Simultaneous determination nucleosides in marine organisms using ultrasound-assisted extraction followed by hydrophilic interaction liquid chromatography-electrospray ionization time-of-flight mass spectrometry.

A new method has been developed based on ultrasound-assisted extraction (UAE) followed by hydrophilic interaction chromatography (HILIC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) for the simultaneous determination of 16 nucleosides and nucleobases in medicinal extracts of various marine organisms. The separation was achieved on a Venusil HILIC column (250×4.6 mm id, 5 µm) and gradient elution using a solution of acetonitrile and buffer (0.20% formic acid and 20 mmol/L ammonium acetate) as the mobile phase. Identification of the 16 target nucleosides and nucleobases was based on the retention time, UV spectra, and mass measurements of the protonated molecules ([M+H](+)) and main fragment ions (ESI-TOF/MS). In addition, non-target compounds of 2'-deoxyinosine and four other amino acids were also tentatively identified by ESI-TOF/MS. The 16 target compounds were quantified by HILIC-ESI-TOF/MS under optimized mass conditions. All calibration curves showed good linearity (r(2)>0.9951). The recoveries were 84.72-124.10%, and the limits of detection of the 16 target compounds were 0.6-130.0 ng/mL. The developed method was applied to quantify the target compounds in 15 batches of various marine organisms. The method has potential applicability for the identification and determination of highly polar and low-concentration active compounds in marine organisms.

Multi-residue method for the confirmation of four avermectin residues in food products of animal origin by ultra-performance liquid chromatography-tandem mass spectrometry.

A confirmatory method was developed for the rapid determination of abamectin, ivermectin, doramectin and eprinomectin residues in various food products of animal origin, such as pork muscle, pork liver, fish and milk. Samples were homogenized, extracted and de-proteinized by acetonitrile, cleaned via two-step cleaning procedure using Bond Elut C(18) SPE columns and then alumina-N cartridges. All the four avermectin residues in different animal-food products were simultaneously separated and determined by ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) within 3.5 min. Data acquisition under positive ESI-MS/MS was performed by applying multiple reaction monitoring (MRM) for both identification and quantification, and mass spectrometric conditions were optimized to increase selectivity and sensitivity. The matrix-matched calibration curves for different matrices, such as pork muscle, pork liver, fish and milk, were constructed and the interference effect of different sample matrices on the ionization was effectively eliminated. The UPLC-MS/MS method was validated with satisfactory linearity, recovery, precision and stability. Matrix-matched calibration curves of abamectin, ivermectin, doramectin and eprinomectin in four different matrices were linear (r(2)( )≥ 0.990, goodness-of-fit coefficients ≤12.8%) in the range 2.5-200 µg kg(-1). The limits of detection and quantification for the four avermectins were in the range 0.05-0.68 and 0.17-2.27 µg kg(-1), respectively. Recoveries were 62.4-104.5% with good intra- and inter-day precision. The method was rapid, sensitive and reliable, and can be applied to the quantitative analysis of avermectin residues in different animal-food products.

Rapid screening and identification of the antioxidants in Hippocampus japonicus Kaup by HPLC-ESI-TOF/MS and on-line ABTS free radical scavenging assay.

In this paper, a method based on HPLC coupled with ESI-TOF/MS and on-line to a stable free radical scavenging detection system has been developed to rapidly screen and identify the major antioxidants in the water extract of Hippocampus japonicus Kaup, a kind of marine medicinal organism. The developed on-line stable free radical scavenging detection method of water extract from H. japonicus Kaup revealed the presence of three radical scavenging compounds, which were simultaneously identified as hypoxanthine, phenylalanine and tryptophane by high-resolution TOF-MS. To the best of our knowledge, it is original to demonstrate the rapid and successful use of HPLC-ESI-TOF/MS and on-line 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt free radical scavenging assay to directly and simultaneously screen and identify the antioxidants in the water extracts of H. japonicus Kaup without any purification. The present method provides a useful improvement on the reducing of workload and a powerful tool for rapid screening and identification of free radical scavenging compounds in complex natural products.

GC-MS fingerprints for discrimination of Ligusticum chuanxiong from Angelica.

A GC-MS fingerprinting technique based on the essential oil components has been developed for the discrimination of chuanxiong against Chinese Angelica (Angelica sinensis (Oliv.) Diels) or other herbs with similar compositions. The analytical performance of four different extraction methods for the separation of essential oil components have been compared and these include: ultrasound-assisted extraction (UAE), supercritical fluid extraction (SFE), Soxhlet extraction (SHE) and hydro-distillation extraction (HDE). The results showed that UAE was the most effective extraction method, and the operational parameters of UAE were optimized. 3-Butylphthalide, Z-butylidenephthalide, senkyunolide I, senkyunolide H, E-butylidenephthalide, senkyunolide A, neocnidilide, Z-ligustilide and E-ligustilide were tentatively identified in chromatograms of chuanxiong based on their GC-EI-MS data. Similarity coefficient calculations based on correlation methods have been performed on the GC-MS fingerprints. Using an authentic standard Chuanxiong as the reference, the similarity coefficients between the standard and all other chuanxiong samples ranged from 0.90 to 1.0 (with 1.0 being the perfect match), which as a group can be readily separated from the Angelica samples for which the similarity index against the chuanxiong standard ranged from 0.75 to 0.77. Conversely, when an authentic Angelica standard was used as the reference, the respective similarity coefficients fall in the range of 0.70-0.75 and 0.98-1.00 for the chuanxiong and Angelica sample groups. Our results thus demonstrate that the fingerprinting technique developed in the study can indeed discriminate the two herbs with high reliability.

Analysis of alkaloids in Coptis chinensis Franch by accelerated solvent extraction combined with ultra performance liquid chromatographic analysis with photodiode array and tandem mass spectrometry detections.

A new method based on accelerated solvent extraction (ASE) followed by ultra performance liquid chromatography (UPLC) analysis has been developed for the identification and quantification of major alkaloids in extracts of Coptis chinensis Franch. The UPLC system consisted of a dual detection system of photodiode array detector (PDA) and positive ion electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in sequential configuration. The operational parameters of ASE including extraction solvent, extraction temperature, static extraction time and extraction cycles were optimized. UPLC analysis was performed on an ACQUITY UPLC BEH C(18) column eluted by a mobile phase of acetonitrile spiked with a buffer solution consisting of 0.50% acetic acid and 20 mmol L(-1) ammonium acetate. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the identification and quantitative analysis of eight major alkaloids in C. chinensis Franch extracts. The samples were also analyzed on a high-performance liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) system to confirm the identification results. Three of the eight major alkaloids, berberine, palmatine and jatrorrhizine were quantified by UPLC-PDA and UPLC-MS/MS. The results indicated that both UPLC-PDA and UPLC-MS/MS methods were simple, sensitive and reliable for the determination of alkaloids in C. chinensis Franch. Seven Huanglian samples from different locations were analyzed using the established methods. UPLC fingerprints based on the distribution of the eight major alkaloids can serve as a rapid and reliable method for the authentication and quality evaluation of traditional Chinese medicine (TCM) herbs.

Analysis of major alkaloids in Rhizoma coptidis by capillary electrophoresis-electrospray-time of flight mass spectrometry with different background electrolytes.

CE-based techniques with DAD and detection ESI-TOF-MS have been developed for the analysis of seven protoberberine alkaloids and one aporphinoid alkaloid in Huanglian (Rhizoma coptidis), a well-known traditional Chinese herbal medicine. One aqueous BGE and one nonaqueous BGE were developed for CE-DAD and CE-MS analyses, and the CE-ESI-TOF-MS conditions including nebulizer gas pressure, the sheath-liquid composition, its flow rate, etc. were optimized. Eight main alkaloids in R. coptidis could be separated with baseline resolution by CE-DAD with these two different BGEs, and identified by TOF-MS analysis. Moreover, three major alkaloids (berberine, palmatine, and jatrorrhizine) could be quantified accurately by CE-DAD and CE-MS with the BGE system consisting of 50:50 v/v water and ACN containing 50 mM ammonium acetate at pH 6.8. Both techniques provided similar LODs and could be applied with confidence within similar linear dynamic range. However, reproducibility and speed of analysis were better using CE-DAD. When the CE technique was compared with the RP-HPLC method, the CE-DAD and CE-MS methods provided greater efficiency and faster analysis speed, i.e., achieving baseline resolution for all the eight main basic compounds in less than 14 min. The CE method, as a viable alternative to HPLC, is suitable for use as a routine procedure for the rapid identification and quantification of basic compounds in herbal or natural product applications.

HPLC fingerprinting and LC-TOF-MS analysis of the extract of Pseudostellaria heterophylla (Miq.) Pax root.

High-performance liquid chromatographic (HPLC) was developed for fingerprint analysis of Pseudostellaria heterophylla (Miq.) Pax. Liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (LC-TOF-MS) technique was first employed to identify the components of the fingerprint. Twelve major peaks in chromatographic fingerprint were analyzed by on-line LC-TOF-MS analysis; one cyclic peptide was unequivocally identified and five cyclic peptides were tentatively assigned based on their MS data. These cyclic peptides served as the marker peaks in the HPLC fingerprints. The chromatographic fingerprints have been analyzed by similarity index calculations and hierarchical clustering analysis (HCA). The result showed that the HPLC fingerprints could be used to determine the optimal harvest time for P. heterophylla (Miq.) Pax and to authenticate the species of the herb.

Optimization of HPLC-APCI-MS conditions for the qualitative and quantitative determination of total solanesol in tobacco leaves.

HPLC coupled online with atmospheric pressure chemical ionization MS (APCI-MS) technique was evaluated for the qualitative and quantitative determination of solanesol in extracts of tobacco leaves. The solanesol and other compounds in the extract were separated on an Alltima C(8) (4.6 mm x 250 mm) column with methanol and water (98:2 v/v) as mobile phase, with flow rate of 0.8 mL/min and UV detection wavelength of 211 nm. In the APCI(+) mode, abundant stable [M-H(2)O + H](+) ion (m/z at 613.5) was observed, with low abundance of other fragmentation ions. A comparison of APCI-MS and ESI-MS techniques showed that APCI mode is more sensitive than ESI mode, and thus better suited for solanesol analysis. When comparing UV 211 nm and APCI-MS in SIM for solanesol quantification, the former offered better precision and reproducibility, but the latter was more than 200 times sensitive in detection. The developed method has been successfully applied to the analysis and comparison of solanesol concentration in different tobacco leaf samples.

[Application of accelerated solvent extraction technique for analysis of active components in traditional Chinese medicinal herbs].

The application of accelerated solvent extraction (ASE) technique for the Analysis of active components in traditional Chinese medicinal herbs was introduced by using two kinds of herbs as examples. The conditions including extraction temperature, static extraction time, the ratio of material to solvent and solvent of ASE for extraction of salvianolic acid B in Salvia miltiorrhiza were optimized by orthogonal experiments, and the optimal conditions were obtained. Different extraction methods (ASE, steam distillation, ultrasonic wave and Soxhlet extraction) were used to extract volatile oil in Aucklandia lappa Decne. Results of the comparative experiments indicated that ASE was the most effective method in this case. All the results from these studies demonstrate that ASE is indeed a powerful tool in the preparation of herbal extracts for downstream chromatographic analysis.

[Excitation-emission-matrix spectral property of dissolved organic matter in seawater of Jiaozhou Bay, China].

Excitation-emission matrix spectroscopy (EEMs) was applied to measure the fluorescence properties of dissolved organic matter (DOM) in seawater collected from Jiaozhou Bay in June, 2005. The study areas include central Bay area, receptor areas of Dagu River, Licun River and Haibo River. The aim was to investigate the influence of discharges from several typical rivers to the receiving seawater. Dagu River discharges mostly freshwater, while the other two rivers discharges include more wastewaters from factories and sewages treatments. The results indicate that there is a redshift of the position of humic-like fluorescence peaks due to the discharges from sewages and wastewaters from factories. The intensities of protein-like and humic-like peaks were the strongest in the samples from area near Haibo River and Licun River, while the weakest fluorescence intensities of both protein-like and humic-like were observed in the samples collected from central bay area. This suggests that river inputs are the mains sources of DOM in seawater of Jiaozhou Bay, while DOM produced by bioactivity in situ dominates those in central Bay area. The relationships of protein-like and humic-like fluorescence intensities vary depending on the water samples. The DOM in seawater of Jiaozhou Bay may be polluted by protein-like organic matter produced by human activity, as indicated by higher ratios of protein-like and humic-like fluorescence intensities. The results suggest the potential utility of EEMs technique for trace DOM and land-based pollution sources.

Determination of four major saponins in the seeds of Aesculus chinensis Bunge using accelerated solvent extraction followed by high-performance liquid chromatography and electrospray-time of flight mass spectrometry.

A new method based on accelerated solvent extraction (ASE) followed by a reliable high-performance liquid chromatography-diode array detector (HPLC-DAD) and positive ion electrospray-time of flight mass spectrometry (ESI-TOF/MS) analysis has been developed for the characterization and quantification of four major saponins in extracts of the seeds of Aesculus chinensis Bunge (semen aesculi). The saponins escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted from seeds of A. chinesis Bunge via ASE, and the operational parameters of ASE were optimized, such as extraction solvent, extraction temperature, static extraction time and extraction cycles. The optimized procedure employed 70% MeOH as extraction solvent, 120 degrees C of extraction temperature, 7 min of static extraction time, 60% flush volume and the extraction recoveries of the four compounds were nearly to 100% for two cycles. The HPLC conditions are as follows: SinoChrom ODS BP C18 (4.6 mm x 200 mm, 5 microm) column, acetonitrile and 0.10% phosphoric acid solution as mobile phase, flow rate is 1.0 mL min(-1), detection length of UV is 203 nm, injection volume is 10 microL. The results indicated that the developed HPLC method is simple, sensitive and reliable for the determination of four major saponins in seeds of A. chinesis Bunge with a good linearity (r2 > 0.9994), precision (relative standard deviation (R.S.D.) < 1.5%) and the recovery ranges of 95.2-97.3%. The limits of detection (LOD) of the four compounds were in the range of 0.40-0.75 microg mL(-1). This assay can be readily utilized as a quality control method for semen aesculi and other related medicinal plants.

Analysis of chloroacetanilide herbicides in water samples by solid-phase microextraction coupled with gas chromatography-mass spectrometry.

A solid-phase microextraction (SPME) method has been developed for the determination of 3 chloroacetanilide herbicides in both fresh and seawater samples. The extracted sample was analyzed by gas chromatography with mass spectrometry detection (GC-MS), and parameters affecting SPME operation including fibre type, sample pH, sample temperature, mixing speed and extraction time have been evaluated and optimized. The amount of dissolved organic matter (DOM) and the salt content both affected SPME extraction efficiency, but the presence of other competitive extractants such as organochlorine pesticides (OCPs) in the matrix showed no insignificance interference. The limit of detection (LOD) for acetochlor, metolachlor and butachlor were 1.2, 1.6 and 2.7 ng L(-1), respectively. The recoveries for the herbicides ranged from 79 to 102%, and the linear dynamic range was from 10 to 1000 ng L(-1). The developed method has been used to monitor herbicides contaminations in coastal water samples collected around Laizhou bay and Jiaozhou bay in Shandong peninsula, China. The concentrations of acetochlor and metolachlor ranged from undetectable to 78.5 ng L(-1) and undetectable to 35.6 ng L(-1), respectively. Butachlor was not observed but in only one sample and the concentration is lower than the limit of quantification (LOQ). The concentrations of the three herbicides in this study are low compared to most of the other places reported.

Rapid determination of total solanesol in tobacco leaf by ultrasound-assisted extraction with RP-HPLC and ESI-TOF/MS.

A reliable and rapid method based on high-performance liquid chromatography (HPLC-UV) and positive ion electrospray-time of flight mass spectrometry (ESI-TOF/MS) has been developed for the characterization and quantification of solanesol in extracts of tobacco leaves from different sources. The solanesol was extracted from tobacco leaf via saponification and ultrasonic-assist extraction, and the extraction conditions were optimized. The HPLC conditions are as following: Hypersil C(4) (4.6 mm x 150 mm, 5 microm) column, acetonitrile and water as mobile phase, flow-rate is 0.8 ml/min, detection length of UV is 202 nm, injection volume is 10 microl. The results indicated that the developed HPLC method is simple, sensitive and reliable for the determination of solanesol in tobacco leaves with a linear dynamic range of 3.65-4672 ng, a detection limit of 1.83 ng, and an average recovery of 98.7%. The method has been applied to analyze and compare different tobacco samples. The results show that the solanesol content in samples of different geographic locations varies widely from 0.20 to 1.50%. When different parts of the tobacco plant are compared, the top parts of the leaves are more abundant in solanesol content than those of lower parts.

Isolation and purification of Pseudostellarin B (cyclic peptide) from Pseudostellaria heterophylla (Miq.) Pax by high-speed counter-current chromatography.

Pseudostellarin B (cyclic peptide) was isolated and purified from the herbs of Pseudostellaria heterophylla (Miq.) Pax for the first time by high-speed counter-current chromatography (HSCCC) using a two-phase solvent system consisting of n-butanol-ethyl acetate-water (0.6:4.4:5, v/v). The technique can isolate mg levels of the target compound per run with purity better than 96%. The chemical structure of the compound has been positively confirmed by electrospray ionization time of flight (TOF) MS, (1)H-NMR, (13)C-NMR and (1)H-(13)C-COSY analyses.

Fingerprint chromatogram analysis of Pseudostellaria heterophylla (Miq.) Pax root by high performance liquid chromatography.

A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the fingerprinting of extracts from the root of Pseudostellaria heterophylla (Miq.) Pax. HPLC with gradient elution was performed on an authentic reference standard of powdered P. heterophylla (Miq.) Pax root and 11 plant samples of the root were collected from different geographic locations. The HPLC chromatograms have been standardized through the selection and identification of reference peaks and the normalization of retention times and peak intensities of all the common peaks. The standardized HPLC fingerprints show high stability and reproducibility, and thus can be used effectively for the screening analysis or quality assessment of the root or its derived products. Similarity index calculations based on cosine angle values or correlation methods have been performed on the HPLC fingerprints. As a group, the fingerprints of the P. heterophylla (Miq.) Pax samples studied are highly correlated with closely similar fingerprints. Within the group, the samples can be further divided into subgroups based on hierarchical clustering analysis (HCA). Sample grouping based on HCA coincides nicely with those based on the geographical origins of the samples. The HPLC fingerprinting techniques thus have high potential in authentication or source-tracing types of applications.

Separation and identification of water-soluble salvianolic acids from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography and ESI-MS analysis.

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been applied to separate and purify salvianolic acids from the water extract of Danshen, Salvia miltiorrhiza Bunge. High efficiency HSCCC separation was achieved on a two-phase solvent system composed of a mixture of n-hexane-ethyl acetate-water-methanol (1.5:5:5:1.5, v/v) by eluting the lower mobile phase at a flow-rate of 1.7ml/min and a revolution of 850rpm. A total of five well separated peaks were obtained in the HSCCC chromatogram, and their purities determined by HPLC-UV absorption. These peaks were characterized by UV-vis spectra and ESI-MS, and the data compared with the reference standards. Salvianolic acid B was positively identified as one of the major peaks. Three of the remaining four peaks were also tentatively identified as rosmarinic acid, lithospermic acid, and salvianolic acid E, an isomer of salvianolic acid B, all are members of the salvianolic acids group. In a typical run, tens of milligrams of samples can be separated with high efficiency to yield tens of milligrams of purified materials with over 98% purity. HSCCC thus provides a cost-effective alternative to preparative scale HPLC for the semi-preparative scale separation and purification of salvianolic acids in Danshen. With appropriate modifications, the technique should also be applicable to other herbs in general.

Application of microemulsion thin layer chromatography for the fingerprinting of licorice (Glycyrrhiza spp.).

Microemulsion thin layer chromatography (ME-TLC) has been developed for the fingerprinting of aqueous extract of licorice (Glycyrrhiza spp.). The separation conditions and operational processes of the method have been optimized, and its chromatographic characteristics compared with conventional TLC. The ME-TLC system is easier to operate, and with higher resolution and better reproducibility than the conventional TLC. The separation mechanism and retention behavior of ME-TLC are found to differ significantly from conventional TLC. The technique has been applied to the analysis of different licorice species including G. uralensis, G. glabra and G. inflata; and to monitor the dynamic accumulation of active ingredients in licorice plant harvested at different times during its growing cycle in a Good Agriculture Practice (GAP) research farm. Results show that without post-chromatographic derivatization, the ME-TLC fingerprinting images of different species appear as clear, well resolved bands and with strong intensities to reveal distinctively different compositional features of the samples. The technique has also been applied successfully to monitor the dynamic accumulation of active components in licorice plant as a function of growing time in an experimental licorice farm. The study demonstrates the potential of ME-TLC technique as a rapid fingerprinting tool for the authentication and quality assessment of licorice as well as other herbs.

Isolation and purification of inflacoumarin A and licochalcone A from licorice by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been applied to isolate and purify bioactive flavone compounds from the ethanol extract of Glycyrrhiza inflata Bat., a particular plant species of licorice. HSCCC separation was performed with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (5:6:3:2, v/v) by eluting the lower mobile phase at a flow rate of 1.8 ml/min and a revolution speed of 800 rpm. Purification was performed with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (1.5:6:3:2, v/v) by eluting the lower mobile phase at a flow-rate of 1.5 ml/min and a revolution speed of 800 rpm. Two major flavone peaks: inflacoumarin A and licochalcone A were collected and the respective yields of the peaks amount to 6 mg (8.6%, w/w) and 8 mg (11.4%, w/w) from 70 mg of the crude extract sample. The purities of inflacoumarin A and licochalcone A reached 99.6% and 99.1%, respectively, after a sequential purification run. The structures of inflacoumarin A and licochalcone A were positively confirmed by 1H NMR and 13C NMR, 1H-13C-COSY, UV, FT-IR and electron ionization MS analyses.

[Characterization method for metallothioneins by reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry].

Mammalian metallothioneins (MTs) usually have two major isoforms, MT-1 and MT-2, existing in tissues because of the genetic polymorphism. Metallothionein isoforms and sub-isoforms were characterized with a hyphenated technique of reversed-phase liquid chromatography (RPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). The MT-isoform separation was carried out using a C8 narrow-bore column Vydac C8 (250 mm x 2.1 mm i.d., 5 microm, 30 nm). Buffer A was 5 mmol/L ammonium acetate in water (pH 6.0) and buffer B was 5 mmol/L ammonium acetate in methanol-water (1:1, v/v) (pH 6.0). A linear gradient elution was used by increasing buffer B from 10% to 37.5% within 40 min. The flow rate was set at 0.20 mL/min. The tentative formulas of MT isoforms and subisoforms were interpreted with the measured masses at the vicinity of apexes in total ion current chromatogram of ESI-MS and the reference data reported previously. The method was validated with the standard reference materials of MT-1 and MT-2. The results indicated that many MT species interpreted were in good agreement with the reference data reported. The MT sample from rabbit liver was measured with the established method. The results, of which the apo-MT was used by acidification of the sample for simplifying the mass spectra, showed that there were species of MT isoforms and subisoforms in rabbit livers.

[Characterization of Hg-binding metallothioneins in tissues of rat after oral intake of mercury chloride by size-exclusion chromatography coupled with inductively coupled plasma-mass spectrometry].

Hg concentrations, total metallothioneins (MTs) and metal-binding MTs in tissues of the rat after oral intake of HgCl2 were determined by the inductively coupled plasma-mass spectrometry (ICP-MS), a modifield mecurry saturarion assay and the size-exclusion chormatography (SEC) coupled with ICP-MS, respectively. The results indicated that Hg accumulation in all of the rat tissues, including kidney, liver, testicle, brain, heart, serum and blood plasma, in HgCl2 group significantly increased compared with that in the control group, especiallyin kidney,liver and testicle. A large amount of MTs was inducted in these exposed rat tissues, especially in their kidneys and livers. The metal species binding with MTs obtained with SECICP-MS showed that the fractions of Hg-MTs in both kidney and liver of exposed rat were significantly higher compared with those of the control. In addition, higher amounts of Cu-MTs observed in exposed rat liver and kidney demomstrated the effect of MTs in the homeostatis of Cu and in the detoxification of Hg.