| High performance liquid chromatography (HPLC) has become an essential analytical tool of carbohydrates analysis, due to the lack of fluorescent or ultraviolet group, carbohydrates need to be derivatized before HPLC analysis. A lot of derivatization reagents have been used to saccharide analysis, however, some of these derivatizations suffered from the problems including tedious reaction procedures or a signal decreasing by biological mass spectrometry detection that did not meet the demands of modern trace level analyses with regard to sensitivity and/or selectivity. Hence, it's significant for us to employ a high sensitive derivatization reagent for saccharide analysis in actual work. In our paper, we established a new pre-column derivatization method for saccharides analysis, employing 3-amino-9-ethylcarbazole as a derivatizing reagent. The derivatives were separated by reverse-phase HPLC and identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. We optimized the derivatization reaction as well as the HPLC conditions and validated the described method. Many samples including monosaccharides and oligosaccharides were successfully analyzed by the described method. Finally, we have got these results and achievements as fellows:1. The standard monosaccharides were well detected and analyzed by HPLC and MALDI-TOF-MS, previously derivatized with AEC. We have optimized the derivatization reaction and investigated the stability, sensitivity and detection limits of the method. The results showed that the reaction condition was mild (70℃, 60 min). The derivatives could be directly detected by MALDI-TOF-MS without desalted procedure, avoiding lose of sample. Detection limits for the samples (D-glucose, Z-xylose, D-mannose, I-arabinose, and L-rhamnose) ranged from 0.06 to 1.97 ng/ml (S/N=3) showed that the method is high sensitive. The derivatives was stable enough for analysis that stability analysis showed R.S.D. was < 3% within five days, and the accuracy analysis was good that recovery ratios were > 98.62% in all cases.2. The HPLC conditions were optimized and a method for monosaccharides analysis was developed. As a result, the sugar compositions of three oligosaccharides SPPA-1, SPPB-1 and SPPC-1 of Spirulina platensis were successfully analyzed by this method. 3. We have optimized the HPLC conditions and established a mode for HPLC and MALDI-TOF-MS analysis of oligosaccharides. Chitosan was firstly pre-column derivatized with AEC and analyzed by HPLC and MALDI-TOF-MS. The massage of main compoment composition of sample was recorded.4. We are also applying this derivatization method to uronic acid analysis in order to evaluate and expand the use of method. |
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