"ANALYTI(~AL BIOCHEMISTRY 71, 597--600 (1976)

Methane- and n-butaneboronates: New Derivatives for Gas-Chromatographic Analysis of 3-Methoxy4- Hyd roxyphe nylethyleng lycol Since it has been shown that 3-methoxy-4-hydroxy-phenylethylenglycol (MHPG) is the major metabolite of brain norepinephrine in various animal species (1), a method of determination of this substance in biological fluids is of evident importance. Gas-chromatographic methods have improved the analysis in these respects (2,3), and recently the mass-fragmentographic technique, which represents the latest advance in the analysis of catecholamine metabolites, has been shown to have the highest specificity and sensitivity (4). However, the procedures so far described suitable to gas chromatography for obtaining volatile derivatives of MH PG did not employ very specific reagents. In fact, silylating (e.g., 5) or acylating (e.g., 6) agents are single protecting group donors. Thus, they do not exploit the distinctive moieties of polyfunctional natural compounds which could allow the use of more specific reagents involving proximal groups. The presence of the 1,2 diol group in the molecule of M H P G permits the use of boronic acids, RB(OH)2, introduced by Brooks and Watson for gas-chromatographic analysis (7) and already employed on similar compounds (8-10). Therefore, for the determination of urinary M H P G we used cyclic boronates by reaction with methaneboronic, MeB(OH)2, and n-butaneboronic, BuB(OH)2, acids. A preliminary study was run with standard solutions of MHPG-Hemipiperazinsalz (E GA, Chemie) in methanol. Aliquots containing 5, 10, 20, 30, and 40/xg of the pure compound were evaporated under a stream of nitrogen and 0.3 ml of 2,2-dimethoxypropane (DMP) containing 300/xg of MeB(OH)2 (Applied Science Lab.) or BuB(OH)2 (Serva Feinbiochemica), and 15 /xg of phenanthrene, as internal standard, were then added to the residues. The samples were allowed to stand 15 min at room temperature and then 1/xl of each reaction mixture was injected into the gas chromatograph together with 0.2 /xl of N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA). The apparatus was a Fractovap Model GV (Carlo Erba) equipped with flame ionization detector. The U-shaped glass column (2 m x 2.5 ram) was filled with 3% OV-101 on Gas Chrom Q (100-120 mesh; Applied Science Lab.). The flow rates of nitrogen, hydrogen, and air were 33, 35, and 400 ml/min, respectively. Injector and detector temperatures were 200 and 240°C; the column temperature was programmed from 130 to 230°C at 15°C/rain and kept at 230°C for 3 min, 597 Copyright © 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.

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Mass spectra were recorded on an LKB-9000 instrument: ionizing voltage was 70 eV, source temperature 290°C and accelerating voltage 3.5 kV. The solvents tested in the treatment with boronic acids were N,Ndimethylformamide, pyridine, acetone, and D MP: the last proved to be the most efficient as it prevents the hydrolysis of boronates owing to moisture. In fact, DMP can react with water yielding acetone and methanol. By the ratio of peak areas it has been shown that the reaction was complete within 10-15 rain and it was independent of the temperature between 25°C and 100°C. Both derivatives showed considerable stability: in fact, the peak areas of the boronates stored 2 days at room temperature did not decrease. MHPG boronates bearing a free phenolic group showed tailed peaks unsuitable for gas-chromatographic analysis of urine samples; in order to obtain sharp peaks we had to protect the phenolic group, and for this purpose we carried out the silylation using BSTFA. At first we added BSTFA in the reaction mixture, but after 1 hr the MHPG-boronate peaks began to decrease and the MHPG-tri-TMS peak appeared, ThereI 1004

MHPG-MeB-TMS CH2-O~` IH_o/B-CH3

~oc~

50

250

?

CH3-Si-CH 3 CH3

w u z < c} z m
.J w 100 cc

MHP6-BuB-TMS CH2~O-., H_O/B-(CH2)3-CH3

~ O C H

50 ¸

0 I CH3--Si-CH 3 CH3

3

d

292

I

loo

307 d,

2oo m/e

FIG. 1. Partial mass spectra of M H P G - M e B - T M S

and M H P G - B u B - T M S .

322 h

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8O

6O

u~ 40

b

20

6

8

i 2

4

6

8

Minutes

FIG. 2. Typical gas chromatograms of urinary extracts subjected to the complete procedure using MeB(OH)2 (A) or BuB(OH)2 (B); l/zl containing 0.05/zg of I.S., phenanthrene, of each sample was injected.

fore, the presence of BSTFA and its by-products increases the solvolysis of the boronates. Then, in order to store the reaction mixture for a longer time before analysis, a direct silylation at the injection step was found to be advisable. The calibration curves obtained with standard aliquots between 5 and 40 /xg treated by this procedure were linear and equal to those for the boronates silylated before injection. The relative weight response calculated from the peak area ratios relative to phenanthrene are 0.73 for M H P G - M e B - T M S and 0.63 for MHPG-BuB-TMS. The mass spectra of the two derivatives showed parent peaks higher (15%) than that of MHPG-tri-TMS ( = l ) reported elsewhere (l 1). The typical fragments arose from the loss of - C H 3 (M-15) and CH20 (M-30) (Fig. 1). The stability of molecular ions ofboronates was noted in the work of Brooks and Watson (7). With regard to biological samples, the different retention times relative to phenanthrene of M H P G - M e B - T M S (0.89) and M H P G - B u B - T M S (1.35) permit the choice of the most suitable derivative to gas-chromatographic analysis in order to obtain well separated peaks. For instance, in our experiments on urine we found that the peaks ofn-butylic derivatives were better separated from each other than the methylic ones (Fig. 2). The urine samples examined were hydrolyzed, extracted, and purified

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in lipophilic Sephadex LH-20 according to the method previously reported (11). The fractions collected from chromatographic column were treated with BuB(OH)2 by the procedure described for standard aliquots. From five different 24 hr urine pools of normal human subjects the obtained output of M H P G was in the range 0.98-1.45 mg/24 hr. In conclusion, we can state that in order to obtain volatile derivatives available for gas-chromatographic analysis of M H P G in urine, the procedure employing boronic acids is rapid and specific; our results are preliminary data from which an effective analytical method might be developed. The sensitivity could be increased either by developing gas chromatography with an electron capture detector on alogenated derivatives or by applying the mass fragmentography to the boronates. ACKNOWLEDGMENT The authors are indebted to Dr. Vincenzo Zovei, Istituto di Richerche Farmacologiche "Mario Negri", for assistance with mass spectrometry.

REFERENCES 1. Maas, J. W., Dekirmenjian, H., and Fawcett, J. A. (1974) Int. Pharmacopsychiat. 9, 14-26. 2. O'Keeffe, R. and Brooksbank, B. W. L. (1973) Clin. Chem. 49, 1031-1035. 3. Dekirmenjian, H. and Maas, J. W. (1974) Clin. Chim. Acta 52, 203-210. 4, Sjoquist, B., Linstrom, B., Angaard, E. (1975)J. Chromatogr. 105, 309-316. 5,-Karoum, F., Anah, C. O., Ruthven, C. R. J. and Sandier, M. (1969) Clin. Chim. Acta 24, 341-348. 6, Fellows, L., Riederer, P., and Sandier M. (1975) Clin. Chim. Acta 59, 255-257. 7, Brooks, C. J. W. and Watson, J. (1967) Chem. Comm., 952-953. 8, Anthony, G. M., Brooks, C. J. W., Maclean, I. and Sangster, I. (1969)J. Chromatogr. Sci. 7, 623-631. 9. Brooks, C. J. W., and Maclean, I. (1971)J. Chromatogr. Sci. 9, 18-24. 10. Pace-Asciak, C., Wolfe, L. S. (1971) J. Chromatogr. 56, 129-133. 11. Cagnasso, M. and Biondi, P. A. (1974) Ital. J. Biochem. 5, 345-355. M . CAGNASSO

P. A. BIONDI lnstituto di Fisiologia Generale e Speciale degli Animali Dornestici e Chimica Biologica Via Celoria 10 20133 Milano Italy Received July 29, 1975; accepted November 20, 1975