Gas-chromatographic and mass-spectrometric analysis of the odor of human feces

GASTROENTEROLOGY 1987;93:1321-9

Gas-Chromatographic and Mass-Spectrometric Analysis of the Odor of Human Feces J. G. MOORE,

L. D. JESSOP,

and D. N. OSBORNE

Section of Gastroenterology, Department of Medicine, Salt Lake Veterans Administration Medical Center, and Division of Gastroenterology, Department of Medicine, University of Utah School of Medicine, Salt Lake City, Utah; and Scientific Resources, Sandy, Utah

Gas-chromatographic and mass-spectrometric analysis of human fecal samples was performed to identify the volatile compounds responsible for fecal odor. The compounds identified with fecal odor were the methyl sulfides methanethiol, dimethyi disulfide, and dimethyl trisulfide. Skatole and indole, the benzopyrrole volatiles believed to be responsible for fecal odor, in fact elaborated a napthalenelike “mothball” odor in the crystalline state as well as after purging from feces. A small amount of hydrogen sulfide gas was also identified in fecal samples. The components responsible for fecal odor are complex and may be influenced by dietary and endogenous contributions. However, the major components are methyl sulfide compounds rather than skatole and indole as is currently believed. It is taught that the odor of feces is due primarily to the presence of the benzopyrrole compounds skatole and indole, which are end products of anaerobic metabolism by colonic microflora (l-4). It is also stated that hydrogen sulfide, ammonia, volatile amines, mercaptans, and short-chain fatty acids contribute to the odor, although quantitative data have never been reported (l-5). In contrast, there is a large body of data on the nonodorous fraction of intestinal gas, believed to comprise >99% of the entire intestinal gaseous mixture (5-7). The detection of diet-related odorous compounds in ancient and modern human fecal samples by vapor-phase (headspace) gas-chromatographic (GC) Received August 19, 1986. Accepted July 27, 1987. Address requests for reprints to: J. G. Moore, M.D., G.I. Section (lllG), Veterans Administration Medical Center, 500 Foothill Drive, Salt Lake City, Utah 84148. The authors acknowledge the Veterans Administration Department of Research for study support. 0 1987 by the American Gastroenterological Association 0016-5085/871$3.50

analysis has recently been reported (8). The odor of licorice, found in both modern and ancient fecal samples, was chemically characterized by massspectrometric (MS) analysis as anethole [l-methoxyJ-(1-propenyl)benzene] (9). Thus, with combination G&odor-MS analysis of human fecal samples, it is possible to isolate and characterize the chemical(s) responsible for specific odors that would ordinarily be dominated by the offensive, foul odor of feces and remain undetected by human olfaction. This report provides information on the GC-odor-MS analysis of the volatiles in human feces responsible for the foul odor.

Materials and Methods Subjects The fecal samples were provided by healthy adult subjects on unrestricted diets representative of western cuisine. Dietary records were kept by all subjects. All diets contained a variety of meats, fruits, vegetables, spices, condiments, pastries, desserts, and drinks. Gas-Chromatographic

Odor

Analysis

The equipment used for GC odor analysis is shown in Figure 1. For each analysis, 5-25 g of frozen feces was placed in 250-ml Erlenmeyer flasks containing 150 ml of 0.5% Na3P04. The solution was kept at room temperature (24%) for 24-72 h. Just before purging, 45 g of (NH&SO4 was added to the sample. The volatile compounds were then purged by bubbling helium gas through the solution at 50 ml/min for 40 min while the solution was magnetically stirred. The volatiles were trapped on a stainless steel tube-collector (2 mm ID X 300 mm) packed with 100 mg of Tenax adsorbent. The collector-trap was then connected to the GC column and desorbed of trapped components by heating to 220°C for 2 min. The eluted volatiles were swept onto the head of the GC column by helium carrier gas. The Abbreviations

used in this paper: GC, gas-chromatographic;

MS, mass-spectrometric.

GASTROENTEROLOGYVol. 93, No. 6

1322 MOORE ET AL.

He Sourc7 Collector With Packed Tenax

Hydrated Sample Flame Ionization Detector

iniffina Port

Flow Control

G-C Column

Integrator-Recorder

&

Figure 1. Schematic of gas-chromatographic-odor-analytic apparatus. 0 Fecal sample is placed in bubble chamber containing 0.5% Na3P0, and (NH&SO,. 0 Organic volatiles are absorbed on a Tenax collector which is then desorbed at 220°C 0 onto the head of a packed chromatographic column 0. The GC column in 8 is then heated from 40°C to 250°C at 8”Cimin to allow for separation of organic volatiles. The effluent stream is split; one-half of the flow is diverted to a flame ionization detector, the other half to the “sniffing” port 0. The human “sniffer” records odors on a chart recorder. Helium is used as the carrier gas.

GC glass column measured 2 mm x 180 mm and was packed with 10% SP-2100 on 80/100 Supelcoport. The column outlet was provided with a 1:l splitter; one branch of the splitter connected to a flame ionization detector, the other to a sniffing port. The GC column was kept isothermal at 40°C for 4 min and then heated at 8”C/min to 250°C and held at the upper temperature for 10 min. The retention time of each eluted sample component peak and the characteristics of the odor emerging from the sniffing port were recorded by the operator. Only fecallike odors were recorded in this study, although many others, including odors that could be directly traced to ingested dietary items, were detected. Analytic instrumentation included a Hewlett-Packard 5700A gas chromatograph (Hewlett-Packard Co., Palo Alto, Calif.) and a C-R3A Shimadzu strip chart recorder/integrator (Shimadzu Scientific Instruments, Columbia, Md.). The above analytic procedures were adapted from the method of Jarke et al. (10). Gas-chromatographic-odorgram analysis was performed on 89 frozen and fresh fecal samples obtained from 9 subjects.

Mass-Spectrometric

Analysis

For mass-spectrometric analysis the same initial procedure was performed on the fecal samples as for GC

odor analysis. The stainless steel collector-trap measured 4 mm (ID) x 70 mm and was packed with 200 mg of Tenax absorbent. The GC glass column measured 2 mm (ID) x 1500 mm and was packed with 10% SP 2100 on 80/100 Supelcoport. Column temperature began at 40°C and was then increased at B”C/min to 25O’C and held at the upper temperature for 10 min. Analytic instrumentation included a DuPont DP-102 gas chromatograph/mass spectrometer (DuPont Instruments, Wilmington, Del.) with a scan range/rate of 33-350 atomic mass units at 250 atomic mass units per second and 70 eV electron impact ionization. A Hewlett-Packard 1000 computer utilized software for instrument control and the search capabilities of an Environmental Protection Agency/National Institutes of Health data base containing spectra of 37,000 compounds. Fifty GC/MS analyses were performed on 14 fecal samples obtained from 5 subjects.

Analysis A collected sis. They analyzed

of Frozen

Fecal Samples

total of 78 fecal samples from 6 subjects were and frozen days to several months before analywere then thawed in the Na3P04 solution and as above.

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1987

FECAL ODOR ANALYSIS

Analysis

GC-odor and GUMS columns and analyzed as outlined above. The data provided reference standards for odor, retention time, and mass spectra for these pure compounds.

of Fresh Fecal Samples

A total of 11 fresh fecal samples from 5 subjects were analyzed within 4 h of collection. From 22 to 30 g of sample was placed in the flask without Na3P04 solution or added (NH&S04, unlike the frozen samples. The samples were then purged with helium at 50 ml/min for 40 min. The analysis was performed thereafter as outlined above.

Direct Injection of Feces Two grams 0.5% Na3P04 and homogenate was dichloromethane. down to a volume of the extract was Direct

of Dichloromethane

Analysis

of Pure

Fecallike

Odorants

Ten microliters of methanethiol (Eastman Kodak, Rochester, N.Y.; 99+% purity), 1 ~1 of dimethyl disulfide (Aldrich; 99+% purity], and 1 ~1 of dimethyl trisulfide (Pfaltz and Bauer Inc., Waterbury, Conn.; 97% purity) were each directly injected onto the GC-odor and GUMS columns and analyzed as outlined above. The data provided reference standards for odor, retention time, and mass spectra for these pure compounds.

Direct Injection

of Skatole and Indole

Standard solutions of skatole (Carl Roth; 98+% purity) and indole (Aldrich; 99+% purity) were prepared by dissolving 10 mg of each compound in 100 ml of dichloromethane. Five microliters of the skatole and 5 ~1 of the indole standards were directly injected, onto the

Table

of H2S Production

From

Stool

One-gram aliquots of a frozen fecal sample were placed into each of three 40-ml screw-capped vials equipped with Teflon-faced septums. Two milliliters of distilled water was added to one vial, 2 ml of Na3P04 buffer was added to a second vial, and nothing was added to the third vial. The same procedures were repeated with 5-g aliquots of a fresh fecal sample obtained from a different subject. The fresh samples were kept at room temperature (24°C) and sampled immediately. In each analysis, 1.0 ml of gas overlying the sample was withdrawn, using a gas-tight syringe, and injected directly onto the GUMS column system. The GC column measured 2 mm (ID) x 1500 mm and was packed with 0.1% SP 1000 on Carbopak C. The Carbopak C column was chosen for its ability to resolve sulfur-containing volatiles. The 10% SP 2100 column does not produce adequate resolution between HzS and methanethiol. The analysis was done at 35°C isothermal. The mass spectrometer was scanned from 33 to 150 atomic mass units at 200 atomic mass units per second. The mass spectrum was plotted for mass 34 ion (H,S). The detection limit for H2S by this method is -1 ppm. In addition, a O.l-ml sample of 1000 ppm H2S in air (Matheson Gas Products, East Rutherford, N.J.; 99.9% purity) was directly injected to provide a reference standard for retention time and mass spectra for this compound.

Extract

of frozen stool was thawed in 10 ml of homogenized in a vortex mixer. The extracted three times with 5 ml of The extracts were pooled and dried of 0.5 ml with helium. Two microliters directly injected onto the GC column.

Injection

1323

1. Odor Panel Results Compound and dilution

MeSH undiluted

MeSH

Me&

M+%

Me&

1o-6

undiluted

1o-6

undiluted

Me&

MeSH, Me&, and Me&

undiluted

10-6

1o-6

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

Do you think this

4

6

5

40

0

10

2

70

2

8

2

8

0

10

5

5

odor could possibly represent the odor of a fresh, human fecal sample? 2. Do you think this odor could possibly represent a component of a fresh, human fecal sample?

5

5

7

2”

4

6

4

5”

6

4

6

4

4

6

7

3

Question 1.

No

MeSH, Me&, and Me&

Ten healthy adult subjects were asked to describe the odor characteristics of the above compounds and answer the listed questions. Each sample was smelled directly from vials containing the undiluted compound, a 1Om6dilution of the compound, or undiluted and diluted mixtures of the compounds. The smelling time was -15 s in an odor-free environment. a One subject was unable to detect the odor of this compound at lo-” dilution.

1324

MOORE ET AL.

Panel Testing

GASTROENTEROLOGY

of Odorants

Ten healthy adult subjects (6 women, 4 men; aged 30-51

yr) were asked to describe the odor characteristics of

methanethiol, dimethyl disulfide, and dimethyl trisulfide from vials containing the undiluted compound, lo-"dilutions of the compound in H20, and undiluted and diluted mixtures of the compounds. The subjects smelled uncapped vials of the compounds in an odor-free atmosphere for -15 s. They were asked to describe the odor and to respond to the two questions listed in Table 1.

Results Analysis

of Frozen

Fecal

Samples

The GC odor analytic pattern of frozen fecal samples demonstrated several fecallike odors as illustrated in Figure 2A. Fecallike odors were detected at retention times of
Vol. 93, No. 6

demonstrated, indicating that the compound is probably a dimeric form of dimethyl disulfide (Figure 2C, peak 4). Thus, the major fecal odorants detected in human fecal samples were methanethiol, dimethyl disulfide, and dimethyl trisulfide. At least one of these compounds was detected by GC odor analysis at the appropriate retention time in all 78 frozen samples obtained from 6 healthy subjects on varying dietary intake. Dimethyl trisulfide, dimethyl disulfide, and methanethiol were detected by GC odorgram in 75, 41, and 27 samples, respectively. By mass spectral analysis, compound identification of dimethyl trisulfide, dimethyl disulfide, and methanethiol was confirmed in, respectively, six, nine, and eight of a total of 10 fecal samples examined.

Gas-Chromatographic-Odor-Mass-Spectrometric Analysis for Indole and Skatole The GC odor analytic pattern of fresh and frozen fecal samples consistently revealed two napthalenelike “mothball” odors at I 7 and 19 min as illustrated in Figure 3A (GC odorgram). Directly injected pure indole and skatole produced flame ionization detector responses at retention times of 17 and 19 min, respectively, and elaborated a “mothball” odor identical with that obtained from the fecal sample (Figure 3D, GC odorgram). Mass spectral chemical identity of directly injected pure indole and skatole with the elaborated compounds from feces at 17 and 19 min was demonstrated (Figure 3C, peaks 5 and 6). Thus, although present in feces, indole and skatole do not contribute to the foul odor characteristic of feces. One or both of these compounds were detected by GC odor analysis at the appropriate retention time in 63 of 78 frozen fecal samples obtained from 6 healthy subjects on varying dietary intake. Skatole was detected in 55 and indole was detected in 32 of the 78 samples. By mass spectral analysis, compound identification of indole and skatole was confirmed in, respectively, one and two of a total of two fecal samples examined. >

Figure

2. Gas-chromatographic odor and GUMS analysis of human fecal samples. (A). The GC-odor and GUMS analysis of a single fecal sample. The GC odorgram, left, contains six fecal-smelling peaks at retention times of
FECAL ODOR ANALYSIS

December 1987

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MOORE ET AL.

1326

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GASTROENTEROLOGY

10 15 Rotontlon Tlmr (mlnutod

Vol. 93, No. 6

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Analysis

of Fresh Fecal Samples

Eleven fresh fecal samples from 5 subjects were analyzed (Figure 4). Methanethiol was detected in 10 samples, dimethyl disulfide in six samples, and dimethyl trisulfide in nine samples at the appro-

D.

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priate retention times. All samples had at least one of these compounds by GC odorgram analysis. Skatole was detected in 10 samples and indole in eight samples. All samples had either skatole or indole, or both, at the appropriate retention times. Mass spectral chemical identity of methanethiol elaborated from

December 1987

FECAL ODOR ANALYSIS

1327

47

34

34

20.700

30

60

70

30

m

Retention Time hinutarl

A. GC odorgram fresh stool

B. Single ion chromatogramr H,B and methanethiol

of

C. Mass spectrum peak II

D. Mass spectrum peak #2

Figure 4. Gas-chromatographic-odor-MS analysis of a fresh fecal sample. A. The GC odorgram. B, C, and D. Data obtained from the GUMS analytic systems. A fecal odor, corresponding to the retention time of methanethiol, is obse’rved at the beginning of the run in A. B. Single ion chromatograms obtained from a 0.1% SP 1000 on a Carbopak C column system. The mass spectrum of peak 1 corresponds to H2S (C). The mass spectrum of peak 2 corresponds to methanethiol (D). The mass spectra of H2S and methanethiol have a correlation of -90% with standards.

two fresh fecal samples with the directly injected standard compound was demonstrated (Figure 4). H2S Production

From Feces

A low level of H2S (Cl ppm) was detected only in those frozen or fresh samples placed in water or Na3P04 solution (Figure 4). Panel Testing of Odorants All odorant samples were described by all subjects as foul and disagreeable and all subjects described a fecallike odor for at least one compound (Table 1).However, there was mixed agreement as to whether the odor of some compounds or their mixtures represented the odor of feces or components of feces. In general, the diluted samples were described as more fecallike in odor when compared with the undiluted samples. The odors of methanethiol and dimethyl trisulfide were described as more fecallike

than the odor of dimethyl disulfide. One subject did not detect the odor of diluted methanethiol; a second subject did not detect the odor of diluted dimethyl disulfide.

Discussion The results of this study indicate that volatile methyl sulfides are probably the major chemical constituents responsible for the foul and disagreeable odor of feces. All of these compounds elaborate a foul odor in their pure state although they differ from each other in their qualitative odor characteristics. Thus, the odor of methanethiol was described as “rotten cabbage-gas”, dimethyl disulfide as “pungent-sulfurous,” and dimethyl trisulfide as “rank and nauseating” by one of the authors (L. D. J.). The panel response also indicated different qualitative descriptions of the three odorants. Thus, it is possible that these three volatile compounds, in varying relative concentrations, sufficiently account for the

< Figure 3. Gas-chromatographic-odor and GUMS analysis for indole and skatole in human fecal samples. A. The GC odorgram of a single fecal sample. Note six fecal-smelling peaks and two napthalenelike “mothball” odors detected at 17 and 19 min. B. The 17and IS-min peaks after pure indole and skatole were added to the fecal sample. A stronger “mothball” odor was detected at both retention times. C, left. The GC odorgram of a methylene chloride fecal extract. Note the “mothball” odor detected at 17 and 19 min. The total ion chromatogram, middle, and mass spectrum, right, of the same stool extract show that the “mothball’ odor at 17 min is due to indole (peak 5, mol wt = 117) and the “mothball” odor at 19 min is due to skatole (peak 6, mol wt = 131). D, left. A composite GC odorgram after pure indole and skatole were separately injected onto the GC column. Note the appearance of the “mothball” odor at 17 and 19 min, identical to the odor and retention times of these compounds detected in the fecal sample GC odorgram in A. The total ion chromatogram, middle, and mass spectrum, right, confirm the identity of these compounds with indole (peak 1, mol wt = 117) and skatole (peak 2, mol wt = 131). The more rapid appearance of the compounds in the total ion chromatogram, compared with the GC odorgram, is attributed to a difference in the dimensions of the collector and GC columns employed in the separate analyses (see text).

1328

GASTROENTEROLOGY Vol. 93, No. 6

MOORE ET AL.

varying odor of feces encountered in health, between and within subjects, on varying dietary intakes. It is also possible that H2S gas may make a contribution to the overall foul order of feces because a small amount (Cl ppm) was generated from both fresh and frozen fecal samples. However, the retention time of methanethiol and H2S gas are so similar on the 10% SP 2100 column that any H2S odor generated would probably be dominated by, and indistinguishable from, the odor of methanethiol, which appears to be present in larger concentrations. Other fecallike volatiles, detected by the human olfactory system but undetected by the GC-odor-MS system employed, may contribute to the odor of human feces. Indeed, in Figures 2A and 3A, fecallike odors are observed at retention times other than those corresponding to methanethiol, dimethyl disulfide, and dimethyl trisulfide. These volatiles were not chemically characterized by mass spectrometry. It is known that the human olfactory system is more sensitive than currently available GC/MS analytic systems by at least an order of magnitude (10-12). Hydrogen sulfide, for example, can be olfactorily detected in concentrations as low as 5 x 10-l parts per billion in air, whereas the limit of detection of the GC/MS analytic system described herein is on the order of 1 x lo3parts per billion in air (12). It is possible, therefore, that volatiles other than H2S and the methyl sulfides, undetected by the GC-odor-MS system employed, contributed to the overall odor of feces. The panel response also indicates that there are probably other odorants contributing to fecal odor because, although all agreed that the methyl sulfides were foul in odor, not all agreed that these compounds were fecallike in odor either alone or in combination. The results of this study also indicate that indole and skatole make little or no contribution to the foul order of feces, contrary to the widely held belief that they do (1-4).Both indole and skatole elaborate a napthalenelike “mothball” odor in the pure state and after purging from feces by the GC-odor-MS analytic system described. This odor is distinct from the fecallike odors of the methyl sulfides. It is also unlikely that either ammonia or volatile amines contribute to the odor of urine-uncontaminated fecal samples, because none of the samples elaborated an ammonia odor or a “fishy” odor characteristic of volatile amines, either before or after purging. However, ammonia and volatile amines were not directly analyzed in the GC/MS system described because of the requirement of a strong base on the column phase for trapping and detection of these compounds. Butyric acid elaborates a “rancid-butter” odor that was not detected in the fecal samples by either human olfaction or the MS compound identi-

fication techniques employed in this study. Nevertheless, it is still possible that these compoundsammonia, short-chain fatty acids, and volatile amines-may contribute to the odor of feces under conditions in which they might be expected to be present in high concentrations (e.g., the “rancid” odor of steatorrheic feces or the ammonia odor of urine-contamined feces). The major fecal odorants identified-methanethiol, dimethyl disulfide, and dimethyl trisulfideand H2S arise from both endogenous and exogenous sources. Many enteric bacteria, e.g., Bacteroides species, elaborate a fecal odor in pure culture. Foods provide an exogenous source. Onions, leeks, garlic, peas, beans, cauliflower, cabbage, carrots, potatoes, sprouts, parsnip, rutabaga, tomatoes, corn, cocoa, beer, and coffee have all been found to contain at least one of these compounds (13,14). Using the GC odor analytic system described herein for feces, methanethiol, dimethyl disulfide, and dimethyl trisulfide were detected in cabbage and cauliflower, and methanethiol and dimethyl trisulfide were detected in broccoli. The GC-odor-MS system described represents a largely unexplored method by which fecal samples may be analyzed for specific odors related to dietary intake and, as described herein, for the identification of specific odorants responsible for fecal odor itself. As such, the method has potential application to the forensic and clinical sciences.

References 1. Orton JM, Neuhaus

OW. Nutrition: digestion, absorption and energy metabolism. In: Orton JM, Neuhaus OW, eds., Human biochemistry. 9th ed. St. Louis: C.V. Mosby, 1975:471-Z. 2. Bauer JD. Stool analysis. In: Bauer JD, ed. Clinical laboratory methods. 9th ed. St. Louis: C.V. Mosby, 1982:790. 3. Beeler MF, Freeman JA. Fecal analysis. In: Freeman JA, Beeler MF, eds. Laboratory medicine urinalysis and medical microscopy. 2nd ed. Philadelphia: Lea & Febiger, 1983266 4. Monroe LS. Fecal analysis. In: Berk JE, ed. Bockus gastroenterology. 4th ed., Volume 1. Philadelphia: WB Saunders, 1985:350.

5. Roth JLA. Gaseousness. In: Berk JE, ed. Bockus gastroenterology. 4th ed., Volume 1. Philadelphia: WB Saunders, 1985:146. 6. Levitt MD. Intestinal gas production: recent advances in flatology. N Engl J Med 1980;302:1474-5. 7. Levitt MD, Bond JH. Volume, composition and source of intestinal gas. Gastroenterology 1970;59:921-9. 8. Moore JG, Krotoszynski BK, O’Neill HJ. Fecal odorgrams. A method for partial reconstruction of ancient and modern diets. Dig Dis Sci 1984;29:907-11. 9. Moore JG, Straight RC, Osborne DN, Wayne AW. Olfactory, gas chromatographic and mass spectral analyses of fecal volatiles traced to ingested licorice and apple. Biochem Biophys Res Commun 1985;131:339+6. 10. Jarke F, Dravnieks A, Gordon SM. Organic contaminants in

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1987

indoor air and their relation to outdoor contaminants. Trans Am Sot Heating, Refrigeration, Air Conditioning Engineers 1981:87:153-66. 11. Wright RH. Why is an odour? Nature 1966;209:551-4. 12. Leonardos G, Kendall D, Barnard N. Odor threshold determinations of 53 odorant chemicals. Air Pollut Control Assoc J 1969;19:91-5. 13. Schutte L. Precursors of sulfur-containing flavor compounds.

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In: Furia TE, Bellanca N, eds. Fenaroli’s handbook of flavor ingredients. 2nd ed. Volume 1. Boca Raton, Fla.: CRC Press, 1975:136-7. 114. Shankaranarayana ML, Raghavan B, Abraham KO, Natarajan CT. Volatile sulfur compounds in food flavors. In: Furia TE, Bellanca N, eds. Fenaroli’s handbook of flavor ingredients. 2nd ed. Volume 1. Boca Raton, Fla.: CRC Press, 1975, 186.