Identification of Crassostrea ariakensis and related oysters by multiplex species-specific PCR

Genetic markers are needed for rapid and reliable identification of oysters. In this study, we developed multiplex genus- and species-specific PCR markers for the identification of oysters from China. We used the mitochondrial cytochrome oxidase I (COI) and nuclear 28S ribosomal RNA genes for marker development. DNA sequences from different species were obtained from GenBank or by direct sequencing. Sequences were aligned, and genus- and species-specific nucleotides were identified. Primers were designed for genus/species-specific amplification to generate fragments of different sizes. A multiplex set of genus- and species-specific primers from the 28S gene was able to separate C. ariakensis and C. hongkongensis from other species and assign oysters to four genera. A set of species-specific COI primers provided positive identification of all five Crassostrea species from China, C. ariakensis, C. hongkongensis, C. angulata, C. gigas, and C. sikamea in a single PCR. The multiplex PCR assays do not require fluorescence-labeling or post-PCR enzyme digestion, providing a simple, fast and reliable method for the identification of oysters from China.

Its length polymorphism in oysters and its use in species identification

In an effort to develop genetic markers for oyster identification, we studied length polymorphism in internal transcribed spacers (ITS) between major ribosomal RNA genes in 12 common species of Ostreidae: Crassostrea virginica, C. rhizophorae, C. gigas, C. angulata, C. sikamea, C. ariakensis, C. hongkongensis, Saccostrea echinata, S. glomerata, Ostrea angasi, O. edulis, and O. conchaphila. We designed two pairs of primers and optimized PCR conditions for simultaneous amplification of ITS 1 and ITS2 in a single PCR. Amplification was successful in all 12 species, and PCR products were visualized on high-resolution agarose gels. ITS2 was longer than ITS 1 in all Crassostrea and Saccostrea species, whereas they were about the same size in the three Ostrea species. No intraspecific variation in ITS length was detected. Among species, the length of ITS I and ITS2 was polymorphic and provided unique identification of 8 species or species pairs: C. ariakensis, C. hongkongensis, C. sikamea, O. conchaphila, C. virginica/C. rhizophorae, C. gigas/C. angulata, S. echinata/S. glonzerata, and O. angasi/O. edulis. The ITS assay provides simple, rapid and effective identification of C. ariakensis and several other oyster species. Because the primer sequences are conserved, the ITS assay may be useful in the identification of other bivalve species.

Sensitisation identification of stainless steel to intergranular stress corrosion cracking by atomic force microscopy

A new technique was developed for characterisation of stainless steel to intergramilar stress corrosion cracking by atomic force microscopy. The technique proved to be effective in sensitisation identification of AISI 304 stainless steel and might be promising in sensitisation identification of other stainless steels. (c) 2007 Elsevier B.V. All rights reserved.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (Triticum aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Analysis of carbohydrates in a tibetan medicine using new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone, by HPLC with DAD detection and ESI-MS identification

A new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP), coupling with liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) for the detection of carbohydrates from a famous Tibetan medicine is reported. Carbohydrates were derivatized to their bis-NMP-labeled derivatives. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose, and fucose could be successfully detected by UV and ESI-MS. Derivatives showed intense protonated molecular ion at m/z [M+H]+ in positive ion mode. The mass to charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative identification of carbohydrates; this characteristic fragment ion was from the cleavage of C2-C3 bond in the carbohydrate chain giving the specific fragment ions at m/z [MH-CmH2m+1Om-H2O](+) for pentose, hexose, and glyceraldehydes, and at m/z [MH-CmH2m-1Om+1-H2O](+) for alduronic acids, such as galacturonic acid and glucuronic acid (m=n-2, n is carbon atom number of carbohydrate). Compared with the traditional 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent, currently synthesized NMP show the advantage of higher sensitivity to carbohydrate compounds with UV and ESI-MS detection.

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C-18 column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2 x 10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N = 3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain. (C) 2008 Elsevier B.V. All rights reserved.

Analysis of primary aromatic amines using precolumn derivatization by HPLC fluorescence detection and online MS identification

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and 2-(9-acridone)-acetic acid (AAA), two novel precolumn fluorescent derivatization reagents, have been developed and compared for analysis of primary aromatic amines by high performance liquid chromatographic fluorescence detection coupled with online mass spectrometric identification. PPIA and AAA react rapidly and smoothly with the aromatic amines on the basis of a condensation reaction using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC) as dehydrating catalyst to form stable derivatives with emission wavelengths at 380 and 440 nm, respectively. Taking six primary aromatic amines (aniline, 2-methylaniline, 2-methoxyaniline, 4-methylaniline, 4-chloroaniline, and 4-bromoaniline) as testing compounds, derivatization conditions such as coupling reagent, basic catalyst, reaction temperature and time, reaction solvent, and fluorescent labeling reagent concentration have also been investigated. With the better PPIA method, chromatographic separation of derivatized aromatic amines exhibited a good baseline resolution on an RP column. At the same time, by online mass spectrometric identification with atmospheric pressure chemical ionization (APCI) source in positive ion mode, the PPIA-labeled derivatives were characterized by easy-to-interpret mass spectra due to the prominent protonated molecular ion m/z [M + H](+) and specific fragment ions (MS/MS) m/z 335 and 295. The linear range is 24.41 fmol-200.0 pmol with correlation coefficients in the range of 0.9996-0.9999, and detection limits of PPIA-labeled aromatic amines are 0.12-0.21 nmol/L (S/N = 3). Method repeatability, precision, and recovery were evaluated and the results were excellent for the efficient HPLC analysis. The most important argument, however, was the high sensitivity and ease-of-handling of the PPIA method. Preliminary experiments with wastewater samples collected from the waterspout of a paper mill and its nearby soil where pollution with aromatic amines may be expected show that the method is highly validated with little interference in the chromatogram.

Determination of free fatty acids in bryophyte plants and soil by HPLC with fluorescence detection and identification by online MS

A method for the determination of long and short chain free fatty acids (FFAs), using 1-[2-(ptoluenesulfonate)-ethyll-2-phenylimidazole-[4,5-f-9,10-phenanthrene (TSPP) as labeling reagent, has been developed. Identification of FFA derivatives was carried out by HPLC-MS with atmospheric pressure chemical ionization (APCI) in positive ion mode. Gradient elution on an Agilent Eclipse XDB-C-8 column gave good separation of the derivatives. Excellent linear responses were observed and good compositional data could be obtained from as little as 200 mg of bryophyte plants and soil samples. Facile TSPP derivatization coupled with HPLC-APCI-MS analysis allowed the development of a highly sensitive method for the quantitative analysis of trace level of FFAs from biological and natural environmental samples.

Identification of phenolics and nucleoside derivatives in Gastrodia elata by HPLC-UV-MS

An HPLC-UV-MS method for simultaneous identification of predominant phenolics and minor nucleoside derivatives in Gastrodia elata was developed, which was based on their UV and MS characteristics summarized through a series of homemade reference standard experiments. Phenolics showed characteristic UV lambda(max) at 267 nm, [M + NH4](+) base peak in positive mode and [M - H](-) base peak in negative mode while nucleosides exhibited UV lambda(max) at 255 nm, [M + H](+), [M - H + 2H(2)O](-) or [M - H + CH3COOH](-). Phenolics conjugates mainly underwent the consecutive loss of gastrodin residue (- 268 U) and the combined loss of H2O and CO2 from the citric acid unit under negative MS/MS conditions whereas nucleosides simply lost the ribose (- 132 U) under positive MS/MS conditions. According to these characteristics, a special pattern under MS/MS conditions and reported compound data for G. elata in the literature, not only 15 phenolics were identified but also 6 nucleoside derivatives were identified. Among these compounds, seven phenolics and three nucleoside derivatives have not been reported yet from G. elata.

Determination of amines using 2-(11H-benzo[a]carbazol-11-yl) ethyl chloroformate (BCEC-Cl) as labeling reagent by HPLC with fluorescence detection and identification with APCI/MS

A pre-column derivatization method for the sensitive determination of amines using a labeling reagent 2-(11H-benzo[a]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) followed by high-performance, liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by LC/APCI/MS in positive-ion mode. The chromophore of 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) reagent was replaced by 2-(11H-benzo[a]-carbazol-11-yl) ethyl functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEC-Cl. BCEC-Cl could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M+ H](+) under APCI/MS in positive-ion mode. The collision-induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 261.8 and m/z 243.8 corresponding to the cleavages of CH2O-CO and CH2-OCO bonds. Studies on derivatization demonstrated excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% were observed with three- to four-fold molar reagent excess. In addition, the detection responses for BCEC-derivatives were compared to those obtained using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) and 9-fluorenyl methylchloroformate, (FMOC-Cl) as labeling reagents. The ratios I-BCEC/I-BCEOC = 1.94-2.17 and I-BCEC/I-FMOC = 1.04-2.19 for fluorescent (FL) responses (here, I was relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C-8 column. Detection limits calculated from 0.50 pmol injection, at a signal-to-noise ratio of 3, were 1.77-14.4 fmol. The relative standard deviations for within-day determination (n = 11) were 1.84-2.89% for the tested amines. The mean intra- and inter-assay precision for all amines levels were < 3.64% and 2.52%, respectively. The mean recoveries ranged from 96.6% to 107.1% with their standard deviations in the range of 0.8-2.7. Excellent linear responses were observed with coefficients of > 0.9996. (C) 2006 Elsevier B.V. All rights reserved.

Mass spectrometric identification of multihydroxy phenolic compounds in Tibetan herbal medicines

A highly selective and accurate method based on derivatization with dansyl chloride coupled with liquid chromatography-mass spectrometry has been developed for identification of natural pharmacologically active phenolic compounds in extracts of Lomatogonium rotatum plants (Tibetan herbal medicine) obtained by solid-phase extraction. The number of hydroxyl groups on the dansylated phenols was estimated by LC-MS-MS analysis in positive-ion mode. Dansyl derivatization of the compounds introduced basic secondary nitrogen into the phenolic core structures and this was readily ionized when acidic HPLC mobile phases were used. MS fragmentation of the derivatives generated intense protonated molecular ions of m/z [MH](+) (phenol aglycones were transformed into the corresponding free phenols by cleavage of an aglycone bond). Collision-induced dissociation of the protonated molecule generated characteristic product ions of m/z 234 and 171 corresponding to the protonated 5-(dimethylamino)naphthalene sulfoxide and 5 -(dimethylamino) naphthalene moieties, respectively. Selected reaction monitoring based on the m/z [MH](+) to 234 and 171 transitions was highly specific for these phenolic compounds. Characteristic ions with m/z values of [MH - 234](+), [MH 2 x 234](+), and [MH - 3 x 234](+) were of great importance for estimation of the presence of multihydroxyl groups on the phenolic backbone.

Determination of long-chain fatty acids in bryophyte plants extracts by HPLC with fluorescence detection and identification with MS

A sensitive method for the determination of long-chain fatty acids (LCFAs) (>C20) using 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4.5-f]-9,10-phenanthrene (TSPP) as tagging reagent with fluorescence detection and identification with post-column APCI/MS has been developed. The LCFAs in bryophyte plant samples were obtained based on distillation extraction with 1: 1 (v/v) chloroform/methanol as extracting solvent. TSPP could easily and quickly label LCFAs at 90 degrees C in the presence of K2CO3 catalyst in DMF. Eleven free LCFAs from the extracts of bryophyte plants were sensitively determined. Maximal labeling yields close to 100% were observed with a five-fold excess of molar reagent. Separation of the derivatized fatty acids exhibited a good baseline resolution in combination with a gradient elution on a reversed-phase Eclipse XDB-C-8 column. Calculated detection limits from 1.0 pmol injection, at a signal-to-noise ratio of 3, were 26.19-76.67 fmol. Excellent linear responses were observed with coefficients of >0.9996. Good compositional data were obtained from the analysis of the extracted LCFAs containing as little as 0.2 g of bryophyte plant samples. Therefore, the facile TSPP derivatization coupled with HPLC/APCI/MS analysis allowed the development of a highly sensitive method for the quantitation of trace levels of LCFAs from biological and natural environmental samples. (c) 2006 Elsevier B.V. All rights reserved.

Determination of 30 free fatty acids in two famous Tibetan medicines by HPLC with fluorescence detection and mass spectrometric identification

A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free Fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) in N,N-dimethylformamide (DMF) at 90 degrees C with anhydrous K2CO3 as catalyst. A mixture Of C-1-C-30 fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C-8 column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines Gentiana straminea and G. dahurica was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were > 0.9991. Relative standard deviations (RSDs, n = 6) for the fatty acid derivatives were < 3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1-38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.

HPLC-APCI-MS determination of free fatty acids in Tibet folk medicine Lomatogonium rotatum with fluorescence detection and mass spectrometric identification

A simple and sensitive method for the determination of free fatty acids (FFAs) using acridone-9-ethyl-p-toluenesulfonate (AETS) as a fluorescence derivatization reagent by high performance liquid chromatography (HPLC) has been developed. Free fatty acid derivatives were separated on an Eclipse XDB-C-8 column with a good baseline resolution and detected with the fluorescence of which excitation and emission wavelengths of derivatives were set at lambda(ex) 404 and lambda(em) 440 nm, respectively. Identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with an atmospheric pressure chemical ionization (APCI) source under positive-ion detection mode. Nineteen FFAs from the extract of Lomatogonium rotatum are sensitively determined. The results indicate that the plant Lomatogonium rotatum is enriched with an abundance of FFAs and FFAs of higher contents, which mainly focus on even carbon atoms, C-14, C-16, and C-18. The validation of the method including linearity, repeatability, and detection limits was examined. Most linear correlation coefficients for fatty acid derivatives are > 0.9989, and detection limits (at signal-to-noise of 3: 1) are 12.3-43.7 fmol. The relative standard deviations (RSDs) of the peak areas and retention times for 19 FFAs standards are < 2.24% and 0.45%, respectively. The established method is rapid and reproducible for the separation determination of FFAs from the extract of Lomatogonium rotatum with satisfactory results.

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and its application for determination of amines by high performance liquid chromatography with fluorescence detection and identification with mass spectroscopy/atmospheric pressure chemical ionization

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCI) as the dehydrant with fluorescence detection has been developed. Amines were derivatized to their acidamides with labeling reagent 2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA). Studies on derivatization conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at lambda(ex) 260nm and an emission maximum at lambda(em) 380nm. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by high-performance liquid chromatography. Identification of derivatives was carried out by online post-column mass spectrometry (LC/APCI-MS/MS) and showed an intense protonated molecular ion corresponding m/z [MH](+) under APCI in positive-ion mode. At the same time, the fluorescence properties of derivatives in various solvents or at different temperature were investigated. The method, in conjunction with a gradient elution, offered a baseline resolution of the common amine derivatives on a reversed-phase Eclipse XDB-C-8 column. LC separation for the derivatized amines showed good reproducibility with acetonitrile-water as mobile phase. Detection limits calculated from 0.78 pmol injection, at a signal-to-noise ratio of 3, were 3.1-18.2 fmol. The mean intra- and inter-assay precision for all amine levels were < 3.85% and 2.11%, respectively. Excellent linear responses were observed with coefficients of > 0.9996. The established method for the determination of aliphatic amines from real wastewater and biological samples was satisfactory. (c) 2006 Elsevier B.V. All rights reserved.