(7) D. Hoffman, W. E. Bondinell, and E. L. Wynder, Science, 183, 215 (1974). ( 8 ) M. Novotny, M. L. Lee, and K. D. Bartle, J. Chromatogr. Sci., 12, 606 (1974). (9) K. D. Bartle, M. L. Lee, and M. Novotny. lnt. J. Environ. Anal. Chem.. 3.349 (1974). (IO M. L. Lee, K. D. Bartle, and M. Novotny, Anal. Chem., 47, 540 (1975). (11) G. Grimmer and H. Bohke, Fresenius' 2.Ana/. Chem,, 261,310 (1972). (12) T. Doran and N. G. McTaggart, J. Chromatogr. Sci., 12, 715 (1974).
(13) M. L. Lee, M. Novotny and K. D.Bartle, Anal. Chem., 48, 405 (1976). (14) M. Novotny and R. Farlow, J. Chromatogr., 103, 1 (1975). (15) R. A. Hites and K. Biemann, Anal. Chem., 43, 681 (1971).
RECEIVEDfor review January 30,1976. Accepted June 3,1976. This work was supported by Grant No. MPS 75-04932 from the National Science Foundation.
Qualitative and Quantitative Analyses of Bansyl Derivatives of Dopamine and Some of Its Metabolites in Urine Samples by Electron Impact and Field Desorption Mass Spectrometry W. D. Lehmann," H. D. Beckey, and H.-R. Schulten Institute of Physical Chemistry, University of Bonn, 5300 BONN, Wegelerstr. 12, West Germany
The derivatives of dopamlne, 3-O-methyldopamlne, and 40-methyldopamine produced by reactlon wlth 5-di-n-butylamlnonaphthalene-1-sulfonylchloride (BANS-CI) were separated by thln-layer chromatography and identifiedby electron impact and field desorption mass spectrometry. The base peaks In the electron impact spectra were formed by loss of C3H7 from the molecular ion. In contrast, the field desorption spectra showed only the molecular Ions. The BANS derlvatlves were isolated from urine samples by thln-layer chromatography. Two novel mass spectrometric methods were used for their quantltation: 1) A twln dlrect Introduction system for calibration in the electron impact mode (external standard) and 2) stable Isotope dilution in connectionwlth electron Impact and field desorption mass spectrometry (Internal standard). The advantages and drawbacks of both technlques are descrlbed and thelr utlllty for physiologicaland pharmacokinetic studies is discussed.
Although there are a great number of biologically active amines (biogenic amines), in recent years analytical interest has been focused on a relatively small number of these compounds, in particular, on those amines which act as inhibitors or transmitters of signals in the nervous system. These include small aliphatic amines (respectively, quaternary ammonium bases) such as acetylcholine, and phenolic and catecholic amines such as tyramine and dopamine. In general, two procedures are used for the identification and quantitation of aromatic amines in body fluids or tissue: 1)Extraction and separation of the amines by chromatography and formation of highly fluorescent derivatives which are quantitated by fluorometry (1, 2). 2) Relatively volatile derivatives of the amines are produced which are subsequently estimated by a coupled gas chromatograph-mass spectrometer unit (3, 4 ) . The sensitivity of both methods is sufficignt for determination of biogenic amines in naturally occurring concentrations (Le., in the range 1 to 100 ng/ml). Possible sources of error are: for method 1, the relatively unspecific identification by thin-layer chromatography (TLC) and fluorometry; for method 2 ions with m / e values considerably below the molecular weight must conventionally be used for the identification of these derivatives, since these compounds undergo strong fragmentation under electron impact. In quantitative determination, problems may arise from background and column bleeding. 1572
In this paper, a new method for qualitative and quantitative analysis of dopamine (DA), 3-0-methyldopamine (3-MDA), and 4-0-methyldopamine (4-MDA) is introduced. Defined, fluorescent derivatives of high molecular weight were generated by reaction with 5-di-n-butylaminonaphthalene-1-sulfonyl chloride (BANS-Cl) ( 5 , 6 ) .The BANS derivatives were isolated by TLC followed by identification and quantitation by two independent methods: electron impact (EI) and field desorption (FD) mass spectrometry (MS) (7).
EXPERIMENTAL Dopamine and 4-0-methyldopamine as hydrochlorides were purchased from Aldrich Chemical Co., Milwaukee; 3-0-methyldopamine hydrochloride was obtained from Regis Chemical Co., Ill. Dopamine-(a-d&d*) hydrochloride was obtained from Merck, Sharp, and Dohme, Canada. All compounds were of analytical grade. BANS-Cl was kindly supplied by N. Seiler, Max-Planck-Institut fur Hirnforschung, Frankfurt, W.-Germany. The reaction of the amines with BANS-CI was carried out according to a procedure described by Seiler et al. for 5-dimethylaminonaphthalene-1-sulfonyl chloride (DANS-Cl) (8). An acidified aqueous solution (sulfuric acid, pH 1) of the amine (about 10 pg/ml) was mixed with 6 ml of a solution of BANS-C1 in acetone (about 1mg/ml) and saturated with sodium carbonate. After 3 h at room temperature, the reaction mixture was decanted from the salt, which was washed once with 1ml of acetone. Saturating of the reaction mixture with potassium dihydrogen orthophosphate was followed by the evaporation of acetone under a stream of nitrogen. The residual aqueous phase was mixed with 3 ml of methanol and extracted with 2 ml of n-heptane or toluene. The extract was concentrated to dryness under a stream of nitrogen and the residue redissolved in 100 pl of ethyl acetate. Aliquots of this solution were submitted to TLC. Thin-layer chromatography was carried out on glass plates 20 X 20 cm coated with 300 Mm Silica gel G layers (Merck AG, Darmstadt) using ascending chromatography in a solvent vapor-saturated atmosphere. The thin-layer plates were developed using cyclohexane/ ethyl acetate (4:l) as eluent. Fluorescence excitation a t 364 nm was used for visualization of the BANS derivatives. The fluorescent spots were scraped off and extracted with 500 p1 of ethyl acetate. After appropriate concentration under a stream of nitrogen, the extract was transferred into the microcrucible of the direct introduction system of the E1 mass spectrometer. Alternatively, for FD analysis, the extract was applied to a high temperature activated wire emitter by the syringe technique (7). The E1 mass spectra were recorded on a modified CH-4 mass spectrometer under standard conditions (70 eV electron energy, 20 FA emission, 150 "C ion source temperature). In order to obtain the complete E1 mass spectrum of the tris-BANS-DA derivative (mol wt 1104), the acceleration voltage was lowered to 2700 V. As shown in Figure 1, the ion source of the E1 mass spectrometer was equipped
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
ion source 1
DANS-
microoven
BANS- d e r i v Q t i v e
derivative
Figure 1. Twin direct introductionsystem used for quantitative measurements with ex4ernal standard in the electron impact mode
Figure 2. Fragmentation processes leading to the base peaks in the El mass spectra of DANS and BANS derivatives
with a twin direct introduction system. Both sliding rods are fitted in adjustable mounts. Thus, by proper positioning, approximately equal sensitivity for both introduction systems can be achieved. This instrumental modificatiion enabled quantitative measurements with external standards to be made. The FD spectra were run on a modified double-focusing CEC21llOB mass spectrometer. Field anodes employed were 10 pm tungsten wires activated a t high temperature (9). The average length of the microneedles was about 30 pm. The ions were recorded on vacuum evaporated AgBr photoplates (Ionomet, Waban, Mass.). All spectra were produced by emission-controlled FD-MS (IO, I I ) at a threshold of 1 X 10+ A.
atives, the base peak is formed by a cleavage of C3H7from the molecular ion (5). In addition, the relative intensities of the molecular ions for DANS derivatives are in the order of a few percent only (preferably when multiple substitution takes place), the BANS derivatives, however, show molecular ions of 30 to 40% relative intensity on the average. In Figure 2, the significant fragmentation in the E1 mass spectra of DANS and BANS derivatives of a primary amine is illustrated. Clearly, the base peaks of the BANS derivatives contain the information about the biogenic amine, whereas this information is lost in the typical fragmentation of the DANS derivative. However, it has been reported that the derivatives of the reagents BANS-Cl and DANS-C1 are formed with similar reaction velocities and show approximately the same fluorescence values (5).Previously Durden et al. ( 1 5 ) described the sensitivity of the mass spectrometric determination of some noncatecholic biogenic amines as their dansyl derivatives and found a linear response over the range 10-9 to mol. Dopamine. The result of the reaction of DA with BANS-C1 is a mixture of four different derivatives: one monobansyl-DA, two constitutionally isomeric bisbansyl-DA derivatives, and one trisbansyl-DA. Because of their differences in polarity, the reaction products are easily separated by TLC and are clearly located by their strong UV fluorescence. Under the conditions selected, the tris-BANS-DA represents the main product. As shown in Figure 3, the E1 spectra of all four de-
RESULTS AND DISCUSSION It is well known that DANS-Cl reacts very easily with molecules possessing primary or secondary amino groups or phenolic hydroxyl functions. The reaction with aliphatic hydroxyl functions proceeds at a considerably slower rate. The DANS-Cl reagent was originally introduced by Gray and Hartley (12) in order to achieve an improvement in the determination of N-terminal groups in peptides, which had been performed mainly using 2,4-dinitrofluorobenzene. BANS-C1 is a modified DANS-C1 containing n-butyl groups in place of the methyl substituents on the amino group. If the derivatives formed by these reagents are identified by E1 mass spectrometry, the use of BANS-C1 offers an essential advantage: In the mass spectra of the DANS derivatives, the base peak is a nonspecific fragment ion at m/e 170 (13,14).In contrast, in the mass spectra of the BANS deriv-
thin-layerseparation
N (C,H,),
BANS:
@
/H ‘BANS
o.s.0 MW 1104
0 \BANS
i 1104
0 0 100 ‘io
1
MW 787
I
707
I
0
I
A70
&i
CL-CHe-N
R -006
4-
mono-BANS-DA MW 470
@
‘
/H \BANS
OH
OH
start
Figure 3. Thin-layer separation of the four derivatives that are formed by reaction of BANS-CI with DA and partial El and FD mass spectra of the separated reaction products. The mass range from m/e 300 to 1200 was recorded electrically with El-MS. For the photoplate detection of the FD spectra, the mass ranige from m/e 40 to 1400 was registered ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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thin-layerseparation
I
BANS dbis-BANS-L-MDA R,=0,3L ybis-BANS-3-MOA
0-BANS OC ,H3
I
CHfCH,-N
/H
'BANS
@O-CH3 @ ,' ANS
/H
P '\CHB +--start
Figure 4. Thin-layer separation of the derivatives that are formed by reaction of BANS-CI with a mixture of 3-MDA and 4-MDA and partial El and FD mass spectra of the separated reaction products. Experimental conditions as described in Figure 3
rivatives yield the [M - 43]+ ion as the base peak. The molecular ion is recorded with approximately 40% relative intensity. In contrast, the FD spectra exhibit only the molecular ion groups. When the thin-layer chromatograms of 3-MDA and 4-MDA (see Figure 4) are compared with the bisbansy1-DA derivatives in Figure 3, it is suggested that the spot with the higher R f value (0.30) is bis(3-0, N)-bansyl-DA and the spot with Rf = 0.24 is bis(4-O,N)-bansyl-DA. The bisbansyland trisbansyl-derivatives have been isolated from human urine samples which were spiked with DA at a level of 10 pg/ml. This value is in the order of magnitude of the concentration of unconjugated DA which is excreted in the urine of patients with Parkinsons disease undergoing L-Dopa therapy (16).
3-0-Methyldopamine a n d 4- 0-Methyldopamine. The two isomeric methyl ethers of DA, 3-MDA and 4-MDA are important products in the human metabolic pathway for LDopa (17). The methylation of DA is accomplished by different enzymes (catecholamine-0-methyltransferases) which attack DA preponderately a t the 3 - 0 - or 4-0-position (18). A method for the separation and identification of 3-MDA and 4-MDA as their isothiocyanate/trimethylsilyl derivatives by GC-MS has been described (19). The reaction of 3-MDA or 4-MDA with BANS-C1 produces a mixture of two different derivatives: one monobansyl and one bisbansyl derivative. The bisbansyl derivatives of 3-MDA and 4-MDA can be separated on TLC nearly completely ( R f = 0.31,0.34, respectively), whereas their monobansyl derivatives exhibit identical R f values (0.17). The derivatization was performed with a large excess of BANS-C1 and yields the bisbansyl derivatives as the main products. The E1 spectra of the four derivatives (Figure 4) display the [M - 43]+ ion as the base peak and the molecular ions with relative abundances between 30 and 47%. The higher stability of the molecular ions upon electron impact is always observed for the derivatives of 3-MDA. Using FD-MS, only the molecular ions and no fragment ions are observed. The occurrence of [MI2+ions of minor intensity is useful for the correct assignment of the molecular weight (20). After reaction with BANS-Cl, the bisbansyl derivatives of 3-MDA and 4-MDA have been iso1574
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
-c m
direct iniet(2)
2
direct inlet(1)
2'
2 -2 r
m
e m .
OI
c r n l e 758
___c
E
x
-
I
-c 'A E
bis-8ANS-3-M
I
100
ris-BANS-DA-do tris-BANS-DA-d&
0
+
108
1100
I io5 mle
L 1110
Figure 6. Field desorption mass spectrum of trisbansyl-DA and trisbansyl-DAd4 (molar ratio 1:0.95). Total amount about 200 ng; emission controlled desorption, threshold 1 X A, exposure time of the photoplate 19 min
sensitivity for both introduction systems was obtained by adjusting the sliding rods (see Figure 1).The system was calibrated by determining the relative sensitivity coefficient [sensitivity direct inlet (l)/sensitivity direct inlet (2)] in 20 measurements to be 1.17 f 0.10. For the estimation of DA, 3-MDA, and 4-MDA, the samples were split into two halves and to one of them a known amount of the compound to be quantitated was added as external standard. Both parts of the sample, the authentic sample (1)and the one to which the standard was added (2) were subjected to the same workup procedure. The isolated BANS derivatives of sample (1)and (2) werie transferred into the micro-ovens of direct inlet system (1)and (2). After mounting the sliding rods into the previously adjusted position, the samples were evaporated consecutively. During evaporation, the mass spectrometer is set on a fixed m/e value, e.g., the [M - 43]+ ion of a BANS derivative (single ion monitoring). The evaporation profiles of bisbansyl-3-MDA, selected ion m/e 758 (corresponding to [M -- 43]+), for a quantitation are shown in Figure 5 . Since the relative sensitivity coefficient of direct inlet (1)and (2) is known, the amount of 3-MDA present in the sample can be calculated in the following way.
__
peak area (1).-sample amount (1) X 1.17 (1). peak area (2) sample amount (2) Because the ratio (peak area (l)/peak area (2)) is 0.66 (in Figure 5 ) , it follows that the ratio [sample amount (l)/sample amount (2)] equals 0.56. As the quantity of standard added to sample (2) is known, the unknown amount of 3-MDA present in sample (1)can be calculated. This method using an external standard yields quantitative results for concentrations between 0.1 pg and 10 pg/ml urine with an error of f10% on the average. The twin direct introduction system shortens the time conmmption of the measurements and minimizes possible errors caused, for example, by drifting of the magnetic field or thie focusing potentials. Stable Isotope Dilution. The method of stable isotope dilution in connection with mass fragmentography has been introduced as the method of choice for the quantitation of
drugs (21,22). For the estimation of DA in urine samples, we have used the trisbansyl derivatives of DA-do and DA-d4. In order to obtain reliable and efficient quantitative results, two independent methods have been used. Either the molecular ion group of the bansyl derivatives was scanned with the electric detection system in the E1 mode or high resolution FD-MS with photoplate detection was employed. With EI-MS at low resolution, the estimation of the trisbansyl derivative in the range as described above could be performed with an error of about f5%. Photographic detection of the molecular ion group of a mixture of trisbansyl-DA-do/dd in the ratio 1: 0.95 gave the spectrum displayed in Figure 6. At a resolution of 10 000 ((at half peak width for log (blackening)),the sample consumption was about 200 ng (-180 pmol). The theoretical molar ratio was reproduced within an error of 5%. This is consistent with a determination of cyclophosphamide-do/dc with FD-MS and photoplate detection reported previously (23). Since 3-MDA-d3 and 4-MDA-ds are easily generated according to a procedure of Karoum et al. ( 2 4 ) ,the determination of these metabolites by stable isotope dilution appears feasible.
ACKNOWLEDGMENT The authors are grateful to N. Seiler for his generous gift of BANS-C1. LITERATURE CITED (1) A. H. Anton and D. F. Sayre, J. Pharmacal. Exp. Tber., 145, 326 (1964). (2) S. H. Snyder and K. M. Taylor in "Research Methods in Neurochemistry", N. Marks and R. Rodnight, Ed., Plenum Press, New York, 1972, Voi. 1, pp 287-315. (3) S.H. Koslow, F. Cattabeni, and E. Costa, Science, 176, 177 (1972). (4) E. Anggard and G. Sedvall, Anal. Cbem., 41, 1250 (1969). (5) N. Seiler, T. Schmidt-Glenewinkei, and H. H. Schneider, J. Cbromatogr., 84, 95 (1973). (6) N. Seiler and H. H. Schneider, Biomed. Mass Spectrom., 1, 381 (1974). (7) H. D. Beckey and H A . Schulten, Angew. Cbem. lnt. Ed. Engl., 14, 403 (1975). (8) N. Seiler, in "Methods of Biochemical Analysis", D. Glick, Ed., Vol. 18, Interscience-Wiiey, New York, 1970. (9) H.-R. Schulten and H. D. Beckey, Org. Mass Spectrom., 6, 885 (1972). (10) H.-R. Schulten and U. Schurath, Atmos. Environ., 9, 1107 (1975). (11) H.-R. Schuiten and H. D. Beckey, Recent Advances in Field Desorption Mass Spectrometry, Twenty-Third Annual Conference on Mass Spectrometry and Allied Topics, Houston, Texas, May 25-30, 1975. Conf. Proceedings 6-1. (12) W. R. Gray and B. S. Hartley, Biocbem. J., 89, 59P (1963). (13) C. R. Creveiing, K. Kondo, and J. W. Daly, Clin. Cbem. (Winston-Salem, N.C.), 14, 302 (1968). (14) N. Seiler, H. Schneider, and K.-D. Sonnenberg, Fresenius' Z.Anal. Cbem., 252, 127 (1970). (15) D. A. Durden, B. A. Davis, and A. A. Boulton, Biomed. Mass Spectrom., 1, 83 (1974). (16) R. L. Bronaugh, R. J. McMurtry, M. M. Hoehn, and C. 0. Rutledge, Biocbem. Pbarmacol., 24, 1317 (1975). (17) N. Seiler, L. Demisch, and H. Schneider, Angew. Cbem. lnt. Ed. Engl., IO, 51 (1971). (18) C. R. Creveling, N. Morris, H. Shimizu, H. H. Ong, and J. Daly, Mol. Pbarmacol., 8, 398 (1972). (19) N. Narashimhacchari and P. Vouros, J. Cbromatogr., 70, 135 (1972). (20) H.-R. Schulten, in "Methods of Biochemical Analysis", D. Glick, Ed., Vol. 24, Interscience-Wiiey, New York, in press. (21) C. C. Sweeley, W. H. Elliot, I. Fries, and R. Ryhage, Anal. Cbem., 38, 1549 (1966). (22) C. G. Hammar, B. Holmstedt, and R. Ryhage, Anal. Biocbem., 25, 532 (1968). (23) H.-R. Schulten, Cancer Treat. Rep., 60, 501 (1976). (24) F. Karoum, J. C. Gillin, R. J. Wyatt, and E. Costa, Biomed. Mass Specfrom., 2, 183 (1975).
RECEIVEDfor review April 12,1976. Accepted June 4,1976. This work was supported by the Deutsche Forschungsgemeinschaft, Ministerium fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen and the Fonds der Deutschen Chemischen Industrie.
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