Resolution and Detection of a-, p-, and y-Hydroxybutyric Acids in Nanomole Quantities by Gas Chromatography John B. Brooks and Cynthia C. Alley Cenrer for Disease Control, Health Services, and Mental Health Administration. Public Health Service, U.S. Department of Health, Education, and Welfare. Atlanta. Ga. 30333
Lemoigne and coworkers ( I , 2) reported the isolation of a polymer consisting of 6-hydroxybutyric acid from the lipid inclusion of Bacillus spp. Since the initial report of the existence of these polymers several investigators have studied the chemical and physical properties of the polymer ( 3 ) ,its role in storage ( 4 ) , its enzymatic synthesis and breakdown (5, 6), its granular structure ( 7 ) ,and the characterization of poly-@-hydroxybutyrate(8). In addition to their importance as cellular constituents, hydroxy acids are produced by microorganisms during the various processes of metabolism. Brooks e t al. (9) introduced a technique for the preparation and analysis of double derivatives of a variety of metabolically important hydroxy acids by gas-liquid chromatography (GLC). It was demonstrated that hydroxy acids produced by some species of Neisseria in culture media were valuable for distinguishing some members of the genus. Recently M . P. Nuti (personal communications) suggested that a GLC technique capable of detecting and resolving m - , @-, and ?-hydroxybutyric acids in small quantities would be of value for the study of these acids in soil fungi. This study was undertaken to develop GLC techniques suitable for analysis of these acids.
EXPERIMENTAL P r e p a r a t i o n of Acid Standards. Four standards were prepared in a total volume of 2 ml each of Pesticidequality (Matheson, Coleman and Bell) diethyl ether. Mixture No. 1 contained 4.32 X mole of tu-hydroxybutyric acid. 3.43 X mole of $hydroxybutyric acid. and 1.18 X 1 0 ~ mole of y-butyrolactone. Mixture No. 2 contained 4.32 X mole of a-hydroxybutyric acid, 3.43 X 10-a mole of' &hydroxybutyric acid, and 1.18 X mole of y-butyrolactone. Mixture No. 3 contained 2.16 X 10-5 mole of a-hydroxybutyric acid, 1.72 x 10- mole of $-hydroxybutyric acid, and 5.90 X mole of y-butyrolactone. Mixture No. 4 contained 1.18 X n;ole of 7-butyrolactone. P r e p a r a t i o n of Butyl Esters. One-tenth milliliter of the standard acid or mixture of acids was placed in a 12 X 75 m m test tube. One-tenth milliliter of a boron trifluoride (14%) butanol reagent (Applied Science Laboratories. 1nc.j was added and the tube was gently shaken; then most of the ether was removed by evaporation with clean dry air. The tube containing the test mixture was sealed with a cork stopper and tape. Some of the butyl esters were prepared as described ( 1 0 ) ;others were prepared as described ( 1 0 ) except that the butyl ester was extracted with 0.1 M. Lemoigne,Ann. Inst. Pasteur. Paris, 41. 148 (1927) M. Lemoigne. B. Delaporte, and M. Croson, Ann. inst. Pasteur. Paris, 70, 224 ( 1944) D. H . Williamson and J. F. Wilkinson, J . Gen. Microbioi.. 19, 198 (1958). M. Doudoroff and R. Stanier, Nature ( L o n d o n ) ,187, 1440 (1959). J. M. Merrick, F. P Delafield, and M. Doudoroff, Fed. Proc.. Fed. Amer. SOC.Exp. Bioi.. 21. 228 (1962). J. M . Merrick and M. Doudoroff, Nature ( L o n d o n ) . 189, 890 (1961). D. G . Lundgren and R. M. Pfister. J. Gen. Microbioi.. 34, 441 (1964). D. G. Lundgren, R . Alper, C. Schnaitrnan, and R . H. Marchessault, J . Bacterioi.. 89, 245 (1965). J. B. Brooks. D. S. Kellogg, L. Thacker, and E. M. Turner, Can. J. Microbioi.. 18. 157 119721. J. 6. Brooks, D. S.'Kellogg. L. Thacker, and E. M. Turner, Can. J , Microbioi., 1 7 , 531 (1971).
ml of a 1:l5 methanol chloroform mixture, and then washed with 1 N N a O H instead of 0.1N NaOH. All butyl esters were washed with 4% HCI following the basic wash as described (9). During all wash procedures, the volume of the chloroform layer was maintained a t least a t 0.1 ml to avoid sample loss. After the acid wash, the chloroform layer was separated from the aqueous layer by pulling the contents of the test tube u p into a disposable pipet, depositing the chloroform layer containing the butyl ester into a second clean dry 12 X 75 m m test tube, and discarding the aqueous layer. The chloroform layer containing the butyl ester was then reduced, if necessary, to about 0.1 ml by gentle evaporation by air. P r e p a r a t i o n of Electron Capturing (EC) Derivatives. Heptafluorobutyric anhydride (HFBA) derivatives of the butyl esters of the hydroxy acids were prepared by a modification of the technique used to form HFBA derivatives of alcohols and amines (11, 12). As a catalyst for the reaction with HFBA, 0.01 ml of a mixture consisting of one part pyridine (Py) to three parts chloroform was added to the chloroform butyl ester mixture. Then 11 p1 of HFBA were added, and the reaction mixture was gently shaken. The test tube containing the reactants was stoppered with a cork and permitted to sit a t room temperature for 10 minutes; then six to eight drops of chloroform were added with a disposable pipet to prevent excessive loss of the derivatives during washing. The mixture was moderately shaken and 0.07 ml of 4% HCI were added. The test tube was thoroughly shaken to remove acid soluble components from the chloroform layer; then the entire contents of the test tube were drawn up into a disposable pipet. The chloroform layer containing the HFBA-butyl esters was dispensed back into the test tube, and the aqueous supernant was discarded. S e x t , 0.07 ml of either a LV NaOH, 0.LV NaOH, or 4% HC1 were added, and the contents of the test tube were thoroughly shaken to remove components (probably heptafluorobutyric acid) that interfere with subsequent GLC analysis. The test tube was stoppered with a cork and permitted to sit for 30 minutes. The entire contents of the test tube were again drawn u p into a disposable pipet; the chloroform layer was dispensed into a clean dry test tube, and the aqueous layer discarded. The chloroform containing the HFBA-butyl esters was evaporated almost to dryness with a stream of clean, dry air. One-tenth milliliter of diethyl ether was added as a final solvent for the HFBAbutyl esters. The contents of the tube were shaken moderately, and cooled in a freezer for a few minutes to avoid excessive loss of ether through evaporation; then 0.2 to 4 pl of the 100-pl sample was injected onto the gas chromatograph for analysis. After the aliquot was removed for GLC analysis, the remainder of the sample, which was contained in a 12 X 75 m m test tube, was immediately corked, taped, and stored in a freezer. Stability of the HFBA-butyl esters of' CY-, 3 - . and ?-hydroxybutyric acids was checked a t weekly intervals for a s long a s a month. C a s Chromatography Analysis. A Barber-Colman Gas Chromatograph (Model 5000) equipped with a tritium 300-mCi EC detector was used. The instrument contained two glass U-shaped columns (0.6-cm inside diameter by 7.3-m length). One column (nonpolar) was packed with Chromosorb W SO/lOO mesh (AWDMCS H . P . ) coated with 3% OV-1 (Applied Science Laboratories). The other column (polar) was packed with TA 33 Tabsorb (Regis Chemical Co.). The instrument was operated isothermally for 5 minutes a t 90 "C. then programmed for a linear increase of 5 " C per minute to 220 "C. The temperature of both the detector and injector were 220 "C; 32 X l 0 - l " A gave full scale deflection. (11) J . 6. Brooks, C. C. Alley, and Roy Jones, Ana/. Chem., 44, 1881 (1972). (12) J. B. Brooks, W. B. Cherry, L. Thacker. and C. C. Alley, J. infec. Dis., 126, 143 (1972).
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Curve A is a 0.4-pI injection of standard No. 4 and is the response to 15 nrnoles of y-butyrolactone. All four washes were made with 4% HCi. Curve 8 is a 0.4-pI injection of standard No. 4. Curve C is a 4-pl injection of standard mixture No. 3. 1 N NaOH was used in two of the four washes in both B and C. All derivatives were prepared with the same amount of reagent. HFBA = heptafluorobutyric anhydride, E.C. = electron capturing, and h y d . but. = hydroxybutyric
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the response obtained on the TABSORB column from 51.8 nrnoles of a-, 41.2 nmoles of p-, and 14.6 nmoles of y-butyrolactone. Curve B is the response obtained on t h e 3% OV-1 column from 43.2 nmoles of a - , 34.3 nmoies of 3-, and 11.8 nmoles of y-butyrolactone. HFBA = heptafluorobutyric anhydride. E.C. = electron capturing. and hyd. but. = hydroxybutyric Curve A
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and the balance setting was lo4. Nitrogen was used as the carrier gas at a flow rate of 36 cm3/minute. The recorder was operated with a n input signal of 1 mV and a chart speed of 30 inches per hour. Calibration curves were constructed (Figure 1) for the EC detector by plotting peak height as a function of concentration. lMass Spectra. An LKB Model 9000 gas chromatograph-mass spectrometer was used. The resolution of the instrument was about 1000; the temperature of the ion source was 290; the electron energy was 70 eV; the acceleration voltage was 3.5 kV; the 146
scan limits were from 0 to 500; the m / e scan speed was 6 (0 to 500 m/e in 16 seconds); and the UV oscillograph chart speed was 5 cm per second. The gas chromatograph effluent was monitored by a total ion current detector and was equipped with a coiled glass column (0.3-cm i.d. by 3.6 m long). The column was packed with Chromosorb U' 80/100 mesh (AW-DMCS H.P.) coated with 3% OV-1. The gas chromatograph was operated isothermally for 5 minutes at 70 "C; then programmed for a linear increase of 5 "C to 225 "C. Helium was used as the carrier gas with a flow of 36 ml per minute. The recorder was operated with a n input signal of 2 mV (full scale) and a chart speed of 30 inches/hour.
RESULTS AND DISCUSSION Both the 3% OV-1 (nonpolar) column (Figure 2, curve B ) and the Tabsorb (polar) column (Figure 2, curve A ) gave excellent resolution and were highly efficient for the HFBA-butyl esters of a - , /3-, and y-hydroxybutyric acids. The EC detector gave excellent response to the HFBAbutyl esters of these acids. A response of slightly more than one-half scale was obtained when 21.6 nmoles of N hydroxybutyric acid, 17.2 nmoles of P-hydroxybutyric acid, and 5.9 nmoles of y-hydroxybutyric acid were injected onto the gas chromatograph. The response of each acid on the Tabsorb column was about 10% less than that obtained on the OV-1 column. Upon esterification, y-butyrolactone is converted to the butyl ester of y-hydroxybutyric
ANALYTICAL C H E M I S T R Y , VOL. 46, NO. 1 , JANUARY 1974
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acid ( 1 3 ) . We assumed the conversion to be 100%. Since the EC detector responds only to electron contributing compounds, it offers a degree of selectivity because acids must react with HFBA before they are detectable. The EC detector was relatively insensitive to the column bleed from either the 3% OV-1 column or the Tabsorb column, and a flat base line was maintained across the entire temperature span (90-220 "C). The columns were conditioned 12 hours a t 245 "C before their initial use and each morning a t 220 "C for 30 minutes. One of the factors limiting the sensitivity of hydroxy acid detection is the quantity of sample that can be injected onto the gas chromatograph. This quantity is limited not only by the quality of reagents and solvents employed, but by the amount of the reagent used to prepare the derivatives. In the preparation of electron-capturing derivatives with reagents such as HFBA, it is essential not only to control the use of excessive quantities of the reagent but also to be able to clean up the derivative before GLC analysis. A simple wash procedure is desirable for removing components such as heptafluorobutyric acid which is formed not only during the reaction between the anhydride and hydroxyl groups, but also upon exposure of the anhydride to aqueous solutions. In addition to interfering compounds being formed during the reaction, the anhydride may decompose with time if it is exposed frequently to moisture. Old reagent that has been opened many times may not only lose its effectiveness as a derivatizing agent but may also contain components that interfere with GLC analysis. One way to prevent deterioration of the anhydride is to disperse the reagent into small aliquots for general use. Also, the stock reagent should be tested about every two months by preparing derivatives of a known standard and comparing the results against those obtained with reagent that has been recently purchased. An effective washing procedure was devised for removing the interfering agent, a modification of the washing procedure previously described (9, 10). The butyl ester was washed in 1N NaOH instead of 0.1N IVaOH, and the HFBA-butyl ester was also washed with 1N NaOH. We tested the effectiveness of the 1N NaOH wash against a 0.1N wash described (9, 10) and against the same number of washes with 4% HCl. Double derivatives were prepared (13) R. T. Morrison and R. N. Boyd, "Organic Chemistry," 2nd ed., Allyn and Bacon, Boston, Mass., 1966, p 953.
148
from standard solution No. 4 of y-butyrolactone and standard mixture No. 3 consisting of e-,p-, and y-hydroxybutyric acids. The results of the washing procedure are shown in Figure 3, curves A , B, and C. The acid wash (Figure 2, curve A ) was ineffective, and the interfering agent was present when only 0.4 p1 of the sample was injected. The 0.1N NaOH wash (not shown) was better than the acid wash, but not nearly as effective as the 1N NaOH wash. The 1N NaOH wash not only removed the interfering agent from the 0.4-pl sample but also from the 4-p1 injection of the standard mixture No. 3 (Figure 3, curve C). In the 4-pl sample, the quantity of hydroxy acids per volume was reduced, but the amount of reagent per volume has not been diluted. The same amount of reagent was used in all tests. The stability of the double derivative of a - , p-, and y-hydroxybutyric acids was determined by analyzing the derivatives after storage for a month a t -4 "C. The derivatives were stable for a t least this period of time. Graphic representations of the mass spectra are shown in Figure 4, patterns A , B, and C. The presence of the heptafluorobutyryl group is clearly indicative in the double derivatized hydroxy acids by the fragments a t m l e 69, 100, 119, 150, and 169. In like manner, evidence for the esterification with butanol is indicated in the fragment a t m / e 101 which has the possible structure C4HQOC(=O)+. A weak molecular ion peak was present in the double ester of both p- and y-hydroxybutyric acid, but was undetected in the esters of a-hydroxybutyric acid. A weak or missing molecular ion peak is typical of HFBA derivatives of aliphatic alcohols which we have tested and probably indicates the influence of the heptafluorobutyryl group on the fragmentation pattern of the esterified acids. Some of the readily observable differences between the double derivatized acids are: the base peak which is a t m l e 45 in a - and y-hydroxybutyric acids and at m l e 59 in p-hydroxybutyric acid and the fragments a t m / e 57 and 101 in ahydroxybutyric acid which are much stronger in intensity than the 57 and 101 fragments in p- and y-hydroxybutyric acids. Received for review March 19, 1973. Accepted July 25, 1973. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health. Education, and Welfare.
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 1 , J A N U A R Y 1974
