Fuming Xie

In vivo microdialysis/liquid chromatography/tandem mass spectrometry for the on‐line monitoring of melatonin in rat

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been coupled to in vivo microdialysis for on-line monitoring of melatonin in a freely moving rat for a period of 15 hours. A microdialysis probe was surgically implanted into the jugular vein of the rat, and deionized water was used as the perfusion medium at a flow rate of 1.0 microL/min. Microdialysis samples were collected in an on-line injector with sample injection every 30 minutes. Melatonin was dosed by intraperitoneal (i.p.) injection and then monitored by microdialysis/LC/MS/MS. The whole experiment, including the microdialysis sampling and sample injection into the LC/MS system, was fully automated. Metabolites of melatonin were identified off-line by LC/MSn experiments. Two metabolites were identified as 6-hydroxymelatonin and cyclic 2-hydroxymelatonin, consistent with ones found previously in the literature.

Improved detection limit for catecholamines using liquid chromatography-electrochemistry with a carbon interdigitated array microelectrode

The detection limit of catecholamines can be lowered by using a carbon-based interdigitated array (IDA) microelectrode as a detector for liquid chromatography (LC). The IDA electrode is more sensitive than conventional glassy carbon electrodes due to the high current density caused by radial diffusion at each microband, and redox cycling between two microband arrays. Since the number of redox cycles increases at lower flow-rates, the carbon IDA is particularly useful for microbore LC. In an LC system with a 1-mm microbore column and a carbon IDA electrode, the peak height of dopamine (DA) and DOPAC did not decrease with decreasing flow-rate because of this redox cycling. A low detection limit of 5 fg (32 amol) and 9.6 fg (57 amol) was obtained for DA and DOPAC due to the high current density and low background noise level (0.1 pA) at the carbon IDA electrode. The total charge generated by oxidizing DA at the anodic array was more than the value calculated by assuming that all the DA molecules were oxidized.

Liquid chromatography coupled with multi-channel electrochemical detection for the determination of daidzin in rat blood sampled by an automated blood sampling system.

Daidzin, a soy-derived biologically active natural product, has been reported to inhibit mitochondrial aldehyde dehydrogenase and suppress ethanol intake. This paper describes a method for the determination of daidzin in rat blood. After administration of daidzin, blood samples were periodically collected from awake, freely moving animals by a Culex automated blood sampler. Daidzin was extracted from 50 microl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 12 min using a microbore C(18) (100 x 1.0 mm) 3 microm column with a mobile phase containing 20 mM sodium acetate, 0.25 mM EDTA, pH 4.3, 4% methanol and 11% acetonitrile at a flow-rate of 90 microl/min. Detection was attained using a four-channel electrochemical detector with glassy carbon electrodes using oxidation potentials of +1100, 950, 850, 750 mV vs. Ag/AgCl. The limit of detection for daidzin in rat plasma was 5 ng/ml at a signal-to-noise ratio of 3:1. The extraction recovery of daidzin from rat plasma was over 74%. Linearity was obtained for the range of 25-1000 ng/ml. The intra- and inter-assay precisions were in the ranges of 2.7-6.6 and 1.9-3.7%, respectively. This method is suitable to routine in vivo monitoring of daidzin in rat plasma.

Determination of Melatonin in Commercially Available Products by LCEC and LC/MS/MS

Abstract Simple and reliable methods for liquid chromatography/electrochemistry (LCEC) and LC/MS/MS with atmospheric pressure chemical ionization (APCI) have been developed for the determination of melatonin in commercially available products. The dissolution of melatonin from the melatonin-containing formulations was simply obtained by dissolving the tablets and capsules in 20% acetonitrile in 0.1 N perchloric acid with ultrasonication. The filtrate of the resultant solution was separated on a 1 × 150 mm C

ANALYSIS OF ISOFLAVONES IN NATURAL SOURCES AND NUTRITIONAL SUPPLEMENTS BY LIQUID CHROMATOGRAPHY AND MULTI-CHANNEL ELECTROCHEMICAL DETECTION

A simple gradient reverse-phase liquid chromatographic method has been developed for the separation of daidzin, genistin, daidzein, and genistein in various natural sources and dietary supplements. Peak assignment at various retention times was confirmed by comparison of the simultaneous response of sample peaks to standards, at glassy carbon electrodes held at different oxidation potentials, using a multi-channel electrochemical detector. Identification was substantiated by ESI MS/MS fragmentation patterns. The analytes of interest were extracted by sonicating raw materials in cold 80% methanol/water for 10 minutes. The concentration of the named isoflavones in various dry food and nutritional supplements was determined using the present LCEC procedure.

In vivo microdialysis systems coupled with LCEC and LC/MS/MS for monitoring of melatonin metabolism and regulation in rat

Mammalian circadian clock is located in the suprachiasmatic nucleus (SCN). It is known that photic signals strongly entrain the phase of the circadian clock to daily light-dark cycle via glutamate release in the SCN. Glutamate was reported to cause Ca*+ influx and the activation of calmodulin-dependent protein kinase II (CaMKII) folIwed by the induction of synaptic plasticity in other brain areas. Therefore, we examined the role of CaMKII in the photic entrainment of the circadian clock. We observed that calmodulin inhibitors or a CaMKII inhibitor significantly suppressed light pulse-induced phase shift of hamster wheel-running rhythm and glutamate (10 PM)-induced phase shift of neuronal activity rhythm in the SCN. Furthermore, immunohistochemical studies revealed that CaMKII was densely expressed in the ventral part of SCN and that light exposure increased phospho-CaMKlI, an active form of CaMKII, in the same ventral SCN. These results suggest that the phosphorylation of CaMIUI in the SCN may be necessary for the photic entrainment of circadian clock

Determination of Tetrahydrobiopterin and its Analogues in Biological Samples by Microbore Liquid Chromatography

Abstract Tetrahydrobiopterin and seven of its analogues (neopterin, xanthopterin, biopterin, pterin, tetrahydropterin, 6-methyltetrahydropterin, and 6-methylpterin) were separated on a 1 × 150 mm C18 microbore column. These analytes were detected by dual-electrode amperometry and UV absorption. Both a conventional glassy carbon electrode and an interdigitated array microelectrode were used. Low fmol amounts of xanthopterin, tetrahydropterin, tetrahydrobiopterin and 6-methyltetrahydropterin could be determined by electrochemical detection in the oxidative mode, but pmol amounts of the other analogues were determined by electrochemical detection in the reductive mode and with UV detection. Catecholamines and their metabolites do not interfere with the determination of tetrahydrobiopterin and its analogues in biological samples. The developed method was explored for the determination of tetrahydrobiopterin in samples of human urine and rat tissue (brain, liver, and kidney).