Determination of Acyl-Thiolesters by Gas-Liquid Chromatography of Their Sodium Borohydride Reduction Products Edward J. Barron and Larry A. Mooney Virginia Mason Research Center, Seattle, Wash. 98101 THE IDENTIFICATION and quantitative analysis of long chain acyl-thiolesters has usually been accomplished by converting these compounds to their hydroxamic acids. The quantitation is carried out by formation of the ferric-hydroxamate complex and its colorimetric (1, 2) analysis. The identification of the hydroxamates has been obtained by paper chromatography, but these procedures have not been entirely satisfactory in the determination of long chain acid hydroxamates (334).
In order to surmount the problems of identification of thiolesters and to increase the sensitivity of the methods, Vagelos, Vanden Heuvel, and Horning (5) developed a gas chromatographic procedure for derivatives of the hydroxamic acids. In their method, the hydroxamic acids are acetylated and the resulting esters injected into the gas chromatograph where they are converted to the isocyanates by a Lossen type rearrangement. These isocyanates are the derivatives actually separated on the column. We have had difficulty in always achieving quantitative results with this method. In attempting to reduce proposed functional groups in some acyl-CoA derivatives with NaBH4, it was found that NaBH4reduced the thiolesters yielding the long chain alcohol and Coenzyme A. 0
”
RCHZC-SCOA >-
NaBHI
RCHzCHzOH
+ HSCOA
Table I. Relative Retention Time of Alcohols and Derivatives Compound Relative retention time 1.00 Fatty acid-methyl ester 1.5 Alcohol 1.27 Alcohol-acetyl ester 0.48 Alcohol-silyl ether Table 11. Rate of Reduction of Myristoyl-CoA pMoles myristoyl alcohol recovered Time, minutes 2 6 8 10 12 15
Expected value: 0.915
* 0.008
0.592 0.732 0.814 0.915 0.914 0.920
(1) F. Lipmann and L. C . Tuttle, J. Biol. Chem., 159,21(1945). (2) A. Kornberg and W. E. Pricer, Jr., ibid., 204, 329 (1953). (3) E. Wainfan and J. T. Van Bruggen, Arch. Biochem. Biophys., 70, 43 (1957). (4) W. Seubert, G. Greull, and F. Lynen, Angew. Chem., 69, 359 (1957). ( 5 ) P. Roy Vagelos, W. J. A. Vanden Heuvel, and M. G. Horning, Anal. Biochem., 2, 50 (1961).
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ANALYTICAL CHEMISTRY
In general, the explanation for the difference in chemical behavior between the thiolester and 0-ester is based on the knowledge that the divalent sulfur has little tendency to form double bonds and, therefore, resonance structures involving positively charged sulfur are unlikely (6). This results in the carbonylcarbon having significant positive character similar to that found in acyl chlorides-Le., the carbonyl group assumes increased ketone character. Thus, it is not too surprising that NaBH4 reduces the thiolester just as it will reduce acyl chlorides. Because NaBH4 does not reduce normal esters (0-acyl esters), it was apparent that utilization of its reduction of thiolesters, coupled with separation and quantitation of the alcohols by gas-liquid chromatography, could allow the rather specific and sensitive determination of thiolesters. EXPERIMENTAL
Material. The NaBH4 was obtained from Metal Hydrides, Inc. (Beverly, Mass.). The long chain alcohols used for standards were purchased from Applied Science Labs., Inc, (State College, Pa.). The tetrahydrofuran was treated with LiAlH4 (Metal Hydrides, Inc.) and distilled just before use to remove peroxides and a contaminate that chromatographs with approximately the same retention time as a Clz alcohol. The long chain acyl-Coenzyme A derivatives were prepared by the method of Vignais and Zabin (7). The hydroxamates were determined by the method of Goddy, Le Blanc, and Right (8), except 6x neutral hydroxylamine in 9 2 x ethanol was used in forming the hydroxamates from the Coenzyme A derivatives. Myristoyl hydroxamate prepared from either methyl myristate or myristic anhydride was used as the standard. Apparatus. A model 600 Research Specialties gas chromatograph (Warner Chilcott, Inst. Labs., Richmond, Calif.) with a radiation ionization detector utilizing 90Sr was used for the gas-liquid chromatography. The column was a l/r-inch diameter, 4-foot glass column packed with 12% ethylene glycol-succinate on Chromasorb AB (80/90 mesh) and adapted to a direct column injection port. Procedure. The reaction conditions used for a 1-ml aqueous solution (or suspension) of acyl-CoA compounds are as follows: Three-tenths of a ml of tetrahydrofuran is added to help solubilize the long chain acyl-CoA esters. Then, approximately 10 mg of NaBH4 (the amount held on the tip of a small spatula) is added and the sample is reduced for 15 minutes at 38 “C. The reaction is terminated by the slow addition of 0.7 ml of 1N HC1 which destroys the excess NaBH4. The reaction mixture is extracted three times with 1.5 ml portions of CHC1,. (If it is desirous to remove free fatty acids that might be present, the solution is made alkaline before the extraction.) The CHC13 phase is washed one (6) F. Lynen, “Symposium on Enzyme Reaction Mechanisms,” J . Cellular Comp Physiol, Supp. 1, 33 (1959). (7) P. V. Vignais and I. Zabin, Biochim. Biophys. Acta, 29, 263 (1958). (8) R. F. Goddu, N. F. Le Blanc, and C. M. Right, ANAL.CHEM., 27, 1251 (1955).
(1
Alfrnualion
110
I20
I10
Figure 1. Chromatogram o f alcohols obtained by reduction of a biological sample The nmoles of each alcohol in the sample injected is I: myristoyl alcohol, 24.2; 2: palmitoyl alcohol, 4.3; 3: palmitoleoyl alcohol, approximately 0.2; 4: stearoyl alcohol, 2.8; and 5: oleoyl alcohol, approximately 0.7 nmoles
time with water, transferred to a 5-ml test tube and the CHCIJ evaporated off at 40 “C under a stream of NP. The residue is then dissolved in a suitable amount of benzene (depending on the quantities of thiolester originally present and on the dynamic response of the mass detector), and the sample is ready for chromatography. RESULTS AND DISCUSSION Proof of Product. That an alcohol was the product was shown by comparison of the gas-liquid chromatographic behavior of known alcohols and their acetoxy and silyl ether derivatives and that of the products (and their derivatives) of the reduction of the acyl-thiolesters, Table I shows the retention behavior of alcohols and their derivatives relative to the methyl ester of the fatty acid of the same chain length. Selection of Phases and Solvents. Only two solvents, chloroform and ether, were evaluated for extraction of the alcohols. Chloroform was chosen because the partitioning into this solvent appeared to be better with the shorter chain alcohols. Benzene was selected as the carrier solvent for chromatography because the alcohols are readily soluble in it and its boiling point is relatively high. Prevention of evaporation of the carrier solvent is, of course, important in quantitative work where small volumes of solvent (2550 pl) may be used. While there are probably better phases for the gas-liquid chromatography of long chain alcohols than ethylene glycol succinate, this phase was chosen for two reasons: It is the most commonly used phase in laboratories analyzing lipids. It can be used for the simultaneous analysis of alcohols and methyl esters of fatty acids from the same reaction mixture if there are no odd acids present. There is, however, some tailing with this phase. If desired, the tailing characteristics of a column can be improved by injection of the silinating reagent hexamethyldisilazane (9) (Pierce Chemical Co., Rockford, Ill.). Reduction Time. Table I1 shows the rate of reduction of myristoyl-CoA in 30 % tetrahydrofuran. The reduction is complete in about ten minutes. The reduction of myristoyl(9) D. T. Sawyer and J. K. Barr, ibid.,34, 1518 (1962).
Table 111. Comparison of Reduction and Hydroxamate Methods pMolesb Preparations” Reduction Hydroxamate Palmitoyl-Co A 1.04 1.00 Palmitoyl-CoA 0.593 0.660 Myristoyl-CoA 0.970 0.985 Myristoyl-CoA 0.930 0.939 Analysis of different preparations of Coenzyme-A derivatives. pMoles in the volume of sample taken for analysis.
Table IV. Precision Obtained When Different Amounts of Myristoyl-CoA Were Taken for Analysis
Number samples ana1y zed
Mean
A. 8
1.92
B. 5
0.93 14.3
c.
9 D. 6
9.0
Values Found Standard deviation =k 0.047 pmoles 1 0.009 pmoles =!= 0.43 nmoles =k 0.41 nmoles
CoA by NaBH4in water alone takes about two hours. Thus, the use of tetrahydrofuran for solubilizing the long chain acyl-CoA derivatives greatly reduces the time for reduction. We originally used 10% methanol in ether for the reduction solvent, but because it is often preferable to use aqueous media and because of somewhat erratic results, its use was discontinued. Comparison with Hydroxamate Method and Precision of Reduction Method, Table I11 gives a comparison of values obtained by the reduction method and the hydroxamate method on different preparations of acyl-Coenzyme A compounds. The agreement between the two methods is remarkably good in most cases. Data concerning the precision of the method is given in Table IV. The amount of data does not allow a rigorous statistical analysis, but does indicate the possible precision. The mean value applies to the actual amount of the samples taken for reduction and carried through the entire analysis. The area under the peaks was obtained by a planimeter, so in order to reduce the possible error due to this type of integration each reduced sample was chromatographed in duplicate. Sample B was analyzed on the same day and showed that the in run analysis was quite good, yielding a relative standard deviation (IO) of approximately 1 1 % . The other samples were analyzed on separate days, and as would be expected, the precision was less. The relative standard deviation for the A and C samples was approximately =!= 3 %. Sample D, the 9 nmol sample, yielded a relative standard deviation of approximately =k 5 %. Because of the small peaks obtained for the amount injected, a partial explanation for this amount of error may be the inaccuracy of the planimeter. Thus, at the level of about 2 nmol injected sample, the limits of our chromatographic techniques are being approached. Biological Samples. Figure 1 is a chromatogram obtained upon analyzing a biological sample. The enzymes used were a solubilized system from mitochondria which catalyze the chain elongation of fatty acids. Myristoyl-CoA and acetyl-coA were incubated with NADPH and NADH as (10) W. J. Youden, “Statistical Methods for Chemists,” Wiley, New York, N. Y., 1961. VOL. 40, NO. 1 1 , SEPTEMBER 1968
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cofactors in a phosphate buffer pH 7.4. At the end of a 15 minute incubation period, tetrahydrofuran was added to a concentration of 30% by volume, 10 mg of NaBH4 was added, and the reduction was carried out for 1 5 minutes at 38 OC. The extracted alcohols (from basic solution) were taken up in 30 p1 of benzene, and 10 pl were chromatographed. Thus, if tetrahydrofuran in a concentration of 30% or greater will stop the enzymic reaction, a stop analysis of coenzyme A derivatives can be achieved. Limitations. There are several factors which may limit the overall effectiveness of this method. A number of divalent ions, such as Mg2+ and Mn2+, catalyze the decomposition of NaBH4 preventing effective reduction. If they were present in the reaction mixture, the acylWoA derivatives could be precipitated by the addition of acid and the precipitate washed with dilute acid and resuspended in 30% tetrahydrofuran for reduction.
NaBH4 will reduce two functional groups which could be present in the acid moiety of the acyl-thiolesters. If keto groups are present, diols would be produced which do not chromatograph well on EGS ; thus acetoxy derivatives should at least be made initially on the alcohols produced from CoA esters of biological origin. We have found that A2 acylthiolesters are readily reduced to the saturated alcohol under the conditions used. Interestingly enough, the double bond in A2 acids or methyl esters is not reduced under these conditions. Double bonds in fatty acid moiety of the acyl-thiolester further from the carboxyl group are not reduced with these conditions.
RECEIVED for review February 26, 1968. Accepted June 17, 1968. This work was supported by the Department of Health, Education, and Welfare, Grant AM-07806. A portion of this paper was presented at the 154th Meeting, ACS, Chicago, Ill., Sept. 1967.
Determination of Trace Nitrogen in Organic Materials by a Microcombustion Technique J. P. Wineburg' Eastern Laboratory, Explosives Department, E. I . du Pont de Nemours & Company, Gibbstown, N . J .
A MICROCOMBUSTION METHOD for use in the determination of trace nitrogen in organic materials has been described by Norris and Flynn ( I ) . They burned an organic sample over a platinized asbestos catalyst at 550 "C, in a tube swept with oxygen. Combustion products were scrubbed from the gas stream via a helical absorber containing a sulfanilic acid solution. Nitrite, formed during scrubbing, reacted with the sulfanilic acid to form a diazo compound which was subsequently coupled with a solution of 1-naphthylamine to form a red dye (Griess-Ilosvay reaction). The amount of nitrogen in the sample was then determined spectrophotometrically. This paper describes a study of the microcombustion technique. The unmodified method of Norris and Flynn could not be reproduced to yield satisfactory results ; corresponding revisions were made in the colorimetric reagents used, the combustion catalyst, the combustion procedure, and the method of calibration. Nitrogen contents of 0.0005-1 .OO% as nitro, amide, nitrile, amine, and heterocyclic (nicotinic acid) compounds were determined. Errors in the analysis of known mixtures averaged & l o % of the amount of nitrogen added. EXPERIMENTAL
Apparatus. The apparatus is basically the same as that used by Norris and Flynn ( I ) . Platinum combustion boats were A. H. Thomas Cat. No. 8308M. Reagents. Matheson Extra Dry Grade oxygen was used. Ground glass joints were lubricated as indicated with Airco No. 20 lubricant for oxygen service. All other reagents were AR grade or equivalent. Acetanilide and m-nitrobenzoic acid were used as standard organic nitrogen compounds. Sulfanilic acid reagent was prepared by weighing 0.60 ir 0.01 of a gram of sulfanilic acid into a 100-ml volumetric flask and 1 Present address, Fels Research Institute, Department of Chemistry, Temple University, Philadelphia, Pa. 19122
(1) T. A. Norris and J. E. Flynn, ANAL.CHEM., 37, 152 (1965).
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ANALYTICAL CHEMISTRY
dissolving it in 50 ml of water; 20 ml of 3 7 z hydrochloric acid were added, and the solution was diluted to volume with water and mixed thoroughly. The solution was then mixed with 1100 ml of distilled water and 300 ml of 95 ethanol. Naphthylamine hydrochloride reagent was prepared by diluting to volume with water in a 100-ml volumetric 0.60 =k 0.01 of a gram 1-naphthylamine hydrochloride and 1 ml. of 37% hydrochloric acid. Sodium acetate buffer was prepared by weighing 16.4 ir 0.1 grams of anhydrous sodium acetate into a 100-ml volumetric flask, dissolving and diluting to volume with water. Cobalt(I1,III) oxide catalyst was prepared as follows. Eighty-eight grams of oxalic acid dihydrate was mixed with 300 ml of water. This mixture was added slowly with continuous stirring to a solution of 145 grams of cobalt(I1) nitrate hexahydrate in 200 ml of water. The precipitated cobalt oxalate was filtered off using a Buchner funnel and washed thoroughly with water and ethanol. The resulting pasty material was spread over a flat bottom evaporating dish and placed under a stream of air to drive off remaining ethanol. When the odor of ethanol could no longer be detected, the cobalt oxalate was heated in a muffle furnace at 500-600 "C for 2 hours to decompose the oxalate to oxide. The moderately dry powder was then pressed into pellets which were broken carefully to pass through a No. 18 U. S. standard sieve. The cobalt oxide was then transferred to a No. 25 U. S. standard sieve. The combustion tube was packed with the cobalt oxide retained on this sieve (0.7-1.0 mm particles). An 11-cm layer was used. Combustion conditions: long furnace 700 "C, short furnace 600 OC, and oxygen flow 10-25 ml/minute. The packed combustion tube was pretreated by subjecting it to the same procedure described for blanks and samples below but without the absorption coil attached. Blank Determination. Pipet 15 ml of sulfanilic acid reagent into the exit end of the absorption coil and let it drain as far as the coil leading to the inlet joint. Connect the inlet end of the coil to the exit end of the combustion tube without greasing the ball and socket joint. Attach the coil to the flowmeter with rubber tubing. Adjust the oxygen flow rate
z
