| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
SImple and highly sensitive FIA and HPLC methods were developed for the determination of several bioactive amines by using highly selective fluorogenic reagents for each the amine.
Orthoacetylbenzaldehyde (OAB) reacts rapidly with histamine in weekly alkaline medium to form a highly fluorescent adduct. Simple and sensitive HPLC method for the assay of histamine in tissues was developed by employing this fluorescence reaction as postcolumn fluoresence labeling of histamine. On the other hand, FIA method for the rapid assay of histamine was established by using 4-bromo-1,2-benzenedicarbaldehyde (BBDA) which reacts specifically with histamine to form a stable fluorescent adduct.
Glutathione also reacts rapidly with OAB at pH 9.3 to form a stable and highly fluorescent adduct. By employing this reaction as precolumn fluorescence labeling, Simple and ultrasensitive HPLC method with detection limit (S/N=3) of 50 fmol/injection was developed for the assay of glutathione in human erythrocytes an
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d sera.
Spermidine gives a stable fluorescent adduct in the reaction with orthophthalaldehyde in alkaline medium followed by acidification of the solution. This fluorescence reaction was highly selective for spermidine and was used to precolumn fluorescence labeling for HPLC measuremment of spermidine. Serum spermidine was determined by this HPLC method in combination with solid phase extraction using ion exchage minicolumn.
Micromethod for the determination of phenylalamine was established, which consisted of conversion of phenylalanine into trans-cinnamic acid with phenylalanine ammonia-lyase, and measurement of trans-cinnamic acid by reverse phase HPLC with UV detection. This method was applicable to the assay of phenylalanine in urine, serum and blood paper for screening of phenylketourea.
Biocytin, one of metabolites of biotin, was determined by HPLC method after fluorescence labeling with the reagent system of 2,3-naphthalenedicarbaldehyde and KCN,which is highly sensitive for primary amines. Less
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