indicadores e Reagentes , Metanefrina/isolamento & purificação , Microquímica , Normetanefrina/isolamento & purificação , Valores de Referência
12.
Meikai Daigaku Shigaku Zasshi ; 19(2): 212-20, 1990.
Artigo em Japonês | MEDLINE | ID: mdl-2134292

RESUMO

Organic solvent extraction of three metanephrines (MNs) in supernatant and sediment fractions obtained by the addition of methanol to the whole saliva and liquid chromatographic analysis with voltammetric detection are described. Two milliliters of unstimulated whole saliva obtained from 13 healthy males was treated with chilled methanol to separate mucin clots. The mucin clots, supernatant, and whole saliva were then hydrolyzed at 98 degrees C for 40 minutes at pH 0.5-0.8. These hydrolyzed samples were subsequently brought up to pH 10.0 with a 6 N NaOH solution in 0.1 M borate buffer (pH 10.0), and then 5-HT and MNs were extracted into toluene: isoamylalcohol (3:2) from the basic phase. MNs in their organic phase were extracted back into 0.1 M acetic acid and used for the high-performance liquid chromatographic (HPLC) analysis. The supernatant fraction showed a higher distribution for MNs than the sediment and whole saliva fractions. The chromatographic profile obtained from the supernatant and the whole saliva fractions contained some serotonin in a pattern similar to that reported from urinary analysis. The use of methanol for the fractionation of saliva resulted in good differential determination of MNs and revealed their distribution in the three salivary fractions. It is attractive to speculate that patients undergoing restorative treatment possibly manifest their anxiety by salivary excretion containing serotonin and MNs. These compounds in saliva may be useful markers of emotional responses.


Assuntos
Metanefrina/análise , Saliva/química , Cromatografia Líquida de Alta Pressão , Ansiedade ao Tratamento Odontológico/metabolismo , Humanos , Masculino , Metanefrina/isolamento & purificação , Metanol , Serotonina/análise
13.
Clin Biochem ; 13(6): 262-5, 1980 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7214693

RESUMO

A simple, but effective, procedure is described for the concurrent estimation of normetadrenaline (NMA) and metadrenaline (MA) in urine. The 3-methoxycatecholamines are adsorbed by a cation exchange resin, desorbed, separated by paper chromatography, and finally estimated spectrophotometrically after oxidation to vanillin. The mean (+/- 2 standards deviations) urinary outputs of NMA and MA from 123 normals (80 males, 43 females) in 24 hours was 2.15 +/- 1.04 and 1.79 +/- 0.96 mu mol respectively. There was no significant difference in the 24-hour urinary outputs of NMA between the male and female groups. However, the output of MA from the males was significantly higher than that from the females.


Assuntos
Epinefrina/análogos & derivados , Metanefrina/urina , Normetanefrina/urina , Adulto , Cromatografia em Papel/métodos , Feminino , Humanos , Masculino , Metanefrina/isolamento & purificação , Normetanefrina/isolamento & purificação , Valores de Referência , Fatores Sexuais , Espectrofotometria Ultravioleta
15.
Br J Clin Pharmacol ; 4(5): 553-8, 1977 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20916

RESUMO

1 An existing radioenzymatic assay for plasma catecholamines using catechol-o-methyl transferase and [3H]-S-adenosyl-methionine has been modified resulting in a more sensitive assay for the measurement of plasma adrenaline and noradrenaline. 2 The lower limit of sensitivity for this method is 25 pg for adrenaline and 30 pg for noradrenaline/ml of plasma. 3 Resting supine (60 min) plasma adrenaline concentration was (mean +/- s.d.) 124 +/- 76 pg/ml(n=11) in males and 130 +/- 71 pg/ml (n=7) in females; plasma noradrenaline concentrations were respectively 444 +/- 129 pg/ml and 550 +/- 87 pg/ml. 4 The changes in plasma catecholamine concentrations in response to 40 degrees head-up tilt have been determined in a group of healthy normal subjects and have been shown to be related to changes in blood pressure and heart rate.


Assuntos
Epinefrina/sangue , Norepinefrina/sangue , Adulto , Idoso , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino , Metanefrina/isolamento & purificação , Pessoa de Meia-Idade , Normetanefrina/isolamento & purificação
16.
Clin Chem ; 23(7): 1268-74, 1977 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-872373

RESUMO

We describe a sensitive procedure for measuring the basic catecholamine metabolites--normetanephrine, metanephrine, and 3-methoxytyramine--in urine by use of liquid chromatography, with electrochemical detection. These substances are isolated from hydrolyzed urine by passage through small ion-exchange columns and then pre-concentrated by a rapid set of solvent extractions before being injected onto the liquid-chromatographic column. Concentration and instrumental response are linearly related to 2.0 mg/liter for all three compounds, and practical lower detection limits are about 20 micrograms/liter for actual urine samples. The high selectivity that accrues from the combination of the extraction procedure, high-performance liquid chromatography, and electrochemical detection makes this procedure suitable for quantitative studies of catecholamine metabolism. A set of six samples can be taken through the extraction procedure in parallel in less than 2 h; once prepared, the extracts can be analyzed at a rate of three per hour.


Assuntos
Epinefrina/análogos & derivados , Metanefrina/urina , Normetanefrina/urina , Tiramina/análogos & derivados , Adulto , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Dopamina/análogos & derivados , Eletroquímica , Humanos , Masculino , Metanefrina/isolamento & purificação , Normetanefrina/isolamento & purificação , Tiramina/isolamento & purificação , Tiramina/urina
17.
Clin Chem ; 22(9): 1497-502, 1976 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8221

RESUMO

Catecholamines and their metabolites have been separated isocratically by reverse-phase chromatography with aqueous (no organic solvent admixed) eluents. Unlike ion-exchange or ion-pair chromatography, mixtures of both acidic and basic substances can be separated in a single chromatographic run, because the retention is governed by hydrophobic interactions between the nonpolar moiety of the solute molecules and the octadecyl-silica stationary phase. The relative retention values strongly depend on the pH of the eluent, which governs the degree of dissociation of ionogenic solutes. The reproducibility of the results and the stability and efficiency of the chromatographic systems make this approach particularly attractive for use in clinical analysis.


Assuntos
Catecolaminas/isolamento & purificação , Ácido 3,4-Di-Hidroxifenilacético/isolamento & purificação , Catecolaminas/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Desoxiepinefrina/isolamento & purificação , Dopamina/metabolismo , Estudos de Avaliação como Assunto , Ácido Homovanílico/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Ácidos Mandélicos/isolamento & purificação , Metanefrina/isolamento & purificação , Norepinefrina/isolamento & purificação , Concentração Osmolar , Fenilalanina/metabolismo , Temperatura , Urina/análise , Ácido Vanilmandélico/isolamento & purificação
18.
Clin Chim Acta ; 69(2): 175-83, 1976 Jun 01.
Artigo em Alemão | MEDLINE | ID: mdl-6173

RESUMO

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%.


Assuntos
Epinefrina/análogos & derivados , Metanefrina/urina , Normetanefrina/urina , Autoanálise , Cromatografia por Troca Iônica/métodos , Humanos , Concentração de Íons de Hidrogênio , Metanefrina/isolamento & purificação , Normetanefrina/isolamento & purificação , Espectrometria de Fluorescência/métodos , Zinco
19.
Endocrinology ; 98(6): 1497-507, 1976 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1278114

RESUMO

A double-isotope, radioenzymatic assay for measuring dopamine, norepinephrine, and epinephrine in one sample is described. The assay procedure includes incubation, solvent extraction, and thin-layer chromatography. Dopamine, norepinephrine, and epinephrine were incubated with catechol-O-methyl transferase (COMT) and [3H]S-acenosyl methionine ([3H]SAM) and were converted to the O-methylated tritiated derivatives: [3H]methoxytyramine, [3H]normetanephrine, and [3H]metanephrine, respectively. After several extraction steps the O-methylated products were purified by means of two-dimensional, thin-layer chromatography using silica gel. The thin-layer chromatographic system resulted complete separation of the three O-methylated compounds with an overlap of only 1-2%. The assay was linear from 0 to 5 ng for each catecholamine and had a sensitivity of 10-30 pg. The addition of large amounts of plasma reduced the activity of COMT, but increasing the magnesium concentration in the incubation mixture and the addition of EGTA to plasma samples improved the recoveries. Each sample was corrected for losses incurred during extraction and chromatography by using [14C]methoxytyramine, [14C]normetanephrine, and [14Ci1metanephrine that were added at the end of incubation. Several catechol compounds known to be O-methylated by COMT were examined for crossreactivity. Of the substances tested, only dihydroxyphenylalanine (DOPA) exhibited cross-reactivity. However, the apparent 30% cross-reactivity of DOPA with dopamine was due to the presence of decarboxylase activity in the COMT preparation. As little as 50 mul of trunk plasma from decapitated rats was sufficient for the determination of the three catecholamines.


Assuntos
Dopamina/isolamento & purificação , Epinefrina/isolamento & purificação , Norepinefrina/isolamento & purificação , Glândulas Suprarrenais/metabolismo , Animais , Radioisótopos de Carbono , Catecol O-Metiltransferase , Cromatografia em Camada Fina , Dopamina/análogos & derivados , Dopamina/sangue , Epinefrina/sangue , Hipotálamo/análise , Masculino , Metanefrina/isolamento & purificação , Métodos , Norepinefrina/sangue , Normetanefrina/isolamento & purificação , Hipófise/análise , Ratos , S-Adenosilmetionina
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