Homogeneous time-resolved fluoroimmunoassay of bensulfuron-methyl by using terbium fluorescence energy transfer

A sensitive homogenous time-resolved fluoroimmunoassay (TR-FIA) method for bensulfuron-methyl (BSM) based on fluorescence resonance energy transfer (FRET) from a Tb3+ fluorescent chelate with N,N,N',N'-[2,6-bis(3'-aminomethyl-1'-pyrazoly)-4-phenylpyridine] tetrakis(acetic acid) (BPTA-Tb3+) to organic dye, Cy3 or Cy3.5 has been developed. New method combined the use of BPTA-Tb3+ labeled streptavidin, Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody and biotinylated BSM-BSA conjugate (BSA is bovine serum albumin) for competitive-type immunoassay. After BPTA-Tb3+ labeled streptavidin was reacted with a competitive immune reaction solution containing biotinylated BSM-BSA, BSM sample and Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody, the sensitized and long-lived emission of Cy3 or Cy3.5 derived from FRET was measured, and thus the concentration of BSM in sample was calculated. The present method has the advantages of rapidity, simplicity and high sensitivity since the B/F (bound reagent/free reagent) separation steps and the solid-phase carrier are not necessary. The method gives the detection limit of 2.10 ng ml(-1). The coefficient variations of the method are less than 1.5% and the recoveries are in the range of 95-105% for BSM water sample measurement. (C) 2001 Elsevier Science 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.

Rapid authentication of ginseng species using microchip electrophoresis with laser-induced fluorescence detection

Ginseng is one of the most expensive Chinese herbal medicines and the effectiveness of ginseng depends strongly on its botanical sources and the use of different parts of the plants. In this study, a microchip electrophoresis method coupled with the polymerase chain reaction (PCR)-short tandem repeats (STR) technique was developed for rapid authentication of ginseng species. A low viscosity hydroxypropyl methylcellulose (HPMC) solution was used as the sieving matrix for separation of the amplified STR fragments. The allele sizing of the amplified PCR products could be detected within 240 s or less. Good reproducibility and accuracy of the fragment size were obtained with the relative standard deviation for the allele sizes less than 1.0% (n = 11). At two microsatellite loci (CT 12, CA 33), American ginseng had a different allele pattern on the electropherograms compared with that of the Oriental ginseng. Moreover, cultivated and wild American ginseng can be distinguished on the basis of allele sizing. This work establishes the feasibility of fast genetic authentication of ginseng species by use of microchip electrophoresis.

Quantitative and qualitative analysis of flavonoids in leaves of Adinandra nitida by high performance liquid chromatography with UV and electrospray ionization tandem mass spectrometry detection

The extract of Adinandra nitida leaves, named as Shiyacha in China, was studied by high performance liquid chromatography (HPLC)-ultraviolet detection-electrospray ionisation (ESI) tandem mass spectrometry (MS). Under the optimized condition, the analysis could be finished in 45 min on a Hypersil C18 column combined with negative ion detection using information-dependent acquisition (IDA) mode of a Q TRAP (TM) instrument. Six flavonoids were identified as epicatechin, rhoifolin, apigenin, quercitrin, camellianin A, and camellianin B among which rhoifolin was for the first time found in Shiyacha. And the fragment pathways of these flavonoids were elucidated. Furthermore, with epicatechin, rhoifolin, and apigenin as markers, the quality control method for Shiyacha and its relevant product was firstly established. Calibration linearity was good (R-2 > 0.9992) over a three to four orders of magnitude concentration range with an S/N = 3 detection limit of 2.5 ng. (c) 2004 Elsevier B.V. All rights reserved.