693
Anal. Chem. 1984, 56,693-695
Desorption Chemical Ionization Mass Spectrometry of Guanidino Compounds E. L. Esmans* a n d F. C. Alderweireldt Laboratory for Organic Chemistry, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
B. A. Marescau a n d A. A. Lowenthal Department of Neurochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
The desorption chemlcai ionization (DICI) mass spectra of 21 biologically important guanidino compounds were recorded with ammonia as the reagent gas. N-a-Acetylarginine was isolated from the urine of patients suffering from hyperargininemia and identified by comparison wlth its DICI reference mass spectrum.
The clinical symptoms of patients with hyperargininemia are irritability, periods of vomiting, coma, and epilepsy. Since these patients have an arginase deficiency, an arginine accumulation is observed in their body fluids (I-3), together with an increased concentration of arginine catabolites in urine (4) and serum (5). It has also been proven that different guanidino compounds like taurocyamine (6),guanidinoacetic acid (7), y-guanidinobutyric acid (8), N-a-acetylarginine (9),etc. could be epileptogenic. In view of this observation, identification of these compounds from biological fluids is not only important but also necessary. In order to elucidate the structure of guanidino compounds in urine, different techniques can be helpful, such as liquid chromatography on an amino acid analyzer and gas chromatography/mass spectrometry (10,11). In this paper we wish to present the possibilities and limitations of D/CI-MS, for identification of this class of compounds.
EXPERIMENTAL SECTION Products. a-Amino-P-guanidinopropionicacid (I) (Calbiochem), homoarginine (11) (Sigma), canavanine (111) (Sigma), a-amino-y-guanidinobutyric acid (IV) (Calbiochem),arginine (V) (Sigma), P-guanidinoisobutyric acid (VI) (synthese) (12), 0guanidinopropionic acid (VII) (Sigma), e-guanidinocaproic acid (VIII) (synthese), 6-guanidinovaleric acid (IX) (synthese), yguanidinobutyric acid (X) (Sigma), a-guanidinoacetic acid (XI) (Sigma), creatine (XII) (Sigma), a-guanidinobutyric acid (XIII) (synthese), a-guanidinoglutaric acid (XIV) (A. Mori), a-guanidinosuccinic acid (XV) (Sigma), y-guanidino-@-hydroxybutyric acid (XVI) (D. J. Durzan), argininic acid (XVII) (Sigma), aketo-6-guanidinovaleric acid (XVIII) (synthese), y-guanidinobutyramide (XIX) (D. J. Durzan), taurocyamine (XX) (synthese), a-guanidino-P-phenylpropionicacid (XXI) (synthese), and N-aacetylarginine (XXII) (Sigma) were used. Urine samples were collected from three sisters with hyperargininemia by H. G. Terheggen from the Children's Hospital, Neuss, Federal Republic of Germany. Chromatography. Cleanup Procedure. A urine sample was treated with 6 N HCI, until pH 3 was obtained, and three times extracted with ether (v/v) to remove the lipid fraction. This acidic sample was then treated with active coal and centrifuged. The pellet was washed three times with 0.01 N HCl. The supernatant was filtered on a 5-wm filter and about 1.5 mL of the solution was brought on a column. Equipment. See ref 10. Chromatographic Conditions. Chambers 1to 4 of the Autograd System (Technicon) were filled each with 75 cma of 0.4 M pyr0003-2700/84/0356-0693$0 1.50/0
idine/formic acid (pH 4); chambers 5 to 9 were each filled with 75 cm3of 1M pyridine. At the start of the analysis, all chambers were connected and the column was eluted at 0.5 mL/min. The column was a 140-cm jacketed (59 "C)glass column (6.2 mm i.d.) filled to 130 cm with Dowex 50X8,8% cross-linked Technicon Chromobeads, type A, 21-wm particles, Under these conditions N-a-acetylarginine is collected after 230 min. Isolation and Extra Purification of N-a-Acetylarginine (XXII). The volatile formic acid/pyridine buffer solvent was removed under vacuum and N-a-acetylarginine was extra purified on a LiChrosorb 10-NH2column (4.6 mm i.d., 1 = 25 cm) using 0.01 M NH,OOCH (A)/CH,CN (B) effluent in a gradient elution from 78% B to 50% B in 10 min. The product was collected and 10 p L of the solution was brought directly on the filament of the D/CI probe. Mass Spectral Conditions. After the guanidino compounds were dissolved in water, about 3 pg of product was placed upon a 60-wm tungsten wire, by means of a microsyringe. After evaporation of the solvent, the wire was inserted in the plasma of the reagent gas. The ion source was operated at a pressure of approximately 0.8 torr, and all heating connections to the source were interrupted. Then a current of 50 mA was sent through the wire, gradually rising to 400 mA at a rate of 10 m A d . During this heating procedure the quadrupole mass spectrometer was scanned from m/z 100 to m / z 250 at a peak integration time of 3 ms. As soon as ions were detected, either on the screen of the computer terminal or on the oscilloscope of the mass spectrometer, the rate of heating of the tungsten wire was set at 1-2 m A d . Primary ionization of the reagent gas was performed with 70-eV electrons at an emission current of 9 mA. All spectra were run on a Riber 10B-10quadrupole mass spectrometer equipped with a SIDAR data system.
RESULTS AND DISCUSSION It has been established that, although m a s spectral analysis of most underivatized amino acids is possible by direct probe introduction (131,this technique fails for guanidino compounds like arginine due to thermal degradation prior to ionization. In order to avoid this phenomenon, one can convert these molecules to such an extent that they become volatile enough. However this is usually not a one-step procedure because of the presence of several functional groups on the same molecule. In an initial derivatization procedure, the guanidino moiety is treated with acetyl acetone so as to obtain the corresponding 2,6-dimethylpyrimidine ring system. Other functionalities such as carboxyl, hydroxy, and amino, are then protected either by esterification, trimethylsilylation, or trifluoroacetylation. This procedure is not always a "clean" one as illustrated in Figure 1, showing the GC-MS of pure 2oxo-5-guanidinovaleric acid (XVIII), after convertion into the pyrimidyl ring system and subsequent esterification with 1-butanol/HCl. Taking these considerations into account, the characteristics of D/CI-MS as a soft ionization technique were evaluated for a set of underivatized guanidino compounds. It has already been shown that ionization techniques such as laser desorption mass spectrometry (14,15), ion evaporation 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
Table I. Percent Relative Intensities of Ions Detected in the D/CI-MS of a Few Guanidino Compounds with Different Reagent Gases CH, [MHI'-
3"
compound VI1 XI1 XIV
[Mt HI+ 100 87
xv
a
[MHP-
HZO
[MH]'2H,O
[Mt HI'
71
11
100 100 100
23
H,O
[MHl+2H,O
14 52
100 100
12
24
100 100 39 100 100
100 13
100 100
looa XIX Corresponds to [ MH]' - NH, signal.
[Mt HI'
i-C,H, [MHl'H,O
[MH]+2H,O
100
4
Table 11. D/CI-MS (NH,) for Biologically Important Guanidino Compoundsa compounds (R = NH, no.
I I1 I11 IV V VI VI1 VI11 IX X XI XI1 XI11 XIV
xv
XVI XVII XVIII XIX
xx
XXI a
fi-' NH
RNHCH,CH( NH, )COOH RNH(CH, ),CH( NH, )COOH RNHO(CH,),CH(NH,)COOH RNH( CH,),CH( NH,)COOH RNH( CH,),CH( NH,)COOH RNHCH,CH( CH,)COOH RNH(CH,),COOH RNH(CH,),COOH RNH( CH,),COOH RNH(CH,),COOH RNHCH,COOH RN( CH,)CH,COOH RNHCH( C,H,)COOH RNHCH( CH,CH,COOH)COOH RNHCH(CH,COOH)COOH RNHCH,CH( OH)CH,COOH RNH(CH,),CH( 0H)COOH RNH( CH,),COCOOH RNH(CH,),CONH, RNH(CH,),SO,H RNHCH(CH,Phe)COOH
[MHI+
[MHl+- HZO
[MHI'-
2H,O
129 (100)
130 (18) 172 (13)
189 (100) 177 (100)
175 (20) 132 (100) 174 (100) 160 (100) 146 ( 2 ) 118 (100) 132 (87)
162 (19) 176 (6)
143 (8) 157 (22) 128 (100) 114 (71) 156 (5) 142 (47) 128 (100) 100 (85) 114 (100) 128 (100) 172 (100) 158 (100) 144 (12)
[MH]+- NH,
154 (12) 126 (100) 140 (35)
156 (100) 128 (100) 168 (19) 208 (100)
190 (66)
Relative intensities are given in parentheses.
ljm'z=200 z R
~
TIC1
2Hz0, and/or [MH]+ - NHB. These results are not quite unexpected and are within the scope of the gas phase acidbase properties of the respective reagent gases. The effect of the heating rate (21) upon the intensity of the protonated molecular ion species has been evaluated for compounds I, V, VI, and XV. Mass spectra were taken adjusting the heating rate of the emitter to a value of 10 m A d , 15 dd,and 20 d&, respectively. With these conditions, compounds I, VI, and XV showed no protonated molecular ion. For arginine (V), the relative intensity of m/z 175 remained almost constant with a value of 20% at 10 dd,23% at 15 m A d , and 22% at 20 mAas-l. Taking these experimental results into consideration, we decided to record the D/CI-MS of 21 biological important guanidino compounds with ammonia as reagent gas (Table 11). Thirteen of them showed an [MH]+ ion with a relative intensity varying from 100 to 2% at a best emitter temperature of approximately 300 d.In eight spectra, [MH]+was absent. In the mass spectra of the homologous series of the amino acids, a-aminoguanidinopropionic acid (I), a-aminoguanidinobutyric acid (IV), arginine (V), and homoarginine (11),molecular weight information was found only for the last two components. In the series of the acids (VI to XV), practically all compounds were characterized by the presence of [MH]+,except for the branched compounds VI and XI11 and compounds XIV and XV, bearing an additional carboxylic acid function. The absence of [MH]+ in 2-oxo-5-guanidinovalericacid (XVIII) is not surprising, since it exists predominantly as
1 100
200
300 scans
Figure 1. GC/MS analysis of a-keto-6-guanidinovaleric acid (XVI I I) after derhretlzatlon Into the 2,6dimethylpyrimldylb1Ayiester (SE 3 0 , 5 % , 110-300 OC at 15 OC/mln).
mass spectrometry (16,17),D/CI (desorption/chemical ionization) mass spectrometry, and liquid chromatography/mass spectrometry (18-20)were capable handling a difficult molecule like arginine but, to our knowledge, no systematic evaluation of D/CI-MS toward a series of these compounds has been published. In order to evaluate the effect of different reagent gases upon the behavior of these guanidino compounds, the chemical ionization desorption mass spectra were recorded by use of either methane, isobutane, or ammonia for a few test components. From the results, summarized in Table I, it can be seen that ammonia should be preferred since, then, the most intense [MH]+ ions are then produced. Replacing ammonia by isobutane or methane results in a decreasing intensity of [MH]+ in favor of an increasing intensity of fragmentations such as [MH]+ - HzO, [MH]+ -
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
171
i60
110
180
200
220
m/z
Flgure 2. D/CI-MS of N-a-acetylarginine isolated from urine. 57
1
i" ,
,
;
L
200
220
,I
pyrrolidine-l-amidino-2-hydroxy-2-carboxylic acid ( I I),which can easily dehydrate, resulting in the formation of a double bond conjugated with the carboxylic acid function
In order to investigate the practical application of D/CI-MS, a guanidino positive compound was isolated from the urine of patients with hyperargininemia by liquid chromatography on a Dowex 50-X-8 resin using a 0.04 M pyridine/formic acid (p'H 4)solution as eluent. The solvent was removed by lyophilization and extra purification was done by semipreparative HPLC on a LiChrosorb 10-NH2 column with a 0.01 M NH,OOCH (A)/CH,CN (B) eluent (78% B 50% B in 10 min). About 10 WLof the collected solution was then directly brought upon the tungsten filament of the D/CI probe and, after evaporation of the solvent, the D/CI (NHJ spectrum was recorded (Figure 2). Together with the protonated molecular ions at m/z 217 (lo),fragmentations were observed at m/z 199 (100) [ m ] + - HzO, m/z 174 (25) [MH]' - CH3C0, and m/z 157 (74)[MH]+- HzO - CHz===C!=O. By comparison of this spectrum with the reference mass spectrum (Figure 3), the isolated guanidino compound was identified as N-aacetylarginine (XXII). The observed differences in relative intensities of the characteristic ions in both spectra are not so unusual in this technique and are highly dependent upon the temperature of the D/CI filament during the desorption process. Slight temperature differences, between two recordings, can explain a change of relative intensities in the spectra.
-
C0NCLU SIO N Identification of guanidino compounds by D/CI-MS can be done if comparison with available reference spectra is possible. This is necessary since molecular weight assignment by using the highest m / z value is not straightforward. Individual peak identification in an off-line liquid chromatography to D/CI-MS experiment is possible, although time consuming, especially in the analysis of complex mixtures. An improvement could be the future development of a convenient on-line LC/MS system, especially since naturally occurring guanidino compounds are polar components, lacking any significant UV absorbance at 254 nm. ACKNOWLEDGMENT We thank the N.F.W.O. We also thank Y. Luyten, J. Schrooten, and W. Van Dongen for technical assistance. A. Mori (Japan) and D. J. Durzan (Canada) are gratefully acknowledged for providing some of the compounds. Registry No. I, 2462-51-3;11, 156-86-5;111, 543-38-4;IV, 2978-24-7;V, 74-79-3; VI, 1115-83-9; VII, 353-09-3; VIII, 6659-35-4; IX, 462-93-1;X, 463-00-3;XI, 352-97-6;XII, 57-00-1;XIII, 3164-99-6;XIV, 73477-53-9; XV, 6133-30-8;XVI, 7010-89-1; XVII, 157-07-3; XVIII, 3715-10-4; XIX, 4210-97-3; XX, 543-18-0;XXI, 88728-27-2; XXII, 155-84-0;ammonia, 7664-41-7; isobutane, 7528-5;methane, 74-82-8.
m/z
Flgure 3. DICI-MS of N-a-acetylarginine standard.
695
LITERATURE CITED (1) Terheggen, H. 0.; Schwenk, A.; Lowenthal, A,; Van Sande, M.; Colombo, J. P. Z . Klnderheilkd. 1970, 107, 313. (2) Terheggen, H. G.; Lowenthal, A,; Lavinka. F.; Colombo, J. P. Arch. Dis. Chikl. 1975, 50, 57. (3) Marescau, B.; Pintens, J.; Lowenthal, A,; Terheggen, H. G.; Adriaenssens, K. J. Clln. Chem. Clln. Biochem. 1979, 77, 211. (4) Terheggen, H. G.; Lavinka, F.; Colombo, J. P.; Van Sande, M.; Lowenthal, A. J . Genet. Hum. 1972, 2 0 , 69. (5) k r e s c a u , 6.;areshi, I. A.; De Deyn, P.; LeSarte, J.; Lowenthal, A. Proceedings of the International Symposium on Guanidino Compounds, Tokyo 5-7 Sept 1983, in press. (6) Miruno, A.; Mukawa, J.; Kabayashl, K.; Mori, A. IRCS Med. Sci.: Llbr. Compend. 1975, 3 , 385. (7) Jlnnai, 0.; Mori, A.; Mukawa, J.; Ohkusu, M.; Hosotani, M.; Mizuno, A.; Tye, L. C. Jpn. J. Brain Physlol. 1969, 160, 3688. (8) Jinnal, 0.; Samai, A.; Morl, A. Nature (London) 1968, 212, 617. (9) Okusu, H. Osaka-Igakkal-Zasshl 1970, 2 1 , 49. ( I O ) Marescau, 6.; Pintens, J.; Lowenthal, A,; Esmans, E.; Luyten, Y.; Lemigre, G.; Domrnisse, R.; Alderweireldt, F.; Terheggen, H. G. J. Clin. Chem. Clh. Blochem. 1981, 19, 61. (11) Marescau, B.; Lowenthal, A.; Esmans, E.; Luyten, Y.; Alderweireldt, F.; Terheggen, H. G. J . Chromatogr. 1981, 224, 185. (12) Schiifte, E. Z . Physiol. Chem. 1943, 279, 52. (13) Vetter, W. "Biochemical Applications of Mass Spectrometry"; Waller, G. R., Ed.; Wlley-Intsrsclence: New York, 1972; Chapter 14. (14) Posthumus, M. A.; Kistemaker, P. G.; Meuzelaar, H. L. C. Anal. Chem. 1978, 50. 985. (15) Hardln, E. D.; Vestal, M. L. presented at the 29th Annual Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, 1981; Abstracts p 92. (16) Thomson, B. A.; Iribarne, J. V., presented at the 30th Annual Conference on Mass Spectrometry and Allied Topics, Honolulu, HI, 1982; Abstracts p 599. (17) Irlbarne, J. V.; Dzledzlc, P. J.; Thomson, B. A., presented at the 29th Annual Conference on Mass Spectrometry and Allied Topics, Mlnneapoils, 1981; Abstracts p 519. (18) Devant, 0.; Beaugrand, C. Adv. Mess Spectrom. 1980, 8 , 1806. (19) Hunt, D. F.; Shabanowitz, J.; Bok, F. K. Anal. Chem. 1977, 49, 1160. (20) Blakley, C. R.;Carmody, J. J.; Vestal, M. L. Anal. Chem. 1980, 5 2 , 1636. (21) Cotter, R. J. Anal. Chem. 1980, 5 2 , 1589A, and references cited therein.
RECEIVED for review October 17,1983. Accepted December 30, 1983.
