A general method for the quantitative determination of catecholamines in body fluids using off-line sample pretreatment and HPLC-ED analysis

Summary Reversed phase HPLC with electrochemical detection (HPLC-ED) was used for quantitative determination of adrenaline, noradrenaline, and dopamine in several complex biological matrices, including plasma, uremic plasma, and urine. Three different methods of sample preparation for use in this clinical chemistry were tested. These were adsorption of catecholamines on alumina, organic solvent extraction after complex formation with diphenylborate, and adsorption of catecholamines on a cation exchange gel followed by organic solvent extraction of the elute. The selectivity and precision of the three methods were evaluated. The organic solvent extraction proved to be more precise and selective than adsorption on alumina (adrenaline: cv=3.80% vs. 7.58%; noradrenaline: cv=1.70% vs. 4.26%); it also proved suitable for use in the routine quantitative determination of catecholamines in plasma from patients with normal renal function (creatinine <1.2 mg/dl). However when working with uremic plasma or urine, a more selective sample preparation was required. In this case the adsorption of catecholamines on a cation exchange gel followed by organic solvent extraction of the elute was sufficiently selective and precise and thus allowed a reliable quantitative determination of adrenaline and noradrenaline from rather complex biological matrices (adrenaline: cv=6.2%; noradrenaline: cv=2.8%). Use of this specific method showed that basal plasma catecholamine levels in dialysis patients are comparable to those found in patients with normal renal function (adrenaline: 47.7±22.2 pg/ml; noradrenaline: 310.3±121.4 pg/ml).     

Basal plasma-catecholamine-level determination using HPLC-ED and different sample cleanup techniques

Summary Reversed phase HPLC with electrochemical detection was used for the determination of basal adrenaline, dopamine and noradrenaline levels in human plasma. These compounds demonstrated good stability during different stages of collection and long-term storage. Using a new electrochemical detector and improving mobile phase parameters, we obtained a detection limit of 2 pg per injection. Good separation of dihydroxyphenylacetic acid was also attained, which is important in investigations with intensive care patients. Good accuracy and precision, demonstrated in the daily quality control measurements taken over a five month period, verified the reliability of the chromatographic separation. However, there was a decrease in the recovery of very low amounts of catecholamines, added to fresh frozen plasma that had previously been made catecholamine-free. According to the widely-accepted extraction method of Anton and Sayre, it is argued that the un-known affinity of catecholamines to acid-prepared aluminium oxide (in comparison to catecholamine —protein binding constants) explains the low accuracy in measurement at very low plasma levels. We thus compared this sample preparation method to recently published extraction procedures.     

Circulating Levels of Dephosphorylated-Uncarboxylated Matrix Gla Protein in Patients with Acute Coronary Syndrome

Vascular calcification contributes to the pathogenesis of coronary artery disease while matrix Gla protein (MGP) was recently identified as a potent inhibitor of vascular calcification. MGP fractions, such as dephosphorylated-uncarboxylated MGP (dp-ucMGP), lack post-translational modifications and are less efficient in vascular calcification inhibition. We sought to compare dp-ucMGP levels between patients with acute coronary syndrome (ACS), stratified by ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction (NSTEMI) status. Physical examination and clinical data, along with plasma dp-ucMGP levels, were obtained from 90 consecutive ACS patients. We observed that levels of dp-ucMGP were significantly higher in patients with NSTEMI compared to STEMI patients (1063.4 ± 518.6 vs. 742.7 ± 166.6 pmol/L, p < 0.001). NSTEMI status and positive family history of cardiovascular diseases were only independent predictors of the highest tertile of dp-ucMGP levels. Among those with NSTEMI, patients at a high risk of in-hospital mortality (adjudicated by GRACE score) had significantly higher levels of dp-ucMGP compared to non-high-risk patients (1417.8 ± 956.8 vs. 984.6 ± 335.0 pmol/L, p = 0.030). Altogether, our findings suggest that higher dp-ucMGP levels likely reflect higher calcification burden in ACS patients and might aid in the identification of NSTEMI patients at increased risk of in-hospital mortality. Furthermore, observed dp-ucMGP levels might reflect differences in atherosclerotic plaque pathobiology between patients with STEMI and NSTEMI.     

Selektivitätseffekte bei der automatisch-fluorimetrischen Bestimmung von Adrenalin,Noradrenalin und 3,4-Dihydroxyphenylalanin durch Optimierung von Geräteparametern im Analysensystem

Adrenaline, noradrenaline, 3,4-dihydroxyphenylalanine (Dopa) and adrenaline plus noradrenaline as total catecholamines can automaticallyfluorimetrically be determined in one analysis system (according to Wisser) by the trihydroxyindole method. The fluorescence yields for the fluorophores of the single compounds are dependent on the pump speed of the autoanalyzer except for the differentiated determination of adrenaline. Caused by these differences effects of selectivity are demonstrated for the determination of Dopa disturbed by noradrenaline and adrenaline disturbed by noradrenaline. Also the influence of the suction time on the fluorescence yield was investigated. The optimal adjustings for the single methods of determination are given.     

Spectrophotometric and titrimetric determination of catecholamines

Ammonium metavanadate is used to determine adrenaline, noradrenaline, isopropylnoradrenaline and methyldopa by titrimetric and photometric procedures. Oxidation of these catecholamines produces aminochrome derivatives which can be measured spectrophotometrically at 485 nm. Beer's law is obeyed over the ranges 0.09-0.90 mg for adrenaline, 0.07-0.65 mg for noradrenaline, 0.07-0.75 mg for isopropylnoradrenaline and 0.10-0.95 mg for methyldopa.     

Detection of Adrenaline in Blood Plasma as Biomarker for Adrenal Venous Sampling

An amperometric bi‐enzyme biosensor based on substrate recycling principle for the amplification of the sensor signal has been developed for the detection of adrenaline in blood. Adrenaline can be used as biomarker verifying successful adrenal venous sampling procedure. The adrenaline biosensor has been realized via modification of a galvanic oxygen sensor with a bi‐enzyme membrane combining a genetically modified laccase and a pyrroloquinoline quinone‐dependent glucose dehydrogenase. The measurement conditions such as pH value and temperature were optimized to enhance the sensor performance. A high sensitivity and a low detection limit of about 0.5–1 nM adrenaline have been achieved in phosphate buffer at pH 7.4, relevant for measurements in blood samples. The sensitivity of the biosensor to other catecholamines such as noradrenaline, dopamine and dobutamine has been studied. Finally, the sensor has been successfully applied for the detection of adrenaline in human blood plasma.     

Evaluation of GSH adducts of adrenaline in biological samples

The sustained high release of catecholamines to circulation is a deleterious condition that may induce toxicity, which seems to be partially related to the products formed by oxidation of catecholamines that can be further conjugated with glutathione (GSH). The aim of the present study was to develop a method for the determination of GSH adducts of adrenaline in biological samples. Two position isomers of the glutathion-S-yl-adrenaline were synthesized and characterized by HPLC using diode array, coulometric and mass detectors. A method for the extraction of these adducts from human plasma was also developed, based on adsorption to activated alumina, which showed adequate recoveries and proved to be crucial in removing interferences from plasma. The selectivity, precision and linearity of the method were all within the accepted values for these parameters. Furthermore, the sensitivity of this method allows the detection of adduct amounts that are within the range of the expected concentrations for these adducts under certain pathophysiological conditions and/or drug treatments. In conclusion, the development of this method allows the direct analysis of GSH adducts of adrenaline in human plasma, providing a valuable tool for the study of the catecholamine oxidation process and its related toxicity.     

二维超声斑点追踪成像评估老年急性心肌梗死患者左室心功能的价值

急性心肌梗死是临床上常见的急危重症,可造成心功能障碍。为研究二维超声斑点追踪成像对评估老年急性心肌梗死患者左室心功能的应用价值,选取2017年10月~2019年10月于我院就诊的老年急性心肌梗死患者100例作为研究对象,分为前壁心肌梗死组和下壁心肌梗死组,两组患者均给予常规超声和二维超声斑点追踪成像检查,对比检查结果和患者左室心功能。结果显示,相较于下壁心梗组患者,前壁心梗组患者左室舒张末期内径(LVDD)和左室收缩末期内径(LVDS)值明显升高,左心室射血分数(LVEF)值明显降低;基底部、乳头肌水平和心尖部的前间隔和前壁的圆周应变峰值明显减小;基底部和心尖部侧壁的圆周应变峰值明显升高;基底部和乳头肌水平的后壁、下壁的圆周应变峰值明显升高;乳头肌水平和心尖部的后间隔圆周应变峰值明显减小。因此,二维超声斑点追踪成像技术对老年急性心肌梗死患者左室功能评估无创、精准,具有重要临床价值。     

Detection of catecholamines in single specimens of groundwater amphipods

Catecholamines play essential roles in several physiological processes in vertebrates as well as in invertebrates. While several studies have shown the presence of these substances in surface water invertebrates, their occurrence in groundwater fauna is unproven. In the present study, the presence of different catecholamines (i.e., noradrenaline, adrenaline, and dopamine) in individual specimens of groundwater amphipods of the genus Niphargus (mostly Niphargus inopinatus) was investigated via two independent analytical methods: HPLC/EcD and UPLC/TOF-MS. Mean values for catecholamine levels were 533 pg mg^?1 fresh weight for noradrenaline, 314 pg mg^?1 for adrenaline, and 16.4 ng mg^?1 for dopamine. The optimized protocol allowed the detection of CAs in single organisms of less than 1 mg fresh weight. Catecholamine concentration patterns in groundwater invertebrates are briefly discussed here with respect to their evolutionary adaptation to an environmentally stable, energy-poor habitat.

反相高效液相色谱-串联质谱法测定尿液中儿茶酚胺和肾上腺素(英文)

The measurement of urine catecholamine and metanephrine concentrations is important for biochemical screening and diagnosis of pheochromocytoma.The goal of this work was to develop a simple liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for determining catecholamines and metanephrines in urine to replace an existing liquid chromatographic method using electrochemical detection.Urine samples were prepared using Oasis weak-cation-exchange cartridges.The eluate was analyzed on an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column in 3 min.Adrenaline,noradrenaline,dopamine,metanephrine,normetanephrine,and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring(MRM).No evidence of ion suppression was observed.The assay was linear up to 5μmol/L for adrenaline,5μmol/L for noradrenaline,6.1μmol/L for dopamine,5.6μmol/L for metanephrine,and 34.6μmol/L for normetanephrine,with lower limits of quantification of 5,5,12,6 and 7nmol/L,respectively.The intra-day and inter-day precisions for all analytes ranged from 0.59%to 4.64%and1.98%to 4.80%,respectively.External quality assurance samples were assayed and showed excellent agreement with the target values.This simple method provides an improved assay for determining urine catecholamines and metanephrines.     

HPLC separation of catecholamines after derivatization with 9-fluorenylmethyl chloroformate

Summary Optimum conditions for the separation of 9-fluorenylmethyl chloroformate derivatized catecholamines by HPLC are described; three catecholamines (noradrenaline, adrenaline and dopamine) and an internal standard (epinine) were separated in less than 20 minutes under isocratic conditions. This method is 17 to 350 times more sensitive than electrochemical detection, depending on the test compounds. It has been applied to the analysis of catecholamines in urine. The sample was extracted by a metal-loaded silica prior to separation.     

THE CHEMICAL TRANSFORMATION OF THE CATECHOLAMINES INDUCED BY UV IRRADIATION*

Abstract— The conversion of catecholamines exposed to ultraviolet irradiation has been investigated spectrophotometricaly. The UV activation of the molecule is a direct effect and the activation is not dependent upon the presence of metals. The transformation, which includes mdolization and polymerization requkes the presence of oxygen. The rate of the oxidative transformation is of the same order of magnitude in 100 per cent oxygen as in air, provided a metal chelating agent is present.
At neutral pH photoactivated adrenaline is oxidized to adrenochrome and further polymerized to melanin. At alkaline pH U.V. irradiation results in the formation of conversion products of adrenaline, different from those observed at neutral pH, and the melanin production is low.
When noradrenaline is subjected to U.V. irradiation at neutral pH the processes of oxidation and polymerization takes place at a much slower rate than that observed for adrenaline. The radiation effect on noradrenaline at alkaline pH is characterized by increased melanin formation.
Radicals derived from U.V. irradiation of H₂O₂ convert the catecholamines to different products, and no melanin formation can be recorded.
The sequence of reactions leading from the activated catecholamines to indolic structures, and the participation of the indolic structures in melanogenesis from the catecholamines is discussed.     

Determination of Catecholamines by Capillary Electrophoresis-Mass Spectrometry

A method for determination of catecholamines (adrenaline, noradrenaline, and dopamine) by capillary electrophoresis-electrospray ionization-mass spectrometry is described. The electrospray operating mode is optimized as well as the system for capillary electrophoresis.     

Measurement of Conjugated Catecholamines in Human Plasma. A Low Cost HPLC-ECD Method

Abstract

We present an HPLC assay with electrochemical detection of conjugated catecholamine levels in human plasma. This method allows the direct assessment of the conjugated catecholamine values without the subtraction of the free catecholamine amounts: the assay is performed in the residual supernatant after the extraction (and the assay) of free catecholamines.

The results are comparable with the classic enzymatic and acid-neat hydrolysis methods (which yield the sum of free and conjugated catecholamine concentrations). This technique shows good reproducibility (within-run and between-run CVs < 8% in the physiological range), and sensitivity (< 20 pg per each deconjugated catecholamine per extract). This method allows about 30 low cost determinations of plasma free and conjugated catecholamines to be done in a working day.     

A Chemiluminiscence Detector for Trace Determination of Fluorescent Compounds

Abstract

A detector making full use of the advantages offered by chemiluminiscence has been constructed. Light measurement was performed with modern electronic equipment using photon counting and the system was tested using dansyl derivatives of adrenaline, noradrenaline and of some amino acids. The detection limits for the two catecholamines were 6 and 16 fmol, respectively, and less than 0.5 fmol for the amino acids. To reach these low levels all reagents were purified or selected for ultra-high purity. The effects of reagent contaminants and of various optical filters have been studied using adrenaline as the test substance.     

PHOTODECOMPOSITION OF CATECHOLAMINES. PHOTOPRODUCTS, QUANTUM YIELDS AND ACTION SPECTRUM OF ADRENALINE

The photochemical decomposition of several catecholamines in aqueous solution was investigated. The respective aminochromes were found to be products of adrenaline and isoprenaline. Irradiation of adrenochrome and N-isopropylnoradrenochrome produced their respective 5, 6-dihydroxyindoles in addition to the ultimate formation of melanine. No trihydroxyindoles could be detected amongst the photoproducts of catecholamines. The action spectrum of adrenaline suggested that the catecholamine itself, in excited state, initiated the first step in the transformation process. The quantum yields for the decomposition were determined for several catecholamines: N-alkyl compounds. decompose more rapidly under influence of light (λ 254 nm) than those with an unsubstituted amino group. In determining the quantum yields the use of corrections for the inner filter effect could be avoided.     

急性心肌梗死患者D-二聚体、cTnT及心肌酶谱联合检测的临床意义

为探讨急性心肌梗死患者D-二聚体、肌钙蛋白及心肌酶谱联合检测的临床意义,选择30例健康者（对照组）和100例患者（病例组）进行D-二聚体、肌钙蛋白及心肌酶谱（AST、LDH、CK、CK-MB）检测,并把100例患者（病例组）分为3组,分别为36例AMI组,34例不稳定型心绞痛（UAP）组,30例稳定型心绞痛（SAP）组。结果表明,AMI组及UAP组中D-二聚体、肌钙蛋白及心肌酶谱（AST、LDH、CK、CK—MB）的升高程度较SAP组和健康对照组显著增高（P〈0．05）;AMI组肌钙蛋白（cTnT）较UAP组明显增高（P〈0．05）;UAP组肌钙蛋白（cTnT）较对照组明显增高（P〈0．05）;D-二聚体、肌钙蛋白及心肌酶谱（AST、LDH、CK、CK—MB）的升高程度与心肌梗死的面积正相关（P〈0．01）。提示D-二聚体、肌钙蛋白及心肌酶谱（AST、LDH、CK、CK—MB）联合检测有助于急性心肌梗死诊断的提高。     

Synthesis and analysis of aminochromes by HPLC-photodiode array. Adrenochrome evaluation in rat blood

The catecholamine oxidation process induces cardiotoxicity and neurotoxicity. Catecholamines can oxidize to aminochromes through autoxidation or by enzymatic or non-enzymatic catalysis. Although some toxic effects seem to be related to the formation of aminochromes there is still scarce information concerning the identification and evaluation of these compounds in in vivo models. In this study five catecholamines were oxidized to their respective aminochromes: adrenaline/adrenochrome; noradrenaline/noradrenochrome; dopa/dopachrome; dopamine/dopaminochrome; and isoproterenol/isoprenochrome. The evaluation of the catecholamines oxidation profile was performed by HPLC with photodiode array detection and using either enzymatic (tyrosinase) or non-enzymatic [Ag(2)O, CuSO(4), NaIO(4) and K(3)Fe(CN)(6)] catalytic systems. The NaIO(4) was found to be the most efficient oxidant of catecholamines. An isocratic reverse-phase HPLC method was developed to analyse each pair of catecholamine-aminochrome. The analytical system was then applied to the detection of adrenochrome in rat blood at 490 nm. Thus, adrenochrome was administered i.p. to rats and its concentration in whole blood was monitored after 5, 15 and 25 min. Blood treatment for adrenochrome evaluation consists of an acidification for protein precipitation followed by a rapid neutralization. The results showed a rapid decrease of adrenochrome concentration in blood after its administration. The adrenochrome present in blood was characterized by UV and tandem mass spectrometry.     

脑梗塞后血清常量元素钙镁含量的动态研究

为探讨急性脑梗塞后血清钙镁含量变化及其对预防和治疗的临床意义,随机选取３２例患者采用不同时期晨起空腹采血,应用ＭＴＢ法６４００－Ａ型临床电解质分析仪测定血清中Ｃａ＾２＋和Ｍｇ＾２＋的含量。结果表明,病例组发病１周时血清Ｃａ＾２＋和Ｍｇ＾２＋含量均低于对照组（Ｐ〈０．０１）,发病后２４ｈ血清Ｃａ＾２＋,Ｍｇ＾２＋含量逐渐降低,到发病１周后血清Ｃａ＾２＋、Ｍｇ＾２＋含量降低最明显,２周后血清Ｃａ＾２＋     

Selective enrichment of catecholamines using iron oxide nanoparticles followed by CE with UV detection

This study examines the use of unmodified magnetite nanoparticles (Fe₃O₄ NPs) for selective extraction and enrichment of the catecholamines dopamine (DA), noradrenaline (NE), and adrenaline (E), prior to analysis using capillary electrophoresis with UV detection. Coordination between Fe³⁺ on‐the‐surface Fe₃O₄ NPs and the catechol moiety of catecholamines enables Fe₃O₄ NPs to capture catecholamines from an aqueous solution. We obtained maximum loading of catecholamines on the NP surface by adjusting the pH of the solution to 7.0. In addition, catecholamine loading on the Fe₃O₄ NPs increased in conjunction with NP concentrations. H₃PO₄ was found to be efficient for the removal of adsorbed catecholamines on the NP surface. Adding 1.2% poly(diallyldimethylammonium chloride) to the background electrolyte resulted in a baseline separation of the liberated catecholamines within 20 min. Under optimal extraction and separation conditions, the limit of detections at a S/N ratio of 3 for E, NE, and DA were 9, 8, and 10 nM, respectively. Significantly, the combination of a phenylboronate‐containing spin column and the proposed method was successfully applied to the determination of NE and DA in human urine and NE in Portulaca oleracea L. leaves.