Studies on the stability of diester-diterpenoid alkaloids from the genus Aconitum L. by high performance liquid chromatography combined with electrospray ionisation tandem mass spectrometry (HPLC/ESI/MSn)

The stability of diester-diterpenoid alkaloids (DDA) from plants of the genus Aconitum L. has been studied in different solvents and pH buffers. The HPLC/ESIMS method for analysing the concentration of DDA was established and DDA's decomposition products were elucidated by HPLC/ESI-MS/MSn. In different solvents, e.g. dichloromethane, ether, methanol and distilled water, the decomposition pathways of DDA are quite different and their difference in stabilities depends on the difference of their structures, in which substituents at the N atom and substituents at C-3 are different. The pyrolytic products of DDA, such as deacetoxy aconitine-type alkaloids, have been observed in the above solvents, whereas 8-methoxy-14-benzoyl aconitine-type alkaloids have been obtained only in methanol.

Studies on principal components and antioxidant activity of different Radix Astragali samples using high-performance liquid chromatography/electrospray ionization multiple-stage tandem mass spectrometry

The principal components, isoflavonoids and astragalosides, in the extract of Radix Astragali were detected by a high-performance liquid chromatography Couple to electrospray ionization ion trap multiple-stage tandem mass spectrometry (HPLC-ESI-IT-MSn) method. By comparing the retention time (t(R)) of HPLC, the ESI-MSn data and the structures of analyzed Compounds with the data of reference compounds and in the literature, 17 isoflavonoids and 12 astragalosides have been identified or tentatively deduced. By Virtue of the extracted ion chromatogram (EIC) mode, simultaneous determination of isoflavonoids and astragalosides could be achieved when the different components formed overlapped peaks. And this method has been utilized to analyze the constituents in extracts of Radix Astragali from Helong City and of different growth years. Then the antioxidant activity of different samples has been Successfully investigated by HPLC-ESI-MS method in multiple selected ion monitoring(MIM) mode, applying the spin trapping technology, and the Ferric Reducing Antioxidant Power (FRAP) assay was applied to support the result.

Investigation of the hydrolysis of ginsenosides by high performance liquid chromatography-electrospray ionization mass spectrometry

The hydrolysis of ginsenoside standards and the crude extracts of ginseng has been investigated at different pH values (2.4 - 11.2) using high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). The experimental results indicated that the pH value of aqueous solutions is an important factor in changing the composition of ginsenosides. For (20S)-protopanaxadiol ginsenosides, ginsenosides with a large mass hydrolyzed to form hydrolysates (20S)-Rg(3) and (20R)-Rg(3) at pH 4.3. There were more hydrolyzed products observed at pH 3.3: (20S)-F-2, C-25,26 hydrated ginsenoside "C-Y-1" and "C-Y-2" (MW = 802 Da) accompanied with (20S)-Rg(3), (20R)-Rg(3). At pH 2.4, only (20R)-Rg(3), (20S)-F-2, a small quantity of (20S)-Rg(3) and three C-25,26 hydrated ginsenosides were obtained. For (20S)protopanaxatriol Re, no hydrolysates were observed at pH 4.3; it was hydrolyzed at pH 3.3 to form hydrolysates (20S)-Rg, (20R)Rg(2) and hydrated C-25,26 (MW = 802 Da) and at pH 2.4 only C-25,26 hydrated ginsenosides "C-Y-1" and "C-Y-2" (MW = 802 Da) were left in the solution. Similar hydrolysis reactions could be also observed for the crude extracts of ginseng. It showed that HPLC/ESI-MS is a fast and convenient method to study the hydrolysis of ginseng.

Studies on lignan constituents from Schisandra chinensis (Turcz.) Baill. fruits using high-performance liquid chromatography/electrospray ionization multiple-stage tandem mass spectrometry

A reversed-phase high-performance liquid chromatography-diode array detector-electrospray ionization multiple-stage tandem mass spectrometry (RP-HPLC-DAD-ESl-MSn) method has been developed for the detection and analysis of lignan constituents in the methanol extract from the fruits of Schisandra chinensis (Turcz.) Baill. RP-HPLC-DAD-ESI-MSn and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-TCR-MSn) have been applied to investigate the characteristic product ions of four lignan reference compounds. Then, the logical fragmentation pathways of the lignans have been proposed. By comparing the retention time (t(R)) of HPLC, the ESI-MSn data and the structures of analyzed compounds with the data of reference compounds and in the literature, 11 peaks in HPLC have been unambiguously identified and another 5 peaks have been tentatively identified or deduced. Also, in the present paper, the extracted ion chromatograms (EIC) have been used to analyze the lignan isomers. The experimental results demonstrate that RP-HPLC-DAD-ESI-MSn is a specific and useful method for the identification of the lignan constituents and their isomers.

Study on the variation of chemical constituents during combination of ginseng with trogopteroum feces and semen raphani by high performance liquid chromatography-mass spectrometry

A high performance liquid chroatography-electrospray ionization-mass spectrometric method was developed for analysis and identification of ginsenosides from the decoction of ginseng, ginseng with trogopteroum feces and ginseng with semen raphani. Ten ginsenosides were separated and detected. The content variation of these ginsenosides was researched. The experimental results showed, that ginsenosides were less in compatible decoction than in separate one expect Ro. the stripping of ginsenosides were restrained by semen raphani and during combination of ginseng with trogopteroum feces, the precipitates were produced by ginsenosides.

A study of isomeric diglycosyl flavonoids by SORI CID of Fourier transform ion cyclotron mass Spectrometry in negative ion mode

High-resolution Sustained off resonance irradiation (SORI) CID was employed to distinguish four pairs of isomeric diglycosyl flavonoids in the negative mode using the electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). All of these isomers can be distinguished via MS/MS data. For these diglycosyl flavones and flavanones, the deprotonated alpha 1-->6 linkage diglycosyl flavonoids produce fewer fragments than the alpha 1-->2 linkage type compounds and the Retro-Diels-Alder (RDA) reaction in MS/MS only takes place when the aglycone is a flavanone and glycosylated with an alpha 1-->2 intersaccharide linkage disaccharide. The deprotonation sites after collisional activation are discussed according to the high mass accuracy and high-resolution data of tandem spectrometry. Some of these high-resolution SORI CID product ions from alpha 1-->2 linkage diglycosyl flavonoids involve multibond cleavages; the possible mechanism is discussed based on the computer modeling using Gaussian 03 program package at the B3LYP/6-31G level of theory. Unambiguous elementary composition data provides fragmentation information that has not been reported previously.

Analysis of strychnos alkaloids using electrospray ionization Fourier transform ion cyclotron resonance multi-stage tandem mass spectrometry

The fragmentations of four strychnos alkaloids have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the positive ion mode. Experiments using multi-stage tandem mass spectrometry (ESI-FT-ICR-MSn) allowed us to obtain precise elemental compositions of product ions at high mass resolution. The experimental data demonstrated that the nitrogen bridge and the coordinated oxygen atom on the nitrogen bridge in the alkaloid compounds were the active sites in the MS2 fragmentations. The loss of CH3 or the OCH3 group in those alkaloids, which have an OCH3 substituent, was the dominant fragmentation mode in the MS3 fragmentations. Logical fragmentation schemes for strychnos alkaloids have been proposed and these should be useful for the identification of these compounds.

Identification of high-lying odd energy levels of uranium by resonant ionization mass spectrometry

Single-colour and two-colour multiphoton resonant ionization spectra of uranium atom were studied extensively with a Nd: YAG laser-pumped dye laser atomic beam apparatus time-of-flight mass spectrometer in our laboratory. The energy locations of high-lying odd-parity levels in the region 33 003-34 264 cm(-1),mearured by a two-colour three-step ionization technique, were reported here. The angular momentum quantum number J was uniquely assigned for these levels by using angular momentum selection rules.

Determination of ultra-trace rare-earth elements in human plasma by inductively coupled plasma mass spectrometry

Inductively coupled plasma mass spectrometry (ICP-MS),a highly sensitive inorgnic analytic technique,fits to determine ultra-nace rare-earth elements in human plasma. Under the optimized conditions detection limits for 15 rare-earth elements are in the range of 0.7 (for Eu)-5.4 (for Gd) ng.L-1. Indium as an internal standard element is used to compensate for matrix suppression effect and sensitivity drift. Three kinds of preparation methods, diluted with 1% HNO3, digested with HNO3-H2O2 and with HNO3-HClO4, are checked and compared,and the former is the simplest way to be measured. The samples diluted with 1% HNO3, stored in 4 degrees C, are very steady for 16 days. With the method, 11 healthy plasma samples in Changchun area of China are analysed.

Determination of the impurities elements in high-purity cadmium by inductively coupled plasma mass spectrometry

A method for the determination of impurity elements in high purity cadmium by inductively coupled plasma mass spectrometry was developed. The spectral interference arising from Cd was discussed and the magnitude of interference by the polyatomic ion of Cd was compared. The effect of the high concentration Cd on the analyte signal was studied. The results showed that Cd possessed both significant suppression effects on the light mass elements signal and enhancement effects on the heavy mass elements signal,and the effects can be corrected by the use of internal standard elements. In this paper, Y and Bi was employed for the light mass elements and the heavy mass elements,respectively. As, Be, Co, Cu, Ga, Ge, Mn, Mo, Pb, Ni, Sr, Au, Tl, Th, V and U in high purity cadmium were determined. Detection limits are 0.005-0.052 mu g L-1,and the recoveries of standard addition are 82%-108%.

Simultaneous Determination of Multi-Organotin Compounds in Seawater by Liquid-Liquid Extraction-High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry

The hyphenated technique of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was applied to the simultaneous determination of five organotin compounds (trimethyltin, dibutyltin, tributyltin, diphenyltin and triphenyltin) in seawater samples. Agilent TC-C18 column was used for the separation, the mobile phase of HPLC was CH3CN : H2O: CH3COOH=65 : 23 : 12 (phi), 0.05% TEA, and pH value was adjusted to 3.0 by diluent ammonia. The flow rate was 0.6 mL . min(-1). Five mixed organotin compounds in a mix standard solution from 100 to 0.5 mu g . L-1 were applied for the method assessment. The experimental results indicate that the correlation coefficient of calibration curves (R-2) for each organotin compound was over 0.998 and the detection limits of the five organotin compounds were lower than 3 ng . L-1. Different mixed organic solvents including dichloromethane or toluene were used for extraction of organotin and the extraction condition of organotin from seawater was optimized. The 100 mL seawater acidized by hydrochloric acid was extracted by 10 mL carbon dichloride (CH2Cl2) with 2% tropolone for 10 min twice. Extracted organic solvents were mixed And blown to one drop by nitrogen with the rate of 1.7 mL . min(-1), then 1 mL acetonitrile was added to the drop for redissolving the organotin compounds. Finally, the mixed redissolution was filtered by 0.22 mu m organic filter membrane before analysis. it was found that the only organotin compound in seawater was triphenyltin (TPHT) and the content was 53.2 ng . L-1. The recoveries test from the standard addition for diphenyltin (DPHT), dibutyltin (DBT), tributyltin (TBT) and triphenyltin (TPHT) were over 80%. However, the recovery for trimethyltin (TMT) was relatively low and the value was 50%. The reason might be attributed to the decomposition or adsorption of those compounds during the extraction procedure. Further study on this subject is in progress.

Speciation Analysis of Organotin Compounds in Shellfish by Hyphenated Technique of High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry

The hyphenated technique of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry(HPLC-ICP-MS) was applied to the simultaneous determination of five organotin compounds in the shellfish samples. Agilent TC-C-18 column was selected, mobile phase of the HPLC was CH3CN:H2O: CH3COOH = 65:23:12 (V/V), 0. 05% TEA, pH = 3.0 at flow rate 0.4 mL/min. Five mixed organotin standards from 100 mu g/L to 0. 5 mu g/L was used for the method evaluation. The experimental results indicate that the linearity (R-2) for each compound was over 0.998. The shellfish samples were treated by supersonic extraction with mobile phase for 30min. Four organotin compounds including dibutyltin (DBT), tributyltin (TBT), diphenyltin (DphT) and triphenyltin (TPhT) in shellfish samples were detected with method mentioned above. It was found that the domain compounds in the samples were tributyltin (TBT) and triphenyltin (TPhT). The recoveries test from the standard addition for trimethyltin (TMT tributyltin (TBT), and triphenyltin (TPhT) were, over 80%. However, the recoveries for diphenyltin (DPhT) and dibutyltin (DBT) were relatively low, 37.3% and 75.2% respectively. The reason might be attributed to the decomposition of those compounds during the extraction procedure. The further study on this subject is under the progress.

Determination of free fatty acids in soil and bryophyte plants by precolumn derivatization via HPLC with fluorescence detection and tandem mass spectrometry (HPLC-MS/MS)

A sensitive method for the determination of 30 kinds of free fatty acids (FFAs, C-1-C-30) with 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f] 9,10-phenan- threne (TSPP) as labeling reagent and using high performance liquid chromatography with fluorescence detection and identification by online postcolumn mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode (HPLC/MS/APCI) has been developed. TSPP could easily and quickly label FFAs in the presence of K2CO3 catalyst at 90 degrees C for 30 min in N,N-dimethylformamide (DMF) solvent, and maximal labeling yields close to 100% were observed with a 5-fold excess of molar reagent. Derivatives were stable enough to be efficiently analyzed by high performance liquid chromatography. TSPP was introduced into fatty acid molecules and effectively augmented MS ionization of fatty acid derivatives and led to regular MS and MS/MS information. The collision induced cleavage of protonated molecular ions formed specific fragment ions at m/z [MH](+)(molecular ion), m/z [M'+CH2CH2](+)(M' was molecular mass of the corresponding FFA) and m/z 295.0 (the, mass of protonated molecular core structure of TSPP). Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C-8 column (4.6 x 150 mm, 5 mu m, Agilent) with a good baseline resolution in combination with a gradient elution. Linear ranges of 30 FFAs are 2.441 x 10(-3) to 20 mu mol/L, detection limits are 3.24 similar to 36.97 fmol (injection volume 10 mu L, at a signal-to-noise ratio of 3, S/N 3:1). The mean interday precision ranged from 93.4 to 106.2% with the largest mean coefficients of variation (R.S.D.) < 7,5%. The mean intraday precision for all standards was < 6.4% of the expected concentration. Excellent linear responses were observed with correlation coefficients of > 0.9991. Good compositional data could be obtained from the analysis of extracted fatty acids from as little as 200 mg of bryophyte plant samples.Therefore, the facile TSPP derivatization coupled with HPLC/MS/APCI analysis allowed the development of a highly sensitive method for the quantitation of trace levels of short and long chain fatty acids from biological and natural environmental samples.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.