Temperature effect on post column derivatisation reaction with o-phthaladehyde of histidine dipeptides and their components
2004
Ducci, M. (Pisa Univ. (Italy). Dipartimento di Anatomia, Biochimica e Fisiologia Veterinaria) | Longa, A. Della (Pisa Univ. (Italy). Dipartimento di Anatomia, Biochimica e Fisiologia Veterinaria) | Gazzano, A. (Pisa Univ. (Italy). Dipartimento di Anatomia, Biochimica e Fisiologia Veterinaria) | Bobowiec, R. (University School of Agriculture, Lublin (Poland)) | Martelli, F. (Pisa Univ. (Italy). Dipartimento di Anatomia, Biochimica e Fisiologia Veterinaria) | Pacchini, S. | Niccolini, A.
The aim of this research was to carry out the o-phthalaldehyde (OPA) post-column derivatisation of histidine dipeptides (carnosine, anserine), L-histidine and 3-methyl-L-histidine at different temperatures, to establish which temperature is the best to obtain the highest sensibility of detection of this compounds. As regards anserine and carnosine, results showed that the best temperature of derivatisation reaction was 45-50 deg C for anserine and 35-45 deg C for carnosine, with an increase of peak area of 9% compared to 30 deg C. For L-histidine and 3-methyl-L-histidine, this research showed that the optimal temperature was 55-60 deg C, with an increase of peak area at each temperature of derivatisation reaction (R**2 = 0.998 for L-histidine and R**2 = 0,999 for 3-methyl-L-histidine). For this latter, the increase of peak area, compared to 30 deg C, was more than 70%. The reproducibility of the method (for histidine dipeptides, L-histidine and 3-methyl-L-histidine) was very high, with a very low coefficient of variation at all temperatures of derivatisation; the derivatisation reaction was very stable. Therefore, all data showed that the increase of sensibility (associated to increase of temperature) was very high for L-histidine and 3-methyl-L-histidine, while it was reduced for histidine dipeptides. In conclusion, this research showed that the increase of derivatisation temperature permitted to obtain an higher sensibility efficiency of the HPLC method used for detection of histidine dipeptides, that are present in animal tissues at different and very low concentrations
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