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.

Separation and determination of the major active components in Tibetan folk medicinal species Swertia franchetiana by HPLC with DAD

A sensitive and specific reversed-phase high performance liquid chromatography (RP-HPLC) method with diode array detection (DAD) was established for the quantitative determination of the nine active components, namely, swertiamarin (SWM, 1), mangiferin (MA, 2), gentipicroside (GE, 3), sweroside (SWO, 4), isoorientin (IS, 5), swertisin (SWS, 6), swertianolin (SWN, 7), 7-O-[alpha-L-rhamnopyranosyl-1 -> 2)-beta-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone (RX, 8), and bellidifolin (BE, 9) used as the external standard, in Tibetan folk medicinal species Swertia franchetiana. Based on the baseline chromatographic separation of most components from the methanolic extract of Swertia franchetiana on a reversed-phase Eclipse XDB-C8 column with water-acetonitrile-formic acid as mobile phase, the nine components were identified by comparison with standard samples and qualified by using the external standard method with DAD at 254 nm. The correlation coefficients of all the calibration curves were found to be higher than 0.9980. The relative standard deviations (RSDs) of the peak areas and retention times for the nine standards were less than 2.07% and 2.86%, respectively.

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.

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.

Analysis of saturated free fatty acids from pollen by HPLC with fluorescence detection

A sensitive method for the determination of free fatty acids using 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-dimidazol-1-yl) ethyl-p-toluenesuIfonate (ANITS) as tagging reagent with fluorescence detection has been developed. ANITS could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90 degrees C for 40 min in N,N-dimethylformamide solvent. From the extracts of rape bee pollen samples, 20 free fatty acids were sensitively determined. Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post-column APCI/MS in positive-ion detection mode. ANITS-fatty acid derivatives gave an intense molecular ion peak at mlz [M+H](+); with MS/MS analysis, the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at mlz [M-345](+) and mlz 345.0 (here, m/z 345 is the core structural moiety of the ANITS molecule). The fluorescence excitation and emission wavelengths of the derivatives were lambda(ex) = 250 nm and lambda(em) = 512 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are > 0.9999. Detection limits, at a signal-to-noise ratio of 3 : 1, are 24.76-98.79 fmol for the labeled fatty acids.

Comparative analysis of allantoin quercetin, and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in Nitraria tangutorum Bobr. Seed by HPLC-APCI-MS and CE

This paper describes the simultaneous determination of allantoin, quercetin, and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCCA) in Nitraria tangutorum Bobr seed by HPLC-APCI-MS and CE (capillary electrophoresis) methods. The final optimized chromatographic conditions were investigated in a reversed-phase Eclipse XDB-C8 column (150 x 4.6 mm, 5 mu m). A seventeen-minute gradient elution, (A: aqueous acetonitrile 20% (v/v); B: aqueous acetonitrile 60% (v/v); C: pure acetonitrile 100%) at a flow rate of 1.0 mL/min was selected for the separation of three natural products with diode array detection (DAD) at 220 nm. A CE experiment was carried out in a fused silica capillary with 32 mmol/L boric acid (pH 10), 32 mmol/L SDS and acetonitrile (10.0%, v/v). The applied potential and temperature was, respectively, set at 19 kV and 25 degrees C. After development, the validation was performed in parallel for HPLC and CE, with the same standards and sample to avoid differences due to the manipulation. The validation parameters of both techniques were adequate for the intended purpose.

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.

Pre-column derivatization of amines with 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl chloroformate followed by LC-fluorescence and LC-APCI-MS

A pre-column derivatization method for the sensitive determination of amines using the labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl chloroformate (BCIC-Cl) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives is carried out by high performance liquid chromatography/atmospheric pressure chemical ionization (LC-APCl-MS-MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent is replaced by 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl functional group, which results in a sensitive fluorescence derivatizing reagent BCIC-Cl. BCIC-Cl can easily and quickly label amines. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography and show an intense protonated molecular ion corresponding m/z [MH](+) under APCl in positive-ion mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 260 corresponding to the cleavage of CH2-OCO bond. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3 to 4-fold molar reagent excess. In addition, the detection responses for BCIC derivatives are compared with those obtained using CEOC and FMOC as derivatization reagents. The ratios of l(BCIC)/l(CEOC) and l(BCIC)/l(FMOC) are, respectively, 1.23-3.14 and 1.25-3.08 for fluorescent (FL) responses (here, l is relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C-8 column. Detection limits are calculated from 1.0 pmol injection, at a signal-to-noise ratio of 3, are 10.6-37.8 fmol. The mean interday accuracy ranges from 94 to 105% for fluorescence detection with the largest mean %CV < 7.5. The mean interday precision for all standards is < 6.0% of the expected concentration. Excellent linear responses are observed with coefficients of > 0.9997.

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.

太阳耀斑和日食期间电离层行为的统计和模拟研究

The ionosphere is the ionized component of the Earth's upper atmosphere. Solar EUV radiation is the source of ionospheric ionization. Thus the ionosphere is affected strongly by the variations in solar radiation. Solar flares and solar eclipses can induce remarkable short time changes in solar radiation: the solar radiation would increase suddenly during solar flares and decrease significantly during solar eclipses. Solar flare and eclipse events not only affect directly the photochemical processes, but also affect the dynamic processes, and even affect the neutral atmosphere, which is strongly coupled with the ionosphere. The study on the ionospheric response to solar flares and eclipses can advance our knowledge on the ionosphere and its photochemical and dynamic processes and help us to evaluate the ionospheric parameters (such as ion loss coefficients). In addition, the study on the ionospheric responses to solar flares and eclipses is an important part of the ionospheric space weather, which can provide guides for space weather monitoring. This thesis devotes to the study on the ionospheric responses to solar flares and solar eclipses. I have developed two models to simulate the variations of solar EUV radiation during solar flares and solar eclipses, and involved in developing a 2D mid- and low-latitude ionospheric model. On the basis of some observed data and the ionospheric model, I study the temporal and spatial variations of the ionosphere during solar flares and eclipses, and investigate the influences of solar activity, solar zenith angle, neutral gas density, and magnetic dip angle on the ionospheric responses to solar flares and solar eclipses. The main points of my works and results are summarized as follows. 1. The ionospheric response to the X17.2 solar flare on October 28, 2003 was modeled via using a one-dimension theoretical ionospheric model. The simulated variation of TEC is in accordance with the observations, though there are some differences in the amplitude of the variation. Then I carried out a series of simulations to explore the local time and seasonal dependences of the ionospheric responses to solar flares. These calculations show that the ionospheric responses are largely related with the solar zenith angle (SZA). During the daytime (small SZA), most of the increases in electron density occur at altitudes below 300 km with a peak at around 115 km; whereas around sunrise and sunset (SZA>90°), the strongest ionospheric responses occur at much higher altitudes. The TEC increases slower at sunrise than at sunset, which is caused by the difference in the evolution of SZA at sunrise and sunset: SZA decreases with time at sunrise and increase with time at sunset. The ionospheric response is largest in summer and smallest in winter, which is also related to the seasonal difference of SZA. 2. Based on the observations from the ionosondes in Europe and the ionospheric model, I investigated the differences of the ionosphere responses to solar eclipses between the E-layer and F1-layer. Both the observation and simulation show that the decrease in foF1 due to the solar eclipses is larger than that in foE. This effect is due to that the F1 region locates at the transition height between the atomic ion layer and the molecular ion layer. With the revised model of solar radiation during solar flares, our model calculates the radiations from both the inside and outside of photosphere. Large discrepancy can be found between the observations and the calculations with an unrevised model, while the calculations with the revised model consist with the observations. 3. I also explore the effects of the F2-layer height, local time, solar cycle, and magnetic dip angle on the ionospheric responses to solar eclipses via using an ionospheric model and study on the solar zenith angle and the dip dependences by analyzing the data derived from 23 ionosonde stations during seven eclipse events. Both the measured and simulated results show that these factors have significant effect on the ionospheric response. The larger F2-layer height causes the smaller decrease in foF2, which is because that the electron density response decreases with height. The larger dip results in the smaller eclipse effect on the F2 layer, because the larger dip would cause the more diffusion from the top ionosphere which can make up for the plasma loss. The foF2 response is largest at midday and decreases with the increasing SZA. The foF2 response is larger at high solar activity than at low solar activity. The simulated results show that the local time and solar activity discrepancy of the eclipse effect mainly attribute to the difference of the background neutral gas density. 4. I carried out a statistical study on the latitudinal dependence of the ionospheric response to solar eclipses and modeled this latitudinal dependence by the ionospheric model. Both the observations and simulations show that the foF2 and TEC responses have the same latitudinal dependence: the eclipse effects on foF2 and TEC are smaller at low latitudes than at middle latitudes; at the middle latitudes (>40°), the eclipse effect decreases with increasing latitude. In addition, the simulated results show the change in electron temperature at the heights of above 300 km of low latitudes is much smaller than that at the same heights of middle latitudes. This is due to the smaller decrease in photoelectron production rate at its conjugate low heights. 5. By analyzing the observed data during the October 3, 2005 solar eclipse, I find some significant disturbances in the conjugate region of the eclipse region, including a decrease in Te, an increase in foF2 and TEC, and an uprising in hmF2. I also simulated the ionosphere behavior during this eclipse using a mid-low latitude ionospheric model. The simulations reproduce the measured ionospheric disturbances mentioned above in the conjugated hemisphere. The simulations show that the great loss of arriving photoelectron heat from the eclipse region is the principal driving source for the disturbances in the conjugate hemisphere.