CHEMICAL-IONIZATION MASS-SPECTROMETRY OF BENZOYL PEROXIDE - A RADICAL AROMATIC-SUBSTITUTION RESULTING IN BIPHENYLCARBOXYLIC ACID IN THE GAS-PHASE

A radical aromatic substitution resulting in biphenylcarboxylic acid is inferred for the decomposition of benzoyl peroxide from the chemical ionization and collision-induced dissociation mass spectra. The thermolysis of benzoyl peroxide gives rise to a benzoyloxy radical, which undergoes rapid decarboxylation and hydrogen abstraction leading to phenyl radical and benzoic acid, respectively. Attack of the resulting phenyl radical on the benzoic acid results in bipbenylcarboxylic acid. On the other hand, the phenyl radical abstracts a hydrogen atom to yield benzene, which is then subjected to the attack of a benzoyloxy radical, affording phenyl benzoate. This substitution reaction rather than the recombination of benzoyloxy and phenyl radicals is found to be responsible for the formation of phenyl benzoate under the present conditions.

Determination of tetrodotoxin by liquid chromatography coupled with electrospray ion-trap mass spectrometry

Two methods for tetrodotoxin analysis using liquid chromatography coupled with electrospray iontrap mass spectrometry (LC-ESI-MS) have been established with C,, reversed phase column and hydrophilic interaction liquid chromatography (HILIC) column, respectively. Sensitivity and reproducibility of the methods were compared. The method using C-18 column in selected ion monitoring (SIM) mode had a detection limit (S/N = 3) of 120 pg, and a good linearity of the calibration curve was obtained for tetrodotoxin (r = 0. 9992). High reproducibility of the method was observed, with a relative standard deviation (RSD) below 10%. The method using HILIC column in SIM mode and selected reaction monitoring (SRM) mode had detection limits (S/N = 3) of 15 and 3.75 pg, respectively. Good linearity of the calibration curves was obtained for tetrodotoxin (r = 0. 9996 and 0. 9998 in SIM and SRM mode, respectively). T he reproducibility was high in SIM mode but relatively poor in SRM mode. Based on the results, the method using HILIC column in SIM mode was suggested for the analysis of tetrodotoxin with LC-MS system.

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 aliphatic amines from soil and wastewater of a paper mill by pre-column derivatization using HPLC and tandem mass spectrometry (HPLC-MS/MS)

A pre-column derivatization method for the sensitive determination of aliphatic amines using the labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by HPLC with fluorescence detection and APCI/NIS identification in positive-ion mode has been developed. The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by the 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent, BCEOC, that 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](+) with APCI/MS in positive-ion mode. The collision induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 264.1, m/z 246.0 and m/z 218.1, corresponding to the cleavages of CH2CH2O-CO, CH2CH2-OCO, and N-CH2CH2O bonds. Studies on derivatization conditions demonstrated that excellent derivatization yields close to 100% were observed with a 3 to 4-fold molar reagent excess in acetonitrile solvent, in the presence of borate buffer (pH 9.0) at 40 degrees C for 10 min. In addition, the detection responses for BCEOC derivatives were compared with those obtained with CEOC and FMOC as labeling reagents. The ratios I-BCEOC/I-CEOC and I-BCEOC/I-FMOC were, respectively, 1.40-2.76 and 1.36-2.92 for fluorescence responses (here, I was the relative fluorescence intensity). Separation of the amine derivatives had been optimized on an Eclipse XDB-C-8 column. Detection limits calculated from an 0.10 pmol injection, at a signal-to-noise ratio of 3, were 18.65-38.82 fmol (injection volume 10 mu L for fluorescence detection. The relative standard deviations for intraday determination (n = 6) of standard amine derivatives (50 pmol) were 0.0063-0.037% for retention times and 3.36-6.93% for peak areas. The mean intra-and inter-assay precision for all amines were <5.4% and 5.8%, respectively. The recoveries of amines ranged from 96 to 113%. Excellent linear responses were observed with correlation coefficients of >0.9994. The established method provided a simple and highly sensitive technique for the quantitative analysis of trace amounts of aliphatic amines from biological and natural environmental samples.

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.

Human plasma proteome analysis by multidimensional chromatography prefractionation and linear ion trap mass spectrometry identification

A resurgence of interest in the human plasma proteome has occurred in recent years because it holds great promise of revolution in disease diagnosis and therapeutic monitoring. As one of the most powerful separation techniques, multidimensional liquid chromatography has attracted extensive attention, but most published works have focused on the fractionation of tryptic peptides. In this study, proteins from human plasma were prefractionated by online sequential strong cation exchange chromatography and reversed-phase chromatography. The resulting 30 samples were individually digested by trypsin, and analyzed by capillary reversed-phase liquid chromatography coupled with linear ion trap mass spectrometry. After meeting stringent criteria, a total of 1292 distinct proteins were successfully identified in our work, among which, some proteins known to be present in serum in < 10 ng/mL were detected. Compared with other works in published literatures, this analysis offered a more full-scale list of the plasma proteome. Considering our strategy allows high throughput of protein identification in serum, the prefractionation of proteins before MS analysis is a simple and effective method to facilitate human plasma proteome research.

On-line concentration of proteins in pressurized capillary electrochromatography coupled with electrospray ionization-mass spectrometry

Pressurized capillary electrochromatography (pCEC) and electrospray ionization-mass spectrometry (ESI-MS) have been hyphenated for protein analysis. Taken cytochrome c, lysozyme, and insulin as samples, the limits of detection (LODs) for absolute concentrations are 10(-11) mol (signal-to-noise ratio S/N = 3) with relative standard deviations (RSDs) of retention time and peak area, respectively, of less than 1.7% and 4.8%. In order to improve the detection sensitivity, on-line concentration by field-enhanced sample-stacking effect and chromatographic zone-sharpening effect has been developed, and parameters affecting separation and detection, such as pH and electrolyte concentration in the mobile phase, separation voltage, as well as enrichment voltage and time, have been studied systematically. Under the optimized conditions, the LODs of the three proteins could be decreased up to 100-fold. In addition, the feasibility of such techniques has been further demonstrated by the analysis of modified insulins at a concentration of 20 mu g/mL.