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Gas chromatography-mass spectrometry of guanidino compounds in brain
ANALYTICAL
BIOCHEMISTRY
89, 393-399 (1978)
Gas Chromatography-Mass Spectrometry Guanidino Compounds in Brain AKITANE MORI, Department Medical
TAKAMICHI
ICHIMURA,*
AND HISAO
of Neurochemistry. Institute for Neurobiology:y, School, Okayama, and *Institute of Research Yamanouchi Pharmaceutical Company, Azusawa. Tokyo, Japan
of
MATSUMOTO*
Okayama University and Development, Itabashiku,
Received December 28, 1977 Authentic guanidinoacetic acid, P-guanidinopropionic acid, y-guanidinobutyric acid, arginine, and homoarginine were converted into dimethylpyrimidyl derivatives by reaction with acetyl acetone, the carboxyl group was esterified by butyl alcohol, and the trifluoroacetyl derivatives were analyzed by the gc/ms technique. Guanidinoacetic acid, y-guanidinobutyric acid, arginine, and homoarginine were identified in rat and bovine brain material by the same technique. Taurocyamine was not detected by this method in either authentic or animal samples.
Brain guanidino compounds analyzed by paper (1) and liquid chromatography (2,3) have a low sensitivity (of the order of 0.01 pmollg). Shemyakin et al. (4) applied mass spectrometric methods to determine the amino acid sequence in arginine-containing peptides after transforming the arginine residue into a dimethylpyrimidylomithine residue by acetylacetone. This step was performed as arginine is not volatile due to the specific behavior of its guanidino group. Vetter-Diechtl et al. (5) converted arginine in the same way to a dimethylpyrimidylomithine ethyl ester (ethyl 5[2’-(4’, 6’-dimethylpyrimidyl)]-amino-2-aminopentanoate) and obtained a mass spectrum of the compound by the gas chromatography-mass spectrometry (gc/ms) technique. In the present communication, we report on determinations of guanidinoacetic acid, y-guanidinobutyric acid, arginine! and homoarginine in rat and bovine brain. These determinations were performed by gc/ms after conversion of these compounds into dimethylpyrimidyl derivatives by acetylacetone. MATERIALS
AND METHODS
Authentic samples. Guanidinoacetic acid, arginine (Wako Chemicals, Osaka, Japan), and homoarginine (Sigma Chemical Co., St. Louis, MO.) were purchased. P-Guanidinopropionic acid was synthesized from /3393
0003-2697/78/0892-0393$02.00/O Copyright All rights
Q 1978 by Academic F’ress, Inc. of reproduction in any form reserved.
394
MORI,
ICHIMURA,
AND
MATSUMOTO
GC-MSOFGUANIDINOCOMPOUNDS
395
+
396
MORI,
ICHIMURA,
AND
MATSUMOTO
alanine and S-methylisothiourea sulfate. y-Guanidinobutyric acid was synthesized from y-aminobutyric acid and S-methylisothiourea sulfate. Both of these guanidino compounds were prepared according to Zervas and Bergmann (6) by one of the authors (A.M.). Brain extract. A rat brain was homogenized in 3 ml of ice-cold 1% picric acid and centrifuged at 10,000 rpm for 10 min. The supernatant was passed through a column of Dowex 1 x 8 (Cl- form), 1.0 x 2.0 cm, and the colorless eluate was dried in vacua. Bovine cerebral cortex (100 g) was homogenized in 300 ml of 7% trichloracetic acid and filtered. The filtrate was passed through a column of AmberliteCG120(Hfform),3.0 x 15cm,washedbywater,andelutedby 1 N-NH,OH. The fraction containing homoarginine (2) was collected and dried in vacua. Preparation of dimethylpyrimidyl derivatives. Guanidino compound (10 mg) was dissolved in 1.O ml of water, and 1.O ml of pyridine and 1.O ml of acetylacetone were added, with the pH of the mixture being adjusted between 8.0 and 9.0 by the addition of sodium bicarbonate. The mixture was then refluxed at 100°C for 10 hr in a water bath. The reaction mixture was dried in vucuo, dissolved in 10 ml of water, washed with ethyl ether twice, dried in vucuo, dissolved in 10 ml of dried HCl gas-saturated nbutanol, and refluxed for 3 hr in a water bath. The syrup-like substances obtained after drying in vacua were trifluoroacetylated by 10% trifluoroacetic anhydrite in ethyl acetate and then applied to gc/ms. Guanidino compounds in the brain extract were converted to dimethylpyrimidyl derivatives in the same way and analyzed by gc/ms. Gas chromatography -mass spectrometry . An Hitachi RMU-6MG mass spectrograph was used under the following conditions: for gc-column length, 1 m; column packing, OV-1; column temperature, 170°C; carrier gas, 1.2 kg/cm2 He; and for ms-chamber voltage, 40 V; target/total current, 80/100 ,uA; evaporator/ion source temperature, 260”/16O”C. An Hitachi MID unit was used for selected ion recording. RESULTS
Mass Spectra of Guanidino
AND DISCUSSION
Compounds
Figure 1 shows mass spectra of authentic guanidinoacetic acid, pguanidinopropionic acid, y-guanidinobutyric acid, arginine, and homoarginine, in the form of trifluoroacetylated dimethylpyrimidyl derivatives. M+ 333 corresponds to trifluoroacetylated n-butyl 2[2’-(4’,6’-dimethylpyrimidyl)]-aminoacetate; M+ 347 corresponds to trifluoroacetylated nbutyl3[2’(4’,6’-dimethylpyrimidyl)]-aminopropionate; M+ 361 corresponds to trifluoroacetylated n-butyl 4[2’-(4’,6’-dimethylpyrimidyl)]-aminobutyrate; M+ 486 corresponds to trifluoroacetylated (two molecules) n-butyl 5[2’-(4’,6’-dimethylpyrimidyl)]-amino-2-aminopentanoate; and M+ 500
W-MS
OF GUANIDINO
COMPOUNDS
397
corresponds to trifluoroacetylated (two molecules) n-butyl 6[2’-(4’,6’dimethylpyrimidyl)]-amino-2-aminohexanoate. Fragments m/e 390 and m/e 404 (Figs. Id and e) were each mono trifluoroacetylated derivatives. The use of 10% trifluoroacetic anhydride in ethyl acetate for acylation resulted in only mono acylated derivatives being recorded. Fragments m/e 123, 124, and 125 in these mass spectra could represent 2’-amino-4’,6’-dimethylpyrimidine residue involving one, two, or three hydrogen atoms, respectively (5). Fragments m/e 136 and m/e 150 could represent 2’-aminomethylen-4’,6’-dimethylpyrimidine residue and 2’aminoethyl4’,6’-dimethylpyrimidine residue, respectively. Taurocyamine, a normal component of brain, was also tested in the same way, but a mass spectrum was not recorded. Assay of Brain Guanidino
Compounds
by Selected Ion Recording
The five guanidino compounds were analyzed by gas chromatography (Fig. 2) and then determined mass spectrometrically. Combinations of several mass fragments frommle 220,232,236,246,250,264,361,403,431, and 500 in trifluoro derivatives were used for determining guanidino compounds in the brain sample. Table 1 shows the height ratios of fragments from brain and from authentic samples at the same retention time in gc (for instance, mle 232 and mle 236 in guanidinoacetic acid analysis). Mass fragments identical to authentic guanidinoacetic acid, y-guanidinobutyric acid, and arginine were detected in rat brain tissue. On the other hand, the identification of homoarginine was not certain, and P-guanidinopropionic acid was not detected. Then, bovine samples containing a higher concentration of homoarginine were analyzed, and the existence of this substance in brain tissue was confirmed (Table 2). We (3) reported earlier
FIG. 2. Gas chromatogram of guanidino compounds in the form of trifluoroacetylated dimethylpyrimidyl derivatives. (A) Authentic sample: (a) guanidinoacetic acid; (b) /3guanidinopropionic acid; (c) y-guanidinobutyric acid; (d) arginine; (e) homoarginine. (B) Brain samples.
398
MORI, ICHIMURA,
AND MATSUMOTO
TABLE IDENTIFICATION
OF GUANIDINO BY SELECTED ION
Sample Guanidinoacetic Authentic
COMPOUNDS RECORDING
BRAIN
Ratio
232 236 232 236
51.2 14 58.7 16
100 27 100 27
246 250 246 250
14 52.5 Over scale 23
27 100 100
264 361 264 361
100 50 18.3 6
100 50 100 60
220 232 220 232
24 6 44 10
100 25 100 23
403 431 403 431
8 13 Trace Trace
61 100 -
acid
P-Guanidinopropionic Authentic
acid
Rat brain acid
Rat brain Arginine Authentic Rat brain Homoarginine Authentic Rat brain
TABLE IDENTIFICATION
IN RAT
(SIR) Height in SIR chart
mle
Rat brain
y-Guanidinobutyric Authentic
1
OF HOMOARGININE
IN BOVINE
2 BRAIN
BY SELECTED
ION
RECORDING
Ratio of ions (%) Sample Homoarginine Bovine brain
43 l/403
500/403
5cw43 1
51.7 52.7
30.9 29.9
63.5 62.4
GC-MS
OF GUANIDINO
COMPOUNDS
399
using liquid chromatography that homoarginine was observed with a peak having the same elution time as the authentic standard. The sensitivity for guanidino compounds by this gc/ms technique was less than 1 ng. Other unknown gc peaks of the guanidino compounds in Fig. 2B are now under examination. ACKNOWLEDGMENTS We thank Dr. R. A. Davidoff, Department of Neurology, University of Miami School of Medicine, for his valuable discussion and editorial assistance, and Dr. M. Kurono, Central Laboratory, Ono Pharmaceutical Co., for helpful discussion. This study was supported partially by Grant-in-Aid for Scientific Research 148128, Japan Ministry of Education, Science and Culture.
REFERENCES 1. 2. 3. 4.
Blass, J. P. (1960) Biochem. J. 77, 484-489. Mori, A., Hosotani, M., and Tye, L. C. (1974) Eiochem. Med. 10, 8-14. Matsumoto, M., Kishikawa, H., and Mori, A. (1976) Biochem. Med. 16, l-8. Shemyakin, M. M., Ovchinnikov, Y. A., Vionogracova, E. I., Feigina, M. Y., Kiryushkin, A. A., Aladanova, N. A., Alakhov, Y. B., Lipkin, V. M., and Rosinov, B. V. (1%7) Experientia
23, 428-430.
5. Vetter-Diechtl, H., Vetter, W., Rechter, W., and Biemann, K. (1968) Experientia 24, 340-341. 6. Zervas, L., and Bergmann, M. (1928) Ber. d. Deutsch. Chem. Ges. 61, 1195-1203.
BIOCHEMISTRY
89, 393-399 (1978)
Gas Chromatography-Mass Spectrometry Guanidino Compounds in Brain AKITANE MORI, Department Medical
TAKAMICHI
ICHIMURA,*
AND HISAO
of Neurochemistry. Institute for Neurobiology:y, School, Okayama, and *Institute of Research Yamanouchi Pharmaceutical Company, Azusawa. Tokyo, Japan
of
MATSUMOTO*
Okayama University and Development, Itabashiku,
Received December 28, 1977 Authentic guanidinoacetic acid, P-guanidinopropionic acid, y-guanidinobutyric acid, arginine, and homoarginine were converted into dimethylpyrimidyl derivatives by reaction with acetyl acetone, the carboxyl group was esterified by butyl alcohol, and the trifluoroacetyl derivatives were analyzed by the gc/ms technique. Guanidinoacetic acid, y-guanidinobutyric acid, arginine, and homoarginine were identified in rat and bovine brain material by the same technique. Taurocyamine was not detected by this method in either authentic or animal samples.
Brain guanidino compounds analyzed by paper (1) and liquid chromatography (2,3) have a low sensitivity (of the order of 0.01 pmollg). Shemyakin et al. (4) applied mass spectrometric methods to determine the amino acid sequence in arginine-containing peptides after transforming the arginine residue into a dimethylpyrimidylomithine residue by acetylacetone. This step was performed as arginine is not volatile due to the specific behavior of its guanidino group. Vetter-Diechtl et al. (5) converted arginine in the same way to a dimethylpyrimidylomithine ethyl ester (ethyl 5[2’-(4’, 6’-dimethylpyrimidyl)]-amino-2-aminopentanoate) and obtained a mass spectrum of the compound by the gas chromatography-mass spectrometry (gc/ms) technique. In the present communication, we report on determinations of guanidinoacetic acid, y-guanidinobutyric acid, arginine! and homoarginine in rat and bovine brain. These determinations were performed by gc/ms after conversion of these compounds into dimethylpyrimidyl derivatives by acetylacetone. MATERIALS
AND METHODS
Authentic samples. Guanidinoacetic acid, arginine (Wako Chemicals, Osaka, Japan), and homoarginine (Sigma Chemical Co., St. Louis, MO.) were purchased. P-Guanidinopropionic acid was synthesized from /3393
0003-2697/78/0892-0393$02.00/O Copyright All rights
Q 1978 by Academic F’ress, Inc. of reproduction in any form reserved.
394
MORI,
ICHIMURA,
AND
MATSUMOTO
GC-MSOFGUANIDINOCOMPOUNDS
395
+
396
MORI,
ICHIMURA,
AND
MATSUMOTO
alanine and S-methylisothiourea sulfate. y-Guanidinobutyric acid was synthesized from y-aminobutyric acid and S-methylisothiourea sulfate. Both of these guanidino compounds were prepared according to Zervas and Bergmann (6) by one of the authors (A.M.). Brain extract. A rat brain was homogenized in 3 ml of ice-cold 1% picric acid and centrifuged at 10,000 rpm for 10 min. The supernatant was passed through a column of Dowex 1 x 8 (Cl- form), 1.0 x 2.0 cm, and the colorless eluate was dried in vacua. Bovine cerebral cortex (100 g) was homogenized in 300 ml of 7% trichloracetic acid and filtered. The filtrate was passed through a column of AmberliteCG120(Hfform),3.0 x 15cm,washedbywater,andelutedby 1 N-NH,OH. The fraction containing homoarginine (2) was collected and dried in vacua. Preparation of dimethylpyrimidyl derivatives. Guanidino compound (10 mg) was dissolved in 1.O ml of water, and 1.O ml of pyridine and 1.O ml of acetylacetone were added, with the pH of the mixture being adjusted between 8.0 and 9.0 by the addition of sodium bicarbonate. The mixture was then refluxed at 100°C for 10 hr in a water bath. The reaction mixture was dried in vucuo, dissolved in 10 ml of water, washed with ethyl ether twice, dried in vucuo, dissolved in 10 ml of dried HCl gas-saturated nbutanol, and refluxed for 3 hr in a water bath. The syrup-like substances obtained after drying in vacua were trifluoroacetylated by 10% trifluoroacetic anhydrite in ethyl acetate and then applied to gc/ms. Guanidino compounds in the brain extract were converted to dimethylpyrimidyl derivatives in the same way and analyzed by gc/ms. Gas chromatography -mass spectrometry . An Hitachi RMU-6MG mass spectrograph was used under the following conditions: for gc-column length, 1 m; column packing, OV-1; column temperature, 170°C; carrier gas, 1.2 kg/cm2 He; and for ms-chamber voltage, 40 V; target/total current, 80/100 ,uA; evaporator/ion source temperature, 260”/16O”C. An Hitachi MID unit was used for selected ion recording. RESULTS
Mass Spectra of Guanidino
AND DISCUSSION
Compounds
Figure 1 shows mass spectra of authentic guanidinoacetic acid, pguanidinopropionic acid, y-guanidinobutyric acid, arginine, and homoarginine, in the form of trifluoroacetylated dimethylpyrimidyl derivatives. M+ 333 corresponds to trifluoroacetylated n-butyl 2[2’-(4’,6’-dimethylpyrimidyl)]-aminoacetate; M+ 347 corresponds to trifluoroacetylated nbutyl3[2’(4’,6’-dimethylpyrimidyl)]-aminopropionate; M+ 361 corresponds to trifluoroacetylated n-butyl 4[2’-(4’,6’-dimethylpyrimidyl)]-aminobutyrate; M+ 486 corresponds to trifluoroacetylated (two molecules) n-butyl 5[2’-(4’,6’-dimethylpyrimidyl)]-amino-2-aminopentanoate; and M+ 500
W-MS
OF GUANIDINO
COMPOUNDS
397
corresponds to trifluoroacetylated (two molecules) n-butyl 6[2’-(4’,6’dimethylpyrimidyl)]-amino-2-aminohexanoate. Fragments m/e 390 and m/e 404 (Figs. Id and e) were each mono trifluoroacetylated derivatives. The use of 10% trifluoroacetic anhydride in ethyl acetate for acylation resulted in only mono acylated derivatives being recorded. Fragments m/e 123, 124, and 125 in these mass spectra could represent 2’-amino-4’,6’-dimethylpyrimidine residue involving one, two, or three hydrogen atoms, respectively (5). Fragments m/e 136 and m/e 150 could represent 2’-aminomethylen-4’,6’-dimethylpyrimidine residue and 2’aminoethyl4’,6’-dimethylpyrimidine residue, respectively. Taurocyamine, a normal component of brain, was also tested in the same way, but a mass spectrum was not recorded. Assay of Brain Guanidino
Compounds
by Selected Ion Recording
The five guanidino compounds were analyzed by gas chromatography (Fig. 2) and then determined mass spectrometrically. Combinations of several mass fragments frommle 220,232,236,246,250,264,361,403,431, and 500 in trifluoro derivatives were used for determining guanidino compounds in the brain sample. Table 1 shows the height ratios of fragments from brain and from authentic samples at the same retention time in gc (for instance, mle 232 and mle 236 in guanidinoacetic acid analysis). Mass fragments identical to authentic guanidinoacetic acid, y-guanidinobutyric acid, and arginine were detected in rat brain tissue. On the other hand, the identification of homoarginine was not certain, and P-guanidinopropionic acid was not detected. Then, bovine samples containing a higher concentration of homoarginine were analyzed, and the existence of this substance in brain tissue was confirmed (Table 2). We (3) reported earlier
FIG. 2. Gas chromatogram of guanidino compounds in the form of trifluoroacetylated dimethylpyrimidyl derivatives. (A) Authentic sample: (a) guanidinoacetic acid; (b) /3guanidinopropionic acid; (c) y-guanidinobutyric acid; (d) arginine; (e) homoarginine. (B) Brain samples.
398
MORI, ICHIMURA,
AND MATSUMOTO
TABLE IDENTIFICATION
OF GUANIDINO BY SELECTED ION
Sample Guanidinoacetic Authentic
COMPOUNDS RECORDING
BRAIN
Ratio
232 236 232 236
51.2 14 58.7 16
100 27 100 27
246 250 246 250
14 52.5 Over scale 23
27 100 100
264 361 264 361
100 50 18.3 6
100 50 100 60
220 232 220 232
24 6 44 10
100 25 100 23
403 431 403 431
8 13 Trace Trace
61 100 -
acid
P-Guanidinopropionic Authentic
acid
Rat brain acid
Rat brain Arginine Authentic Rat brain Homoarginine Authentic Rat brain
TABLE IDENTIFICATION
IN RAT
(SIR) Height in SIR chart
mle
Rat brain
y-Guanidinobutyric Authentic
1
OF HOMOARGININE
IN BOVINE
2 BRAIN
BY SELECTED
ION
RECORDING
Ratio of ions (%) Sample Homoarginine Bovine brain
43 l/403
500/403
5cw43 1
51.7 52.7
30.9 29.9
63.5 62.4
GC-MS
OF GUANIDINO
COMPOUNDS
399
using liquid chromatography that homoarginine was observed with a peak having the same elution time as the authentic standard. The sensitivity for guanidino compounds by this gc/ms technique was less than 1 ng. Other unknown gc peaks of the guanidino compounds in Fig. 2B are now under examination. ACKNOWLEDGMENTS We thank Dr. R. A. Davidoff, Department of Neurology, University of Miami School of Medicine, for his valuable discussion and editorial assistance, and Dr. M. Kurono, Central Laboratory, Ono Pharmaceutical Co., for helpful discussion. This study was supported partially by Grant-in-Aid for Scientific Research 148128, Japan Ministry of Education, Science and Culture.
REFERENCES 1. 2. 3. 4.
Blass, J. P. (1960) Biochem. J. 77, 484-489. Mori, A., Hosotani, M., and Tye, L. C. (1974) Eiochem. Med. 10, 8-14. Matsumoto, M., Kishikawa, H., and Mori, A. (1976) Biochem. Med. 16, l-8. Shemyakin, M. M., Ovchinnikov, Y. A., Vionogracova, E. I., Feigina, M. Y., Kiryushkin, A. A., Aladanova, N. A., Alakhov, Y. B., Lipkin, V. M., and Rosinov, B. V. (1%7) Experientia
23, 428-430.
5. Vetter-Diechtl, H., Vetter, W., Rechter, W., and Biemann, K. (1968) Experientia 24, 340-341. 6. Zervas, L., and Bergmann, M. (1928) Ber. d. Deutsch. Chem. Ges. 61, 1195-1203.