ANALYTICAL

BIOCHEMISTRY

A Sensitive Acid

and

Method

69, 268-277 (1975)

for

y-Butyrolactone Gas

Quantitation in Brain Chromatography

of Y-Hydroxybutyric by Electron

JOHN D. DOHERTY, 0. CARTER AND ROBERT H. ROTH

Capture

SNEAD,

Departments of Pharmacology, Psychiatry, and Neurology, Yale University School of Medicine, New Haven, Connecticut 06510 Received April 8, 1975; accepted June 4, 1975 A new and sensitive method for the quantitation of y-hydroxybutyric acid (GHBJ and its lactone precursor y-butyrolactone (GBL), has been developed and successfully utilized to determine the endogenous content of these compounds in a single rat brain. The method involves conversion of endogenous GHB into GBL and extracting the GBL with CHCI,. The concentrated CHCI, extract is treated with BF, methanol reagent to produce methyl y-hydroxybutyrate. lntroduction of electron capturing groups was accomplished by further reacting the methyl y-hydroxybutyrate with heptafluorobutyric anhydride in the presence of pyridine. Prior to quantitation by electron capture gas chromatography. the sample was cleaned up by thin layer chromatography (tic) using a preabsorbent plate which removed many extraneous peaks as well as CHCI, used as the solvent. The efficiency of the procedure was evaluated by carrying [ I-W]GBL through the derivatization. This indicated that about 15% of the starting labeled GBL was converted to the final electron capturing product. SValerolactone was used as an internal standard.

y-Butyrolactone (GBL) and its product of hydrolysis, y-hydroxybutyric acid (GHB), are compounds of considerable importance to both microbiologists and neurobiologists. These compounds when administered systemically to humans and test animals exert a variety of pharmacological effects (1) and bacteria appear to have the oxidative enzymes to utilize GHB as a carbon source via conversion to succinate (2). Presently, the methods of analysis for these compounds include the use of the Hestrin reaction (31, which detects esters, and a more specific flame ionization gas chromatographic procedure (4). However, both of these methods have a degree of sensitivity insufficient to estimate the naturally occurring concentrations of GHB in biological samples unless large amounts of material are available. Indeed even the use of flame ionization gas chromatography requires the injection of several hundred nanograms to be quantitative. In spite of the relatively poor sensitivity of existing methods, GHB has been shown to be a naturally occurring metabolite in several mammalian brains present in small but significant 268 Copyright @ 1975 by Academic Press, Inc. All tights of reproduction in any form reserved.

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amounts (l-4 nmoles/g) (4). The possible significance for the presence of GHB as a nervous system regulator in the human has been suggested (15). The development of a more sensitive method of analysis for this compound is desirable since it is essential to demonstrate its presence and regional distribution in human brain before any meaningful hypothesis can be made. Also, a more sensitive method of analysis for GHB would facilitate studies on factors influencing the turnover of this metabolite in experimental animals and the effects of various drugs on its endogenous level. The procedure described here was successfully used to determine the GHB content of a single rat brain and thus is 100 or more times as sensitive as previous methods of analysis (3.4). The method is modified from and superior to a previously suggested procedure developed by Brooks er al. (6) for the detection of various hydroxy acids in spent bacteria culture media. METHODS Preparation of rat brain sample. Rats [male Sprague-Dawley, 300-400 g, obtained from Charles River Co., Boston, Mass.] were sacrificed by decapitation and their brains were rapidly removed and homogenized in ice-cold 1 M HClO, (2 ml/g) and then centrifuged at 17,OOOg for 12 min on a Sorvall centrifuge. The supernatant was heated at SO-85°C for 15 min to convert GHB to GBL (7). After cooling, the solution was adjusted to pH 6 by first adding 1 ml of 0.2 M phosphate buffer (pH 6) and then adding dropwise 2 N NaOH to bring the pH to 6. The solution was then extracted with 2 vol of CHCl, by shaking for 10 min. The two layers were separated and another 2 vol of CHCl, were used for a second extraction. Each time the CHCl, layer was dried by mixing with MgSO, (1 g/l 0 ml of solvent). The pooled CHCl, was condensed to a volume of 500 ~1 by gently blowing N, over the sample maintained at 35-40°C in a water bath. The remaining aqueous phase was reextracted a third time with 4 vol of CHCl, and this extract was carried through the derivatization procedure and used as the sample blank. Preparation qf derivatives. CHCl, extracts from rat brain or GBL standards were adjusted to a volume of 500 ~1 and placed into a 3-ml Microflex tube (Kontes Glass Co., Vineland, New Jersey). To each tube an aliquot containing 2 wg of &valerolactone (DVL) (Aldrich Chemical Co., Milwaukee, Wisconsin) in CHCI, was added. 500 ~1 of 14% BF, methanol reagent (Analabs Inc., North Haven, Conn.) was then added and the tubes were sealed with Teflon lined caps. After standing at room temperature for lo-15 min, the tubes were placed in a water bath at 100°C for 10 additional min. After this time, the tubes were cooled in an

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ice bath and 1 ml of ice-cold distilled water was added to wash away excess methanol and acid catalyst. The vials were vortexed for 15-20 set, the aqueous methanol layer was transferred to a separate tube using a disposable Pasteur pipette, and the CHCl, layer was placed in a clean dry graduated test tube. An additional 0.5 ml of CHCl, was then used to rinse out the original Microflex tube. This same 0.5 ml of CHCl:, was then used to wash the aqueous methanol phase to extract back some of the methyl ester before being pooled with the original CHCl, phase. The pooled CHCl, was condensed to 400 ~1 by gentle N, blowing. Ten microliters of a mixture of 1: 3 parts pyridine to CHCl, was added followed by 10 ~1 of heptaflourobutyric anhydride (HFBA Aldrich Chemical Co., Milwaukee, Wise.). Twenty-five minutes were allowed for this esterification to proceed before the tubes were placed in an ice bath and cooled; 300 ~1 of 1 M NaHCO, were then added and the mixture was washed by mixing on a Vortex mixer for 15-20 sec. The aqueous phase containing excess HFBA and sodium fluorobutyrate and the CHCl, phases were placed into separate tubes and the aqueous phase was washed with 300 ~1 of CHCl,. The pooled CHCl, was condensed by gentle N, blowing to a volume of 200 ~1 before being spotted onto the preabsorbent area of an LQ6DF Quantagram 5 X 20 cm channeled plate (Quantum Industries, Fairheld, New Jersey). CHCl, (50 ~1) was used to wash the tube after all of the final reaction mixture was applied to the plate and this was also spotted. The chromatogram was developed in benzene and ether (4: 1) for 12 cm. In preliminary work in which each 2 cm of this chromatogram was cut out and extracted with ethyl acetate, it was found that only the area about 7 cm from the origin gave positive responses to the presence of GBL. Therefore, in routine work, R, .50 to R, .67 inclusive, were scraped from the plate and placed into a conical centrifuge tube with 1 ml of ethyl acetate and extracted by mixing with a Vortex mixer for 15-20 sec. The tube was then centrifuged at 600- 1000 rpm for 5 min and the clear supernatant was transferred to a graduated test tube. The silica gel was rewashed with 2 additional ml of ethyl acetate, centrifuged, and the supernatant was combined with the first wash, For these studies the ethyl acetate extract was concentrated to 1 ml by gently blowing with a stream of N, and a l-p1 aliquot of this sample assayed gas chromatographically. The ethyl acetate phase can be concentrated to smaller volumes prior to analysis in order to increase the sensitivity of the method and quantitate even lower concentrations than those reported here. A Hewlett-Packard model 57 1OA Gas chromatographic analysis. isothermal gas chromatograph equipped with an electron capture detector that contained a 15-m ci 63Ni plate was used throughout these studies (Hewlett-Packard Co., Avondale, Penn.). Separation was ac-

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complished using a 24-ft long, l/s-in. i.d. glass column packed with 3% OV-1 on Chromsorb-W AW DMCS SO/l00 mesh (Analabs, Inc., North Haven, Conn.). The carrier gas, 5% methane-95% argon (M. G. Scientific, Kearney, N. J.), was set at a flow rate of 36 ml/min. The oven temperature was 125°C and both the detector and injector temperatures were set at 150°C. In order to eliminate drift, the detector may be set at higher temperatures. One-microliter injections were made using a lo-p1 gas-tight Hamilton syringe (Hamilton Co., Reno, Nev.). The column was conditioned at 225°C at least 24 hr prior to use and the temperature of the oven was adjusted to 175°C while the injector and detector temperatures were set at 250°C at the end of the day to recondition the column. Benzene, ether, and ethyl acetate were pretested by injection into the gas chromatograph prior to use. The ethyl acetate was concentrated severalfold but gave no peaks in the critical region of the GBL or DVL response. However, it was often found necessary to redistill the CHCl, prior to extracting the brain tissue in order to effectively remove potentially interfering peaks. Preparation of [‘“Cl GBL from [14C]GHB. [ I-l”C]GHB (SchwarzMann Co., Orangeburg, N. Y.) was obtained as a sodium salt. An aliquot of an aqueous solution was adjusted to pH 1, heated at 80°C for 15 min and then extracted into CHCl,. The CHCl, was condensed to a volume of 3 ml such that 25 ~1 contained 180,000 cpm. The scintillation cocktail used contained 5 g of PPO (2,5-diphenyloxazole), 300 mg of dimethyl POPOP (1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene), 1 liter of Triton X- 100, and 2 liters of toluene. RESULTS Analysis of the procedure using [‘T]GBL and thin layer chroma-tography (tic). Fig. 1 shows that in the chosen tic system [“C]GBL gives a single peak at R, .17 (Fig. 1A) but the mixture following the reaction with BF, methanol reagent gives additional peaks at R, .08 and R, .33 (Fig. IS). The peak at R, .08 is considered to be the methyl ester of

y-hydroxybutyrate based upon the expected polarity and solubility (8) and the fact that it should be the major product resulting from a reaction with an acid (BF,) and excess methanol (9). The small quantity of 14C migrating to R, .33 was not further identified but may be either GHB itself or a y-methyl ether of this compound. Following the reaction with HFBA in pyridine, the peak at R, .08 disappears and a peak at R, .58 appears (part C) and is considered to be the heptafluorobutyric y ester of methyl y-hydroxybutyrate based upon expected migration. Furthermore, in other experiments using unlabeled GBL as starting product, when the chromatogram was cut into 2-cm sections and extracted with ethyl ace-

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Methyl Ester

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: t t FIG. 1. Thin-layer chromatography of the preparation of the electron capturing derivative of y-hydroxybutyrate from [ lJ*C]GBL. Part A shows the migration of [“C]GBL. Part B shows the migration of the products of the reaction resulting from the BF, methanol reaction. Part C shows the migration of radioactivity following derivatization with HFBA. A 25-~1 aliquot of stock [W]GBL was diluted to 500 ~1 and carried through the derivatization procedure as in the Methods section. For each of the two reaction product chromatograms, 50 ~1 of the solution were spotted just after the pooled CHCI, was concentrated. About 5 ~1 of the stock [r*C]GBL were spotted for part A. After developing, 2 cm representing the origin and preabsorbant area were cut out to represent “0”: each l-cm section above the origin was cut out and placed directly into a counting vial with 10 ml of counting solution. In part C of the figure, the unreacted GBL gave its peak at Rf .25 rather than at R, .17 as in parts A and B. In other chromatograms, the [14C]GBL spotted as in part A also peaked at R, .25 leaving a considerable portion tailing in about R, .17. Also for part C, the abscissa shows the response of each 2 cm of the chromatogram indicating the presence or absence of GBL derivative following injection into the electron capture gas chromatograph (-, no response; +, a response).

tate, only sections about R, .58 gave a positive response when analyzed by electron capture gas chromatography. This response was quantitatively related to the amount of GBL originally derivatized. More positive identification of the structure of the electron capturing derivative using mass spectroscopy is in progress. Fig. 1 demonstrates that only about 40% of the GBL was converted to methyl ester, while the second derivatization step appears to be nearly quantitative. Other experiments have demonstrated that as much as 60-650/o of the GBL can be con-

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GBL/DVL

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OF

B. RAT

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Y-BUTYROLACTONE

BRAIN

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BRAIN

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Fro. 2. Gas chromatograms of electron capturing derivatives of GBL, DVL, and rat brain extract and blank. All three represent the injection of 1 ~1 of 1000 PI of ethyl acetate extract from the tic plate. Ah samples contained 2 pg of DVL at the start, therefore. the response shown here to DVL is the equivalent to the injection of less than 2 ng. The GBL standard contained 2 I5 ng GBL at the start of the derivatization. therefore the response shown is the equivalent to the injection of 32 pg (assuming 15% conversion to the final diester product). The attenuation was set at I6 until a few minutes after the GBL peak had passed and then the attenuation was changed to 128, except for the blank in which the attenuation was changed to 64.

verted to the methyl ester if additional heating time is allowed. However the heating time is limited since leakage from the reaction vial results in some loss of product. Studies with [14C]GBL also revealed that 15% of the starting radioactivity could be isolated as final product. The discrepancy between the recovery as final product ( 15%) and the production of methyl ester (40%) likely results from the incomplete recovery of the intermediate reaction products since the reaction with HFBA was shown by tic to be nearly quantitative. Gas chromatographic analysis of standard preparations of GBL and DVL. Fig. 2A, shows a representative chromatogram following the in-

jection of derivatives of GBL and DVL. The DVL derivative gives a peak at 12.5 min and the GBL derivative appears 6.5 min after the first appearance of the solvent front. The peak at approximately 5.5 min is a contaminant dependent upon the lot of HFBA used and appears in the blank (Fig. 2B) also. DVL serves as an ideal internal standard ( IO) since

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I!

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200 GBL

SNEAD, AND ROTH

400

600

600

CONCENTRATION

FIG. 3. Standard curve for the ratio of GBL to DVL. The abscissa represents the quantity in nanograms of GBL in 500 ~1 at the start of the reaction before adding the BF, methanol reagent. All preparations contained 2 Kg of DVL. Each point represents the average of two or four determinations made by injecting only 1 ~1 of 1000 ~1 of the ethyl acetate wash of the tic plate and the vertical lines show the standard deviation (except for the 350 ng preparation where the two experimental values were plotted).

the DVL derivative has a retention time in the gas chromatograph different from the GBL derivative, but has nearly the same migration in the tic precleanup step as does GBL. Figure 3 shows that the ratio for the peak height of the GBL derivative to that of the DVL derivative is linear over the series of concentrations starting at 50 to over 800 ng GBL in 500 ~1 of CHCl,. These concentrations were chosen because the median values shown represent the concentration of GBL in a single rat brain based upon previous reported data (4). Although not shown, the response is linear even at considerably lower concentrations of GBL. When determining the response to a given concentration of GBL, the ratio of the peak heights must always be used because of the inherent errors of the multistep assay and the day-to-day variation in the response of the electron capture detector. In these experiments, the

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peak height response to the derivatives for a given concentration varied, but the ratio provided a reproducible and accurate measure of the concentration of GBL in the initial reaction mixture (Fig. 3). This standard curve was employed in subsequent analyses of samples containing unknown amounts of GBL or GHB. Quantitation of GHB in rat brain. Using the method described above. CHCl, extracts’from single rat brains were carried through the derivatization procedure and a peak corresponding to GBL was obtained (Fig. 2, part C). For the third extraction of the brain sample that was prepared as the blank (see experimental), no peaks that interfered with the quantitation of GHB were present (Fig. 2, part B). Using the response curve in Fig. 3 the GHB content of a rat brain was determined to be 2.06 + 0.14 (mean & standard error) nmoles/g based on seven determinations. This agrees very well with the previously reported value of 1.78 * 0.10 nmoles/g (4). The data using this method were obtained by injecting only 1 ~1 of final 1 ml of the ethyl acetate wash (i.e., l/ 1000 of the extract) whereas for the previous assay method 4-5 ~1 of about 50 ~1 (i.e., about one-tenth of the extract) of concentrated sample were injected (4) and assayed gas chromatographically. DISCUSSION

A modified and much improved method for the estimation of GHB and GBL in nerve tissue has been presented and used successfully to give a value comparable to that previously reported in the literature for the endogenous concentration of this metabolite in rat brain. The method developed here is 100 or more times as sensitive as previous methods including flame ionization gas chromatography combined with isotope dilution techniques. The use of the internal standard (DVL) described here offers a method for quantitating unknown amounts of GHB in a manner that minimizes errors due to the multistep procedure of sample preparation, injection, and other inherent variables associated with electron capture gas chromatography. The tic system introduced here removes many extraneous peaks resulting from both the brain sample and the HFBA as well as eliminates the CHCl, used as the solvent for derivatization. This method offers advantages over the original attempt to prepare a heptafluorobutyric derivative of GBL (6). Firstly the use of methanol in place of butanol provides for the production of more primary ester (methyl y-hydroxybutyrate) to be available for reaction with the fluorinated anhydride. Brown reported previously (8) that in acid media and in the presence of excess alcohol that methanol gave higher yields than other alcohols, but that 100% conversion was never attained. Consistent with this reaction equilibrium, when the synthesis was attempted with

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either methanol, ethanol, propanol, or butanol mixtures of BF,, the methanol derivative always gave the biggest response using the electron capture detector. In one experiment, using butanol as the alcohol, the reaction was evaluated using the flame ionization gas chromatographic procedure for the estimation of unreacted GBL (4). In this instance the response for GBL was nearly the same both before and after treatment with the BF, butanol reagent. The results here and the observations by Brown (8) on the reaction equilibria are in conflict with the assumption of Brooks (6) that the conversion of GBL to butyl y-hydroxybutyrate was complete. The synthesis was also tried using a single step procedure as suggested by Brooks (11) using ethanol as the alcohol, but although a quantitative response to GBL was attained the yield was larger with the two step methanol procedure as described. When the butanol was used, a compound frequently appeared which caused the detector to shoot off scale and require several hours to reequilibrate. It is not known if tic will remove this contaminant. However, when methanol was used as the alcohol no such contaminant was noted whether or not the tic system was used. At present, the disadvantage of the system is the presence of extraneous peaks that apparently result from the individual HFBA preparations depending upon the lot of reagent. One such peak comes about 1 min before the critical time for the GBL derivative. In addition to the peak shown, an additional peak may also appear at the critical time for the response to the GBL derivative, again depending upon the amount and lot of the HFBA used as well as the purity of the CHCI,. Thus, a blank must always be run and any contribution that the blank provides to the response to GBL must be taken into consideration. Studies are now in progress employing the above described method to investigate whether or not GHB occurs endogenously in human and monkey brain and spinal fluid. It is hoped that these studies may provide further insight as to the possible significance and role GHB may play in normal and abnormal brain function. ACKNOWLEDGMENTS This work was supported in part by United States Public Health Service Grant No. MH-14092. The first author is a postdoctoral fellow under training Grant No. P.H.S.-NS-05706.

REFERENCES 1. 2. 3. 4. 5. 6.

Laborit. H. (1973) Progr. Neurobiol. 1, 257-274. Nirenburg, M. W.. and Jakoby, W. B. (196Oj.I. Biol. Chem. 235, 954-960. Bessman. S. P., and Fishbein. W. N. (1963) Nature (London) 200, 1207-1208 Roth, R. H., and Giarman, N. J. (1970) Biochem. Phtrrmucol. 19, 1087-1093. Sprince. H. (1969) Biol. Psychinr. 1, 301-315. Brooks, J. B. and Alley, C. C. (1974) Anal. Chem. 46, 145-348.

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7. Fieser. L. F. and Fieser, M. ( 1961) Advanced Organic Chemistry, p. 574, Reinhold. New York. 8. Brown, H. C., and Keblys, K. (I 966) J. Org. Chrm. 31, 485-487. 9. Metcalfe. L. D., and Schmitz, A. A. (196l)Anul. Chem. 33, 363-364. 0. Dal Nogare. S. and Juvet, R. S.. Jr. (1962) Gas Liquid Chromatography Theory and Practice, pp. 256-257, lnterscience, New York. 1. Brooks, J. B., Alley, C. C., and Liddle, J. A. (1974) Anal. Chem. 46, 1930-1934.