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.

Independence of alpine meadow plants from soil organic matter for nitrogen supply: evidence from stable nitrogen isotopes

Stable nitrogen isotope signatures of major sources of mineral nitrogen ( mineralization of soil organic nitrogen, biological N-2 fixation by legumes, annual precipitation and plant litter decomposition) were measured to relatively define their individual contribution to grass assimilation at the Haibei Alpine Meadow Ecosystem, Qinghai, China. The results indicated that delta N-15 values (- 2.40 parts per thousand to 0.97 parts per thousand) of all grasses were much lower than those of soil organic matter (3.4 +/- 0.18 parts per thousand) and mineral nitrogen ( ammonium and nitrate together,7.8 +/- 0.57 parts per thousand). Based on the patterns of stable nitrogen isotopes, soil organic matter (3.4 +/- 0.18 parts per thousand), biological N-2 fixation (0 parts per thousand), and precipitation (- 6.34 +/- 0.24 parts per thousand) only contributed to a small fraction of nitrogen requirements of grasses, but plant litter decomposition (- 1.31 +/- 1.01 parts per thousand) accounted for 67%.

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.

The effect of land management on carbon and nitrogen status in plants and soils of alpine meadows on the Tibetan plateau

Large-scale grassland rehabilitation has been carried out on the severely degraded lands of the Tibetan plateau. The grasslands created provide a useful model for evaluating the recovery of ecosystem properties. The purposes of this research were: (1) to examine the relative influence of various rehabilitation practices on carbon and nitrogen in plants and soils in early secondary succession; and (2) to evaluate the degree to which severely degraded grassland altered plant and soil properties relative to the non-disturbed native community. The results showed: (1) The aboveground tissue C and N content in the control were 105-97 g m(-2) and 3.356gm(-2), respectively. The aboveground tissue C content in the mixed seed treatment, the single seed treatment, the natural recovery treatment and the severely degraded treatment was 137 per cent, 98 per cent, 49 per cent and 38 per cent, respectively, of that in the control. The corresponding aboveground tissue N content was 109 per cent, 84 per cent, 60 per cent and 47 per cent, respectively, of that in the control. (2) Root C and N content in 0-20 cm depths of the control had an 2 2 average 1606 gm(-2) and 30-36 gm(-2) respectively. Root C and N content in the rehabilitation treatments were in the range of 26-36 per cent and 35-53 per cent, while those in the severely degraded treatment were only 17 per cent and 26 per cent of that in the control. (3) In the control the average soil C and N content at 0-20 cm was 11307 gm(-2) and 846 gm(-2), respectively. Soil C content in the uppermost 20 cm in the seeded treatments, the natural recovery treatment and the severely degraded treatment was 67 per cent, 73 per cent and 57 per cent, respectively, while soil N content in the uppermost 20cm was 72 per cent, 82 per cent and 79 per cent, respectively, of that in the control. The severely degraded land was a major C source. Restoring the severely degraded lands to perennial vegetation was an alternative approach to sequestering C in former degraded systems. N was a limiting factor in seeding grassland. It is necessary for sustainable utilization of seeding grassland to supply extra N fertilizer to the soil or to add legume species into the seed mix. Copyright (c) 2005 John Wiley & Sons, Ltd.