John S. Wishnok and Steven R. Tannenbaum
The Analytical Approach
Department of Nutrition and Food Science Massachusetts Institute of Technology Cambridge, Mass. 02139
Edited by Claude A. Lucchesi
An Unknown Salivary Morpholine Metabolite Identification of the metabolite leads to the discovery of a new biochemical reaction of secondary amines
N-nitrosodialkylamines (nitrosamines) are well-known chemical car cinogens in a large variety of animal species (1,2) and have been implicat ed as potential human health hazards as well (3, 4). Nitrosamines are formed efficiently from secondary amines and nitrite via the active species, N2O3:
R. R·
NH
R.
[N20b]
"N—NO R ^
ion by oral microorganisms (8). Nitrite is a well-known food additive, and ni trate is found in various foods, partic ularly roots and leafy vegetables (9, 10). Consequently, we became inter ested in knowing whether or not nitro samines could be formed under physi ological conditions in the digestive system. Preliminary experiments were car ried out with morpholine, which react ed readily with saliva to form N-nitrosomorpholine:
+ other products This reaction occurs optimally at low pH (5) but will take place under a wide variety of conditions when amines are allowed to come into con tact with nitrite (6, 7). Secondary amines are natural con stituents of many foods, and low levels of nitrite are generally present in human saliva via reduction of nitrate
Ov
Saliva *•
Ν" Η
I NO
A useful analytical system for these experiments is gas chromatography/ mass spectrometry by simultaneous
Ozone Catalytic Pyrolysis Chamber
Optical Filter
From GC Column
total-ion/single-ion detection with the single-ion monitor set at m/e = 30. Simple nitrosamines frequently pro duce a characteristic, prominent frag ment (NO) at mass 30; thus, this method indicates which of the many peaks in the often complex biological extracts mass spectrum are potentially significant. In addition to N-nitrosomorpholine, the extracts from the saliva experi ments contained a number of other compounds, including one that gave an intense signal on the m/e = 30 mass chromatogram. Therefore, we thought it might be a new nitrosamine and placed fairly high priority on the identification of this unknown sub stance. The most straightforward approach to an identification, of course, is sim ply to get as much pure material as possible and then obtain NMR, UV, IR, and mass spectra, which usually prove sufficient. Two major problems prevented this approach with the above saliva extract: The concentra tions were low (/ig/mL range), and it was impractical to run large-scale re actions since this required large amounts of fresh human saliva. At the outset, we knew from the low-resolution mass spectrum that the molecular weight of the compound was 112, which formally corresponds to the loss of four hydrogen atoms from N-nitrosomorpholine (MW = 116). We were intrigued by the possi bility that the compound might be the TV-nitrosooxazine since the parent ring structure has never been synthesized.
Photomultiplier
Ό
Cold Trap To Rotary Pump
-Ν
NO Figure 1 . Schematic diagram of nitrosamine detector (TEA)
iV-nitroeooxazine ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977 · 715 A
This also meant, however, t h a t direct synthesis of an authentic reference sample might be prohibitively diffi cult. Since an a t t e m p t to isolate a small amount of the compound by microscale preparative gas chromatogra phy was unsuccessful, we appeared limited to information t h a t could be obtained in conjunction with gas chro matography. T h e most powerful gas chromato graphic detection technique available to us at t h a t time was high-resolution mass spectrometry, with an instru ment equipped with a movable photoplate detector, at the National Cancer Institute. A concentrated saliva ex tract was examined at NCI by Peter Roller; the exact molecular weight of the unknown (112.0631) ruled out the nitrosooxazine structure (calculated MW = 112.0273) and indicated a mo lecular formula of C ^ H g ^ O rather than C4H4N2O2. Unfortunately, a large number of structures could be drawn for this molecular formula, in cluding simple substitution of CN at any of the three types of hydrogen on the morpholine molecule or substitu tion o f — C H = N H at various posi tions on an unsaturated morpholine ring. It was not even clear t h a t the morpholine structure had not been
disrupted or t h a t the new molecule was in fact a morpholine derivative. (Most of the new substances, e.g., phe nol, found in saliva following addition of morpholine were not formed from morpholine). In addition, because all of the various possible structures seemed equally implausible from a chemical or biochemical viewpoint, there appeared to be no rational way to select a given structure for further study, e.g., independent synthesis.
Thermal Energy Analyzer During this period we obtained a new GC detector—a thermal energy analyzer (TEA)—which is highly sen sitive and specific for the iV-nitroso functionality (11). This detector is based on the catalytic disruption of the Ν — N O bond to release molecular NO. T h e NO is then reacted with ozone to yield O2 and excited NO2*, which decays to the ground state and emits a photon of near-infrared radia tion:
Ru
Cat
^NNO ^ +
NO
R; +
other
fragments
NO NO,*
03 —
• NO, + hv (0.6-2.8 μ)
T h e radiation is then amplified by a photomultiplier (Figure 1). Although simple in operation, the complexity of the reaction sequence assures t h a t there will be few false positives. T h e T E A showed no response for the un known compound, thus indicating t h a t it was not a nitrosamine. Under these circumstances, we did not feel t h a t a major effort to identify the compound could be justified, and ac tive experiments were stopped. How ever, we remained interested and dis cussed possible structures from time to time. T h e original project was gradually expanded to include the reactions of a variety of other amines in saliva, many of which yielded products with structures apparently analogous to the unknown morpholine derivative. We began to realize that structural re straints in some of these new amines could limit the possible structures of the unknown derivatives. About a year after t h e original observation, we car ried out an experiment with diphenylamine and again observed an appar ently analogous derivative:
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716 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
N0 2 * + 0 2
.ο. +
CH2CI2 Extract
Saliva
.ο +
Unknown
GC-TEA
No Response Not a Nitrosamine
GC-MS Low Resolution
MW = 112 Strong mle • 30
GC-MS High Resolution
MW = 112.0631 CsH.N 2 0
Ν' H NO
GC-TEA
Ο
ο,
CH2CI2 Extract
BrCN
-*•
GC-MS Low Resolution
Ν' I CN
Η
GC-MS -*· High Resolution
No Response
MW = 112 Strong m/e = 30 MW = 112.0637
Figure 2. Analytical a p p r o a c h for identification of u n k n o w n m o r p h o l i n e metabolite
for t h e d i p h e n y l a m i n e derivative a n d (Π)
Ν
α
H m/e = 169
Ν'
Saliva
CN II
*-
Ν' NO m/e = 198 + unknown m/e = 194 Since the only active position on diphenylamine is the a m i n e hydrogen, t h e reaction could be considered for mally as loss of H followed by addition of a functional group with mass = 26 (169 - 1 = 168; 194 - 168 = 26). T h e most obvious identity for a moiety with m = 26 is C N , suggesting t h e cyanamide s t r u c t u r e (I)
•Ν"" CN I
for the morpholine derivative. Synthesis of cyanamides from sec ondary amines by reaction with cyano gen bromide is well known, and an au thentic reference sample of Com pound II was prepared and found to be indistinguishable (identical lowresolution and high-resolution mass spectrum and GC retention times) from the saliva metabolite: Ό
•Ν' Η
BrCN
Λ)
I
Saliva
-ο N' H
CN T h e a p p r o a c h t a k e n for t h e identifica tion is shown in Figure 2. T h e formation of cyanamides from secondary amines in saliva is a p p a r ently fairly general, a n d t h e identifica tion of this c o m p o u n d t h u s effectively constituted t h e discovery of a new metabolic pathway for secondary amines.
718 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
T h e morpholinocyanamide was evaluated for biological activity a n d found to be nonmutagenic a n d moder ately toxic. Although t h e use of m u t a genicity as an indicator of carcinoge nicity is controversial, it does a p p e a r to be generally t r u e t h a t n o n m u t a g e n ic c o m p o u n d s are probably noncarcinogenic; therefore, we are no longer actively investigating t h e formation of cyanamides from secondary amines. We are, however, investigating t h e possibility t h a t analogous transforma tions of primary amines m a y lead t o t h e production of carcinogenic sub stances in h u m a n saliva.
References (1) H. Druckrey, R. Preussmann, S. Ivankovic, and D. Schmâhl, Ζ. Krebsforsch., 69, 103 (1967). (2) P. N. Magee and J. M. Barnes, Adv. Cancer Res., 10,163(1967). (3) P. N. Magee, Food Cosmet. Toxicol., 9, 207 (1971). (4) *R. A. Scanlan, Crit. Rev. Food Technoi, 5,357(1975). (5) S. S. Mirvish, Toxicol. Appl. Pharma col., 31,325(1975). (6) T. Y. Fan and S. R. Tannenbaum, J. Agric. Food Chem., 21,967 (1973). (7) S. R. Tannenbaum and T. Y. Fan, Proc. Meat Industry Res. Conf., Chicago, 111., 1973. (8) S. R. Tannenbaum, A. J. Sinskey, M. Weisman, and W. Bishop, J. Nat. Cancer Inst., 53, 79 (1974). (9) W. E. Phillips, J. Agric. Food Chem., 16,88(1968). (10) E. C. Hersler, J. Siciliano, S. Krulick, W. L. Porter, and J. W. White, Jr., ibid., 21,970(1973). (11) D. H. Fine, H. Rufeh, and B. Gunther, Anal. Lett., 6,731 (1973).
