Examination of Volatile Metabolites in Plasma Elizabeth Stoner,' David Cowburn,* and Lyman C. Craig3 The Rockefeller University, New York, N. Y. 10021
In studying the volatile metabolites found in the plasma of patients with the uremic syndrome, we have devised a technique for the isolation and concentration of neutral organic volatiles, prior to their examination by gas chromatography. The method described is quick, efficient, and routinely applicable. The study of volatile metabolites presents the two-fold problem-choosing a suitable gas chromatographic procedure, and extraction and concentration. Excess amounts of water and extraction solvents must he removed, and the minute quantities of volatile material separated from the wide variety of other molecules. Several techniques have been reported for the isolation of volatiles, particularly from urine, and examination by gas chromatography and mass spectrometry. A continuous liquid-liquid extraction of urine was coupled with distillation ( I ). Others have condensed the headspace in a cooled trap ( I - 3 ) , or absorbed volatiles on an inert porous polymer, such as Tenax-GC ( 4 - 7 ) . Such methods raise technical problems in the design of apparatus, and in the possibilities of specific adsorption/ desorption. The method described here eliminates some of these problems, and makes use of either standard laboratory equipment or items easily constructed by a glass blower.
the apparatus. The inner column is removed and the residue removed with a 1-ml glass syringe with polyethylene tubing attached. The concentrate is then transferred to 6-mm by 50-mm glass tubes and further evaporated in a slow stream of dry nitrogen a t 4 "C. A final volume of about 100 11 was used, from which 10-11 samples were applied to the gas chromatograph. An F&M Model 402 gas chromatograph with flame ionization detection was used with glass columns of 2-mm i.d. and 2.5-m length, packed with 10% Carbowax 1500 ( l O O / l Z O mesh) obtained from Applied Science Laboratories, State College, Pa. The columns were conditioned for 2 hours at 115 O C with helium as carrier gas, a t 35 ml/min. Samples were injected at an oven temperature of 55 "C, flash heater 85 "C, detector temperature 95 "C. The temperature program was 1) post-injection isothermal at 55 O C for 20 min, 2) linear increase a t 10 "C/min to 115 "C. 3) isothermal a t 115 "C for 60 min. This program minimizes artifactual peaks which can arise in biological samples at high temperature by degradation. A Packard 3000 scintillation counter measured radioactivity samples dissolved in Scintisol (Iso-Lab). Ether was purchased from Mallinckrodt, and redistilled from lithium aluminum hydride (Alfa Inorganics, Beverly, Mass.) through a Vigreux column. Naphthalene was obtained from Fisher Scientific, and used after drying. All other standards were purchased from various sources, and used as received after purity was monitored by GC. 14C-naphthalene (5.1 mCi/mmol), I4C-acetone (26 mCi/mmol), and I4C-benzyl alcohol (4.66 mCi/mmol) were from Amersham Searle, Arlington Heights, Ill.
EXPERIMENTAL
RESULTS AND DISCUSSION
Blood was collected into heparinized evacuated Vacuatainer tubes (No. 4792). Plasma was separated from the formed elements at 4300 g for 20 minutes at 4 "C. T o C Q . 15 ml of plasma, 150 fig of naphthalene was added as an internal standard. Extractions were tested with and without added concentrations of sodium chloride. The use of sodium chloride in the extraction of urine has been widely reported [e.g., ( I ) ] . A thick emulsion is formed in the presence of salt in the extraction of plasma. In this case, extraction in the absence of salt appears to be the desirable method. Qualitatively, the two methods seem to be comparable, but the salt-free extraction is much quicker and slightly more efficient. Extractions were carried out in 50-ml graduated conical glass-stoppered centrifuge tubes. Fifteen milliliters of plasma were shaken with three 20-ml portions of ether, freshly redistilled, using a Vortex mixer. The phases were left to separate, and the upper phase was removed and pooled. Centrifugation is advantageous for the separation of salt emulsions, but does not speed the procedure in the absence of salt. The ether extracts were concentrated on a fractionation column designed in this laboratory (Figure 1).The outer portion of the apparatus consisted of a modified 250-ml Erlenmeyer flask, to which is attached standard size Pyrex tubing. The lower portion holds approximately 100 ml filled t o the base of the neck. The concentric inner column was wrapped with pure nickel wire (B & S gauge 261, and attached to glass protrusions a t the top and bottom of the column. The inner column fits snugly within the outer, and the nickel wire produces an effective refluxing path of more than 380 cm. A large glass syringe, with about 40 cm of 0.034-in. i.d. polyethylene tubing attached was used to fill the bulbs, with the inner column removed. Approximately two thirds of the lower bulb was immersed in a constant temperature water bath at 41 "C, the whole apparatus standing in a fume hood. Concentration proceeded, reducing the volume to about 0.8 ml within 45 min. The glass apparatus is removed from the water bath, and allowed to cool to room temperature for about 2 min, in order to collect residual vapors in
Figure 2 represents a typical chromatogram for a normal subject and Figure 3, a procedural blank. Approximately 20 subjects have been studied by the method described. When blood is drawn from the same subject a t intervals up to several weeks, the chromatograms are very similar, an observation comparable to those on volatile metabolites of the urine, where dietary changes have apparently little effect on the GC profiles (6). The recovery of materials a t different stages of the procedure was measured using gas chromatography or radioactivity of added standards in outdated plasma. In the initial extraction, we recovered in excess of 95% of added naphthalene in 10 minutes, compared to 85% with a liquid-liquid method in 18 hours ( I ) . Three concentration apparatuses have been made, and each calibrated by measuring the recovery of acetone in ether. Acetone is apparently the component of next lower volatility to ether that may be resolved in the GC program. Thus the recovery represents the efficiency of concentration of the most volatile component measured, and presumably less volatile components will be less readily lost. This technique will therefore not detect components more volatile than ether, including acetaldehyde and formaldehyde. This represents a considerable disadvantage compared to other methods proposed. (2, 5, 7, 8 ) . For a standard 100-micromolar acetone solution in ether, approximately 40% remains in the collection bulb, as measured by GC. Using a 15 cm X 0.8 cm Vigreux column. only 14% of the acetone was retained, in a comparable concentration experiment. Thus, this concentration technique is necessary for tolerable yields of metabolite. The concentration step takes about 1 hour. Several techniques were tried, in order to get a final volume of about 100 p1 from about 800 pl. Of the variables, airhitrogen, ambient/4 "C, the
Current address, Albert Einstein School of Medicine, Bronx, New York. * To whom correspondence should be addressed. Deceased. 344
ANALYTICAL CHEMISTRY, VOL. 47, NO. 2, FEBRUARY
1975
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Figure 1. Apparatus for concentration of voiatiles from ether
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Time ( m i n ) Figure 4. Sensitivity of standards in GC procedure Time
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Figure 2. Chromatogram of volatile components from plasma of normal subject
combination nitrogen a t 4 OC gave apparently the greatest reproducibility and efficiency. Using again an acetone standard, there was about a 50% loss in an eightfold concentration. This step takes about 30 min, so the total time from blood drawing to GC was approximately 2 hours. The overall recovery of a model component, benzyl alcohol, was 61%. It is thought that the concentration step compares favorably with normal methods of fractional distillation under vacuum, especially if convenience and simplicity of routine operation are considerations. In the GC, there is measurable bleed during the temperature program, which becomes greater as the column is reused, presumably due to trace volatility of compounds absorbed a t the start of the column. Although other column systems investigated ( e . g . , Carbowax 20 M) avoided the build-up of bleed after exposure to their upper temperature limits, none of the other systems improved the separation of the very volatile components. Therefore effective separation without bleed problems may be best secured by the use of microcolumns, as previously reported ( 1 - 3 ) . The concentration of components detected in this system is in the range 1-100 nanomoles, in the chromatograms. Peak positions and relative detection sensitivities of some standards are presented in Figure 4.Actual structural determination of the 23 or more volatile components partially resolved by this method, in normal and disease states
Range 100, attenuation 1 on instrument described in text. Ambient temperature (55 " C . ) , program: 10°/min to 115 ' C . limit, isothermal at 115 'C. 100 nanomoles of each compound applied
is currently under way, using mass spectrometry and other methods. Several authors (7, 8 ) have proposed urine or plasma screening as a potential "profile" in health and disease, but complexity of apparatus and method is a considerable drawback to routine examination. While recognizing the potential diagnostic value of screening profiles, we have attempted to simplify the approach by group separations prior to chromatography. The method described here represents the examination of the more volatile elements of the non-polar neutral group of metabolites.
ACKNOWLEDGMENT We thank Annabella Bushra for technical assistance, and Balz Gisin for use of GC equipment. LITERATURE CITED (1) A. Zlatkis and H. M. Liebich, Clin. Chem., 17, 592 (1971). (2) R. Teranishi, T. R. Mon, A. B. Robinson, P. Cary, and L. Pauling, Anal. Chem., 44, 18 (1972). (3) K. E. Matsumoto, 0. H. Partridge, A. B. Robinson, L. Pauling, R . A. Flath, R. Mon, and R. Teranishi, J. Chromatogr., 85, 31 (1973). (4) A. Zlatkis, H. A. Lichtenstein, A. Tishbee, W. Bertsch, F. Shunbo, and H. M. Liebich, J. Chromatogr. Sci., 11, 299 (1973). (5) A. Zlatkis, H. A. Lichtenstein, and A. Tishbee, Chromatographia, 6 , 67 (1973). (6) A . Zlatkis, W. Bertsch, H. A . Lichtenstein. A. Fishbee, F. Shunbo. A. M. Coscia, N. Fleischer. and H. M. Liebich, Anal. Chem., 45, 763 (1973).
ANALYTICAL CHEMISTRY, VOL. 4 7 , NO. 2, FEBRUARY 1975
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(7)A. Zlatkis, w. Bertsch, D. A. Bafus, and H. M. Liebich, J. Chromatogr., 91,379(1974). (8)A. Robinson, D. Partridge, M. Turner, R. Teranishi, and L. Pauling, J, Chromatogr., 85, 19 (1973).
RECEIVEDfor review jUly 23, 1974. Accepted October 3, 1974. The study was supported in part by NIAMDD grant AM-02493 to LCC.
I CORRESPONDENCE
I
Measurement of Dissolved Oxygen Concentrations and Diffusion Coefficients by Electron Spin Resonance Sir: The effect of dissolved oxygen on the Electron Spin Resonance (ESR) spectra of free radicals in solution is a familiar phenomenon to ESR spectroscopists. Because oxygen is paramagnetic, oxygen-radical collisions contribute to the magnetic relaxation of the free radical and cause subsequent ESR line broadening ( I ). The magnitude of the line broadening can be related to the frequency ( u ) of oxygenradical collisions ( 2 ) :
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P, YN'
0
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+
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W = K ~ T R ~ , D , +~ R~ ~ ' ~ ~
20 GAUSS
Figure 1. ESR spectra of 3.4 X 10-4M TMP in air saturated solvents at 25 OC Signal gain is presented at left to help visualize the effect of oxygen broadening on peak height. Oxygen solubility decreases from ethylether (0.455 cm3/cm3)to nitrobenzene (0.075cm3/cm3)
(4)
where K = 3.54 X 10-7 gauss sec. This paper is a preliminary investigation to determine the potential usefulness of oxygen broadening to measure the concentration or diffusion coefficient of dissolved oxygen. The method involves obtaining the ESR spectra of free radical solutions prepared from solvents with different oxygen solubilities.
l
ANALYTICAL CHEMISTRY, VOL. 47, NO. 2 , FEBRUARY
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EXPERIMENTAL The free radical 2,2,6,6-tetramethylpiperidinooxy( T M P ) was purchased from Eastman Organic Chemicals (catalog No. 10992) and used without further purification. The structure of T M P is illustrated in Figures l, 2, and 3. A stock solution of 23.3 mg of T M P in 20.0 m l 9 9 mole % hexane was used to prepare subsequent solutions. One ml of stock T M P solution was mixed with 20.0 ml of various reagent grade solvents. The final concentration of T M P in each solution was 3.4 X 10-4M Table I lists the solvents and their reported oxygen solubilities. Air was bubbled through each solution prior to recording the ESR spectrum to ensure air saturation. The ESR machine was a Jeol Company Model JES ME-X ESR spectrometer with X-band microwave unit, 100-KHz modulation, and a TEoll cylindrical cavity. Samples were placed in the cylindrical cavity via a Jeol Company capillary sample cell Model LC-01. All spectra were obtained a t 346
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Figure 2. ESR linewidth ( W ) in gauss plotted vs the oxygen solubility of the solvents The concentration of TMP in each solution is 3 4 X 10-4M Temperature =
25 "C 1975
