Gas chromatography of volatiles from breath and urine

Gas Chromatography of Volatiles from Breath and Urine Roy Teranishi and T. R. Mon Western Regional Laboratory, US.Department of Agriculture, Albany, Gal$ 94710 Arthur B. Robinson, Paul Cary, and Linus Pauling Department of Chemistry, Stanford University, Stanford, Calif. 94305 A technique of direct gas-chromatographic analysis of volatiles from human breath and urine was developed. A valving system permits trapping of volatiles and later release of the concentrated volatiles to a largebore open tubular column. Programmed columntemperature runs, with flame ionization detectors, show the presence of many volatile components in breath and urine.

Figure 1. Breath trap, made from 0.25-in. o.d., 0.20-in. id., stainless steel tubing, 5 ft long

ANALYSIS OF VOLATILES by gas chromatography (GC) has been shown to be useful in indicating age of stored food products ( I ) , differences in varieties of fruit (2,3), changes in emanations from plants from different exposures to light and temperature (4), etc. Sickness in human beings, whether it be from malfunctions of organs and tissues o r from invasions of viruses or microorganisms, would be reflected in qualitative and quantitative changes in the metabolites emitted. It seems that GC methods are now sensitive enough and have enough resolution and reliability to be useful as a diagnostic tool for indications of malfunctions in human beings. This paper describes the application of some of the techniques developed in flavor research to the analysis of volatiles from human breath and urine. EXPERIMENTAL

Equipment. Dual flame ionization detectors (FID) (5) and dual large-bore open tubular columns (6) were constructed in our laboratories. The columns used in these experiments were dual 1000-ft X 0.03-in. i.d., 0.0625-in. o.d., stainless steel tubing, chromatographic grade from Handy and Harman, coated with methylsilicone oil, S F 96(50). Oven and temperature controls were those of Varian Aerograph Model 2100. The column-temperature programming was commenced a t 20 “C, and the column temperature was increased a t the rate of ‘/z “C/min to a limit of 180 “C. The inlet and valve system was maintained at 100 “C, and the detector temperature was 150 “C. The helium, hydrogen, nitrogen, and air flows were 15, 50, 50, and 600 ml/min, respectively. The gases were purified (7), and flows were regulated with restrictors and surge tanks. The efficiency of these columns was found to be over 100,000 theoretical plates with respect t o n-decane, limonene, n-amyl acetate, n-octanal, and nhexanol. Procedure. Human breath volatiles, 10-1 5 exhalations per sample, were first trapped in a coiled stainless steel tubing, 5-ft x 0.25-in. o.d., 0.20-in. i.d. (see Figure 1). The stainless steel coil was cooled in a n isopropyl alcohol-dry ice bath. Care must be exercised to minimize the amount of isopropyl

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alcohol inhaled, in order to decrease the amount of this material showing up in the analysis. Because there is about 0.8 gram of water per 10-exhalation sample, the exhalations should be introduced from the coiled side of trap A, Figure 1, The trap quickly plugs with ice if the exhalations are introduced to the tubing which goes straight down into the coolant B, Figure 1. The volatiles from the large coil were transferred to the gas chromatographic system, as in techniques used in flavor research (3). Because of the large amount of water in breath and urine samples, a larger trap than those usually used in flavor studies was constructed; see Figure 2. With vapor introduction to the 0.10-in. i.d. tube, it is possible to introduce samples with several tenths of a gram of water without plugging. As soon as the volatiles were transferred, the volatiles in the double-helix trap were flashed into the column with a heat gun. Urine volatiles were analyzed directly by passing helium a t 15 ml/min over a 200-ml sample which was magnetically stirred and maintained a t 80 “C; see Figure 3. The volatiles were collected over a 1-hour period in the “double helix” trap maintained at liquid nitrogen temperatures. The volatiles were then flashed into the column as described above.

(1) R. G. Buttery and R. Teranishi, J. A g r . Food Chem., 11, 504 (1963). (2) R . G. Buttery and R. Teranishi, ANAL. CHEM., 33, 1439 (1961). (3) R. A. Flath, R. R. Forrey, and R. Teranishi, J. Food Sci., 34, 382 (1969). (4) A. J. Burbott and W. D. Loomis, Plant Physiol., 42, 20 (1967). (5) R.Teranishi, R. G. Buttery, and R. E. Lundin, ANAL.CHEM., 34, 1033 (1962). (6) R. Teranishi and T. R. Mon, ibid.,36, 1490 (1964). (7) B. 0. Prescott and H. J. Wise, J. Gas Chromatogr., 4 (2), 80 (1966). 18

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ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972

RESULTS AND DISCUSSION

It is recognized that some compounds cannot be successfully analyzed with metal open-tube columns (8). Horning, VandenHeuvel, and Creech (9) and Horning ( I O ) have shown the usefulness of packed glass columns for biochemical research. Novotny and Zlatkis ( I I ) have recently discussed (8) R. A. Flath and R. R. Forrey, J. Agr. Food Chem., 18, 306 (1970). (9) E. C. Horning, W. J. A. VandenHeuvel, and B. G. Creech, “Methods of Biochemical Analysis,” Vol. XI, D. Glick, Ed., Interscience, New York, N.Y., 1963. (IO) M. G . Horning, in “Biochemical Applications of Gas Chromatography,” Vol. 2, Plenum Press, New York, N.Y. 1968, pp 53-86. (11) M. Novotny and A. Zlatkis, Chromatogr. Rev., 14, l(1971).

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Figure 2. "Double-Helix" sample trap in gas chromatograph

Figure 3.

A. Inlet side, 0.125411. o.d., 0.10-in. i.d., stainless steel tubing, wound into helix. B. Silver solder joint from 0.125-in. 0.d. tubing to 0.0625-in. 0.d. tubing. C. Outlet side, 0.0625-in. a d . , 0.040-in. i.d., stainless steel tubing, 10-in. long, wound into helix. D. 3411. E. 1.5-in. F. 0.5411.

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ysis of breath and urine volatiles. The use of the metal columns is a compromise until glass-capillary technology is improved. Use of the flame-ionization detector permits the partial saturation of the carrier gas with water vapor (12). The water vapor reduces the adsorption on the column and there-

the possibilities of glass capillary columns in biochemical analyses, We have used the large-bore open tubular stainless steel columns in these experiments because of their availability, resolution, sample-size tolerance, and general ruggedness. If any method is to be used for many analyses, such as for diagnosis, it must be relatively trouble-free. As glass-capillary technology improves, so that sample size and column life are increased, these columns must be considered for anal-

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Urine volatiles sample holder

A. Urine sample. B. Bar magnet. C. Helium gas in. D. Helium stream out, with urine volatiles. E. Condenser water out. F. Condenser water in. G. Heated water out. H. Heater water in to jacket of sample holder

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Figure 4. Gas chromatogram of breath volatiles from normal human being, 1 0 exhalations. FID. 1000-ft, 0.03-in. i.d., stainless steel open tubular column coated with methylsilicone oil, SF96(50). Temperature programmed from 1 6 "C at 0.05 "/min ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972

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Figure 5. Gas chromatogram of urine volatiles from normal human being, 200 cc sample. FID. 1000-ft, 0.03-in. i.d., stainless steel open tubular column coated with methylsilicone oil, SF 96(50). Temperature programmed from 21 "C at 0S0/min fore reduces tailing and losses. Continuous addition of water also reduces "ghosting"-i.e., the desorption by water injected with a sample of compounds from previous injections that are tightly adsorbed to active sites on the column walls. The reduction of tailing, and therefore the reduction of band broadening, lowers the limit of detection of those compounds that tend to adsorb. However, the large amounts of water in breath and urine present the special problem of introducing the volatiles in a sharp band into the open tubular columns. If excess water is introduced into the double-helix trap, ice will stop the flow of gas. If too much water is introduced into the open tubular column, a plug of water will form and interrupt the flow of carrier gas. Therefore an attempt was made to reduce the amount of water introduced into the gas chromatographic system. This reduction was accomplished by inserting a small condenser between the large breath trap or the urine sample (Figure 3) and the gas chromatographic system. It was noticed in some earlier work with volatiles from oranges that water-soluble compounds of low molecular weight and high volatility condensed with the bulk of water with a cold water condenser. The higher molecular weight compounds,

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ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972

which are less water-soluble, passed along with some water to a colder condenser (13). (Such later observations can be explained thermodynamically.) Forss et al. (14) have shown that volatile compounds from dilute aqueous solutions can be distilled past a condenser which stops most of the water. Figures 4 and 5 show volatiles from human breath and urine, respectively. These chromatograms show considerable detail as to the constituents. These analyses, with those of the neutral-basic fraction and the acidic fraction of urine (IO), should be very useful in following changes in metabolites emitted from hwnan beings. Experiments are under way to establish the identity of the constituents by mass spectrometry and other means of identification.

RECEIVED for review July 19, 1971. Accepted August 12, 1971. (13) T. H.Schultz, R. Teranishi, W. H. McFadden, P. W.Kilpatrick and J. Corse, J. Food Sci.,29,790 (1964). (14) D.A. Forss, V. M. Jacobsen, and E. R. Ramshaw, J. Agr. Food Chem., 15, 1104 (1967).