GC-MSによるアミノ酸の重窒素存在比の測定とその生体試料への応用

Concerning the amino acids which are very important biological materials of nitrogen compound, many works have been performed with ^<14>C and ^3H-labeled radioactive tracers in the fields of biochemistry and medicine. Since half life of ^<13>N compounds is as shortly as 9.96 minutes, its tracer experiment is limited by the facilities. Therefore, it needs to use stable isotope of nitrogen-15 for the study of amino acid metabolism. Conventional method for the analysis of the nitrogen-15 was carried out by the isotope mass spectrometry or the emission spectrometry. In both cases, however, amino acids must be decomposed into nitrogen gas by KJELDAHL and RITTENBERG methods after the separation procedures. These analytical methods require much labor. Recently, a gas chromatography - mass spectrometry (GC-MS) has been developed. The apparatus has been mainly used to study the metabolism of various drugs and identify the biological materials. In spite of many references in which the ^2H and ^<13>C-labeled compounds are analyzed by the GC-MS have been reported, no investigation on the ^<15>N excess ratio of amino acids by the GC-MS has been done. The present study has been carried out on this problem by GC-MS. Derivatives of amino acids were prepared after GEHRKE et al. The derivatives used were N-trifluoroacetyl butyl ester form. A Hitachi GC-MS (RMU-6MG) was employed. The silanized glass columns (1m×3mm i.d.) were packed with 0.5% EGA on 80 to 100 mesh Chromosorb W. Temperature - programmed separations were done at 3℃/min from 70 to 200℃ with helium as the carrier gas (30ml/min). The accelerating voltage, trap current and electron energy were 3.2KV, 80μA and 20eV, respectively. The mass fragment ions used for measuring ^<15>N excess ratio of amino acids were adopted as follows: alanine (m/e 141/140), valine (m/e 169/168), glycine (m/e 128/127), isoleucine (m/e 183/182), leucine (m/e 183/182), proline (m/e 167/166), threonine (m/e 154/153), serine (m/e 140/139), aspartic acid (m/e 241/240), glutamic acid (m/e 199/198, 255/254) and lysine (m/e 181/180). A mass range of the repeat scanning of magnetic field was set in the range of A ±5 (as to aspartic acid A=240). The intensity of mass fragment ion was recorded by the repeat scanning of magnetic field. From the maximum intensity of mass fragment ion, ^<15>N excess ratio was calculated with the formula of VANDEN HEUVEL. The ^<15>N-labeled aspartic acid (50 atom % excess ^<15>N) and glutamic acid (93.5 atom % excess ^<15>N) were employed as to authentic sample of amino acid. The results indicated that the values of 50.2 atom % excess ^<15>N (aspartic acid) and 92.6-92.8 atom % excess ^<15>N (glutamic acid) which could be measured with the coefficient of variation below 0.3 % by the repeat scanning of magnetic field method of GC-MS. Comparing with MID method, more simple and better accuracy in measuring ^<15>N excess ratio of amino acids were found in the repeat scanning of magnetic field method. The ^<15>N excess ratios of amide group in asparagine and glutamine could not be measured with N-TFA butyl ester derivatives. Therefore, asparagine and glutamine were converted into TMS-MT derivatives. The results indicated that the ^<15>N excess ratios of amide group were able to analyze with the specific mass fragment ion by the repeat scanning of magnetic field of GC-MS. This analytical method was applied to the investigation of nitrogen metabolism of amino acids in the ^<15>N tracer experiments. It was demonstrated that the method was able to apply to measuring ^<15>N excess ratio even in tracer free amino acids in the biological materials. This method had a merit to be able to analyze without any separation of each amino acid from amino acids mixture as compared with the conventional method (the isotope ratio mass spectrometry and the emission spectrometry). And also it became clear that the ^<15>N excess ratio of amino acids in trace amounts of biological materials was able to measure with the coefficient of variation below 0.2-0.3%. Its accuracy was a little lower as compared with the isotope ratio mass spectrometry (0.01 atom % excess ^<15>N --- Isotope ratio mass spectrometry, 0.1 atom % excess ^<15>N --- GC-MS). But this method was able to analyze ^<15>N excess ratio of trace amunts of amino acids (10^<-9>10^<-12> g N). Therefore, ^<15>N tracer experiments could be done using the trace amounts of high concentrated ^<15>N-labeled compounds. The present analytical method of the ^<15>N excess ratio in the trace amounts of amino acids by GC-MS will be useful for studying nitrogen metabolism of amino acids and protein in biochemistry and medicine