Anal. Chem. 1988, 60,649-651
649
High-Performance Liquid Chromatography-Fluorescence Detection Method for Endogenous y-Aminobutyric Acid Validated by Mass Spectrometric and Gas Chromatographic Techniques C r i s t i n a Suiiol, Francesc Artigas, Josep Ma. Tusell, a n d Emilio Gelpi* Department of Neurochemistry, CSIC, Jorge Girona Salgado 18-26, E-08034 Barcelona, Spain
Endogenous y-amlnobutyrlc acld (GABA) from aqueous (1 M KCVHCI) or organic (methanol) homogenates of rat brain tlssue has been determlned by reversed-phase lsocratlc hlgh-performance liquid chromatography (HPLC) (0.1 M KH,PO,/acetonltrlle, 4:6 at pH 2.8) wlth fluorometrlc detectlon of the corresponding o-phthalaidehyde derlvatlve at 360 nm (excltatlon) and 440 nm (emlsslon). Callbration standards were prepared with deuterlated GABA and/or aminovaierlc acld (AVA). The precision of the method was 3.2% and the detection limit was establlshed at 64 pg of GABA on column allowing the determination of GABA at the level of 1.7 ng/mg of tissue. The content of GABA In brain was 227.2 f 11.1 ( n = 10) pg/g and in cerebellum 110.3 f 13.9 ( n = 10) pg/g. Average recovery was 91.4 f 11.6%. The method was vaMated by comparison of these results wlth those obtalned by analyzlng braln homogenates of 10 rats by caplilary gas chromatography-mass spectrometry, dlrect thermospray hlghpetformancellquld chromatography-mass spectrometry, and gas chromatography wlth electron capture detectlon.
GABA (y-aminobutyric acid) was described in the vertebrate central nervous system as early as 1950, and its role as an inhibitory neurotransmitter was established some years later. It is concerned with the control of neuronal activity in the mammalian central nervous system. Impaired GABAergic transmission appears to be implicated in certain neurological and psychiatric disorders: Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease, alcoholism, etc. Moreover, drugs such as benzodiazepines and barbiturates are known to exert their effects through the GABAergic system (for a review see ref 1). Since then, various chromatographic methods have been reported for the determination of GABA in brain tissue. Some are based on the use of gas chromatography (GC) with electron capture (ECD) or mass spectrometric (MS) detection (2-4); high-performance liquid chromatography (HPLC) has also been used either with UV (5),electrochemical (EC) (6, 7), or fluorescence (F) detection @-IO), although very recently direct HPLC-MS techniques have also been successfully applied for the first time to the determination of endogenous GABA in rat brain (11). Also, a thin-layer chromatography method with fluorescence detection has been reported (12). In all cases, excepting the HPLC-MS method, GABA requires an appropriate derivatization step to render it in a form suitable for detection. The need to form derivatives lends an added complication to the analytical scheme and may lead to increased result variability and lack of accuracy, especially since none of these methods has been properly validated. Usually, validation of GABA determinations has been carried out by simple comparison of the results obtained with those previously reported in the literature, with the exception
of Schmid et al. (2) who compared the suitability of GC-ECD relative to GC-MS for the same samples. Here, we report on the determination of GABA from aqueous or organic homogenates of brain tissue by isocratic HPLC with fluorometric detection (HPLC-F) of the corresponding o-phthalaldehyde derivative. The method has been cross validated by comparison of the results with those obtained by GC-ECD, GC-MS/SIM (selected ion monitoring), and direct HPLC-MS/SIM methods. EXPERIMENTAL SECTION Chemicals. GABA, 5-aminovaleric acid (AVA), and 4aminobutyric-2,2-dzacid (GABA-dz)were from Sigma, Aldrich Chemical Co., and MSD isotopes, respectively. o-Phthalaldehyde (OPA) was from Carlo Erba, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was from Sigma, and pentafluoropropionic anhydride (PFPA) was from Regis. All other reagents were analytical grade. Methanol and acetonitrile were HPLC grade and were from Scharlau (Ferosa, Barcelona) and Koch-Light, Ltd. Animals. Male Wistar rats weighing 140-170 g were purchased from Iffa Credo (Panlab, Barcelona) and were maintained in a constant dark/light cycle of 12 h, with controlled temperature and humidity. Animals were sacrificed by decapitation and the brains were quickly (C45 s) extracted from the skull onto a cold plate (-10 "C) and frozen in liquid nitrogen. Brains were stored at -80 "C until processing the tissue for analysis. Tissue Preparation. Brains were homogenized during 30 s with a Polytron homogenizer (position 5) in 10 volumes of either methanol or an aqueous medium (1M KC1 adjusted at pH 2 with HC1 plus 0.2% sodium bisulfite) (13). Internal standards were added before homogenization in amounts within the range of endogenous GABA (315.2 pg of AVA and 289.0 pg of GABA-d2 per brain sample). The homogenate was centrifuged at 48000g for 20 min and the supernatant was collected. Ten aliquots of each individual brain homogenate were stored at -80 "C until analysis. The whole process was carried out at low temperature (4 "C). Standard mixtures were prepared by adding different concentrations of unlabeled GABA to a fixed amount of the two internal standards used for quantitation (GABA-d2and AVA). Aliquots were stored at -80 "C for daily calibration purposes. Derivatization Procedures. For HPLC with fluorescence detection, the OPA derivative was prepared in accordance with described procedures (14), as follows: 10 pL of homogenate or equivalent standard preparation was mixed with 50 pL of borate solution (pH 10.5) and 100 KLof a solution containing 4 mg of OPA plus 40 pL of ethanethiol in 4 mL of methanol. The reaction was stopped at 90 s by adding 100 pL of acetic acid. In this case the preparation was quickly injected into the chromatographic system, as the OPA derivative is not stable in an acidic medium (half-life N 8 min). For GC with ECD or MS-SIM detection the pentafluoropropionylhexafluoroisopropyl ester was prepared as follows: 10 pL of homogenate or equivalent standard preparation was dried and derivatized with 100 pL of PFPA and 50 pL of HFIP at 60 "C during 1 h. The reaction mixture was gently evaporated to dryness under a stream of helium and dissolved in 1mL of hexane.
0003-2700/88/0360-0649$01.50/0 @ 1988 American Chemical Society
650
ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988
.
*I,
/I
B
Table I. GABA Determination in Rat Brain by Different Methods"
I
I
Ion
Ion
1 0 4 . 0 0 arnu.
106.00 a m u
[GABA],p g / g
re1 std dev, 70
HPLC-F HPLC-MS/SIM GC-MS/SIM GC-MS/SIM GC-ECD (packed) GC-ECD (capillary)
247.6 f 7.9 255.3 f 26.2
3.2 10.3
233.2 f 5.4 243.2 f 14.9 251.2 f 12.0 241.2 f 16.4
2.3
GABA-d, GABA-d,
6.1 4.8 6.9
AVA AVA AVA
internal std AVA
Six aliquots from each of 10 rat brain homogenates were taken for dudicate assavs bv each one of the six different methods.
5
2.0E5-l
U
I : : -
Ion
/iL
0 118.00
am".
2 . BE5
&
0
method
4
T,me
mi---'"0
2
4
6
( m i n . )
Figure 1. Determination of GABA in brain tissue by HPLC-F (1A) and HPLC-MS/SIM (16): 1, GABA; 2, AVA.
Chromatographic Equipment and Procedures. HPLC with Fluorescence Detection (HPLC-F). The HPLC system consisted of a Waters delivery pump, model 590, a manual injector Waters, Model U6K, and a Perkin-Elmer fluorescence detector, Model 650-10s. The chromatographic elution was carried out with a 10-pm reversed-phase column (Spherisorb ODS-1,30 X 0.39 cm, Tracer Analitica, Barcelona) and a mobile phase containing 0.1 M KH2P04at pH 2.8/acetonitrile, 4/6, at a flow rate of 1.5 mL/min. The fluorescence detector was set at 360 and 440 nm for excitation and emission wavelengths, respectively. HPLC-MSISIM. The effluent from a Waters delivery pump, Model 6000 A, equipped with an U6K manual injector was fed to the inlet of a Hewlett-Packard Model thermospray HPLC-MS probe. This interface fitted onto the thermospray ion source of a Hewlett-Packard Model 5988A mass spectrometer. The chromatographic elution was carried out on a 5-pm reverse-phase column (Spherisorb ODS-1, 15 X 0.46 cm, Tracer Analitica, Barcelona) using a mobile phase containing 0.1 M ammonium acetate adjusted at pH 3.7 with formic acid. The ions monitored for GABA quantification were m / t 104 (endogenousGABA) and 106 (internal standard GABA-d2)(11). GC-ECD. A Perkin-Elmer Model 900or KNK-2000-C (Konik Instruments, Spain) equipped with split/splitless injector were used with packed (5% OV-15 in Gas Chrom Q 100/120) or capillary column (BP-1,0.25 pm, 25 m X 0.22 mm i.d. from S.G.E.), respectively. Carrier gas was nitrogen. Column temperatures were 110 "C and 60-130 "C at 4 "C/min for packed and capillary columns, respectively. Injector and detector temperatures were maintained at 250 and 275 "C, respectively. GC-MS j S I M . A Hewlett-Packard 5995 gas chromatograph/mass spectrometer equipped with a splitless injection system was used with a wall-coated open tubular (WCOT) fused silica column (CP-Sil-8 CB, 0.12 pm, 26 m X 0.22 mm i.d.) from Chrompack. Temperature program was from 90 to 150 "C at a rate of 4 "C/min. The ions monitored for quantification were m / z 232 for GABA, 234 for GABA-d2,and 246 for AVA. Injector, transfer line, mass analyzer, and ion source temperatures were 150, 180, 180, and 150 "C, respectively. Analysis of Data. Results are expressed as mean f standard deviation. Statistical comparisons were made by using the one way analysis of variance (ANOVA).
RESULTS AND DISCUSSION GABA was extracted from brain tissue by homogenization with a KC1 solution in the presence of sodium bisulfite and analyzed by HPLC-F. Figure 1A shows the HPLC profile of
a brain extract with the internal standard (AVA) used for the quantification of GABA. The content of GABA (in micrograms per gram) in brain (without cerebellum and pons + medulla oblongata) and in cerebellum determined by this method was 227.2 f 11.1(n = 10) and 110.3 f 13.9 (n = lo), respectively. Both of these values are in line with previously reported data (2,8,10,15)obtained by different experimental approaches. The recovery of GABA following the process of aqueous homogenization was established by adding different amounts of GAI3A (0-350 pg/g) to aliquots of the homogenate which were then analyzed by HPLC-F. The average recovery was 91.4 f 11.6% (n = 7). For samples homogenized in methanol, as proposed by Balcom et al. (15), the values obtained were identical with those obtained by homogenization in an aqueous medium (0.1 M HC1): the ratio GABA (homogenized in methanol)/GABA (homogenized in 0.1 M HC1) was found to be 1.02 f 0.10 ( n = 4). The absolute detection limit of this chromatographic method was established a t 64 pg of GABA on column at a signal to noise ratio of 2. This would allow in practice the detection of 1.7 ng/mg of tissue which represents a 10-fold improvement over a previously reported GC-MS/SIM method (16). The GABA-AVA response obtained with derivatized 10- or 30-pL aliquots of four GABA-AVA different mixtures was plotted vs the GABAIAVA concentration ratios (0.3-1.3). In both cases, straight lines were obtained (slopes 1.67 and 1.72, correlation coefficients of 0.9950 ( n = 12) and 0.9977 (n = 8), respectively). The identical slopes obtained for both concentration ranges and the high correlation coefficients indicate that both GABA and AVA are derivatized to the same extent in the whole range of concentrations assayed. The quantitative determination of brain GABA obtained by the HPLC-F method described above was validated by direct thermospray HPLC-mass spectrometric (HPLCMS/SIM) detection (Figure 1B). In this case the underivatized homogenates were injected into the HPLC column and the eluent was monitored for GABA by thermospray selected ion mass spectrometry. Both HPLC methods gave the same results as the isotope dilution GC-MS determination using deuteriated GABA as internal standard which was taken as the reference method (see Table I). The linearity of response was the same for HPLC-MS/SIM and GC-MS/SIM, as shown by the equations of the best fitted calibration lines (y = 1.00~ - 0.03, n = 16, and y = 1 . 0 0 ~- 0.01, n = 16, respectively, being y the ratio of GABA to GABA-d2 injected and x the response ratio in peak height (HPLC) or area (GC-MS) units) (see also Table 11). The only difference between both mass spectrometric methods was found on the greater relative standard deviation of the HPLC-MS/SIM vs GC-MS/SIM determination (see Table I). This effect could be explained by the greater peak height variability caused by an insufficient dampening of the HPLC pump pulses to which the thermospray interface is particularly sensitive, especially a t low m / z values such as those produced by GABA and GABA-d2. With an appropriate restrictor in front of the HPLC column
ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988
Table 11. Relative Linearity of Response of Different Analytical Methods for GABA
linearity regression
method
internal std used
range of the ratio GABA/int std
HPLC-F HPLC-MS/SIM GC-MS/SIM GC-MS/ SIM GC-ECD (packed) GC-ECD (capillary)
AVA GABA-d2 GABA-dz AVA AVA AVA
0.3-1.3 0.3-1.4 0.3-1.4 0.3-1.3 0.3-0.9 0.3-0.9
0.998 (8) 0.988 (16) 0.982 (16) 0.966 (15) 0.899 (16)
A
B
r2
0.764 (12)
2
:0 0 0 0 9000 8000
1 7000
1
6000
1246)
12321
results showed a significant difference between the methods in Table I (F = 2.614; df = 5, 54; p < 0.05). However, the Scheffe test for the multiple comparison between methods was unable to detect such a difference at the 95% level of confidence. I t must be taken into account that the experimental differences observed between these methods were less than 10% of the corresponding mean values. The linearity of the response for the GABA relative to the internal standard was determined for all methods. Table I1 shows the results obtained. All methods yielded regression coefficients above 0.95 except for GC-ECD. A possible explanation for this fact may lie in the shorter absolute linearity range of this GC detector. Whereas linearity was lost above 240 pg when using the GC-ECD procedure, in the other cases no effort was made to define the higher limit of linearity beyond the actual working range. Furthermore, to check for interassay variability, one extra aliquot of each of the 10 brain samples used to obtain the data shown in Table I was analyzed by HPLC-F with identical results (249.8 f 6.6 pg/g, n = 10) 7 months after the first determinations had been completed, thus proving the reproducibility and precision of this method. Also, as stated above, the accuracy is demonstrated by the excellent intermethod correlation of endogenous values as shown in Table I.
ACKNOWLEDGMENT
ll
&
1
I _ _
0
4
4
8
Time
651
6
(rnin.:
Figure 2. Determinationof GABA in brain tissue by GC-ECD (1A) and GC-MS/SIM (le): 1, GABA; 2, AVA. Numbers in parentheses are ions monitored. The ion at m l z 234 is attenuated by 4-fold.
The skillful assistance of C. Cleries, R. Alonso and J. Abidn is gratefully acknowledged. Registry No. GABA, 56-12-2.
LITERATURE CITED (1) Benzodiazepine IGABA Receptors and Chloride Channels. Structursf and Functional Properties; Olsen, R. W., Venter, J. C., Eds.; Alan R. Liss, Inc.: New York, 1986. (2) Schmid, R.; Karobath, M. J. Chromatogr. 1977, 139, 101-109. (3) Bertilsson, L.; Costa, E. J. Chromatogr. 1978, 118, 395-402. (4) Kapetanovic, I. M.; Yonekawa, W. D.; Kupferberg, H. J. J. Chromatogr. 1987, 414,265-274. (5) Pahuja, S. L.; Albert, J.; Reid, T. W. J. Chromatogr. 1981, 225, 37-45.
the direct HPLC-MS technique could be a useful alternative reference method since it obviates the need for prior sample derivatization as in GC-MS. On the other hand, the relative standard deviation of HPLC-F was of the same order as that of the GC-MS/SIM method. The comparison of the complete mass spectral patterns of endogenous and standard GABA proved that no interfering substances were coeluting with endogenous GABA in the HPLC-MS method. Table I summarizes the results of the determination of brain GABA in a different group of 10 rats by five different methods (HPLC-F (Figure 1A); CG-ECD with packed and capillary columns (Figure 2A); GC-MS/SIM with capillary columns (Figure 2B), and HPLC-MS/SIM (Figure 1B)). For this comparison between different analytical methods, the brain samples were homogenized in methanol, the reason being that a methanol homogenate can be more readily taken to dryness than a purely aqueous medium, thus minimizing losses of GABA upon extended evaporation. GABA was quantitated by using either AVA or deuteriated GABA as internal standards, as indicated in Table I. The ANOVA of these
(6) Caudiil; W. L.; Houck, G. P.; Wightman, R. M. J. Chromatogr. 1982, 227, 331-339. (7) Lasley, S. M.; Greenland, R . D.; Michaelson, I.A. Life Sci. 1984, 35, 1921-1930. (8) van der Heyden, J. A. M.; Korf, J. J. Neurochem. 1978, 31, 197-203. (9) Griesmann, G. E.; Chan, W.-I.; Rennert, 0. M. J. Chromafogr. 1982, 230, 121-124. (10) Herranz, A. S.; Lerma, J.; Martin del Rio, R. J. Chromatogr. 1984, 309, 139-144. (11) Artigas, F.; Gelpi, E. J. Chromatogr. 1987, 394, 123-143. (12) Sharan, S.; Seiler, N.; Grove, J.; Bink, G. J. Chromafogr. 1979, 162, 561-572. (13) Artigas, F.; Gelpi, E. Anal. Biochem. 1979, 92,233-242. (14) Lingeman, H.; Underberg, W.J. M.; Takadate, A,; Hulshoff, A. J. Liq. Chromatogr 1985, 8 789-874. (15) Balcom, G. J.; Lenox, R. H.; Meyerhoff, J. L. J. Neurochem. 1975, 24, 609-613. (16) Holdiness, M. R.; Justice, J. B.; Salamone. J. D.; Neill, D. B. J. Chromafogr. 1981, 225, 283-290.
.
RECEIVED for review June 29, 1987. Accepted November 23, 1987. This work has beep carried out under the Programa Movilizador de Toxicologia of the Spanish Research Council (CSIC) and was supported by CSIC (Grant No. 618/581). Presented at the International Symposium on Pharmaceutical and Biomedical Analysis, Barcelona, Sept 23-25, 1987.
