ANALYTICAL
BIOCHEMISTRY
187,234-239
(1990)
The Measurement of Dimethylamine, Trimethylamine, and Trimethylamine A/-Oxide Using Capillary Gas Chromatography-Mass Spectrometty Kerry-Ann
daCosta,*
J. James
Vrbanac,?
and Steven
*Nutrient Metabolism Laboratory, Departments of Pathology 85 East Newton Street, Room M1002, Boston, Massachusetts The Upjohn Company, Kalamazoo, Michigan 49001
Received
December
5,1989
We have developed a method for measuring dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO) in biological samples using gas chromatography with mass spectrometric detection. DMA, TMA, and TMAO were extracted from biological samples into acid after internal standards (labeled with stable isotopes) were added-p-Toluenesulfonyl chloride was used to form the tosylamide derivative of DMA. 2,2,2-Trichloroethyl chloroformate was used to form the carbamate derivative of TMA. TMAO was reduced with titanium(II1) chloride to form TMA, which was then analyzed. The derivatives were chromatographed using capillary gas chromatography and were detected and quantitated using electron ionization mass spectrometry (GC/MS). Derivative yield, reproducibility, linearity, and sensitivity of the assay are described. The amounts of DMA, TMA, and TMAO in blood, urine, liver, and kidney from rats and humans, as well as in muscle from fishes, were determined. We also report the use of this method in a pilot study characterizing dimethylamine appearance and disappearance from blood in five human subjects after ingesting [13C]dimethylamine (0.5 wmol/kg body wt). The method we describe was much more reproducible than existing gas chromatographic methods and it had equivalent sensitivity (detected 1 pmol). The derivatized amines were much more stable and less likely to be lost as gases when samples were stored. Because we used GC/MS, it was possible to use stable isotopic labels in studies of methylamine metabolism in humans. 0 1990 Academic Press, Inc.
Dimethylamine (DMA)l and trimethylamine (TMA) are important precursors of N-nitrosodimethylamine 1 Abbreviations used: DMA, dimethylamine; TMAO, trimethylamine N-oxide; FID, flame
234
H. Zeisel*
& Pediatrics, Boston University School of Medicine, 02118, and TDrug Metabolism Research,
TMA, trimethylamine; ionization detector.
(l-4), which is a potent carcinogen in a wide variety of animal species (5). These two aliphatic amines are found in human and rat urine, blood, and gastric fluid (1,6,7). DMA is minimally metabolized in mammals and is excreted intact in the urine (8). TMA is normally oxidized within liver and is excreted in the urine as trimethylamine N-oxide (TMAO) (9). The TMA content of seafoods is commonly used to assessfreshness, as TMA is formed by bacteria from TMAO in fish muscle and imparts a characteristic “fishy” odor (10-14). Various procedures exist for the analysis of these methylamines, including thin-layer chromatography (9,15), ion chromatography (16,17), calorimetric assays (18,19), gas chromatography (12,20-27), and high-performance liquid chromatography (13,28,29). These existing methods either lack sensitivity, specificity, or reproducibility. DMA and TMA are gases at room temperature and are easily lost during sample storage. In addition, these amines tend to adhere to many surfaces and many chromatographic procedures are subject to severe “tailing” and “ghosting” (6). We have developed a method for the measurement of DMA, TMA, and TMAO in tissues and biological fluids using capillary gas chromatography with mass spectrometric detection. This assay is preferable to existing methods because the derivatized amines are stable during storage, there is no ghosting or tailing, and internal standards labeled with stable isotopes can be used to correct for variations in recovery and for metabolic tracer studies. MATERIALS
AND
METHODS
Reagents. All reagents were obtained from Fisher Chemicals (Springfield, NJ) unless otherwise noted. Stock solutions of DMA and TMA (HCl salts, Sigma Chemical Co., St. Louis, MO), TMAO (Sigma), di0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHIC
W,),-N
MEASUREMENT
Cd-0-CH,
-Ccl,
2,2,2-trlchloro~thylchloroformate
trlmethylamlne
OF
METHYLAMINES
(CH,),-NH
cl-O-4
235
oCH3
dlmsthylamlne+chlo
rt de
electron
(CH&
-N-C-0-CH,
loniratlon
-CCl,
N,Kdtmathyl-2,2,2-trlchloroethylcarbsmate
electron Ionization
0+ (CH,),-N
-O-@H,
o+ N,N-dimethyl-ptOluen0
ii
(CH,),-N-C FIG. 1. Derivatization dimethyl-2,2,2-trichloroethyl
(m/z 72)
(m/z
sultonamlde
199)
of TMA and DMA. DMA and TMA were derivatized so as to form N,N-dimethyl-p-toluene carbamate. These were isolated using gas chromatography and fragmented using electron
methyl[‘HG]amine HCl (DMA-ds, 98 at.% D, Aldrich Chemical Co., Milwaukee, WI), dimethyl[13C,]amine HCl ([13C]DMA, 99.2 at.% 13C, MSD Isotopes, Montreal, Canada), trimethyl[2HG]amine HCl {TMA-ds, (CD,H),N; 99 at.% D, MSD Isotopes}, and TMAO-dG (prepared from TMA-d, with hydrogen peroxide (30)) were made up in 0.1 N HCl and stored at 4°C. These solutions were stable over several months. The derivatization agents, p-toluenesulfonyl chloride (2 M solution diluted in toluene) and 2,2,2-trichloroethyl chloroformate, were purchased from Aldrich Chemical. Methanolic alkali was prepared by dissolving 2.8 g KOH in 100 ml methanol/water (3/l, v/v). The reducing reagent was prepared with titanium(II1) chloride (Aldrich), 1 N HCl, and 10% formalin (l/1/2, v/v; must be prepared fresh under a nitrogen atmosphere). Experimental. Male Sprague-Dawley rats (300-400 g, Charles River Breeders, Wilmington, MA) were housed in individual metabolic cages in a controlled environment (24”C, 12 h of light from 6 AM to 6 PM). They were offered food (Ralston Purina rat chow No. 5001, Farmers Exchange, Framingham, MA) and water ad Zibiturn. Urine was collected, over a 24-h period, into acid (so that the final concentration was approximately 0.1 N HCl). The volumes were recorded, and aliquots were stored at -20°C. After urine collection was completed, the rats were anesthetized with diethyl ether, and blood was drawn by intracardiac puncture. Blood samples were collected into tubes containing heparin and kept on ice. Liver and kidney samples were collected by freezeclamping the specimen between tongs that had been precooled in liquid nitrogen. They were stored at -95°C until used.
sulfonamide and N,Nimpact ionization.
Control samples of urine (24-h collections) and blood were collected from humans eating a normal diet. Five normal humans (male, ages 24-36 years) were also given an oral dose of [13C]DMA (0.5 hmol/kg body wt), and urine was collected at timed intervals after the dose. Urine was collected and stored as described above for rat urine. Fresh fish was purchased locally and analyzed that day. A sample was also stored at -20°C for several months before analysis. Sample preparation and extraction of amines. Deuterated internal standards (DMA-ds, TMA-d,, TMAO$; as appropriate) were added to all samples to be analyzed. Before urine samples were assayed, they were filtered with a Sep Pak (Waters Associates, Milford, MA; 92-94% of DMA, TMA, or TMAO were recovered). Tissue samples were sonicated (setting 5, 50% pulse; Heat Systems Ultrasonics, Inc., Model W-225R, Plainview, NY) in ice-cold 0.9% NaCl (l/5, w/v). An aliquot of sonicate containing 100 mg tissue was added to 0.6 ml 0.1 N HCl and 3 ml chloroform to precipitate protein. Aliquots of whole blood (1 ml) were added to 0.6 ml 0.1 N HCl and 3 ml chloroform to precipitate protein. Fish (500 mg) was added to 5 ml 0.1 N HCl and mixed thoroughly (sonication was not required due to the soft consistency of fish). Samples were mixed and then subjected to centrifugation at 2000g for 20 min at 4°C. The supernatants were aspirated and used for analyses. Derivatization and analysis of DMA, TMA, and TMAO (see Fig. 1). For DMA analysis, N,N-dimethylp-toluene sulfonamide was formed (10). A portion of the acidified biological fluid or extract was placed into a glass test tube sealed with a Teflon-lined septum (WISP sep-
236
DACOSTA, Jon
72 amu
3.OES-
t
4 1.2ES-
0’
5
2.5E5-
l.OES-
2
2.0ES-
6.OE4 -
a
l.SES-
B.OE4-
1 .OES-
4.OE4-
5.OE4-
3.OES
2
2SE5
;
(CH,)
2-N-!
-12
2.OE41 3.1
Q)
VRBANAC,
II
1 4.5
I
I
60
80
100
120
111
i
I
140
160
I
180
I
I
IL 1
200
220
180 200
220
71’
(C’H,H),-N-C
2.OE5 5
n u
1.5E5 l.OE5 5.OE4
3.1
4.5
a Ion
199
60
amu
5
o
-
1.5E5-
100
120
140
160
4
1.8E5-
b
80
0+ (CH,),-N
9’\
-O-&H, 199
4.5E5-
1.2E5-
5 n a
#.OE4-
3.OE5-
b.OE4-
I\
3.OE4-
1.5E5,
I
5.2
6.7
I.
60
-
I
80
I-
IL
r-l
100
120
1
I
140
160
I
+
180
200
180
200
0' Q)
1.6E5
ti
1.5E5
g
1.2E5
5
0.OE4
9
&OE4 3.OE4
6.2
,lon
6.7
205
60
amu
80
100
120
140
160
d
1.6E5-
0’ (C%JiN-O-!+H,
9’1
8 5
1.5E5-
w
1.2E5-
5
9.OE4-
3.OE5-
s
6-oE4e 3.OE4
1.5E5-
205
4.5E5-
[\ I
I
5.2
6.7
Time
(min)
60
80
I
1
100
120
!A ,
140
.I I
160
I
I
180
IJ
200
Mass/Charge
FIG. 2. Mass spectra obtained from fragmentation of DMA and TMA. Methylamines were isolated and derivatized (TMAO and its deuterated standard were first reduced to TMA) as described in the text. They were separated by GC/MS using a silica-bonded capillary column and a temperature gradient program. The compounds were fragmented by electron ionization mass spectrometry.
AND
ZEISEL
turn, Waters Associates, Milford, MA). Toluene (1 ml for urine and fish, 0.5 ml for all other samples) and ptoluenesulfonyl chloride (38 mg for urine and fish; 3.8 mg for liver, kidney, and blood) were added. KOH (1 ml; 65% in water, w/v) was injected into the tube, bringing the pH above 9. Tubes were incubated at 90°C for 2.5 h, vigorously shaken, and then subjected to centrifugation at 1OOOg for 5 min. An aliquot (1~1) of the toluene layer was injected onto the gas chromatograph/mass spectrometer (GC/MS). In order to measure TMA, N,N-dimethyl-2,2,2-trichloroethyl carbamate was formed from DMA and TMA (31). A portion of the acidified biological fluid or extract was placed into a glass test tube sealed with a Teflonlined septum (Waters Associates). Toluene (1 ml for urine samples, 0.5 ml for all other samples) was then added. KOH (1 ml; 65% in water, w/v) was injected into the tube. Tubes were incubated at 90°C for 1 h, vigorously shaken, and then subjected to centrifugation at 1OOOg for 5 min at 4°C. An aliquot (0.5 ml) of the cooled toluene layer was added to a sealed vial containing 50 ~1 2,2,2-trichloroethyl chloroformate and 10 mg anhydrous sodium carbonate. Tubes were incubated at 110°C for 30 min. Methanolic alkali (1 ml) was added after cooling, and the reaction mixture was mixed. Water (2 ml) was then added, and the sample mixed and subjected to centrifugation at 1OOOg for 5 min at 4°C. An aliquot (1 ~1) of the toluene layer was injected onto the GC/MS. TMAO in the acidified samples was reduced to TMA (32). A portion of the acidified biological fluid or extract was placed into a glass test tube sealed with a Teflonlined septum (Waters Associates). An equal volume of reducing reagent was added under an atmosphere of nitrogen and the tube was sealed and incubated at room temperature for 1 h. The TMA formed was analyzed as described earlier. Gas chromatography. We used a Hewlett-Packard 5890/5970 GC/MS equipped with an automatic liquid sampler. This operates in the electron ionization mode. A fused silica capillary column (12.5 m X 0.2 mm i.d. dimethyl silicone bonded-phase HP-l; film thickness, 0.33 pm; Hewlett-Packard, Andover, MA) was used in these analyses. GC conditions were as follows: 0.75 min, splitless mode; He, carrier gas; temperature gradient, from 80 to 280°C at 25”C/min. Fragments formed by electron impact ionization were quantitated using selected ion monitoring. The molecular ions at m/z 199 (DMA), m/z 201 (formed from [13C]DMA, and m/z 205 (formed from DMA-de) and the fragments m/z 72 (formed from TMA), m/z 76 (formed from TMA-dG), and m/z 78 (formed from DMA-&) were monitored (see Figs. 1 and 2). Calculations. DMA concentrations were determined directly from analysis of the tosylamide derivatives. TMA concentrations were calculated using
CHROMATOGRAPHIC
MEASUREMENT
OF
237
METHYLAMINES TABLE
2
Concentrations of DMA, TMA, and TMAO Tissue
in Various
Tissues TMA
DMA
TMAO
nmol/g
Theoretical
Rat blood Rat liver Rat kidney Human blood
pmoles
of assays for DMA, TMA, and TMAO. Authentic FIG. 3. Linearity standards of DMA, TMA, and TMAO were derivatized and quantitated using GC/MS as described under Materials and Methods. Correlation coefficients were calculated using linear regression analysis and were equal to 0.9960 for all three compounds.
TMA
Rat urine Human urine
27 6
{ DM&,,
x
area(DMA)
* ml/nmol} 9
area(TMA)
* ml/nmd
where = area at mass 72 DMAtos = concentration (nmol/ml) of DMA in sample as determined using the toluenesulfonyl chloride method = area generated by 1 nmol/ml internal area(DMA) standard of isotopically labeled DMA that was converted to the carbamate derivative = area generated by 1 nmol/ml internal =alTMA) standard of isotopically labeled TMA that was converted to the carbamate derivative. arqcarbamate)
TMAO concentrations were calculated as the differences in TMA content after and before reduction. AND
body
DISCUSSION
The assays as described were able to detect as little as 1 pmol of each amine and remained linear up to 250 pmol TABLE
Shrimp Tuna (canned) Cod Cod (frozen)
10 84 97 381
wt/24
h 14 10
2 1
fimol/lOO -
92 633 0 21
40 437 531 40 rmol/kg
(nmol/ml) = are%rbamate)
RESULTS
4 136 161 4
wet wt
g wet wt 171 57 3400 1958
9 247 44 218
Note. DMA, TMA, and TMAO were measured as described Data are expressed as means of triplicate determinations.
in text.
(Fig. 3). Coefficients of variation for intrasample reproducibility were 1.26, 1.96, and 1.26% for DMA, TMA, and TMAO, respectively. Coefficients of variation for intersample reproducibility were 3.30, 1.31, and 1.78% for DMA, TMA, and TMAO, respectively. When authentic standards of DMA, TMA, and TMAO were added to samples of urine, blood, and rat liver, recoveries of 93103% were observed (Table 1). The peak contours from derivatized DMA, TMA, and TMAO exhibited modest tailing and no ghosting (Fig. 2; common problems in other gas chromatography assays
1
Recovery of Methylamine Standards from Biological Fluids and Tissue Percentage Urine DMA TMA TMAO
100 f 2.1 95 f 1.7 100 f 0.01
recovery
Blood 99 t 1.8 103 + 1.7 101 f 0.3
Liver 100 t 1.7 101 f 0.4 93 * 1.0
Note. Authentic standards were added to biological samples (250 nmol of DMA or TMAO was added to 1 ml blood or urine or 100 mg liver; 100 nmol of TMA was added to 1 ml of blood or urine; 400 nmol of TMA was added to 100 mg liver). Methylamines were determined as described in text. Data are expressed as the mean recoveries f standard deviation of triplicate determinations.
10 20 30 40 Time After Dose (hr) FIG. 4. Kinetics of oral dose (0.5 Gmol/kg normal humans. Urine to the intervals noted methods described in dard error of the mean.
50
excretion of [‘%]DMA in urine of humans. An body wt) of [‘%]DMA was administered to five was collected into acid in blocks corresponding in the figure. [‘aCIDMA was analyzed using the the text. Data are expressed as means f stan-
238
DACOSTA,
VRBANAC,
for these amines). Once the derivatives were formed, they were stable at room temperature or at -20°C for 2 weeks. Ninety-nine percent of the DMA was converted to the tosylamide, and 95% of the TMA was converted to NJ-dimethyl-2,2,2-trichloroethyl carbamate. The reducing reagent used in TMAO analyses did not alter the efficiency of conversion of TMA to the carbamate derivative. Betaine, ethylamine, phosphatidylcholine, and ammonia did not interfere with the assay. However, at concentrations 10 times higher than those of the amines of interest, betaine aldehyde, phosphocholine, and choline formed some dimethyltrichloroethyl carbamate (co.1 % conversion), while 0.5% of carnitine was converted. Dimethylglycine was the only compound that formed some N,N-dimethyl-p-toluene sulfonamide (~0.2% converted). Choline, which is also present in biological fluids and tissues, will break down to form TMA when heated above 110°C. Therefore, it is important that this temperature limit is not exceeded during derivatization. DMA, TMA, and TMAO concentrations were measured in human urine and blood and in rat urine, blood, liver, and kidney (Table 2). Our values for DMA, TMA, and TMAO in human and in rat urine were consistent with those published in the literature (7,11,18,33-35). Some of the tissues analyzed had not been previously examined for methylamines or TMAO. Our DMA values were lower than those which we and others have previously reported in blood (6,36), and our TMA values were higher (1). We believe these differences reflect improved calculation of recovery with our method. We compared an existing gas chromatography method (6) which uses a packed column and FID detection with the GC/MS method. The two methods gave comparable results for DMA and TMA, but the packed column method was much more variable for TMAO determinations (data not shown). This occurred because, in the GC-FID method, there was a variable loss of internal standard (isopropylamine) during the reduction of TMAO. In the GC/MS method we used TMAO-dG as an internal standard. Seafoods are a rich source of dietary amines, and we analyzed several species for their DMA, TMA, and TMAO contents (Table 2). We found that the DMA and TMA concentrations in fresh fish were two to three times lower than those present in processed (canned) or frozen fish. As would be expected, fresh fish had higher concentrations of TMAO than did canned or frozen fish. Our methods for measuring DMA, TMA, and TMAO are suitable for studying the metabolism of these amines in humans because we can use a nonradioactive isotopic label. In a pilot experiment, in which five humans ingested [13C]DMA, we observed that DMA was rapidly absorbed from the intestine and excreted into urine (Fig. 4). Peak enrichment of urine was observed within 30 min
AND
ZEISEL
after the dose, and most of the isotopic label was excreted in urine over a 48-h period. In summary we have developed a GC/MS assay for DMA, TMA, and TMAO which is sensitive, specific, and reproducible. Because we used mass spectrometry, isotopically labeled internal standards could be used to correct for losses during extraction, derivatization, and gas chromatography. In addition, the assay is suitable for metabolic studies in humans using isotopically labeled methylamines. ACKNOWLEDGMENTS We thank Drs. John Otis and Daniel R. Knapp work was supported by grants from the National (CA26731, RR-00533).
for their advice. This Institutes of Health
REFERENCES 1. Zeisel, S. H., daCosta, 9,179-181. 2. Lijinsky, J. Natl.
W., Keefer, Cancer Inst.
3. Lijinsky, 269-274.
K., and LaMont, L., Conrad, 49,1239-1249.
W., and Taylor,
4. Ohshima, 153.
H., and
6. Zeisel, S. H., dacosta, 403-408. 7. Al-Waiz, Toxicology
Mitchell,
J. G. (1985)
11. Zeisel, 6138.
and
S. H.,
W. (1976)
dacosta,
K.
and Lai, C-C.
(1980)
14. Lundstrom, R. C., Correia, Food Biochem. 6,229-241.
R. L. (1987)
Biochim.
Biophys. R. L. (1987)
Food
Chem.
Cancer
Res.
46,
L. D. (1983) Anal.
Chem.
F. F., and
S. (1985)
Anal.
S. A. (1977)
18. Blau,
K. (1961)
Biochem.
19. Dyer,
W. J. (1945)
Biochem. Anal.
J. Assoc.
6136-
Off
Anal.
K. A. (1982)
D., andzikova,
J.
Z. (1972)
149,572-574.
Chem.
49,401-403.
J. 80,193-200.
J. Fish Res. Board
Canad
6,351-358.
A., III, Scanlan, R. A., Lee, J. S., and Libbey, 20. Miller, J. Agric. Food Chem. 20,709-711. M. A., and Schedewie,
22. Lowis, S., Eastwood, togr. 278,139-143.
24,
52,630-635.
Wilhelm,
15. Churacek, J., Pechova, H., Tocksteinova, J. Chromatogr. 72,145-152.
21. Brewster, 20-24.
J. 232,
J. Agric.
(1986)
12. Lundstrom, R. C., and Racicot, Chem. 66,1158-1163.
16. Bagnasco,
5, 1381-
Biochem.
M. L. (1965)
15,
19,143-
Curcinogenesis
S. C., Idle, J. R., and Smith,
and Lijinsky,
17. Bouyoucos,
Z’onicol.
17,551-558.
10. Singer, G. M., 550-553.
13. Lin, J-K.,
R. (1972)
Sci. Publ.
S. C., Idle, J. R., and Smith,
8. Asatoor, A. M., and Simenhoff, Acta 111,384-392. 9. Al-Waiz, M., Xenobiotica
Cosmet.
ZARC
R. (1984)
K., and Fox,
M., Mitchell, 43,117-121.
Food
T. (1978)
Montesano,
Carcinogenesis
E., and Van de Bogart,
H. W. (1977)
H., and Kawabata,
5. Bartsch, 1398.
J. T. (1988)
H. (1983)
M. A., and Brydon,
Ann.
L. M. (1972)
Clin. Lab. Sci.
W. G. (1983)
13,
J. Chroma-
CHROMATOGRAPHIC 23. Dalene, M., Mathiasson, togr. 207,37-46. 24. Brand, 685.
J. M.,
25. Dunn, Chem.
S. R., Simenhoff, 48,41-44.
L., and Jonsson,
and Galask,
R. P. (1986)
J. A. (1981) Obstet.
M. L., and Wesson,
26. Zeisel, S. H., daCosta, K., Edrise, Carcinogenesis 7, 775-778. 27. DiCorcia,
A., and Samperi,
28. Elskamp,
C. J., and Schultz,
R. (1974)
MEASUREMENT
B. M., Anal.
G. R. (1986)
J. Chroma-
Gynecol.
68,
L. G., Jr. (1976) and Chem. Amer.
Fox,
682Anal.
J. G. (1986)
46,977-981. Znd. Hyg.
Assoc.
J. 47,41-49. 29. Baba,
257.
S., and
Watanabe,
Y. (1988)
Anal.
Biochem.
175, 252-
OF
METHYLAMINES
239
30. Hickinbottom, W. J. (1957) Reactions of Organic Compounds, p. 420, Longmans, Green, New York. 31. Hartvig, P., Ahnfelt, N. O., and Karlsson, K. E. (1976) Acta Pharm. Suet. 13,181-189. 32. Cohen, J., Krupp, M., and Chidsey, C. (1958) Amer. J. Physiol.
194,229-235. 33. Al-Waiz, M., Ayesh, R., Mitchell, S. C., Idle, J. R., and Smith, R. L. (1987) Clin. Pharmacol. Ther. 42,608-612. 34. Lowis, S., Eastwood, M. A., and Brydon, W. G. (1985) &it. J. Nutr. 54,43-51. 35. Zeisel, S. H., Gettner, S., and Youssef, M. (1989) Food Chem. Tonicol. 27,31-34. 36. Ishiwata, H., Iwata, R., and Tanimura, A. (1984) Food Chem. Toxicol. 22.649-653.
BIOCHEMISTRY
187,234-239
(1990)
The Measurement of Dimethylamine, Trimethylamine, and Trimethylamine A/-Oxide Using Capillary Gas Chromatography-Mass Spectrometty Kerry-Ann
daCosta,*
J. James
Vrbanac,?
and Steven
*Nutrient Metabolism Laboratory, Departments of Pathology 85 East Newton Street, Room M1002, Boston, Massachusetts The Upjohn Company, Kalamazoo, Michigan 49001
Received
December
5,1989
We have developed a method for measuring dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO) in biological samples using gas chromatography with mass spectrometric detection. DMA, TMA, and TMAO were extracted from biological samples into acid after internal standards (labeled with stable isotopes) were added-p-Toluenesulfonyl chloride was used to form the tosylamide derivative of DMA. 2,2,2-Trichloroethyl chloroformate was used to form the carbamate derivative of TMA. TMAO was reduced with titanium(II1) chloride to form TMA, which was then analyzed. The derivatives were chromatographed using capillary gas chromatography and were detected and quantitated using electron ionization mass spectrometry (GC/MS). Derivative yield, reproducibility, linearity, and sensitivity of the assay are described. The amounts of DMA, TMA, and TMAO in blood, urine, liver, and kidney from rats and humans, as well as in muscle from fishes, were determined. We also report the use of this method in a pilot study characterizing dimethylamine appearance and disappearance from blood in five human subjects after ingesting [13C]dimethylamine (0.5 wmol/kg body wt). The method we describe was much more reproducible than existing gas chromatographic methods and it had equivalent sensitivity (detected 1 pmol). The derivatized amines were much more stable and less likely to be lost as gases when samples were stored. Because we used GC/MS, it was possible to use stable isotopic labels in studies of methylamine metabolism in humans. 0 1990 Academic Press, Inc.
Dimethylamine (DMA)l and trimethylamine (TMA) are important precursors of N-nitrosodimethylamine 1 Abbreviations used: DMA, dimethylamine; TMAO, trimethylamine N-oxide; FID, flame
234
H. Zeisel*
& Pediatrics, Boston University School of Medicine, 02118, and TDrug Metabolism Research,
TMA, trimethylamine; ionization detector.
(l-4), which is a potent carcinogen in a wide variety of animal species (5). These two aliphatic amines are found in human and rat urine, blood, and gastric fluid (1,6,7). DMA is minimally metabolized in mammals and is excreted intact in the urine (8). TMA is normally oxidized within liver and is excreted in the urine as trimethylamine N-oxide (TMAO) (9). The TMA content of seafoods is commonly used to assessfreshness, as TMA is formed by bacteria from TMAO in fish muscle and imparts a characteristic “fishy” odor (10-14). Various procedures exist for the analysis of these methylamines, including thin-layer chromatography (9,15), ion chromatography (16,17), calorimetric assays (18,19), gas chromatography (12,20-27), and high-performance liquid chromatography (13,28,29). These existing methods either lack sensitivity, specificity, or reproducibility. DMA and TMA are gases at room temperature and are easily lost during sample storage. In addition, these amines tend to adhere to many surfaces and many chromatographic procedures are subject to severe “tailing” and “ghosting” (6). We have developed a method for the measurement of DMA, TMA, and TMAO in tissues and biological fluids using capillary gas chromatography with mass spectrometric detection. This assay is preferable to existing methods because the derivatized amines are stable during storage, there is no ghosting or tailing, and internal standards labeled with stable isotopes can be used to correct for variations in recovery and for metabolic tracer studies. MATERIALS
AND
METHODS
Reagents. All reagents were obtained from Fisher Chemicals (Springfield, NJ) unless otherwise noted. Stock solutions of DMA and TMA (HCl salts, Sigma Chemical Co., St. Louis, MO), TMAO (Sigma), di0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHIC
W,),-N
MEASUREMENT
Cd-0-CH,
-Ccl,
2,2,2-trlchloro~thylchloroformate
trlmethylamlne
OF
METHYLAMINES
(CH,),-NH
cl-O-4
235
oCH3
dlmsthylamlne+chlo
rt de
electron
(CH&
-N-C-0-CH,
loniratlon
-CCl,
N,Kdtmathyl-2,2,2-trlchloroethylcarbsmate
electron Ionization
0+ (CH,),-N
-O-@H,
o+ N,N-dimethyl-ptOluen0
ii
(CH,),-N-C FIG. 1. Derivatization dimethyl-2,2,2-trichloroethyl
(m/z 72)
(m/z
sultonamlde
199)
of TMA and DMA. DMA and TMA were derivatized so as to form N,N-dimethyl-p-toluene carbamate. These were isolated using gas chromatography and fragmented using electron
methyl[‘HG]amine HCl (DMA-ds, 98 at.% D, Aldrich Chemical Co., Milwaukee, WI), dimethyl[13C,]amine HCl ([13C]DMA, 99.2 at.% 13C, MSD Isotopes, Montreal, Canada), trimethyl[2HG]amine HCl {TMA-ds, (CD,H),N; 99 at.% D, MSD Isotopes}, and TMAO-dG (prepared from TMA-d, with hydrogen peroxide (30)) were made up in 0.1 N HCl and stored at 4°C. These solutions were stable over several months. The derivatization agents, p-toluenesulfonyl chloride (2 M solution diluted in toluene) and 2,2,2-trichloroethyl chloroformate, were purchased from Aldrich Chemical. Methanolic alkali was prepared by dissolving 2.8 g KOH in 100 ml methanol/water (3/l, v/v). The reducing reagent was prepared with titanium(II1) chloride (Aldrich), 1 N HCl, and 10% formalin (l/1/2, v/v; must be prepared fresh under a nitrogen atmosphere). Experimental. Male Sprague-Dawley rats (300-400 g, Charles River Breeders, Wilmington, MA) were housed in individual metabolic cages in a controlled environment (24”C, 12 h of light from 6 AM to 6 PM). They were offered food (Ralston Purina rat chow No. 5001, Farmers Exchange, Framingham, MA) and water ad Zibiturn. Urine was collected, over a 24-h period, into acid (so that the final concentration was approximately 0.1 N HCl). The volumes were recorded, and aliquots were stored at -20°C. After urine collection was completed, the rats were anesthetized with diethyl ether, and blood was drawn by intracardiac puncture. Blood samples were collected into tubes containing heparin and kept on ice. Liver and kidney samples were collected by freezeclamping the specimen between tongs that had been precooled in liquid nitrogen. They were stored at -95°C until used.
sulfonamide and N,Nimpact ionization.
Control samples of urine (24-h collections) and blood were collected from humans eating a normal diet. Five normal humans (male, ages 24-36 years) were also given an oral dose of [13C]DMA (0.5 hmol/kg body wt), and urine was collected at timed intervals after the dose. Urine was collected and stored as described above for rat urine. Fresh fish was purchased locally and analyzed that day. A sample was also stored at -20°C for several months before analysis. Sample preparation and extraction of amines. Deuterated internal standards (DMA-ds, TMA-d,, TMAO$; as appropriate) were added to all samples to be analyzed. Before urine samples were assayed, they were filtered with a Sep Pak (Waters Associates, Milford, MA; 92-94% of DMA, TMA, or TMAO were recovered). Tissue samples were sonicated (setting 5, 50% pulse; Heat Systems Ultrasonics, Inc., Model W-225R, Plainview, NY) in ice-cold 0.9% NaCl (l/5, w/v). An aliquot of sonicate containing 100 mg tissue was added to 0.6 ml 0.1 N HCl and 3 ml chloroform to precipitate protein. Aliquots of whole blood (1 ml) were added to 0.6 ml 0.1 N HCl and 3 ml chloroform to precipitate protein. Fish (500 mg) was added to 5 ml 0.1 N HCl and mixed thoroughly (sonication was not required due to the soft consistency of fish). Samples were mixed and then subjected to centrifugation at 2000g for 20 min at 4°C. The supernatants were aspirated and used for analyses. Derivatization and analysis of DMA, TMA, and TMAO (see Fig. 1). For DMA analysis, N,N-dimethylp-toluene sulfonamide was formed (10). A portion of the acidified biological fluid or extract was placed into a glass test tube sealed with a Teflon-lined septum (WISP sep-
236
DACOSTA, Jon
72 amu
3.OES-
t
4 1.2ES-
0’
5
2.5E5-
l.OES-
2
2.0ES-
6.OE4 -
a
l.SES-
B.OE4-
1 .OES-
4.OE4-
5.OE4-
3.OES
2
2SE5
;
(CH,)
2-N-!
-12
2.OE41 3.1
Q)
VRBANAC,
II
1 4.5
I
I
60
80
100
120
111
i
I
140
160
I
180
I
I
IL 1
200
220
180 200
220
71’
(C’H,H),-N-C
2.OE5 5
n u
1.5E5 l.OE5 5.OE4
3.1
4.5
a Ion
199
60
amu
5
o
-
1.5E5-
100
120
140
160
4
1.8E5-
b
80
0+ (CH,),-N
9’\
-O-&H, 199
4.5E5-
1.2E5-
5 n a
#.OE4-
3.OE5-
b.OE4-
I\
3.OE4-
1.5E5,
I
5.2
6.7
I.
60
-
I
80
I-
IL
r-l
100
120
1
I
140
160
I
+
180
200
180
200
0' Q)
1.6E5
ti
1.5E5
g
1.2E5
5
0.OE4
9
&OE4 3.OE4
6.2
,lon
6.7
205
60
amu
80
100
120
140
160
d
1.6E5-
0’ (C%JiN-O-!+H,
9’1
8 5
1.5E5-
w
1.2E5-
5
9.OE4-
3.OE5-
s
6-oE4e 3.OE4
1.5E5-
205
4.5E5-
[\ I
I
5.2
6.7
Time
(min)
60
80
I
1
100
120
!A ,
140
.I I
160
I
I
180
IJ
200
Mass/Charge
FIG. 2. Mass spectra obtained from fragmentation of DMA and TMA. Methylamines were isolated and derivatized (TMAO and its deuterated standard were first reduced to TMA) as described in the text. They were separated by GC/MS using a silica-bonded capillary column and a temperature gradient program. The compounds were fragmented by electron ionization mass spectrometry.
AND
ZEISEL
turn, Waters Associates, Milford, MA). Toluene (1 ml for urine and fish, 0.5 ml for all other samples) and ptoluenesulfonyl chloride (38 mg for urine and fish; 3.8 mg for liver, kidney, and blood) were added. KOH (1 ml; 65% in water, w/v) was injected into the tube, bringing the pH above 9. Tubes were incubated at 90°C for 2.5 h, vigorously shaken, and then subjected to centrifugation at 1OOOg for 5 min. An aliquot (1~1) of the toluene layer was injected onto the gas chromatograph/mass spectrometer (GC/MS). In order to measure TMA, N,N-dimethyl-2,2,2-trichloroethyl carbamate was formed from DMA and TMA (31). A portion of the acidified biological fluid or extract was placed into a glass test tube sealed with a Teflonlined septum (Waters Associates). Toluene (1 ml for urine samples, 0.5 ml for all other samples) was then added. KOH (1 ml; 65% in water, w/v) was injected into the tube. Tubes were incubated at 90°C for 1 h, vigorously shaken, and then subjected to centrifugation at 1OOOg for 5 min at 4°C. An aliquot (0.5 ml) of the cooled toluene layer was added to a sealed vial containing 50 ~1 2,2,2-trichloroethyl chloroformate and 10 mg anhydrous sodium carbonate. Tubes were incubated at 110°C for 30 min. Methanolic alkali (1 ml) was added after cooling, and the reaction mixture was mixed. Water (2 ml) was then added, and the sample mixed and subjected to centrifugation at 1OOOg for 5 min at 4°C. An aliquot (1 ~1) of the toluene layer was injected onto the GC/MS. TMAO in the acidified samples was reduced to TMA (32). A portion of the acidified biological fluid or extract was placed into a glass test tube sealed with a Teflonlined septum (Waters Associates). An equal volume of reducing reagent was added under an atmosphere of nitrogen and the tube was sealed and incubated at room temperature for 1 h. The TMA formed was analyzed as described earlier. Gas chromatography. We used a Hewlett-Packard 5890/5970 GC/MS equipped with an automatic liquid sampler. This operates in the electron ionization mode. A fused silica capillary column (12.5 m X 0.2 mm i.d. dimethyl silicone bonded-phase HP-l; film thickness, 0.33 pm; Hewlett-Packard, Andover, MA) was used in these analyses. GC conditions were as follows: 0.75 min, splitless mode; He, carrier gas; temperature gradient, from 80 to 280°C at 25”C/min. Fragments formed by electron impact ionization were quantitated using selected ion monitoring. The molecular ions at m/z 199 (DMA), m/z 201 (formed from [13C]DMA, and m/z 205 (formed from DMA-de) and the fragments m/z 72 (formed from TMA), m/z 76 (formed from TMA-dG), and m/z 78 (formed from DMA-&) were monitored (see Figs. 1 and 2). Calculations. DMA concentrations were determined directly from analysis of the tosylamide derivatives. TMA concentrations were calculated using
CHROMATOGRAPHIC
MEASUREMENT
OF
237
METHYLAMINES TABLE
2
Concentrations of DMA, TMA, and TMAO Tissue
in Various
Tissues TMA
DMA
TMAO
nmol/g
Theoretical
Rat blood Rat liver Rat kidney Human blood
pmoles
of assays for DMA, TMA, and TMAO. Authentic FIG. 3. Linearity standards of DMA, TMA, and TMAO were derivatized and quantitated using GC/MS as described under Materials and Methods. Correlation coefficients were calculated using linear regression analysis and were equal to 0.9960 for all three compounds.
TMA
Rat urine Human urine
27 6
{ DM&,,
x
area(DMA)
* ml/nmol} 9
area(TMA)
* ml/nmd
where = area at mass 72 DMAtos = concentration (nmol/ml) of DMA in sample as determined using the toluenesulfonyl chloride method = area generated by 1 nmol/ml internal area(DMA) standard of isotopically labeled DMA that was converted to the carbamate derivative = area generated by 1 nmol/ml internal =alTMA) standard of isotopically labeled TMA that was converted to the carbamate derivative. arqcarbamate)
TMAO concentrations were calculated as the differences in TMA content after and before reduction. AND
body
DISCUSSION
The assays as described were able to detect as little as 1 pmol of each amine and remained linear up to 250 pmol TABLE
Shrimp Tuna (canned) Cod Cod (frozen)
10 84 97 381
wt/24
h 14 10
2 1
fimol/lOO -
92 633 0 21
40 437 531 40 rmol/kg
(nmol/ml) = are%rbamate)
RESULTS
4 136 161 4
wet wt
g wet wt 171 57 3400 1958
9 247 44 218
Note. DMA, TMA, and TMAO were measured as described Data are expressed as means of triplicate determinations.
in text.
(Fig. 3). Coefficients of variation for intrasample reproducibility were 1.26, 1.96, and 1.26% for DMA, TMA, and TMAO, respectively. Coefficients of variation for intersample reproducibility were 3.30, 1.31, and 1.78% for DMA, TMA, and TMAO, respectively. When authentic standards of DMA, TMA, and TMAO were added to samples of urine, blood, and rat liver, recoveries of 93103% were observed (Table 1). The peak contours from derivatized DMA, TMA, and TMAO exhibited modest tailing and no ghosting (Fig. 2; common problems in other gas chromatography assays
1
Recovery of Methylamine Standards from Biological Fluids and Tissue Percentage Urine DMA TMA TMAO
100 f 2.1 95 f 1.7 100 f 0.01
recovery
Blood 99 t 1.8 103 + 1.7 101 f 0.3
Liver 100 t 1.7 101 f 0.4 93 * 1.0
Note. Authentic standards were added to biological samples (250 nmol of DMA or TMAO was added to 1 ml blood or urine or 100 mg liver; 100 nmol of TMA was added to 1 ml of blood or urine; 400 nmol of TMA was added to 100 mg liver). Methylamines were determined as described in text. Data are expressed as the mean recoveries f standard deviation of triplicate determinations.
10 20 30 40 Time After Dose (hr) FIG. 4. Kinetics of oral dose (0.5 Gmol/kg normal humans. Urine to the intervals noted methods described in dard error of the mean.
50
excretion of [‘%]DMA in urine of humans. An body wt) of [‘%]DMA was administered to five was collected into acid in blocks corresponding in the figure. [‘aCIDMA was analyzed using the the text. Data are expressed as means f stan-
238
DACOSTA,
VRBANAC,
for these amines). Once the derivatives were formed, they were stable at room temperature or at -20°C for 2 weeks. Ninety-nine percent of the DMA was converted to the tosylamide, and 95% of the TMA was converted to NJ-dimethyl-2,2,2-trichloroethyl carbamate. The reducing reagent used in TMAO analyses did not alter the efficiency of conversion of TMA to the carbamate derivative. Betaine, ethylamine, phosphatidylcholine, and ammonia did not interfere with the assay. However, at concentrations 10 times higher than those of the amines of interest, betaine aldehyde, phosphocholine, and choline formed some dimethyltrichloroethyl carbamate (co.1 % conversion), while 0.5% of carnitine was converted. Dimethylglycine was the only compound that formed some N,N-dimethyl-p-toluene sulfonamide (~0.2% converted). Choline, which is also present in biological fluids and tissues, will break down to form TMA when heated above 110°C. Therefore, it is important that this temperature limit is not exceeded during derivatization. DMA, TMA, and TMAO concentrations were measured in human urine and blood and in rat urine, blood, liver, and kidney (Table 2). Our values for DMA, TMA, and TMAO in human and in rat urine were consistent with those published in the literature (7,11,18,33-35). Some of the tissues analyzed had not been previously examined for methylamines or TMAO. Our DMA values were lower than those which we and others have previously reported in blood (6,36), and our TMA values were higher (1). We believe these differences reflect improved calculation of recovery with our method. We compared an existing gas chromatography method (6) which uses a packed column and FID detection with the GC/MS method. The two methods gave comparable results for DMA and TMA, but the packed column method was much more variable for TMAO determinations (data not shown). This occurred because, in the GC-FID method, there was a variable loss of internal standard (isopropylamine) during the reduction of TMAO. In the GC/MS method we used TMAO-dG as an internal standard. Seafoods are a rich source of dietary amines, and we analyzed several species for their DMA, TMA, and TMAO contents (Table 2). We found that the DMA and TMA concentrations in fresh fish were two to three times lower than those present in processed (canned) or frozen fish. As would be expected, fresh fish had higher concentrations of TMAO than did canned or frozen fish. Our methods for measuring DMA, TMA, and TMAO are suitable for studying the metabolism of these amines in humans because we can use a nonradioactive isotopic label. In a pilot experiment, in which five humans ingested [13C]DMA, we observed that DMA was rapidly absorbed from the intestine and excreted into urine (Fig. 4). Peak enrichment of urine was observed within 30 min
AND
ZEISEL
after the dose, and most of the isotopic label was excreted in urine over a 48-h period. In summary we have developed a GC/MS assay for DMA, TMA, and TMAO which is sensitive, specific, and reproducible. Because we used mass spectrometry, isotopically labeled internal standards could be used to correct for losses during extraction, derivatization, and gas chromatography. In addition, the assay is suitable for metabolic studies in humans using isotopically labeled methylamines. ACKNOWLEDGMENTS We thank Drs. John Otis and Daniel R. Knapp work was supported by grants from the National (CA26731, RR-00533).
for their advice. This Institutes of Health
REFERENCES 1. Zeisel, S. H., daCosta, 9,179-181. 2. Lijinsky, J. Natl.
W., Keefer, Cancer Inst.
3. Lijinsky, 269-274.
K., and LaMont, L., Conrad, 49,1239-1249.
W., and Taylor,
4. Ohshima, 153.
H., and
6. Zeisel, S. H., dacosta, 403-408. 7. Al-Waiz, Toxicology
Mitchell,
J. G. (1985)
11. Zeisel, 6138.
and
S. H.,
W. (1976)
dacosta,
K.
and Lai, C-C.
(1980)
14. Lundstrom, R. C., Correia, Food Biochem. 6,229-241.
R. L. (1987)
Biochim.
Biophys. R. L. (1987)
Food
Chem.
Cancer
Res.
46,
L. D. (1983) Anal.
Chem.
F. F., and
S. (1985)
Anal.
S. A. (1977)
18. Blau,
K. (1961)
Biochem.
19. Dyer,
W. J. (1945)
Biochem. Anal.
J. Assoc.
6136-
Off
Anal.
K. A. (1982)
D., andzikova,
J.
Z. (1972)
149,572-574.
Chem.
49,401-403.
J. 80,193-200.
J. Fish Res. Board
Canad
6,351-358.
A., III, Scanlan, R. A., Lee, J. S., and Libbey, 20. Miller, J. Agric. Food Chem. 20,709-711. M. A., and Schedewie,
22. Lowis, S., Eastwood, togr. 278,139-143.
24,
52,630-635.
Wilhelm,
15. Churacek, J., Pechova, H., Tocksteinova, J. Chromatogr. 72,145-152.
21. Brewster, 20-24.
J. 232,
J. Agric.
(1986)
12. Lundstrom, R. C., and Racicot, Chem. 66,1158-1163.
16. Bagnasco,
5, 1381-
Biochem.
M. L. (1965)
15,
19,143-
Curcinogenesis
S. C., Idle, J. R., and Smith,
and Lijinsky,
17. Bouyoucos,
Z’onicol.
17,551-558.
10. Singer, G. M., 550-553.
13. Lin, J-K.,
R. (1972)
Sci. Publ.
S. C., Idle, J. R., and Smith,
8. Asatoor, A. M., and Simenhoff, Acta 111,384-392. 9. Al-Waiz, M., Xenobiotica
Cosmet.
ZARC
R. (1984)
K., and Fox,
M., Mitchell, 43,117-121.
Food
T. (1978)
Montesano,
Carcinogenesis
E., and Van de Bogart,
H. W. (1977)
H., and Kawabata,
5. Bartsch, 1398.
J. T. (1988)
H. (1983)
M. A., and Brydon,
Ann.
L. M. (1972)
Clin. Lab. Sci.
W. G. (1983)
13,
J. Chroma-
CHROMATOGRAPHIC 23. Dalene, M., Mathiasson, togr. 207,37-46. 24. Brand, 685.
J. M.,
25. Dunn, Chem.
S. R., Simenhoff, 48,41-44.
L., and Jonsson,
and Galask,
R. P. (1986)
J. A. (1981) Obstet.
M. L., and Wesson,
26. Zeisel, S. H., daCosta, K., Edrise, Carcinogenesis 7, 775-778. 27. DiCorcia,
A., and Samperi,
28. Elskamp,
C. J., and Schultz,
R. (1974)
MEASUREMENT
B. M., Anal.
G. R. (1986)
J. Chroma-
Gynecol.
68,
L. G., Jr. (1976) and Chem. Amer.
Fox,
682Anal.
J. G. (1986)
46,977-981. Znd. Hyg.
Assoc.
J. 47,41-49. 29. Baba,
257.
S., and
Watanabe,
Y. (1988)
Anal.
Biochem.
175, 252-
OF
METHYLAMINES
239
30. Hickinbottom, W. J. (1957) Reactions of Organic Compounds, p. 420, Longmans, Green, New York. 31. Hartvig, P., Ahnfelt, N. O., and Karlsson, K. E. (1976) Acta Pharm. Suet. 13,181-189. 32. Cohen, J., Krupp, M., and Chidsey, C. (1958) Amer. J. Physiol.
194,229-235. 33. Al-Waiz, M., Ayesh, R., Mitchell, S. C., Idle, J. R., and Smith, R. L. (1987) Clin. Pharmacol. Ther. 42,608-612. 34. Lowis, S., Eastwood, M. A., and Brydon, W. G. (1985) &it. J. Nutr. 54,43-51. 35. Zeisel, S. H., Gettner, S., and Youssef, M. (1989) Food Chem. Tonicol. 27,31-34. 36. Ishiwata, H., Iwata, R., and Tanimura, A. (1984) Food Chem. Toxicol. 22.649-653.
