HPLC-electrochemical Detection Research Articles

In Situ Formation of a Relatively Transparent Ion-Associate Liquid Phase from an Aqueous Phase and Its Application to Microextraction/High-Performance Liquid Chromatography-Fluorescence Detection of Bisphenol A in Water.

The design of a simple approach enabling the detection of bisphenol A (BPA) in water samples without the need for large amounts of solvents is of utmost importance. This paper reports a simple method for the separation, concentration, and quantification of BPA in water samples using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after its microextraction into an in situ formed organic ion-associate (IA) liquid phase (LP). Novel IA phase components without conjugated double bonds, such as benzene rings, were investigated. Ethylhexyloxypropylamine hydrochloride and sodium dodecyl sulfate solutions were added to the water samples to form IAs. The aqueous phase and ion-associate liquid phase (IALP) were separated by centrifugation. The aqueous phase was removed, and the liquid phase was recovered and measured using HPLC-FLD or HPLC-electrochemical detection (ECD). The concentrated phase (IALP) had a relatively low viscosity and could be injected directly into the chromatograph without dissolving it in organic solvents. The detection limits for BPA by HPLC-FLD and HPLC-ECD were 0.009 and 0.3 µg L-1, respectively.

Serotonin secretion by blood platelets: accuracy of high-performance liquid chromatography-electrochemical technique compared with the isotopic test and use in a clinical laboratory

BackgroundMild secretion defects are the most frequent and challenging blood platelet disorders to diagnose. Most δ-granule secretion tests lack validation, are not quantitative, or have unreliable response to weak platelet agonists. ObjectivesTo compare platelet serotonin secretion by HPLC-electrochemical detection technique (HPLC-ECD) with the reference isotopic test (3H-5-HT), evaluating its performance in clinical laboratories. MethodsThe assay validation followed STARD-2015 recommendations. HPLC-ECD measured the nonsecreted serotonin remaining in platelet pellets after aggregation, comparing it with the reference 3H-5-HT assay. We studied subjects with inherited and aspirin-induced blood platelet disorders and assessed the HPLC-ECD operation for routine clinical diagnosis. ResultsCalibration curves were linear (R2 = 0.997), with SD for residuals of 3.91% and analytical sensitivity of 5ng/mL. Intra- and interassay imprecision bias ranged between −8.5% and 2.1% and −9% and 3.1%, respectively. Serotonin recovery and stability were >95%, and the variability range of measurements was −5.5% to 4.6%. Statistical differences detected between tests were biologically irrelevant, with bias of 1.48% (SD, 8.43) and CI agreement of −18% to 15%. Both assays distinctly detected platelet secretion induced by 10 μM epinephrine and 4 μmM adenosine diphosphate. However, HPLC-ECD is quantitative and more sensitive to low serotonin content in blood platelets. Reference cutoffs for each agonist were determined in 87 subjects. Initially, the HPLC-ECD requires relatively expensive equipment and trained operators but has remarkably cheap running costs and a turn-around time of 24-36 hours. We have used this diagnostic tool routinely for >8 years. ConclusionHPLC-ECD assay for platelet serotonin secretion is highly accurate, has advantages over the reference 3H-5-HT test, and is suitable as a clinical laboratory technique.

Review of the Analytical Methods Based on HPLC-Electrochemical Detection Coupling for the Evaluation of Organic Compounds of Nutritional and Environmental Interest

This review would like to show the state of the art regarding the coupling of High-Performance Liquid Chromatography (HPLC) with Electrochemical Detection (ED). Since a universal detector for HPLC is not available, the electrochemical detection methods, thanks to their versatility and specificity, are competitive with respect to the detectors currently used. The papers present in literature on HPLC-ED technique are analyzed and discussed: for example, they regard the development of analytical determinations of resveratrol, rosmarinic acid, aromatic heterocyclic amines and glyphosate in food matrices such as meat, aromatic plants, vegetables, fruit and tomato juices. These papers show that electrochemical sensors used as detectors for HPLC can offer better sensitivity values than other detectors. Furthermore, the use of specific working potentials allows avoid matrix interferences to be avoided by almost exclusively determining the analytes of interest. It should be underlined that HPLC-ED methods have a selectivity that allows for limitation of the sample preparation and clean-up procedures to a minimum, making them quick and easy to apply. In addition, these methods offer advantages such as the possibility of direct analysis, that derivatization is often not necessary, the cost-effectiveness of the instrumentation and the possibility of regenerating the electrodes which allows numerous analyses in succession.

Changes in the antioxidative properties of honeys during their fermentation

Abstract The aim of this preliminary study was to investigate whether the fermentation process affects the antioxidative properties of honeys. Therefore, the total antioxidant potential (TAP) of eleven meads was examined. TAPs were estimated using DPPH, hydroxyl radicals, and amperometric measurements in the flow-through HPLC system. The results were correlated with the total content of polyphenols and anthocyanins. Additionally, the concentrations of ethanol, sugars, and hydrogen peroxide were measured. The influence of side reactions and the presence of hydrogen peroxide, glucose, ethanol, and anthocyanins on the free radicals generation were also tested. The use of HPLC electrochemical detection for TAP measurements is particularly preferred. Depending on the potential used, different antioxidants can be measured. It turned out that fruit and species meads are characterized by much higher TAPs measured at high potential of the working electrode than at lower potential due to the high concentration of weak antioxidants which do not provide signals at low potentials. It was found that dark honeys (buckwheat, honeydew) are characterized by much higher TAP values than light ones (acacia, linden, multi-floral). The concentration of anthocyanins decreased during fermentation. They are removed together with the sediment.

HPLC electrochemical detection and quantification of monoamines and their metabolites in rat brain tissue samples.

The monitoring of monoamines and their metabolites in CNS samples can be very valuable in pharmaceutical and biomedical research. A specific high performance liquid chromatography, coupled to a coulometric electrochemical detection method, for the assay of monoamines (dopamine, norepinephrine, epinephrine and serotonin) and their metabolites in rat brain tissue samples was developed. The chromatographic separation was achieved on a C8 reversed phase column with a mobile phase consisting of 0.1 M sodium formate buffer, 5 mM sodium 1-heptanesulfonate, 0.17 mM ethylenediaminetetraacetic acid disodium salt and 5% v/v acetonitrile (pH ±4.0). The detection was achieved through electrochemical detection, with a coulometric cell potential setting of +650 mV. The flow-rate was at 1 ml/min and the total run time was 50 min. The method was validated according to validation guidelines. The method was found to be linear (R2 > 0.99) over the analytical range (5 to 200 ng/ml) for all monoamines and their metabolites. All the other validation parameters were acceptable and within range. The method was applied to three rat brain areas (pre-frontal cortex, hippocampus and striatum), where the monoamines (except for epinephrine) and their metabolites were easily detected.

RTP801 is a critical factor in the neurodegeneration process of A53T α-synuclein in a mouse model of Parkinson's disease under chronic restraint stress.

Recently, the incidence of Parkinson's disease has shown a tendency to move to a younger population, linked to the constantly increasing stressors of modern society. However, this relationship remains obscure. Here, we have investigated the contribution of stress and the mechanisms underlying this change. Ten-month-old α-synuclein A53T mice, a model of Parkinson's disease (PD), were treated with chronic restraint stress (CRS) to simulate a PD-sensitive person with constant stress stimulation. PD-like behavioural tests and pathological changes were evaluated. Differentiated PC12-A53T cells were treated with corticosterone in vitro. We used Western blot, microRNA expression analysis, immunofluorescence staining, dual luciferase reporter assay and HPLC electrochemical detection to assess cellular and molecular networks after stress treatment. In vivo, stereotaxic injection of shRNA lentivirus was used to confirm our in vitro results. The protein RTP801 is encoded by DNA-damage-inducible transcript 4, and it was specifically increased in dopaminergic neurons of the substantia nigra after CRS treatment. RTP801 was post-transcriptionally inhibited by the down-regulation of miR-7. Delayed turnover of RTP801, through the inhibition of proteasome degradation also contributed to its high content. Elevated RTP801 blocked autophagy, thus increasing accumulation of oligomeric α-synuclein and aggravating endoplasmic reticulum stress. RTP801 inhibition alleviated the symptoms of neurodegeneration during this process. RTP801 is a promising target for the treatment of PD, especially for PD-sensitive patients who live under increased social pressure. Down-regulation of RTP801 could inhibit the current tendency to an earlier onset of PD.

Нейропротекторные эффекты антител к глутамату при нарушениях памяти, индуцированных олигомерами провоспалительного белка S100A9, у стареющих животных

Цель исследования - изучение эффектов хронического интраназального введения антител к глутамату совместно с полученными in vitro олигомерами провоспалительного белка S100A9 на процесс воспроизведения пространственной памяти, а также на содержание нейромедиаторных аминокислот и биогенных аминов в релевантных структурах мозга - гиппокампе и префронтальной коре у 12-месячных мышей С57Bl/6. Методика. В поведенческих экспериментах у всех животных проводили выработку условного рефлекса пассивного избегания и тестировали воспроизведение памятного следа, после этого в нейрохимическом исследовании в гиппокампе и префронтальной коре методом ВЖХ проводили определение концентрации нейромедиаторных аминокислот и биогенных аминов. Результаты. Показано, что введение белка S100A9 олигомеров индивидуально приводило к нарушению воспроизведения памятного следа, а в сочетании с антителами к глутамату данного эффекта выявлено не было. Обнаружено значительное повышение концентрации глутамата в обеих церебральных структурах стареющих животных при действии S100A9 олигомеров и снижение содержания аминокислоты при совместном введении с антителами к глутамату до нормы. Показано существенное снижение содержания дофамина в гиппокампе и префронтальной коре в условиях влияния S100A9 олигомеров, а также повышение уровня его метаболитов в гиппокампе с нормализацией обмена дофамина в присутствии антелел к глутамату. Заключение. Выявленные антиамнестические эффекты антител к глутамату и нормализация нейрохимического профиля в условиях вызванной центральной токсичностью S100A9 олигомерами могут быть использованы в разработке подходов нейропротективной коррекции в том числе при болезни Альцгеймера. The aim of this study was to investigate effects of chronic intranasal administration of glutamate antibodies with in vitro -generated proinflammatory S100A9 protein oligomers, on spatial memory of 12-month old C57Bl/6 mice. In addition, the brain content of neurotransmitter amino acids and biogenic amines was monitored in memory-relevant brain structures (hippocampus and prefrontal cortex) of these animals. Methods. In behavioral experiments, all animals were conditioned in a passive avoidance reflex test and the memory trace was evaluated. In a simultaneous neurochemical study, HPLC-electrochemical detection analysis was performed to measure concentrations of neurotransmitter amino acids and biogenic amines in the hippocampus and prefrontal cortex. Results. Administration of S100A9 oligomers alone resulted in disruption of the memory trace retrieval whereas their combination with glutamate antibodies abolished this memory disorder. Significant increases in glutamate concentration were observed in both of the cerebral structures of ageing animals in response to S100A9 oligomers alone, and there was a reduction of the amino acid levels when coadministered with glutamate antibodies. S100A9 oligomers also evoked a decrease in hippocampal and prefrontal cortical dopamine and synchronously elevated dopamine metabolite concentrations, both of these actions being normalized by glutamate antibody coadministration. Conclusion. Disclosure of the antiamnesic effects of glutamate antibodies, along with their neurochemical stabilizing activity to S100A9 oligomer neurotoxicity might be utilized in the development of neuroprotective approaches in Alzheimer’s disease.

1-Methyl-4-Phenylpyridinium-Induced Death of Differentiated SH-SY5Y Neurons Is Potentiated by Cholesterol

Background/Aims: Hypercholesterolemia is recently considered a risk factor for Parkinson’s disease (PD), the most consistent neurodegenerative movement disorder. The study aimed to investigate the effect of exogenous cholesterol on 1-methyl-4-phenylpyridinium (MPP⁺) parkinsonian neurotoxin-induced cell death, loss of mitochondrial membrane potential, and dopaminergic deficiency in a cellular model of PD. Methods: Cholesterol (50 μM) when added in the culture media alone or in combination with MPP⁺ was studied in SH-SY5Y neuroblastoma cells. There were 4 groups that were studied; SH-SY5Y cells treated with vehicle (control), cells that were treated with 1 mM MPP⁺ (MPP⁺), or cholesterol (chol) or both (M + chol). The loss of cell survival was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Dopamine depletion, microtubule-associated protein 2 (MAP-2), and tyrosine hydroxylase (TH)-positive neuronal loss were determined by HPLC-electrochemical detection and TH immunocytochemistry respectively. Mitochondrial membrane potential in cells stained by tetramethylrhodamine methyl ester dye was analysed by flow cytometry. Results: Cholesterol treatment potentiated a reduction of neuronal viability with loss of TH-positive neurons in cultures. MPP⁺-induced depletion of dopamine level in the post-mitotic MAP-2 immunoreactive neurons and loss of mitochondrial membrane potential were also heightened by cholesterol. Conclusion: Apparently, changes in neuronal cholesterol content significantly influenced the neurotoxicity and the direct mitochondrial mechanisms involved in MPP⁺-induced cell death. Our observations demonstrate that high cholesterol incorporated into the differentiated human neuroblastoma cells worsened dopaminergic neuronal survivability through increased depolarization of mitochondrial membrane potential, which is a known mechanism of dopaminergic cell death by MPP⁺. The present findings support the hypothesis that hypercholesterolemia could be a risk factor for PD.

Determination of urinary 15α-hydroxyestrogen levels via immunoaffinity extraction.

15α-Hydroxyestrogens (15α-OHEs) are metabolites of the female hormone estradiol. In this study, to discover physiological markers that can be utilized for monitoring fetal conditions and estrogen-induced cancers, we established a method for quantifying 15α-OHEs in rat urine via immunoaffinity column extraction and HPLC-electrochemical detection, and detected 15α-OHEs in urine obtained male rats treated with estradiol. Notably, the standard curves for quantification obtained using the column were linear over a range of 0.5-50ng 15α-OHEs. The accuracy of the analytical method with cleanup was 97-109% for the three kinds of 15α-OHEs examined, and the intra-assay precision of the measured values had a coefficient of variation of ≤20.6%. Therefore, the theoretical limit of quantification was 0.5ng. However, the actual measured values obtained from the urine of male rats indicated that the detection limits were 0.425, 0.103, and 0.047ng for estetrol, 15α-hydroxyestradiol, and 15α-hydroxyestrone, respectively. Our newly established method for measuring 15α-OHE concentrations in urine could facilitate characterization of the in vivo metabolic profile of 15α-OHEs in mammals under various physiological conditions, which could comprise clinical markers for monitoring human fetal health conditions in mammals.

Attention deficit-hyperactivity disorder suffers from mitochondrial dysfunction

BackgroundPathophysiology of attention-deficit hyperactivity disorder (ADHD) is not known, and therefore the present study investigated mitochondrial defects, if any in cybrids created from patients and control population. MethodsTo investigate mitochondrial pathology in ADHD, cybrids cell lines were created from ADHD probands and controls by fusing their platelets with ρ0-cells prepared from SH-SY5Y neuroblastoma cell line. Cellular respiration, oxidative stress, mitochondrial membrane potential and morphology were evaluated employing oxygraph, mitochondria-specific fluorescence staining and evaluation by FACS, and immunocytochemistry. HPLC-electrochemical detection, quantitative RT-PCR and Blue Native PAGE were employed respectively for assays of serotonin, mitochondrial ATPase 6/8 subunits levels and complex V activity. ResultsSignificantly low cellular and mitochondrial respiration, ATPase6/8 transcripts levels, mitochondrial complex V activity and loss of mitochondrial membrane potential and elevated oxidative stress were observed in ADHD cybrids. Expression of monoamine oxidizing mitochondrial enzymes, MAO-A and MAO-B levels remained unaffected. Two-fold increase in serotonin level was noted in differentiated cybrid-neurons. ConclusionsSince cybrids are shown to replicate mitochondrial defects seen in post-mortem brains, these observed defects in ADHD cybrids strongly suggest mitochondrial pathology in this disorder. General significanceMitochondrial defects are detected in ADHD cybrids created from patients' platelets, implying bioenergetics crisis in the mitochondria could be a contributory factor for ADHD pathology and/or phenotypes.

Anti-depressant effects of Gastrodia elata Blume and its compounds gastrodin and 4-hydroxybenzyl alcohol, via the monoaminergic system and neuronal cytoskeletal remodeling

Ethnopharmacology relevanceGastrodia elata Blume is a highly valuable traditional Chinese medicine used in the treatment of depression. However, compounds with antidepressant effects in water extracts of G. elata Bl. (WGE) have not been identified. The aims of this study were to determine the major antidepressant compound in WGE and to evaluate the antidepressant effects of WGE and its active compounds which involved the monoaminergic system and neuronal cytoskeletal remodeling. Materials and methodsGastrodin (GAS) and 4-hydroxybenzyl alcohol (HBA) in WGE, were analyzed with high-performance liquid chromatography (HPLC)-ultraviolet detection.The forced swimming test (FST) was used to induce depression-like symptoms in 9 weeks old male Sprague-Dawley rats. The open field test (OFT) was used to measure anxiety after WGE, GAS, and HBA treatments. The levels of monoamine such as serotonin (5-HT), dopamine (DA), and their metabolites 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured using HPLC-electrochemical detection. Western blotting was used to examine the 5-HT1A receptor and the neuronal cytoskeleton remodeling-related proteins, Slit, dihydropyrimidinase-related protein 2 (DPYSL2, also called CRMP2), Ras homologous member A (RhoA), and profilin 1 (PFN1) in vivo. Slit1 expression was evaluated in Hs683 cell line after treated with WGE (0.5mg/mL), GAS (50, 100 and 100μM), and HBA (50, 100 and 100μM). ResultsOral administration of WGE (500mg/kg bw), GAS (100mg/kg bw), and HBA (100mg/kg bw) exhibited the anti-depressant effect by significantly reducing the immobility time in FST, monoamine metabolism including the 5-HT to 5-HIAA in the hippocampus and DA to DOPAC and HVA ratios in the frontal cortex, amygdala, and hippocampus. In the hippocampus, the expression of the neuronal cytoskeleton remodeling-related negative regulators Slit1 and RhoA were significantly down-regulated. In addition, the positive regulators CRMP2 and PFN1 were significantly up-regulated following GAS, HBA, and WGE treatments. Moreover, WGE, GAS, and HBA were directly down-regulated Slit1 expression in Hs683 cells. ConclusionWGE, GAS, and HBA exhibited potential anti-depressant effects in rats by decreasing monoamine metabolism and modulated cytoskeleton remodeling-related protein expression in the Slit-Robo pathway. These results suggest that WGE can be used as agent for depressive prevention.

Reassessment of the antioxidative mixture for the challenging electrochemical determination of dopamine, noradrenaline and serotonin in microdialysis samples.

In recent years, the simultaneous monitoring of the monoamine neurotransmitters dopamine, noradrenaline and serotonin in vivo is advancing due to innovations in miniaturized and fast chromatographic techniques. However, the determination of the most hydrophilic compound, noradrenaline, in microdialysis samples by (ultra-)high performance liquid chromatography ((U)HPLC) with electrochemical detection (ECD) is impeded by a broad solvent front, caused by the addition of antioxidative agents. Hence, an elaborate reassessment of currently used antioxidative mixtures is necessary for further analytical improvements. The proposed mixture, containing 100mM acetic acid, 0.27mM Na2EDTA and 12.5μM ascorbic acid (pH 3.2), is less complex than previously described mixtures and shows minimal ECD interference. It stabilizes the three monoamines in standard solutions and in microdialysis samples, considering both autosampler stability at 4°C for 48h and long term stability at -20°C for a duration of six months. An in vivo microdialysis experiment demonstrates the possibility to monitor changes in extracellular levels of the three monoamines simultaneously in the rat hippocampus with UHPLC-ECD using the optimized antioxidative mixture.

Boron doped diamond microelectrodes arrays for electrochemical detection in HPLC

Boron doped diamond microelectrodes arrays (MEA) have been prepared in order to be used as new amperometric sensors in electrochemical cells for HPLC detectors. The following parameters were studied: number and diameter (15–40µm) of the electrodes, distance between them (50–240µm), and effect of the flow rate (0.1–3mL/min). It was thus possible to find the optimum value of the parameters which give a good signal/noise ratio in the chronoamperometric responses, with a size of the electrochemical sensors as small as possible.

A Ferrocene‐Tagged Amyloid‐β Fragment for Rapid Screening of Aggregation Inhibitors from Natural Compounds by HPLC‐Electrochemical Detection

AbstractAggregates of amyloid beta (Aβ) peptides are believed to be responsible for the neuropathology of Alzheimer’s disease. In this work, ferrocene (Fc) is attached to the aggregating core of the Aβ peptides, KLVFFAE. Inhibition of Fc‐KLVFFAE aggregation by curcumin, a natural compound, was monitored by HPLC‐electrochemical detection (HPLC‐EC). The Fc oxidation current is dependent on the incubation condition and curcumin can retain Fc‐KLVFFAE in its monomeric form. We demonstrate that tagging Fc to KLVFFAE affords a cost‐effective and electroactive mimicry of Aβ(142) and HPLC‐EC is suitable for sensitive, reproducible, and facile screening of drugs for inhibiting the aggregation of Aβ peptides.

Title: Effect of Insulin‐like Growth Factor 1 on Cobalamin Composition in Neuronal Cells

Cobalamin is an essential co‐factor for methionine synthase (MS), which catalyzes the conversion of homocysteine to methionine, however, oxidization of cobalamin halts MS activity. In cultured neuronal cells reactivation of MS requires the antioxidant glutathione (GSH) to produce the active cofactor methylcobalamin (MeCbl). Hydroxocobalamin is rapidly and irreversibly converted to glutathionylcobalamin (GSCbl) in the presence of glutathione, then to MeCbl in the presence of S‐adenosylmethionine. Cysteine is the rate‐limiting precursor for GSH synthesis, and in neurons uptake via excitatory amino acid transporter 3 (EAAT3) provides the predominant source of cysteine. Insulin growth factor 1 (IGF‐1) increases cellular cysteine uptake by promoting cellular surface expression of EAAT3. We investigated the influence of IGF‐1 on cellular redox status and cobalamin composition in SH‐SY5Y human neuroblastoma cells. HPLC separation and electrochemical detection were used to measure six intracellular cobalamin species, as well as the levels of GSH and its oxidized GSSG form. We found that GSCbl is a prominent intracellular cobalamin species in SH‐SY5Y cells. IGF‐1 treatment shifted the cellular environment to a more reduced state by increasing intracellular levels of GSH and the GSH/GSSG ratio, leading to an increased level of GSCbl. This novel action of IGF‐1 allows it to regulate cellular redox status.

A novel HPLC-electrochemical detection approach for the determination of d-penicillamine in skin specimens

d-penicillamine is a thiol drug mainly used for Wilson's disease, rheumatoid arthritis and cystinuria. Adverse effects during normal use of the drug are frequent and may include skin lesions. To evaluate its toxic effects in clinical cases an original method based on high performance liquid chromatography coupled to amperometric detection in a specific biological matrix such as skin has been developed.The chromatographic analysis of d-penicillamine was carried out on a C18 column using a mixture of acid phosphate buffer and methanol as the mobile phase. Satisfactory sensitivity was obtained by oxidizing the molecule at +0.95V with respect to an Ag/AgCl reference electrode. A chemical reduction of d-penicillamine-protein disulphide bonds using dithioerythritol combined with microwaves was necessary for the determination of the total amount of d-penicillamine in skin specimens. A further solid-phase extraction procedure on C18 cartridges was implemented for the sample clean-up. The whole analytical procedure was validated: high extraction yield (>91.0%) and satisfactory precision (RSD<6.8%) values were obtained. It was successfully applied to skin samples from a patient who was previously under a long-term, high-dose treatment with the drug and presented serious d-penicillamine-related dermatoses. Thus, the method seems to be suitable for the analysis of d-penicillamine in skin tissues.

Comparative study on effect of three preparations of Xiaoyao formula on soothing liver and strengthening spleen

To compare the effect of three preparations of xiaoyao formula (xiaoyao capsule, xiaoyao pill and Xiaoyao decoction) on soothing liver and strengthening spleen. The liver depression and spleen deficiency rat model were established by forcing swimming, confining movement test and feeding on alternate days. The rats were randomly divided into eight groups, namely the blank group, the model group, the Cisapride group (2.7 mg x kg(-1)), the xiaoyao decoction group (1.62 g x kg(-1)), the xiaoyao pill group (1.62 g x kg(-1)) and the Xiaoyao capsule groups of high dose (3.24 g x kg(-1)), medium dose (1.62 g x kg(-1)) and low dose (0.81 g x kg(-1)), with intragastric administration for 3 weeks. The contents of NE, DA, 5-HT in serum were measured by HPLC-electrochemical detection method. The contents of motilin (MTL) and somatostation (SS) in plasma was determined by radioimmunassay. The expressions of MTL and SS in tissue were determined by immunohistochemical method. The contents of NE and MTL in the xiaoyao decoction group was significantly higher than that in the model group, while the content of 5-HT and SS was significantly lower than that in the model group (P < 0.01). The expression of MTL in the xiaoyao capsule group (1.62 g x kg(-1)) was significantly higher than that in the model group and the contents of 5-HT and SS was significantly lower than that in the model group (P < 0.05). The mean optical density of MTL in gastrointestinal tissue of rats in the xiaoyao decoction group and the xiaoyao capsule group (1.62 g x kg(-1)) was remarkably higher than that in the model group, while the expression of SS was notably lower than that in the model group (P < 0.05). All of the three preparations of Xiaoyao formula have the effect on soothing liver and strengthening spleen. Xiaoyao decoction shows better effect than xiaoyao capsule with same dosage, while xiaoyao capsule shows better effect than xiaoyao pill with same dosage.

Tear fluid small molecular antioxidants profiling shows lowered glutathione in keratoconus

Keratoconus (KC) is a non-inflammatory disease of the cornea involving structural changes. Oxidative stress is reported to be parts of its pathology, yet the tear antioxidant status contributed by smaller molecule antioxidants can be indicative of the disease. Therefore this study is an attempt to present the status of small molecule antioxidants in KC condition as well as the influence of contact lens wear (CLW) in KC as evaluated in the tear specimen. Tear fluid was collected using schirmer strips from KC with and without CLW (n = 40) with age matched controls (n = 29). Tear fluid antioxidants cysteine, ascorbic acid, glutathione, uric acid and tyrosine were determined by HPLC electrochemical detection. Tear reactive oxygen species was estimated by fluorescence detection using dichlorodihydrofluorescein diacetate (DCF-DA) method. The corneal epithelial mRNA expression of the enzymes, gamma-glutamine cysteinyl synthase (γ-GCS) and gamma-glutamyl transpeptidase (γ-GT) by semiquantitative RT-PCR. Among the five antioxidant molecules estimated, GSH decreased significantly 50.9 ± 9.4 μM in control and 16 ± 5.7 μM in KC (p = 0.015) with increase in tyrosine 13.9 ± 2.6 μM in control, 30 ± 6.4 μM in KC cases (p = 0.022) and uric acid 162 ± 18 μM in control and 210 ± 32 μM (p < 0.00) in KC compared to the controls. The ROS levels were increased significantly, 55.7 ± 16.7 AU in KC and 23.2 ± 5.8 AU in controls (p = 0.023). No significant change in the tear antioxidants studied was observed in KC cases with and without CL wear. However tyrosine levels were increased significantly in CL wear amongst healthy controls compared to controls (p < 0.000). γ-GCS and γ-GT showed no significant change in KC epithelial cells. Though variations were seen in other antioxidants analysed, they had no statistical significance. Tear specimen in KC can indicate the antioxidant status. KC is associated with increased tear levels of tyrosine, uric acid and decrease in GSH apart from increased ROS. Glutathione decreases with increase in oxidative stress and this emphasises the need for antioxidants to balance the redox status in disease management of KC.

Ionic Cross Talk Can Lead to Overestimation of 3-Methoxytyramine during Quantification of Metanephrines by Mass Spectrometry

To the Editor: Measurements of urine or plasma metanephrine (MN)1 and normetanephrine (NMN) are considered pivotal tests in the diagnosis of pheochromocytoma and paraganglioma. There has also been increasing interest in measuring 3-methoxytyramine (3MT), the corresponding O-methylated metabolite of dopamine. 3MT measurement might allow earlier diagnosis of rare cases of pheochromocytoma and paraganglioma that secrete predominately dopamine (1,–,3). 3MT measurement might also enable stratification of familial cases by secretion patterns and play a role in predicting the malignant potential of pheochromocytoma and paraganglioma (2). Eisenhofer and others have used HPLC–electrochemical detection (HPLC-ECD) to measure 3MT in plasma (1,–,4). de Jong et al. developed an online solid-phase extraction liquid chromatography–tandem mass spectrometry (LC-MS/MS) method that simultaneously measures NMN, MN, and 3MT (5). We attempted to reproduce the latter method for plasma 3MT measurement and were initially pleased with its analytical performance. When we attempted to establish a reference interval for a healthy population, however, it became apparent that our upper reference limit of <73 pg/mL (<0.437 nmol/L) was much higher than the 1–14 pg/mL (0.006–0.084 nmol/L) interval for the published HPLC-ECD method (1). de Jong et …

High-Performance Anion-Exchange Chromatography Coupled with Pulsed Electrochemical Detection as a Powerful Tool to Evaluate Carbohydrates of Food Interest: Principles and Applications

Specific HPLC approaches are essential for carbohydrate characterization in food products. Carbohydrates are weak acids with pKa values in the range 12–14 and, consequently, at high pH can be transformed into oxyanions, and can be readily separated using highly efficient anion-exchange columns. Electrochemical detection in HPLC has been proven to be a powerful analytical technique for the determination of compounds containing electroactive groups; pulsed amperometric detection of carbohydrates is favourably performed by taking advantage of their electrocatalytic oxidation mechanism at a gold working electrode in a basic media. High-performance Anion Exchange Chromatography (HPAEC) at high pH coupled with pulsed electrochemical detection (PED) is one of the most useful techniques for carbohydrate determination either for routine monitoring or research application. This technique has been of a great impact on the analysis of oligo- and polysaccharides. The compatibility of electrochemical detection with gradient elution, coupled with the high selectivity of the anion-exchange stationary phases, allows mixtures of simple sugars, oligo- and polysaccharides to be separated with high resolution in a single run. A few reviews have been written on HPAEC-PED of carbohydrates of food interest in the last years. In this paper the recent developments in this field are examined.