Electrodeposited acetaminophen as a bifunctional electrocatalyst for simultaneous determination of ascorbic acid, glutathione, adrenaline and tryptophan

In this study, an acetaminophen-modified glassy carbon electrode (ACMGCE) was fabricated for the purpose of investigating its electrochemical behavior by cyclic voltammetry (CV). ACMGCE serves as an excellent bifunctional electrocatalyst for the oxidation of ascorbic acid (AA) and glutathione (GSH) in a phosphate buffer solution (pH 7.0). The kinetic parameters such as the electron transfer coefficient, α, and the heterogeneous electron transfer rate constant, k', for the oxidation of AA and GSH at the ACMGCE surface were determined by CV. In addition, detection limits of 3.89 μM for AA and 0.37 μM for GSH were obtained at the ACMGCE using a differential pulse voltammetric (DPV) method. In DPV, the bifunctional modified electrode could separate the oxidation peak potentials of AA, GSH, adrenaline (AD) and tryptophan (Trp) present in the same solution, though the peak potentials were indistinguishable at a bare GCE. Finally, the modified electrode was successfully applied to the determination of AA in a pharmaceutical preparation and GSH in a human plasma sample.     

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high-resolution mass spectrometry

Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high-resolution mass spectrometry operated in positive-ion mode. The metabolites were characterized by accurate masses, MS² spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human-specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron-rich 1,4-benzenediamine to a 1,4-diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.     

Identification of primary and sequential bioactivation pathways of carbamazepine in human liver microsomes using liquid chromatography/tandem mass spectrometry

Carbamazepine (CBZ)-induced idiosyncratic toxicities are commonly believed to be related to the formation of reactive metabolites. CBZ is metabolized primarily into carbamazepine-10,11-epoxide (CBZE), 2-hydroxycarbamazepine (2-OHCBZ) and 3-hydroxycarbamazepine (3-OHCBZ), in human liver microsomes (HLM). Over the past two decades, the 2,3-arene oxidation has been commonly assumed to be the major bioactivation pathway of CBZ. Recently, CBZE has been also confirmed to be chemically reactive. In order to identify other possible primary and sequential CBZ bioactivation pathways, individual HLM incubations of CBZ, CBZE, 2-OHCBZ and 3-OHCBZ were conducted in the presence of glutathione (GSH). In the CBZ incubation, a variety of GSH adducts were formed via individual or combined pathways of 10,11-epoxidation, arene oxidation and iminoquinone formation. In the CBZE incubation, the only detected GSH adducts were CBZE-SG1 and CBZE-SG2, which represented the two most abundant conjugates observed in the CBZ incubation. In the incubation of either 2-OHCBZ or 3-OHCBZ, a number of sequential GSH adducts were observed. However, none of the 2-OHCBZ-derived GSH adducts were detected in the CBZ incubation. Meanwhile, several GSH adducts were only observed in the CBZ incubation. In conclusion, CBZ can be bioactivated in HLM via 10,11-epoxidation, 2,3-arene oxidation, and several other pathways. In addition, the sequential bioactivation of 3-OHCBZ appeared to play a more important role than that of either CBZE or 2-OHCBZ in the overall bioactivation of CBZ in HLM. The identification of several new bioactivation pathways of CBZ in HLM demonstrates that possible CBZ bioactivation can be more complex than previously thought.     

Sensitive assay for catecholamines in pharmaceutical samples and blood plasma using flow injection chemiluminescence analysis.

A rapid and sensitive chemiluminescence (CL) method using flow injection analysis was described for the determination of three catecholamines: dopamine, adrenaline and dobutamine, based on their greatly enhancing effects on the CL reaction of luminol-potassium periodate in basic solutions. Under the optimized conditions, the calibration graphs relating the increase of CL intensity to the concentration of the analytes were linear. The present method allows for the determination of dopamine, adrenaline, and dobutamine over the range of 1.0 x 10(-10) - 1.0 x 10(-7) g/ml. The relative standard deviations for measurements (n=11) of dopamine, adrenaline and dobutamine were 2.9, 2.3 and 1.8% when the concentrations of three catecholamines were at 1.0 x 10(-9) g/ml, respectively. The detection limits of the method were 2.0 x 10(-11) g/ml dopamine, 1.0 x 10(-11) g/ml adrenaline and 4.0 x 10(-11) g/ml dobutamine. The method was successfully applied to the determination of three catecholamines in pharmaceutical samples and blood plasma.     

Metabolism of a sulfur-containing heteroarotionoid antitumor agent, SHetA2, using liquid chromatography/tandem mass spectrometry

SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methanethione], NSC 726189}, a sulfur-containing heteroarotinoid, selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective of this study was to investigate its in vitro metabolism in rat and human liver microsomes and in vivo metabolism in the mouse and rat using liquid chromatography-ultraviolet/multi-stage mass spectrometry (LC-UV/MS(n)) on an ion-trap mass spectrometer coupled with a photo-diode array (PDA) detector. In vitro, in the absence of glutathione (GSH), oxidation of the four aliphatic methyl groups of SHetA2 yielded one mono-, two di-, and one tri-hydroxylated SHetA2 metabolites, which were identified based on their UV and multi-stage mass spectra. In the presence of GSH, in addition to these primary oxidative metabolites, four GSH adducts of SHetA2 and a novel rare form thioether GSH adduct was detected and characterized. In vivo, the monohydroxylated SHetA2 metabolites were also detected in mouse and rat plasma and two GSH adducts were detected in rat liver following intravenous (i.v.) bolus administration of SHetA2 at 40 mg/kg.     

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).     

The significance of plasma catecholamine levels in the pathogenesis of arteriosclerosis

Summary Experiments, with animals and with cultured vascular wall cells as well as clinical observations on patients suffering from pheochromocytoma, favour the hypothesis that catecholamines could play an important role in the pathogenesis of arteriosclerosis. As the catecholamines, adrenaline and/or noradrenaline, promote metabolic dysfunctions in vascular wall cells, which are important in the pathogenesis of arteriosclerosis, these compounds may well be acting as chemical mediators during atherogenesis. Therefore investigations on plasma catecholamine levels in patients, exposed to atherogenic risk factors, with different stages of arteriosclerosis, and in patients suffering from thrombotic complications of an arteriosclerotic vascular disease, may lead to an answer for the question of whether or not plasma catecholamine levels are of clinical importance in the pathogenesis of arteriosclerosis in man.In individuals subjected to the atherogenic risk factors, smoking, essential hypertension, and mental stress, plasma catecholamine levels were statistically significantly elevated. In patients suffering from diabetes mellitus plasma adrenaline and noradrenaline concentrations however were similar to those of healthy controls. In dialysis patients, who often develop arteriosclerosis which progresses with great rapidity, plasma adrenaline and noradrenaline concentration showed a positive correlation with different stages of arteriosclerosis; i.e. plasma catecholamine concentration increases with the severity of this disease. Arteriosclerotic vascular diseases sometimes give rise to thrombotic complications, the most prominent ones being myocardial infarction and stroke. Compared with healthy controls, even one year after infarction, 70% of the patients who had had a myocardial infarction and 50% of the stroke patients had significantly elevated plasma adrenaline and/or noradrenaline levels. Further investigations on patients suffering from coronary heart disease showed that elevated plasma catecholamine concentration is more probably related to the pathogenesis of the vascular disease than to the incurrence of myocardial infarction.From these data we conclude that catecholamines are substances that can act as chemical mediators during the pathogenesis of arteriosclerosis in man and thus may contribute to the development and subsequent complications of this important vascular disease.     

Second-order calibration of excitation-emission matrix fluorescence spectra for determination of glutathione in human plasma

A rapid non-separative spectroflourimetric method based on the second-order calibration of the excitation-emission data matrix was proposed for the determination of glutathione (GSH) in human plasma. In the phosphate buffer solution of pH 8.0 GSH reacts with ortho-phthaldehyde (OPA) to yield a fluorescent adduct with maximum fluorescence intensity at about 420 nm. To handle the interfering effects of the OPA adducts with aminothiols other than GSH in plasma as well as intrinsic fluorescence of human plasma, parallel factor (PARAFAC) analysis as an efficient three-way calibration method was employed. In addition, to model the indirect interfering effect of the plasma matrix, PARAFAC was coupled with standard addition method. The two-component PARAFAC modeling of the excitation-emission matrix fluorescence spectra accurately resolved the excitation and emission spectra of GSH, plasma (or plasma constituents). The concentration-related PARAFAC score of GSH represented a linear correlation with the concentration of added GSH, similar to that is obtained in simple standard addition method. Using this standard addition curve, the GSH level in plasma was found to be 6.10 ± 1.37 μmol L⁻¹. The accuracy of the method was investigated by analysis of the plasma samples spiked with 1.0 μmol L⁻¹ of GSH and a recovery of 97.5% was obtained.     

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.     

Biosensors for the Rapid Repetitive Detection of Adrenaline Using Stabilized Bilayer Lipid Membranes (BLMs) with Incorporated Calix[4]resorcinarene Receptor

This work uses lipid film based biosensors with incorporated calix[4]resorcinarene receptor (lipophilic macrocyclic host molecule) for the rapid electrochemical detection of adrenaline. Freely‐suspended and metal supported BLMs (composed of egg phosphatidylcholine (PC) and 35% (w/w) dipalmitoyl phosphatidic acid) modified with the resorcin[4]arene receptor were used as one shot sensors to rapidly detect this catecholamine. The interactions of this compound with freely‐suspended BLMs were found to be electrochemically transduced in the form of a transient current signal with duration of seconds, which reproducibly appeared about 14 s after exposure of the membranes to adrenaline. The response time for these BLMs without incorporated receptor for adrenaline was about 1.5 min. The magnitude of the transient current signal was related to the concentration of adrenaline in bulk solution in the micromolar range. Differential scanning calorimetric (DSC) experiments were performed to explore the mechanism of interactions of BLMs with incorporated receptor with adrenaline. The interactions of adrenaline with surface‐stabilized bilayer lipid membranes (sBLMs) with incorporated receptor produced electrochemical ion current increases, which reproducibly appeared within a few seconds after exposure of the membranes to the stimulant. The use of the receptor in sBLMs increased the sensitivity of the method 6‐fold. The current signal increases were related to the concentration of adrenaline in bulk solution in the micromolar range. Stabilized lipid membranes formed by polymerization on glass fiber microfilters were used as practical chemical biosensors for the rapid detection of adrenaline. The interactions of polymerized lipid films with adrenaline were also found to provide transient current signals similar to those of freely‐suspended BLMs. The magnitude of the transient current signal was also related to the concentration of the stimulating agent in bulk solution in the micromolar range and these stabilized lipid films can be used again after storage in air. No interferences from ascorbic acid were noticed because of the negatively charged lipids in membranes. The effect of other compounds such as proteins and other compounds closely related to adrenaline was also investigated. Results of recovery experiments using human urine have shown recoveries ranged between 94 to 105%, which shows no interferences from matrix effects due to the presence of urine constituents. The present sensor based on stabilized lipid films can be used for the rapid repetitive detection of this pharmaceutical substance and keep prospects for the selective determination of catecholamines in biofluids.     

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.     

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.     

Spectrophotometric determination of catecholamines with metaperiodate by flow-injection analysis

A flow-injection Spectrophotometric method for the determination of adrenaline and isoprenaline, based on the reaction with metaperiodate, is described. The calibration graphs are linear up to 2 × 10⁻⁴ M. Flow injection allows the measurement of 120 samples per hour. The method was successfully applied to the determination of both catecholamines in pharmaceuticals.     

Characterization of the metabolites of rosmarinic acid in human liver microsomes using liquid chromatography combined with electrospray ionization tandem mass spectrometry

Rosmarinic acid (RA) is a phenolic acid originally isolated from the herb medicine Rosmarinus officinalis. The purpose of this study was to identify the metabolites of RA. RA was incubated with human liver microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate tetrasodium salt and/or uridine diphosphate glucuronic acid using glutathione (GSH) as a trapping agent. After 60-min incubation, the samples were analyzed using high-resolution liquid chromatography tandem mass spectrometry. Under the current conditions, 14 metabolites were detected and identified. Our data revealed that RA was metabolized through the following pathways: the first pathway is the oxidation of catechol to form ortho-quinone intermediates, which react with GSH to form mono-GSH adducts (M1, M2, and M3) and bis-GSH adducts (M4 and M5); the second pathway is conjugation with glucuronide to yield acylglucuronide (M7), which further reacts with GSH to form RA-S-acyl-GSH adduct (M9); the third pathway is hydroxylation to form M10, M11, and M12, which further react with GSH to form mono-GSH adducts (M13 and M14); the fourth pathway is conjugation with GSH through Michael addition (M6); the fifth pathway is conjugation with glucuronidation, forming M8, which is the major metabolic pathway of RA.     

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.     

Validation of a high performance liquid chromatography analysis for the determination of noradrenaline and adrenaline in human urine with an on-line sample purification

A high performance liquid chromatography (HPLC) method with fluorescence detection including an on-line purification was established for determination of catecholamines in human urine. The method was evaluated using samples of pooled urine spiked with catecholamines and validated for measurements of catecholamines in urine of healthy individuals in a field study. The laboratory method evaluation study showed that the recovery of the method was 0.82 (confidence interval (CI): 0.79-0.86) and 0.92 (CI: 0.89-0.95) for noradrenaline and adrenaline, respectively. Thus, correction factors of 0.82(-1) and 0.92(-1) were applied to correct the measurement results for this systematic effect. Furthermore, an uncertainty budget was generated for the analytical method using the BIPM-approach recommended by the International Organization for Standardization. The relative uncertainty of the method was estimated to be 10-12%, which was consistent with the observed relative variability found in the method evaluation. The method was evaluated in accordance with EURACHEM Guidance Document No 1 concerning accreditation for chemical laboratories with respect to accuracy and precision. The field study showed that the standard deviation of the method was sufficiently low to distinguish between persons working with two different processes of garbage collection, i.e. collection using four wheeled containers versus collection using bins.     

Identification and quantification of glutathione adducts of clozapine using ultra-high-performance liquid chromatography with orthogonal acceleration time-of-flight mass spectrometry and inductively coupled plasma mass spectrometry

The application of sulphur-specific detection via ultra-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (UPLC/ICPMS) to detect and quantify the glutathione (GSH)-adducts produced via the in vitro formation of reactive metabolites is demonstrated. The adducts were formed in human liver microsomes supplemented with unlabelled GSH for clozapine. The calculation of adduct concentration was performed via comparison of the peak areas to calibration curves constructed from omeprazole, a sulphur-containing compound over the range of 0.156 to 15.62 μM of sulphur with a detection limit of 1.02 ng of sulphur on-column. Identification of the adducts was performed using conventional UPLC/time-of-flight (TOF)-MS with the calculation of clozapine intrinsic clearance carried out by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). The use of ICPMS in this way appears to offer a novel, rapid and sensitive means of determining the quantity of GSH conjugates with the combined adducts producing 0.9 μM of reactive metabolite out of a total of 3.5 μM of metabolites. The GSH adduct therefore represents 26% of this total produced as a result of the metabolism of drug to reactive species.     

Improved detection of reactive metabolites with a bromine‐containing glutathione analog using mass defect and isotope pattern matching

Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug‐induced toxicity. Reactive metabolite detection using in vitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N‐(2‐bromocarbobenzyloxy)‐GSH (GSH‐Br), for the in vitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. In vitro rat microsomal incubations were performed with GSH and GSH‐Br for each drug with subsequent analysis by liquid chromatography/high‐resolution mass spectrometry on an electrospray time‐of‐flight (ESI‐TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug‐specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH‐Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH‐Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations. Copyright © 2010 John Wiley & Sons, Ltd.     

Solid Phase Reactors as an Analytical Tool in the Determination of Urinary Noradrenaline and Adrenaline

Abstract

The possibility of performing the oxidation of the catecholamines noradrenaline and adrenaline inside a PbO₂- or a MnO₂- solid phase reactor (SPR), which is integrated in the chemical reaction detector of the trihydroxyindole (THI) method is demonstrated. The adaptation of the SPR to the chromatographic conditions and the optimization of the different reaction parameters is described. A comparison of the two SPRs to the ferricyanide oxidation in the homogenous system is made with respect to accuracy, sensitivity and precision of the results. The new SPR-technique enables simplification and low cost running of the THI-system in routine analysis of urinary catecholamines.     

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.