New Insights into the Brown Carbon Chromophores and Formation Pathways for Aqueous Reactions of α-Dicarbonyls with Amines and Ammonium.

Aqueous-phase reactions of α-dicarbonyls with ammonium or amines have been identified as important sources of secondary brown carbon (BrC). However, the identities of most chromophores in these reactions and the effects of pH remain largely unknown. In this study, the chemical structures, formation pathways, and optical properties of individual BrC chromophores formed through aqueous reactions of α-dicarbonyls (glyoxal and methylglyoxal) with ammonium, amino acids, or methylamine at different pH's were characterized in detail by liquid chromatography-photodiode array-high resolution tandem mass spectrometry. In total, 180 chromophores are identified, accounting for 29-79% of the light absorption of bulk BrC for different reactions. Thereinto, 155 newly identified chromophores, including 76 imidazoles, 57 pyrroles, 10 pyrazines, 9 pyridines, and 3 imidazole-pyrroles, explain additionally 9-69% of the light absorption, and these chromophores mainly involve four formation pathways, including previously unrecognized reactions of ammonia or methylamine with the methylglyoxal dimer for the formation of pyrroles. The pH in these reactions also shows remarkable effects on the formation and transformation of BrC chromophores; e.g., with the increase of pH from 5.0 to 7.0, the light absorption contributions of imidazoles in identified chromophores decrease from 72% to 65%, while the light absorption contributions of pyrazines increase from 5% to 13% for the methylglyoxal + ammonium reaction; meanwhile, more small nitrogen heterocycles transformed into oligomers (e.g., C9 and C12 pyrroles) via reaction with methylglyoxal. These newly identified chromophores and proposed formation pathways are instructive for future field studies of the formation and transformation of aqueous-phase BrC.

Occurrence, spatial distribution, and partitioning behavior of marine lipophilic phycotoxins in the Pearl River Estuary, South China.

The occurrence, spatial distribution, and partitioning behavior of 17 marine lipophilic phycotoxins (MLPs) in surface and bottom seawater, particulate organic matter (POM), and surface sediment from the Pearl River Estuary (PRE) were investigated to understand current contamination and the potential risks to marine ecosystems in this region. Nine MLPs were detected, including azaspiracid1-3, gymnodimine, okadaic acid, dinophysistoxin 1-2, pectenotoxin2 (PTX2), and homoyessotoxin, with Σ17MLP concentrations ranging 545-12,600 pg L-1 and 619-8,800 pg L-1 in surface and bottom seawater, respectively; 0-294 ng g-1 and 0.307-300 ng g-1 dry weight (dw) in surface and bottom POM, respectively; and 3.90-982 pg g-1 dw in surface sediment. Lower Σ17MLP levels in the seawater were found at the mouth of the PRE, and gradually increased with increasing distance offshore. According to the calculated partition coefficient, the affinity of MLPs for the aquatic environment components was as follows (from highest to lowest): POM > seawater > sediment. Overall, the distribution and migration of MLPs in the PRE may depend on partition coefficients, the organic carbon fraction, and environmental factors.

Direct determination of nosiheptide residue in animal tissues by liquid chromatography-tandem mass spectrometry.

Because only very weak signals of fragment ions of nosiheptide can be obtained, nosiheptide is usually detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) via the determination of its hydrolyzed degradation product named HMIA in previous studies. The indirect method suffers from several problems, such as complicated samplepreparation, unavailable commercial HMIA, and the risk of the false-positive result by HMIA. However, we found that nosiheptide could produce several significant fragment ions under high collision energy (CE). Therefore, we developed a method for the direct determination of nosiheptide by LC-MS/MS in animal tissues. The sample was extracted with ACN, then degreased with n-hexane, and purified by an HLB solid-phase extraction (SPE) cartridge. After being filtered through the PTFE filter, it was analyzed by LC-MS/MS in selected reaction monitoring (SRM) mode. The influencing factors, such as mobile phase, SPE cartridge, filter material, and matrix effect, were investigated. Nosiheptide showed a good linear relationship (R2 ≥ 0.999) within the concentration range from 0.3 µg/L to 20 µg/L under optimized conditions. The limit of detection (LOD) was 0.3 µg/kg, while the limit of quantification (LOQ) was 1.0 µg/kg in chicken, bovine muscle, swine muscle, and swine liver. The average recoveries at spiked levels of 1.0, 2.0, and 10 µg/kg ranged from 83% to 101%, with the relative standard deviations (RSDs) <12%. Compared with the methods previously reported, our newly developed method was more simple, convenient, and sensitive. Moreover, it was successfully applied for the determination of nosiheptide residue in medicated chicken samples.

Water-Insoluble Organics Dominate Brown Carbon in Wintertime Urban Aerosol of China: Chemical Characteristics and Optical Properties.

The chromophores responsible for light absorption in atmospheric brown carbon (BrC) are not well characterized, which hinders our understanding of BrC chemistry, the links with optical properties, and accurate model representations of BrC to global climate and atmospheric oxidative capacity. In this study, the light absorption properties and chromophore composition of three BrC fractions of different polarities were characterized for urban aerosol collected in Xi'an and Beijing in winter 2013-2014. These three BrC fractions show large differences in light absorption and chromophore composition, but the chromophores responsible for light absorption are similar in Xi'an and Beijing. Water-insoluble BrC (WI-BrC) fraction dominates the total BrC absorption at 365 nm in both Xi'an (51 ± 5%) and Beijing (62 ± 13%), followed by a humic-like fraction (HULIS-BrC) and high-polarity water-soluble BrC. The major chromophores identified in HULIS-BrC are nitrophenols and carbonyl oxygenated polycyclic aromatic hydrocarbons (OPAHs) with 2-3 aromatic rings (in total 18 species), accounting for 10% and 14% of the light absorption of HULIS-BrC at 365 nm in Xi'an and Beijing, respectively. In comparison, the major chromophores identified in WI-BrC are PAHs and OPAHs with 4-6 aromatic rings (in total 16 species), contributing 6% and 8% of the light absorption of WI-BrC at 365 nm in Xi'an and Beijing, respectively.

Uptake and Depuration Kinetics of Pacific Ciguatoxins in Orange-Spotted Grouper (Epinephelus coioides).

Ciguatoxins (CTXs), produced by toxic benthic dinoflagellates, can bioaccumulate in marine organisms at higher trophic levels. The current study evaluated the uptake and depuration kinetics of some of the most potent CTXs, Pacific CTX-1, -2, and -3 (P-CTX-1, -2, and -3), in orange-spotted grouper (Epinephelus coioides) exposed to 1 ng P-CTXs g-1 fish daily. Over a 30 d exposure, P-CTX-1, -2, and -3 were consistently detected in various tissues of exposed fish, and the concentrations of the total P-CTXs in tissues generally ranked following the order of liver, intestine, gill, skin, brain, and muscle. Relatively higher uptake rates of P-CTX-1 in the groupers were observed compared with those of P-CTX-2 and -3. The depuration rate constants of P-CTX-1, -2, and -3 in different tissues were (0.996-16.5) × 10-2, (1.51-16.1) × 10-2, and (0.557-10.6) × 10-2 d-1, respectively. The accumulation efficiencies of P-CTX-1, -2, and -3 in whole groupers were 6.13%, 2.61%, and 1.15%, respectively. The increasing proportion of P-CTX-1 and the decreasing proportion of P-CTX-2 and -3 over the exposure phase suggest a likely biotransformation of P-CTX-2 and -3 to P-CTX-1, leading to higher levels of P-CTX-1 in fish and possibly a higher risk of CTXs in long-term exposed fish.

Ultra-Performance Liquid Chromatography Hyphenated with Quadrupole-Orbitrap Mass Spectrometry for Simultaneous Determination of Necine-Core-Structure Pyrrolizidine Alkaloids in Crotalaria sessiliflora L. without all Corresponding Standards.

INTRODUCTION: Crotalaria sessiliflora L. is a Chinese traditional herb for treatment of cutaneum carcinoma and cervical carcinoma. In addition to monocrotaline, coexisting pyrrolizidine alkaloids (PAs) also require further quantification for quality control and pharmaceutical uses of the herb. OBJECTIVE: To establish a UPLC-Q-Orbitrap/MS method of simultaneous determination of coexisting PAs with same parent structure for quality control and comprehensive researches of Crotalaria sessiliflora L. METHODOLOGY: PAs in Crotalaria sessiliflora L. were analysed by UPLC-Q-Orbitrap/MS method. Coexisting PAs were identified by mass data of full MS-dd-MS2 based on the characteristic fragmentation pattern and necine-core structure. Moreover, quantification of PAs was conducted by parallel reaction monitoring (PRM) mode using m/z 138, m/z 120 and m/z 94 from identical necine-core structure as quantitative ions with single monocrotaline standard for accurate calibration. RESULTS: Five PAs, named monocrotaline, retrorsine, senecionine, integerrimine, O-9-angeloylretronecine, were indentified and confirmed. Quantitative ions of m/z 138, m/z 120 and m/z 94 were used for quantification of PAs containing the necine-core structure in Crotalaria sessiliflora L. The results demonstrated that contents, precision and recoveries of the five PAs mentioned earlier were respectively 3.307-30.35 µg/g, 1.1-4.5% and 88.91-92.33% while using m/z 120 as the best quantitative ion. CONCLUSION: The UPLC-Q-Orbitrap/MS method was established for simultaneous determination of five PAs in Crotalaria sessiliflora L. without all corresponding standards, and was proved that it was simple, convenient and effective for comprehensive quality control and pharmaceutical uses. Copyright © 2017 John Wiley & Sons, Ltd.

Composition, distribution and risk of total fluorine, extractable organofluorine and perfluorinated compounds in Chinese teas.

To explore the residual characteristics of fluorine and perfluorinated compounds (PFCs) in tea, the total fluorine (TF), extractable organofluorine (EOF) and PFCs in 19 Chinese commercial teas of five categories were measured using cyclic neutron activation analysis combined with HPLC-MS/MS. The results showed that fluorine mainly existed as inorganic fluorine in teas, and identified fluorine (∑PFCs quantified as F) only accounted for 0.023-0.41% of EOF, indicating that most EOF in tea were still unknown. 50-99% of ∑PFCs in tea were short-chain (C⩽6), while perfluorooctanoic acid was the typical PFCs residual species. Less fermented teas contained significantly higher PFCs (mean, 20ng/g) than more fermented teas (3.0ng/g, p<0.01), suggesting that microbe may degrade PFCs during fermentation. The highest TF content was discovered in Hubei brick tea, which poses risk of fluorosis, whereas PFCs residues in teas caused no immediate harm.

[Residue Characteristics of Perfluorinated Compounds in the Atmosphere of Shenzhen].

In order to explore the residual characteristics of perfluorinated compounds (PFCs) in the atmosphere of Shenzhen, passive air samplers consisting of polyurethane foam (PUF) disks impregnated with XAD-4 power were deployed at 12 sites in Shenzhen from November 2014 to February 2015. Seven volatile and fifteen ionic PFCs were analyzed. The results indicated that ΣPFCs concentration ranged from 23.7 pg x m(-3) to 157 pg x m(-3) (mean: 79.0 pg x m(-3)), dominated by volatile PFCs, with 8:2 FTOITI, 6:2 FTOH, PFPeA, PFOA being the dominant compounds. The spatial distribution of volatile and ionic PFCs concentrations was the same, displaying the characteristic of "the northwest being higher than the southeast ". Furthermore, the concentrations of 6:2 FTOHs, 8:2 FTOHs, PFPeA, PFHxA and PFOA had positive correlations with PM2.5 and PM10 (P < 0.05, P < 0.01) and were more positively correlated with the levels of PM10 than those of PM2.5.

Determination of four paraben-type preservatives and three benzophenone-type ultraviolet light filters in seafoods by LC-QqLIT-MS/MS.

For the first time, an efficient and sensitive analytical method based on liquid chromatography-quadrupole linear ion trap-tandem mass spectrometry (LC-QqLIT-MS/MS) was developed for the simultaneous determination of four paraben-type preservatives and three benzophenone-type ultraviolet light filters in both plant (Sargassum fusiforme, porphyra, kelp) and animal (hairtail, yellow croaker, shrimp) seafood. The samples were extracted in methanol by pressurized liquid extraction (PLE), and the extracts were then cleaned up by mixed-mode cationic exchange (MCX) solid-phase extraction cartridges. Both isotope-labeled internal standards and matrix-matched calibration standards were used to alleviate and correct for the matrix effects, and the limits of quantification (LOQs) were 10.0µg kg(-1) for all target compounds. The average recoveries were in the range of 80.6-107.8% at three spiked concentration levels (10, 50 and 100µgkg(-1)) with relative standard deviations (RSDs) below 8.5%. The results suggest that very limited contamination of these seven emerging contaminants, mainly associated with PCPs, occurred in these common seafoods.

[Pollution fingerprints and sources of perfluorinated compounds in surface soil of different functional areas].

In order to explore the effect of regional functions on the pollution fingerprints and levels of perfluorinated compounds (PFCs), principal components analysis was combined with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and solid phase extraction enrichment in this research to analyze the levels of 14 PFCs species in 76 surface soil samples, which were collected from 4 functional regions, namely parks, communities, crossroads and surroundings of factories, as well as the urban background of Shenzhen. The results showed that regional functions had significant effect on total PFCs (sigma PFCs), which ranked as the following: surroundings of factories (P < 0.001) >> crossroads (P < 0.01) >> communities (P < 0.05) >> parks > urban background. The distribution of PFCs was in the order of medium- (P < 0.05) >> long- > short-chain PFCs in surroundings of factories, communities and crossroads, while in the order of medium- approximately short- (P < 0.05) >> long chain PFCs in crossroads. Over 81% of sigmaPFCs in the surface soil of Shenzhen could be attributed to the three principal components, represented by perfluorooctane sulfonate, perfluorooctane acid and perfluorotridecanoic acid, respectively. PFCs fingerprints were clearly shown in crossroads, as well as in surroundings of electroplating and paint factories.

[Distribution of perfluorinated compounds in surface water of Shenzhen reservoir groups].

In order to study the concentrations of 14 perfluorinated compounds (PFCs) in 25 surface water samples collected from 12 Shenzhen reservoirs in November of 2012 and January of 2013, high performance liquid chromatography-tandem mass spectrometry was combined with solid phase extraction enrichment in this research. The results indicated that perfluorohexane sulfonate and long-chain (C > or = 11) PFCs were below the detection limit in all samples and perfluorooctane acid was the primary species. No significant difference in concentration was found between samples from the center of the reservoir and the outlet. Heavy precipitations diluted PFCs concentrations in surface water, but also led to PFOA input. PFCs concentrations in surface water of the reservoir were mainly affected by water inlet, source environment and geography. Although the water temperature had positive correlations with sigma PFCs concentration, the influence of heavy precipitations was stronger than that of water temperature.

[Influence of tap water treatment on perfluorinated compounds residue in the dissolved phase].

To study the perfluorinated compounds (PFCs) residues through water treatments including flocculation, sedimentation, sand filtration, ozonation with activated carbon and chlorination, as well as the seasonal variation of PFCs in the raw water of waterworks, 13 PFCs species in the dissolved phase of raw water, finished water, as well as the water samples after flocculation, sedimentation, sand filtration, and ozonation with activated carbon filtration were measured by the high performance liquid chromatography-tandem mass spectrometry combined with solid phase extraction. Results indicated that sigma PFCs residue in water was higher in spring and summer than that in fall and winter. The vast majority of PFCs in samples were of short and medium chains (C < or = 10), and perfluorooctane sulfonate was the most typical residue species. Among the five water treatment stages, sedimentation, sand filtration and ozonation with activated carbon filtration can remove PFCs, while flocculation and chlorination significantly raise the levels of short- (C < or = 6) and medium-chain (10 > or = C > or = 7) PFCs, respectively, causing sigma PFCs increase in finished water by 10%-44% compared to raw water. However, the PFCs residues in finished water are still far below their limit values, posing no threat against human health.

[Residue of organic fluorine pollutants in hair and nails collected from Tianjin].

In order to explore the residue and distributions of organic fluorine pollutants in hair and nails, the residue levels of total fluorine (TF), extractable organic fluorine (EOF) and perfluorinated chemicals (PFCs) in hair and nails collected from Tianjin adults were measured by the cyclic neutron activation analysis (CNAA) combined with the high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The results showed that inorganic fluorine (mean: 2.0 mg.kg-1, 4.5 mg.kg-1) was the primary fluorine in TF while EOF(mean: 0.7 mg.kg-1, 1.8 mg.kg-1) was minor. The average amount of identified fluorine (IF) was 0.038 mg.kg-1 in hair and 0.047 mg.kg-1 in nails, accounting or 7.1% (2.6%-16%) and 3.5% (1.1%-11%) of EOF, respectively, which indicated that more than 84% of EOF was unknown. The major residue in hair and nails were medium-and short-chain PFCs,in which perfluorooctane sulfonate, perfluorooctanoic acid and perfluorononanoic acid were the main species. TF, EOF and IF levels in dyed and permed hair were significantly higher than untreated hair (P <0.05), and the concentrations of Sigma PFCs in hair and nails showed no difference between genders. With significantly higher levels of sigma PFCs and PFOS residues than hair (P <0.01), nails could potentially become a more sensitive bioindicator for the exposure level of PFCs in human.

[Residue characteristics and distributions of perfluorinated compounds in surface seawater along Shenzhen coastline].

In order to explore the residue characteristics and distributions of 15 perfluorinated compounds (PFCs) in 18 surface seawater samples along Shenzhen coastline, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) combined with solid phase extraction enrichment was applied in this research. The results indicated that residue level of PFCs in coastal surface seawater samples was significantly affected by human activities. Sigma PFCs residue levels in surface seawater from Shenzhen west coast, which locates below the estuary of Pearl River and Donghao River, are much higher than those from the east coast, which has low development and sparse population (P<0.05). Under natural conditions, sigma PFCs residue levels in coastal surface seawater samples from Shenzhen Bays are higher than those out of bays. The major residue species in surface seawater samples along Shenzhen coast were medium- and short-chain PFCs, including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexanoic acid and perfluoropentanoic acid. Their similar environmental behavior (P<0.05, P<0.01) is likely associated with the production process of PFCs-related products. Furthermore, cluster analysis results show that PFOS (R2 = 0.4092) level can be used as a representative parameter for evaluating PFCs contamination status in surface seawater along Shenzhen coast.

[Determination of three estrogens in animal liver and kidney tissues by liquid chromatography-tandem mass spectrometry].

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of estrone, 17beta-estradiol, estriol in animal liver and kidney tissues. The sample was extracted by tert-butyl methyl ether, and the extract was evaporated by nitrogen at 45 degrees C. The residue was redissolved in hexane-dichloromethane (6:4, v/v), then purified on a silica solid-phase extraction column. The eluant was evaporated by nitrogen, dissolved in acetonitrile-water (7:3, v/v) and then analyzed by LC-MS/MS. The separation of estrogens was performed on a Poroshell 120 EC-C18 column with the mobile phases of acetonitrile and water with a gradient elution. The identification and quantification of estrogens were carried out by negative electrospray ionization in the multiple reaction monitoring mode using external standard method. The calibration curves showed good linearity in the range of 1.0-20.0 microg/kg with the correlation coefficients above 0.99. The limit of quantification was 1.0 microg/kg for each estrogen. The average recoveries of the estrogens spiked at 1.0, 2.0, 10.0 microg/kg levels in different matrices were between 70.2% and 114%, and the relative standard deviations were between 2.01% and 14.5%. The method is simple, rapid, sensitive, good in purification effect. It is suitable for the confirmation and quantification of estrogens in animal liver and kidney tissues.

[Determination of 41 hormones in cereal feeds by liquid chromatography-tandem mass spectrometry].

A method was developed for the determination of 10 androgens, 11 progesterones, 10 glucocorticoids, 5 estrogens and 5 resorcylic acid lactones in cereal feeds by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ethyl acetate was used to extract the sample, and the mixed QuEChERS sorbents were dispersed for purification. The extract was separated by a C18 column and detected in positive or negative multiple reaction monitoring (MRM) mode. With the optimal conditions, the linear ranges of 41 hormones were 5.0 - 200.0 microg/kg with the correlation coefficients above 0.99 and the limits of quantification (LOQs, S/N > or = 10) were 0.123- 2.72 microg/kg. The method validation was carried out at three spiking levels, and the recoveries were 70.3% - 118.1% for soybean meals with the relative standard deviations (RSDs) of 0.82% - 19.5%, and 76.1% - 122.8% for corn meals with the RSD of 1.3% - 15.0%. This method is simple, fast and credible, can be applied to simultaneous screening and determination of estrogens, progesterones, androgens, glucocorticoids and resorcylic acid lactones belonging to the illegal additive of hormones in cereal feeds.

Quantification of acetylcholine in microdialysate of subcutaneous tissue by hydrophilic interaction chromatography/tandem mass spectrometry.

It has recently been suggested that acetylcholine plays an important role in the modulation of tissue inflammation. In order to further understand the newly discovered cholinergic anti-inflammatory pathway, tracking the concentration changes of acetylcholine in tissue is required. This paper describes the development of a method coupling hydrophilic interaction chromatography with electrospray ionization tandem mass spectrometry (HILIC/ESI-MS/MS) for the separation and quantification of acetylcholine in microdialysis samples of normal rats and of rats with local inflammation. The separation of acetylcholine from interferential endogenous compounds and inorganic cations was achieved with a zwitterionic stationary phase column using isocratic elution. Low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis was carried out in the positive ion mode using multiple reaction monitoring (MRM) of the following mass transitions: m/z 146 --> 87 for acetylcholine and m/z 155 --> 87 for the internal standard acetylcholine-D9. The limit of detection for acetylcholine was found to be 0.075 fmol on-column with a signal-to-noise ratio of 3:1. The lower limit of quantification was 0.25 fmol on-column. The calibration curves obtained for acetylcholine in blank microdialysates were linear in the ranges of 0.025-50 nM and 0.025-0.5 nM, with correlation coefficients equal to or greater than 0.9994 and 0.9969, respectively. The recoveries of acetylcholine for high (2 nM) and low (0.5 nM) concentrations were in the ranges of 90-96% and 95-109%, respectively. The coefficients of variation for intra-day and inter-day reproducibility were equal to or less than 7.3% and 10.4%, respectively. The method has been successfully applied in the measurement of acetylcholine in microdialysates from normal and inflamed rat tissue.

[Analysis of three metabolite residues of dipyrone in bovine muscle and pork muscle using high performance liquid chromatography].

A method for the determination of metabolite residues of dipyrone, 4-formylaminoantipyrine (FAA), 4-aminoantipyrine (AA) and 4-methylaminoantipyrine (MAA) in bovine muscle and pork muscle has been developed. Homogenized muscle sample was extracted with Na2SO4-Na2SO3 solution. After filtration, the extract was cleaned-up by a C18 solid phase extraction cartridge. Prepared sample was determined using high performance liquid chromatography under the following conditions: Inertsil ODS-3 column, methanol and water as mobile phase with gradient elution, detection wavelength of 265 nm. FAA was quantified by external standard method, while AA and MAA were quantified by internal standard method using isopropylaminoantipyrine as internal standard. Limits of detection of FAA was 12.5 microg/L, while those of AA and MAA were 15.0 microg/L and 20.0 microg/L, respectively. The limit of detection of this method was 50 microg/L for all the three metabolites. The recoveries of FAA, AA and MAA were 81.3% - 92.5%, 82.0% - 96.0%, and 80.4% - 90.6%, respectively, at the spiked levels of 50 - 400 microg/kg. The relative standard deviations of the method were less than 7%.

Investigation of zinc binding metallothioneins' polymerization in tris(hydroxymethyl)-aminomethane buffer by coupling of size exclusion chromatography with electrospray ionization mass spectrometry.

Polymerization of metallothioneins (MTs) is one of the commonly encountered puzzles in researching the structure and function of metallothioneins. In this work, a method involving SEC coupled with negative ion electrospray ionization mass spectrometry (ESI-MS) detection has been developed for the study of zinc binding MTs' polymerization in tris(hydroxymethyl)-aminomethane (TRIS) acetate buffer at physiological pH. This hyphenated technique allows separating the different polymeric states of MTs by SEC, followed by on-line identification of the individual MT subisoforms in each polymeric peak by ESI-MS detection. Purified MT subisoforms (MT-2d and MT-2a), MT-2d and MT-2a mixture and rabbit liver MT complexes were investigated in the experiments to confirm the results obtained. From the results, both oxidative polymerization and non-oxidative oligomerization were found. The cystein-dependent oxidation results in the tetrameric peak as shown in the chromatograms of oxidized MT-2d, and stable dimeric and monomeric of MT were detected in this peak by MS. For the dimeric and trimeric peaks, different MT subisoforms were detected. In the five major subisoforms detected in rabbit liver MT complexes, MT-2a and MT-2c exist primarily as trimer, while MT-2e, MT-2d and MT-1a exist mainly as dimer. Our results suggest that in the three kinds of polymers, dimer, trimer and tetramer that were found in samples, the tetramer comes from the oxidation of MT molecular; for the dimer and trimer resulting from cystein independent oligomerization, they are closely associated with the charge of subisoform.

[Separation and identification of metallothionein isoforms and sub-isoforms by liquid chromatography].

A method has been established for the separation and identification of metallothionein (MT) isoforms and sub-isoforms by liquid chromatography. A mixture of rabbit liver MT was separated on a DEAE A-25 weak anion-exchange chromatographic (AEC) column. Inductively coupled plasma-mass spectrometer (ICP-MS) was used for off-line detection of Zn in each fraction. The two main MT isoforms, MT-1 and MT-2, were isolated. The collected MT isoform fractions were then desalted by a homemade Sephadex G-25 size exclusion chromatographic (SEC) column. Afterwards, the sample was separated on a C18 reversed-phase column with UV detection at 210 nm. Different separation conditions were discussed and several sub-isoforms of MT were well separated at pH 2.0. The sub-isoforms were finally characterized by on-line HPLC-ESI-MS. The isoforms and sub-isoforms were well separated on a reversed-phase column under optimized chromatographic conditions and the detection results of ESI-MS were in agreement with the data found in literature. The method developed can be well used for the separation of metallothionein isoforms and sub-isoforms.