Profile of Volatile Metabolites in Urine by Gas ChromatographyMass Spectrometry Albert Zlatkis, Wolfgang Bertsch, H. A. Lichtenstein, Arye Tishbee, and Farid Shunbo Chemistry Department, University of Houston, Houston, Texas
H. M. Liebich Medizinische Universitaetsklinik, Tuebingen, Germany
A. M. Coscia and N. Fleischer Baylor College of Medicine, Houston, Texas
Profiles of volatile metabolites of 150 urine samples from normal individuals and 40 samples from subjects with diabetes mellitus have been studied by gas chromatography and mass spectrometry. The technique involves adsorption of the urinary headspace volatiles on Tenax GC, heat desorption with helium, trapping on a cooled precolumn, and chromatography on 100-m X 0.50-mm i.d. nickel columns. Individual profiles were observed over a period of 2 months. Characteristic constituents in normal urines are 2-butanone, 2-pentanone, 4-heptanone, dimethyl disulfide, several alkyl furans, pyrrole, and carvone. In subjects with diabetes mellitus under insulin treatment, high concentrations of pyrazines, cyclohexanone, lower aliphatic alcohols, and octanols were found.
Studies of volatile metabolites in human urine have been undertaken with the object of examining qualitative and quantitative variations in the patterns of the metabolites. Gas-liquid chromatography (GLC) using high-efficiency open tubular columns and sensitive specific detectors allows the separation of highly complex samples of urinary components and the monitoring of trace constituents (1). Mass spectrometry (MS) adds information on the qualitative nature of changes in the profile of the metabolites. Among the techniques used to isolate organic volatiles and to prepare samples suitable for gas chromatographic and mass spectrometric analysis, liquid-liquid extraction (2), condensation of the headspace in a cooled trap (3) and adsorption of the volatiles on an inert adsorbent ( I , 4 ) are frequently used. For routine analyses and screening, rapid sampling and analytical procedures are desirable. This paper describes the analysis of volatiles in normal urines and in urines of individuals with diabetes mellitus.
EXPERIMENTAL Sample Preparation. The volatile urinary constituents were trapped on Tenax GC, a porous polymer of 2,6-diphenyl-paraphenylene oxide according to the procedure described by Zlatkis et al. ( 4 ) . A mixture of 100 ml of a 24-hr urine and 30 grams of (NH4)2S04 in a 500-ml flask was heated in a boiling water bath and stirred vigorously with a magnetic stirrer. Lower temperatures yield less volatiles. Helium was passed over the urine at a flow rate of 20 ml/min for 1 hour. After the sampling period, the (1) A . Zlatkis. H. A . Lichtenstein. A . Tishbee. W. Bertsch. F. Shunbo, and H . M . Liebich, J. Chrornatogr. Sci., in press. ( 2 ) A . Zlatkis and H. M . Liebich, Clin. Chern., 17, 592 ( 1 9 7 1 ) . (3) R . Teranishi, T . R. M o n . A . B. Robinson, P. Cary. and L. Pauiing, Anal. Chern.. 44, 18 ( 1 9 7 2 ) . ( 4 ) A . Zlatkis, H . A . Lichtenstein, and A . Tishbee, Chrornatographia, in
press.
glass tube with the volatiles adsorbed on the Tenax GC was stored in the freezer a t -10 "C until the sample was chromatographed. Gas Chromatography. A Perkin-Elmer Model 900 gas chromatograph with flame ionization detectors (FID) was used in this work. The columns were 100-m X 0.50-mm i.d. nickel tubing. A capillary pre-column 3-m x 1-mm i.d. between injector block and analytical column was used as a trap. Both analytical column and pre-column were coated with Emulphor O S 4 7 0 (polyoxyethylated fatty alcohol, Supelco Inc., Bellefonte, Pa.). The sample tube was inserted into the modified injector block which was held at a temperature of 300 "C. The volatiles were desorbed from the adsorbent with helium at a flow rate of 20 ml,'min and trapped on the pre-column using a four-port valve ( 4 j . After a trapping period of 20 min with dry ice as the coolant, the gas chromatographic separation was begun a t room temperature after the container with the dry ice had been removed. The flow rate of the carrier gas (helium) was 3 ml/min. After 15 min. the column temperature was raised to 80 "C and held for 15 min, then programmed up to 190 "C at 2 "C/niin. Hydrogen and air flows for the FID were adjusted to 14 and 50 psi, respectively. All the chromatograms were recorded at an attenuation of 256. Sulfur chromatograms were obtained using a Shimadzu GC-SAPS gas chromatograph. Mass Spectrometry. The GLC/MS analyses were made on a Model 9000 gas chromatograph-mass spectrometer (LKB-Produkter AB, S-161 25 Bromma 1, Sweden) at 70 eV, with an ion source temperature of 250 "C and a separator temperature of 220 "C. The scan speed was 4 seconds for a mass range of 15-200. Mass spectral interpretation was obtained by comparisons with the spectra of known compounds which were available in our laboratory. Sampling, trapping, and GLC conditions were the same as on the Perkin-Elmer instrument except that no valve was used between pre-column and analytical column. The connections were changed manually.
RESULTS AND DISCUSSION Approximately 150 urine samples were studied by this procedure. The technique involves less than 4 hours for sample preparation and GLC separation and can be considered suitable for routine analyses. The good reproducibility of the method has been described previously ( I , 4 ) . Figures 1, 2, and 3a show chromatograms of normal urines. The variance between the urinary profiles of different, individuals is significant, whereas for t,he same individual the profile in urines collected on different days remains remarkably constant (Figure 1). These profiles were compared over a period of 2 months involving dietary changes, however, no characteristic variations in the chromatograms could be observed. Similar, highly constant, and reproducible results were also found in the profiles of sulfur components detected with a specific flame photometric sulfur detector. The chromatograms of the volatile urinary metabolites can be considered characteristic for the individual. In order for long term comparisons to be meaningful, the analytical conditions must be constant, in particular the condition and performance of the column. ANALYTICAL CHEMISTRY, VOL. 45,
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Table 1. Volatile Components in Normal Human Urine Peak 1 2
3
4
5 6 7
a 9
11
12
13
14
16
Compound Acetone 2-Butanone ,Ethanol Propionaldehyde 3-Methyl-2-butanone 2,3-Butanedione 2,3-Dimethylfuran 2-Pentanone 2,4-Dimethylfu:an Chloroform 4- Meth y I- 2-pent anone 3-Methyl-2-pentano~e 2 (?)-Methyl4 (?)-ethylfuran To1uene Dimethyl disulfide 3-Hexanone 2,3,5-'Trimethylturan 5-Methyl-3-hexanone 3-Penten-2-one 1-Methylpyrrole 1-Butanol 4-Methyl-3-penten-2-one (tent.) 4-Heptanone &-Furan Thiolan-2-one (tent.) 2-Heptanone 3-Methylcyclopentanone 6 (?)-Methyl-3-heptanone 3-Octanone Methylpyrazine 2-n-Pentyifuran 2,6- or 2,5-Dimethyipyrazine Allyl isothiocyanate 2,3-Dimethylpyrazine Furfural 2,3,5-Trimethylpyrazine Vinylpyrazine 2-Mzthyl-6-ethylpyrazine Pyrrole 2-Nonanone Acetylf ur an p-Methylpropenylbenzene (tent.) Benzaldehyde P-Pinene (tent.) 2-Methyl-6 (?)-vinylpyrazine 2-Methylpyrrole Dimethyl pyrrole 1-Butylpyrrole (tent.) Carvone Piperitone p-Cresol
Found in distillate of ether extract
+ + +
Peak
1 2
4-
+ + + + + + + +
3
+
A
+
7
+ +
9
K 4
5
B
+
B 11
t
+
+
L
C
+
+ + +
Peak numbers refer to number in the Figures. Identified compounds without number assignments appear between two numbered peaks in the chromatogram in the order gven in the Table. Numbers were not assigned to these components because in some parts of the chromatograms, the correlation between GLC analysis and GLC/MS analysis was not uneauivocal. Sampling a n d analysis techniques applied in t h i s study include adsorption of volatiles a t t h e surface of t h e polym e r a t room temperature a n d desorption a t 300 "C. Possib l e chemical changes of compounds d u r i n g t h e procedure have n o t yet been extensively investigated. However, for comparison of GLC profiles, possible artifacts generated d u r i n g the procedure could be tolerated as long as t h e ana l y t i c a l conditions r e m a i n strictly constant a n d as long as t h e artifacts are reproducibly generated. For M S studies a n d identifications of the metabolites, complementary methods such as liquid-liquid extraction are required t o
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Table I t . Volatile Components in Urine of Subjects with Diabetes Mellitus
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12 M
N N 13 14 D
0 P
a R
16
Compound Acetaldehyde Acetone 2-Butanone Ethanol 2-Propanol Propionaldehyde 3-Methyl-2-butanone 2,3-Dimethylfuran 2,3-Butanedione 2-Pentanone 2,4-Dimethylfuran Chloroform 4-Methyl-2-pentanone 3-Methyi-2-pentanone Toluene 2 (?)-Methyl-5 (?)-ethylturan 7 -Propanol Dimethyl disulfide 3-Hexanone 2,3,5-Trimethylfuran 2-Methyl- 7-propanol 3-Penten-2-one 1-Methylpyrro\e 1 -Butanol 4-Heptanone 3-Heptanone Pyridine P yrazine 2-Methyl- 7-butanol 3-Methyl- 7-butanol 2-Heptanone 3-Methyicyclopentanone Dimethylbenzene 3-Octanone 7 -Pen tan0l 6 (?)-Methyl-3-Heptanone Methylpyrazine 3-Hydroxy-2-butanone 2-n-Pentylfuran Cyclohexanone P icoiine 2.6- or 2,5-Dimethy/pyrazine Allyl isothiocyanate 2,3-Dirnethy/pyrazine Furfural 2-Me th yl-6-ethylpyrazine Vin ylp yrazine 2,3,5-Trimethy/pyrazine Pyrrole 2-Nonanone Benzaldehyde Several isomers with moi wt 730 and a characteristic fragment at 112, probably octanois 1-Octanol Dime th yle th ylp yrazine 2-Methyl-6 (?I -vinylpyrazine C4-Pyrazine C4-Pyrazine Cj-Pyrazine Dimeth ylvin ylp yrazine (tent.) 2-Methylpyrrole 1-Butyipyrroie (tent.) Carvone Piperitone Phenol p-Cresol
13('
Figure 1. Volatile urinary (top); 1190 mi (bottom)
components in
two 24-hr
samples of a male individual collected 4 days apart. Total urine volume, 1210 mi
Figure 2. Profiles of urinary volatiles in 24-hr urines of a normal male, total volume, 960 mi (top): and a normal female, total volume, 900 ml (bottom)
prove that compounds investigated by the adsorption technique are originally urinary components and are also found in samples prepared by a method less likely to involve chemical alterations. Constituents identified in normal urines by mass spectrometry are listed in Table I. The identities of the com-
pounds indicated by the mass spectra were confirmed by comparing the gas chromatographic retention times of the components with those of the authentic compounds, by using reference spectra from the literature, and in some cases by measuring the spectra of the authentic compounds. Most of the substances of Table I were found in ANALYTICAL CHEMISTRY, VOL. 45, NO. 4. A P R I L 1973
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Figure 3. Profiles of 24-hr urines of a normal female, total voiurne, 810 ml (3a, top): and a male subject with adult diabetes on insulin, total volume, 2680 ml (3b, bottom)
lgo
80
varying concentrations in all qf the normal urines investigated. The comparison of the volatiles found by the adsorption method and by the previously described extraction and distillation technique (2) shows similar results for both techniques. Discrepancies can be explained mainly by the higher sample concentration in the adsorption technique, Key components in the profiles of normal urines are ketones, especially 2-butanone, 2-pentanone, and 4-heptanone; also, dimethyl disulfide, several alkyl furans, pyrrole, and carvone. Pyrazines are present in trace quantities. Dimethyl sulfone, a major constituent identified in urine extracts, was not found by the adsorption procedure. A number of isomeric hydrocarbons with molecular weight 134 And a wide range of retention times ( e . g . , peak 10 and peak 15) were not found in other extracts. Approximately 40 urine samples from subjects with diabetes mellitus were investigated. These subjects all had overt carbohydrate intolerance. Some were on no therapy and the remainder were receiving either sulfonylurea or insulin. Several types of urinary profiles were recognized. Figures 36, 4, and 5 depict the profiles of urinary volatiles of diabetics treated with insulin, Figure 6, a-c represent urines of subjects on therapy with sulfonylureas or no therapy. Figures 3b and 4 exhibit in many respects obvious similarities whereas Figure 6, a-c represent a distinctly different group. The chromatogram in Figure 5 is
6p
4
32 MIN.
Figure 4. Profileof a 24-hr urine of a subject with adult diabetes on insulin, total volume, 1640 ml
Figure 5. Profile of a 24-hr urine of a female subject with juvenile diabetes on insulin, total volume, 1240 ml 766
0
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B
F6 I1
so
20
MIN
Figure 6. Profile of 24-hr urines of 3 subjects with diabetes and not on insulin. Total volumes: 840 ml (a, top); 2980 ml ( b , mid-
dle); and 3900 ml (c, bottom)
unique in the sense that most of the regular urinary volatiles are completely covered by new substances. MS analyses of the samples of insulin treated diabetics reveal several characteristic classes of compounds. Table I1 lists all the substances identified. The italicized components must be considered typical of insulin-treated patients either because these substances are not found in normal urines or because they are present in concentrations higher by one or several orders of magnitude in diabetics than in normals. The characteristic components can be classified essentially into four groups with varying ratios: the pyrazines, cyclohexanone, the lower aliphatic alcohols and the compounds with fragment m / e 112, the octanols. Only one subject had large amounts of pyrazines which may be due to other complications. Cyclohexanone and most of the alcohols were also identified in the distillates of urine-extracts of patients with diabetes. In none of the normals or subjects with other diseases which we studied, were the high concentrations of the four groups of compounds found. Aliphatic alcohols could also be identified in samples of diabetic patients who were not treated with insulin, however in smaller amounts (see Figure 6). Further studies are required to investigate the correlations between the volatile metabolites produced in the different forms and stages of diabetes mellitus and in the different forms of treatment. Simpler chromatographic profiles can be obtained by selective detectors (flame photometric, electron capture) and subtractive techniques. This work is now in progress and could lead to easier interpretations of the characteristics of different metabolic states. Received for review December 4, 1972. Accepted January 10, 1973. The authors gratefully acknowledge the support of the National Aeronautics and Space Administration, Life Sciences Directorate, Manned Spacecraft Center, Houston, Texas.
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