Determination of Choline and Acetylcholine in Distinct Rat Brain Regions by Stable Isotope Dilution and Field Desorption Mass Spectrometry? W. D. Lehmann and H.-R. Schulten Institut fur Physikalische Chemie, Universitat Bonn, Wegelerstr. 12, 5300 Bonn, W. Germany
N. Schroder Max-Planck-Institut f u r Hirnforschung, Neurobiologische Abteilung, Deutschordenstr. 46, 6000 Frankfurt, W. Germany
The determination of choline and acetylcholine by field desorption mass spectrometry from rat brain tissue samples has been demonstrated. Essential points of the assay are the use of stable isotope labelled internal standards, a simple ion pair extraction procedure, direct analysis of the quaternary ammonium ions without derivatization, and accumulation of the electrically recorded field desorption ion signals with a multi-channel analyser. The analysis of sample amounts in the picogram range gave quantitative data of good precision (0.6-10% standard deviation).
INTRODUCTION The determination of choline and of its physiologically most important ester, acetylcholine, in tissue samples is a broad area of biomedical research and a large number of biological and chemical assays have been developed for this purpose.'.' Focal points of these efforts are, for example, the regional distribution of these compounds in brain and peripheral nerve tissue, the study of the effects of drugs on the tissue concentrations or t h e measurement of turnover rates. The coupling gas chromatograph mass spectrometer is a powerful tool for quantitative organic trace analysis on biological samples, especially when the technique of internal standardization by stable isotope labelled compounds is applied.'-5 Investigations of quaternary ammonium ions using GCMS, however, require a derivatization of these organic cations to obtain neutral derivatives. Demethylation with benzenethiolate, for example, is a recommended derivatization procedure6.' as a result of which tertiary amines are produced that can be separated by GC and analysed by conventional EIMS. Further, for a simultaneous determination of choline and acetylcholine, choline is usually derivatized to form an alkyl e ~ t e r before ~ . ~ demethylation in order to improve the extraction yield and its gas chromatographic properties. In general such a procedure is tedious and time-consuming. Among the novel ionization techniques in mass spectrometry, field de~orption"-'~offers the unique advantage that organic onium ions can be analysed directly without derivatization and that the FD mass spectra show the onium ion as the signal of highest relative intensity.14 This characteristic feature of FDMS stimuI'Quantitative Field Desorption Mass Spectrometry: Part IX; for Part VIII, see Ref. 38).
lated a number of investigations concerning the FD mass spectra of these corn ounds and their tendency for cluster ion formation.R-2 1 The extraordinarily intense FD ion currents of the onium ions enabled field desorption-collisional activationz2 studies to be performed." This study is the first attempt to make quantitative measurements of choline and acetylcholine in brain tissue using a simple ion pair extraction procedure and direct analysis of the ammonium cations by FDMS and stable isotope dilution.
~~~~~
~
EXPERIMENTAL Tissue samples A half-wild rat of 280 g weight was killed by decapitation and the brain was quickly removed. Tissue samples of about 2 0 m g from distinct brain regions were prepared, weighed, and transferred into homogenizators at O'C, each containing 1 ml perchloric acid (4'/0), 10.74 nmol ['H2]choline and 10.11 nmole ['Hg]acetylcholine as internal standards. The sample sizes were as follows: frontal cortex: 38.90 mg; cerebellum: 25.65 mg; colliculus: 26.30 mg; hippocampus superior: 19.00 mg; hippocampus inferior: 22.35 mg; striatum caudatum: 20.25 mg. The samples were homogenized at 0 "C in an ice bath for 30 min. The homogenizate then was spinned in a centrifuge for 20min at 5000 rev min-', 350 p1 of the supernatant was transferred into a 5 ml reacti-vial, and 1.5 ml aqueous Na2HP04 solution (0.5 molar)+ 1 ml solution of dipicrylamine in CH2C12was added and stirred for 20 min with a magnetic stirrer. After spinning to obtain clearly separated phases, the upper aqueous phase was discarded and 1 ml of carbonate buffer solution of pH 9 was added and magnetically stirred for 5 min. The magnetic
CCC-0306-042X/78/0005-0591$02.50 @ Heyden & Son Ltd, 1978
BIOMEDICAL MASS Sf'ECTROMETRY, VOL. 5, NO. 10, 1978 591
W. D. LEHMANN, H.-R. SCHULTEN AND N. SCHRODER
stirrer was removed and the mixture centrifuged for separation of the two phases. Then 700 p l of the lower phase were transferred into a 1 ml reacti-vial, the solvent was blown off under a stream of nitrogen and the residue dried under vacuum conditions. The residue was redissolved in 50p1 absolute methanol and 5 pI aliquots were used for quantitative analysis by FDMS. The steps of the work-up procedure employed, which correspond to the first steps of an assay developed by Eksborg and P e r ~ s o n , ’ are ~ displayed schematically in Fig. 1.
10 p m tungsten wires activated at high temperature.26 The length of the carbon microneedles was 20 pm on average. The F D ion currents were recorded electrically with a secondary electron multiplier and the signals were accumulated by a type CAT-1024 Varian multi-channel analyser. The mass spectrometer multi-channel analyser coupling was achieved by triggering the time averaging computer from the cyclic magnetic scan of the mass ~ p e c t r o m e t e r . ~ ~For ’ ~ ’ all quantitative determinations, the lowest resolution available was adjusted in order to realize maximal transmission and a broad flat-topped peak shape.
Internal standards [2H2]Cholineiodide and [2H9]acetylcholineperchlorate were used as internal standards. Both compounds were prepared following a procedure given by KarlCn el af.” The degree of labelling of the quaternary ammonium ions obtained was determined by FDMS and the use of a multi-channel analyser. The following isotopic distributions were found: [’H2]choline: 1.2% -do; 1.7% d l ; 97.1% - dZ. [2H9]Acetylcholine: 4.9%- d 8 ; 95.1% -d9.
Mass spectrometry The studies were performed on a type 731 Varian MAT double focusing mass spectrometer equipped with a combined EI/FI/FD ion source. F D emitters used were
Brain tissue (about 20 mg)+ 1 ml HC1044% +internal standards at 0°C
Homogenization (30 min) Spinning (20 min)
Supernatant (350p1)+1.5 m10.5 m Na2HP04 (HzO)+dipicrylamine (CH,CI,)
RESULTS A N D DISCUSSION A direct quantitative analysis of choline and acetylcholine in a crude tissue extract appeared promising, because of (i) the ability of FDMS to produce fragment poor or even fragment free mass spectra, (ii) the outstanding sensitivity of FDMS for the detection of organic and inorganic cations” as compared with neutral organic molecules, and (iii) the encoura in results obtained in recent quantitative FD studies.2L-I In these studies it was the use of stable isotope labelled internal standards in particular which enabled quantitative data of good precision and accuracy to be obtained. Furthermore, the frequently observed strong fluctuations of the F D ion currents which hamper the measurement of relative intensities considerably could be compensated by averaging over a large number of cyclic scans. This is most convenient1 accomplished by the use of a multi-channel a n a l y ~ e r ~accumulating ~?~’ the electrically recorded signals of subsequent cyclic scans. This technique was also applied in this study and Fig. 2 shows the beneficial effect of signal accumulation for the determination of FD ion current ratios. The accumulation of such a large number of cyclic scans allows a precise determination of the intensity ratio (m/e 104)/(m/e 106), whereas this cannot be achieved using a single scan plot.
B
Stirring (20 min) Spinning (1 min)
I
Lower phase + 1 mi carbonate buffer pH 9
(b)
Stirring (5 min)
Lower phase (700 pl) dried down under N2and redissolved in 50 pI methanol
FD quantitation of 5 @I aliquots m / e 104, m / e 106for 12Holcholine/[2H21 m / e 146, m / e 155 for [2Ho]acetylcholine/[zH9] Figure 1. Schematic display of the work-up procedure used for the determination of choline and acetylcholine in rat brain tissue by FDMS.
592 BIOMEDICAL MASS SPECTROMETRY, VOL. 5,
NO. 10,
1978
Figure 2. Accumulation of the FD ion currents of choline ( m / e 104) and [2H21choline( m / e 106) with a time averaging computer. (a) Single scati; (b) 200 scans accumulated.
@ Heyden & Son Ltd, 1978
DETERMINATION OF CHOLINE AND ACETYCHOLINE IN RAT BRAIN
The linearity between the molar composition of the sample/standard mixtures and the observed peak height ratios was confirmed by establishing a calibration curve. Two solutions were prepared, containing 107.4 nmol ml-’ choline (solution A ) and 107.4 nmol ml-’ dideuterated choline (solution B) respectively. The calibration curve observed in the investigation of a number of different mixtures of these two solutions is displayed in Fig. 3. The investi ation of model mixtures of [2Ho]acetylcholine and [8H9]acetylcholine also resulted in a linear calibration curve. For the determination of choline and the dideuterated internal standard from the various tissue samples, the mass region from about m / e 100 to m / e 115 was scanned magnetically 50 times for one determination. The mass range was chosen in order to include a signal at m / e 113 corresponding to [2H9]~holine. This species had not been added to the samples but rather originated from hydrolysis of the internal standard [2H9]acetylcholine during the work-up procedure. The extent of hydrolysis observed was finally used to correct the observed intensity for [2Ho]choline for an amount that stemmed from hydrolysis of [2Ho]acetylcholine. Figure 4 shows a typical plot for [2Ho]choline/[2H2]/[2H9] obtained in the analysis of tissue samples. It is remarkable that the detected mass range was free of interfering background ions when an emitter heating current between 10 and 25 mA was used. Under these conditions intense and relatively stable emission of the ammonium ions and, most importantly, constant ion cilrrent ratios were observed. Below 10 mA emitter heating current, some interference of more volatile constituents of the sample mixture occurred and from about 25-30 mA emitter heating current upwards (depending on the diameter of the FD emitter) the observed relative intensities became more or less dependent upon the emitter temperature. In particular,
1.
7H3 H C-N+-CD~--CH~-OH 3 1
1
’i/
CH3 m / e 106
H3
It
HO-CH2-CH2-N-CH 1 CH3
3
m / e 104 I
Figure 4. Molecular ion group of [2H21choline ( m l e 104), [2H21choline( m l e 106). and [2H&holine ( m l e 113) from a rat brain sample (hippocampus superior). Fifty cyclic magnetic scans were accumulated at 1 5 rnA emitter heating current.
the signal at m / e 113 increased rapidly, indicating a thermally induced hydrolysis of the [2H9]acetylcholine standard to [2H9]cholineo n the FD emitter. Table 1 ives the relative abundances for [2Ho]choline/[’H2]/[ Hg] in the tissue samples under investiga-
B
tion all being obtained between 10 and 25 mA emitter heating current. The direct analysis of acetylcholine and its [2H9] analogue also resulted in clear-cut mass spectra. Figure 5 shows a typical plot for the analysis of a tissue extract. Between about 10 and 25 mA emitter heating current intense ion emission and constant ion current ratios could be observed and all determinations were performed in this range. Again, at heating currents greater than 20-25 mA, surface reactions could be observed. In the mass range scanned for [2Ho]acetylcholine/[2H9], an additional signal appeared at m / e 148. This probably corresponds to [2H2]acetylcholine and indicates a thermally induced exchange of the ester moiety between choline and acetylcholine. Table 2 contains the observed relative abundances for acetylcholine and its 12H9]analogue in the same tissue Table 1. Isotopic abundances obtained by FDMS and a multichannel analyser in quantitating choline using [2H,]choline as internal standard in various samples of rat brain tissue Tissue
0
10
20
30
LO
50
50
70
80
90
100
pl solution B added to 100 pI solution A
Figure 3. Calibration curve for the determination of choline by FDMS using [2H2]cholineas internal standard.
@ Heyden & Son Ltd, 1978
Number of measurements
Frontal cortex Cerebellum Colliculus Hippocampus superior Hippocampus inferior Striatum caudatum
2 3 2 3 4 2
Rel. int. m f e 104
24.41t0.9 15.21t1.4 30.21t0.9 19.7~k0.1 22.3+0.8 33.01tl.O
Rel. int. m l e 106
Rel. int. m f e 113
100 100 100 100 100 100
3.9+0.4 5.410.3 12.11t0.6 4.8*0.3 3.1 +0.2 9.61t1.2
BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 10, 1978 593
w.
D. LEHMANN, H.-R. SCHULTEN AND N. SCHRODER ~~
0 H~c-L--CH~-CH
CH
2 1
3
~
Table 2. Isotopic abundances obtained by FDMS and a multichannel analyser in quantitating acetylcholine using [*H,]acetylcholine as internal standard in various samples of rat brain tissue
!
-AH 1 3
~
I
m / e 1L6
Tissue
I
Frontal cortex Cerebellum Colliculus Hippocampus superior Hippocampus inferior Striatum caudatum
Figure5. Molecular ion group of [ZHo]acetylcholine( m / e 146) and [2H9]acetylcholine ( m / e 155) from a rat brain sample (hippocampus inferior). Fifty cyclic magnetic scans were accumulated at 22 mA emitter heating current.
4 3 3 3 5 2
Rel. int. rnle 146
Rel. int. rnle 155
5.8k0.4 1.4kO.05 9.5k 0.6 4.9 0.3 4.9k0.2 5.210.2
*
100 100 100 100 100 100
Table 3. Tissue concentrationsof choline and acetylcholine in various rat brain regions calculated from the ion abundances displayed in Tables 1 and 2 Concentration Tissue
samples as listed in Table 1 for the choline determination. The tissue concentrations for acetylcholine were calculated directiy from the peak height ratios displayed in Table 2. The observed peak heights for [*Ho]choline at m / e 104, however, had to be corrected for a certain amount of choline originating during the work-up procedure as described above. The calculated values for the choline and acetylcholine tissue concentrations are summarized in Table 3. The concentration data found in this FD study generally show good agreement with a number of previous studies using G C and EIMS.42-44For instance, a significantly low value for acetylcholine in the cerebellum was found in all of these investigations. Additionally, the data obtained show a good precision (between 0.6 and 10% on a relative basis), although the ratio sample to standard deviated considerably from unity especially for the acetylcholine determinations. The clear results obtained can be ascribed mainly to the extraordinarily high sensitivity of FDMS for the organic cations under investigation. Using a freshly prepared FD emitter an ion current lasting several hours could be obtained when a 5 pl aliquot of the extract was applied to the FD emitter. Since the accumulation of 50 scans was performed in c. 10 min it can be estimated that the signals for acetylcholine, one of which is shown in Fig. 4, represent between 2 and 10 pg of the quaternary ammonium ion. Thus, the assay shows an impressive sensitivity even when it is compared with novel GCMS procedure^.^^^' Simultaneously, high specificity is achieved by direct analysis of the quaternary ammonium
Number of measurements
Frontal cortex Cerebellum Colliculus Hippocampus superior Hippocampus inferior Striatum caudatum
choline nmol g-’ fresh weight
61.6k2.4 56.6* 5.7 110.8 k 3 . 5 100.4+0.6 97.813.7 161.4k 5.1
Concentration acetylcholine nmol Q fresh weight
14.3*1.0 5.2 i 0.2 34.7 2.2 24.8 i 1.5 21.1 10.8 24.7* 1.1
*
ions avoiding, for example, interference from endogenous tertiary amines which is a possible source of error when analysing the demethylation product of ~ h o l i n e . ~ ~ Finally, the use of two differently labelled internal standards enables the data to be corrected for the influence of hydrolysis during the work-up procedure. In conclusion, the combined use of ion pair extraction, stable isotope dilution and field desorption mass spectrometry is a highly sensitive and specific procedure for the quantitation of choline and acetylcholine in tissue samples. Since the required work-up procedure is simple and the mass spectrometric detection of the ammonium ions by F D is very sensitive, the analysis of much smaller sample sizes or considerably lower concentrations than investigated in this study appears possible.
Acknowledgements This work was financially supported by the Deutsche Forschungsgemeinschaft, the Ministerium fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen, and the Fonds der Deutschen Chemischen Industrie. We wish to thank Dr H. M. Schiebel. Technische Universitat Braunschweig, for his encouraging advice and fine collaboration.
REFERENCES 1. D. J . Jenden and L. B. Campbell, in Methods of Biochemical Analysis, ed. by D. Glick, Vol. 18, p. 183. Wiley, New York (1971). 2. I. Hanin (ed.), Choline and Acetylcholine: Handbook of Chemical Assay Methods, Raven Press, New York (1974). 3. A. P. De Leenheer and R. A. Roncucci, (eds.), Quantitative Mass Spectrometry in Life Sciences, Elsevier Scientific, Amsterdam (1977).
594 BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 10. 1978
B. J . Millard, Quantitative Mass Spectrometry, Heyden, London (1978). 5. W. D. Lehmann and H.-R. Schulten. Angew. Chern. Int. Ed. Engl. 17,221 (1978). 6. D. J . Jenden, I. Hanin and S. I. Lamb, Anal. Chern. 40, 125 (1968). 7. I. Hanin and D. J . Jenden, Biochem. Pharmacol. 18, 837 (1969).
4.
@ Heyden & Son Lid, 1978
DETERMINATION OF CHOLINE AND ACETYCHOLINE IN RAT BRAIN 8. D. J. Jenden, R. Booth and M. Roch, Anal. Chem. 44, 1879 ( 1972).
9. I. Hanin, R. Massaralli and E. Costa, J. fharmacol. Exp. Ther. 181,10 (1972). 10. H. D. Beckey, lnt. J. Mass Spectrom. Ion Phys. 2,500 (1969). 11. H. D. Beckey and H.-R. Schulten, Angew. Chem. Int. Ed. Engl. 14,403 (1975). 12. H.-R.Schulten, in Methods of BiochemicalAnalysis, ed. by D. Glick, Vol. 24, p. 313.Wiley and Sons, New York (1977). 13. H. D. Beckey, Principles of Field Ionization and Field Desorption Mass Spectrometry, Pergamon Press, Oxford (1977). 14. D. A. Brent, D. J. Rouse, M. C. Sammons and M. M. Bursey, Tetrahedron Left. 4127 (1973); M. C. Sammons, M. M. Bursey G. W. Wood, J. and C. K. White, Anal. Chem. 47,1165 (1975); M. Mclntosh and P.-Y. Lau, J. Urg. Chem. 40.636 (1975). 15. G. W. Wood and P. Y. Lau, Biomed. Mass Spectrom. 1, 154 (1974). 16. M. C. Sammons, M. M. Bursey and D. A. Brent, Biomed. Mass Spectrom. 1, 169 (1 974). 17. H.-R. Schulten and F. W. Rollgen, Org. Mass Spectrom. 10, 649 (1975). 18. H.-R. Schulten and F. W. Rollgen,Angew. Chem. Int. Ed. Engl. 14,561 (1975). 19. H. J. Veith, Org. Mass Spectrom. 11,629(1976). 20. G. W. Wood, P.-Y. Lau and G. N. Subba Rao, Biomed. Mass Spectrom. 3, 172 (1976). 21. H. Ogino, T. Matsumura, K. Satouchi and K. Saito, Biomed. Mass Spectrom. 4. 326 (1977). 22. K. Levsen and H. Schwarz, Angew. Chem. lnt. Ed. Engl. 15, 509 (1976) 23. H. H. Gierlich, F. W. Rollgen, F. Borchers and K. Levsen, Urg. Mass Spectrom. 12,387(1977). 24. S. Eksborg and B. A. Persson, Acta Pharm. Suec. 8, 205 (1971). 25. B. Karlen, G. Lundgren, 1. Nordgren and B. Holmstedt, in Choline and Acetylcholine: Handbook of Chemical Assay Methods, ed. by I. Hanin, p. 163. Raven Press, New York
(1974). 26. H.-R. Schulten and H. D. Beckey, Org. Mass Spectrom. 6,885 (1 972). 27. W. D. Lehmann and H A . Schulten, Anal. Chem. 49, 1744 (1977). 28. H.-R. Schulten, Cancer Treat. Rep. 60, 501 (1976). 29. 1. Jardine, M. N. Kan, C. C. Fenselau, R. Brundrett, M. Colvin, G. Wood, P.-Y. Lau and R. Charlton, in Proceedings of the Second International Symposium on Stable Isotopes, Oak Brook Illinois, October 1975, p. 138. National Technical Information Service, US Department of Commerce, Springfield, Virginia, ERDA CONF-751027(1976).
@ Heyden & Son Ltd, 1978
30. W. D. Lehmann, H. D. Beckey and H A . Schulten,Anal. Chem. 48, 1572 (1976). 31. W. D. Lehmann, H. D. Beckey and H.-R. Schulten, in Quantitative Mass Spectrometry in Life Sciences, p. 177.Elsevier, Amsterdam (1977). 32. H A . Schulten, W. D. Lehmann and M. Jarman, in Quantitative Mass Spectrometry in Life Sciences, p. 187.Elsevier Amsterdam (1977). 33. S.Pfeifer, H. D. Beckey and H.-R.Schulten, Z. Anal. Chem. 284,193(1 977). 34. W. D. Lehmann and H.4. Schulten, Angew. Chem. Int. Ed. Engl. 16,184(1977). 35. W. D.Lehmann and H.-R. Schulten, Biomed. MassSpectrom. 5,208 (1 978). 36. W. D. Lehrnann and H . 4 . Schulten, Angew. Chem. Int. Ed. Engl. 16,852(1977). 37. W. D. Lehmann, H A . Schulten and H. M. Schiebel, Z. Anal. Chem. 289,ll (1978). 38. H.4. Schulten, R. Ziskoven and W. D. Lehmann, Z. Naturforsch. Teil C 33,178 (1 978). 39. H . 4 . Schulten, U. Bahr and W. D. Lehmann, Biomed. Mass Spectrom. 5,536 (1 978). 40. H.-R. Schulten and W. D. Lehmann, in Quantitative Mass Spectrometry in Life Sciences 11, ed. by A. P. De Leenheer, R. Roncucci and C. van Peteghem, Elsevier Scientific, Amsterdam (1978). 41. H.-R. Schulten, R. Ziskoven and W. D. Lehmann, Z. Naturforsch. Teil C 33, 178 ( 1978). 42. L. B. Campbell and D. J. Jenden, J. Neurochem. 17, 1697 ( 1970). 43. W. B. Stavinoha, S. T. Weintraub and A. T. Modak, J. Neurochem. 23,885 (1974). 44. S.1.Weintraub, A. T. Modak and W. B. Stavinoha, Brain Res. 105,179 (1976). 45. D.J. Jenden, M. Roch and R. A. Booth.Ana1. Biochem. 55,438 (1973). 46. W. M. Einolf and C. Fenselau, Biomed. Mass. Spectrom. 1, 195 (1974). 47. J. Shabanowitz, P. Brynes, A. Maelicke, D. V. Bowen and F. H. Field, Biomed. Mass Spectrom. 2,164 (1975). 48. D. J. Jenden and I. Hanin, in Choline and Acetylcholine:
Handbookof Chemical Assay Methods, ed. by I. Hanin, p. 135.Raven Press, New York (1974).
Received 6 April 1978 @ Heyden & Son Ltd, 1978
BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 10, 1978 595
N. Schroder Max-Planck-Institut f u r Hirnforschung, Neurobiologische Abteilung, Deutschordenstr. 46, 6000 Frankfurt, W. Germany
The determination of choline and acetylcholine by field desorption mass spectrometry from rat brain tissue samples has been demonstrated. Essential points of the assay are the use of stable isotope labelled internal standards, a simple ion pair extraction procedure, direct analysis of the quaternary ammonium ions without derivatization, and accumulation of the electrically recorded field desorption ion signals with a multi-channel analyser. The analysis of sample amounts in the picogram range gave quantitative data of good precision (0.6-10% standard deviation).
INTRODUCTION The determination of choline and of its physiologically most important ester, acetylcholine, in tissue samples is a broad area of biomedical research and a large number of biological and chemical assays have been developed for this purpose.'.' Focal points of these efforts are, for example, the regional distribution of these compounds in brain and peripheral nerve tissue, the study of the effects of drugs on the tissue concentrations or t h e measurement of turnover rates. The coupling gas chromatograph mass spectrometer is a powerful tool for quantitative organic trace analysis on biological samples, especially when the technique of internal standardization by stable isotope labelled compounds is applied.'-5 Investigations of quaternary ammonium ions using GCMS, however, require a derivatization of these organic cations to obtain neutral derivatives. Demethylation with benzenethiolate, for example, is a recommended derivatization procedure6.' as a result of which tertiary amines are produced that can be separated by GC and analysed by conventional EIMS. Further, for a simultaneous determination of choline and acetylcholine, choline is usually derivatized to form an alkyl e ~ t e r before ~ . ~ demethylation in order to improve the extraction yield and its gas chromatographic properties. In general such a procedure is tedious and time-consuming. Among the novel ionization techniques in mass spectrometry, field de~orption"-'~offers the unique advantage that organic onium ions can be analysed directly without derivatization and that the FD mass spectra show the onium ion as the signal of highest relative intensity.14 This characteristic feature of FDMS stimuI'Quantitative Field Desorption Mass Spectrometry: Part IX; for Part VIII, see Ref. 38).
lated a number of investigations concerning the FD mass spectra of these corn ounds and their tendency for cluster ion formation.R-2 1 The extraordinarily intense FD ion currents of the onium ions enabled field desorption-collisional activationz2 studies to be performed." This study is the first attempt to make quantitative measurements of choline and acetylcholine in brain tissue using a simple ion pair extraction procedure and direct analysis of the ammonium cations by FDMS and stable isotope dilution.
~~~~~
~
EXPERIMENTAL Tissue samples A half-wild rat of 280 g weight was killed by decapitation and the brain was quickly removed. Tissue samples of about 2 0 m g from distinct brain regions were prepared, weighed, and transferred into homogenizators at O'C, each containing 1 ml perchloric acid (4'/0), 10.74 nmol ['H2]choline and 10.11 nmole ['Hg]acetylcholine as internal standards. The sample sizes were as follows: frontal cortex: 38.90 mg; cerebellum: 25.65 mg; colliculus: 26.30 mg; hippocampus superior: 19.00 mg; hippocampus inferior: 22.35 mg; striatum caudatum: 20.25 mg. The samples were homogenized at 0 "C in an ice bath for 30 min. The homogenizate then was spinned in a centrifuge for 20min at 5000 rev min-', 350 p1 of the supernatant was transferred into a 5 ml reacti-vial, and 1.5 ml aqueous Na2HP04 solution (0.5 molar)+ 1 ml solution of dipicrylamine in CH2C12was added and stirred for 20 min with a magnetic stirrer. After spinning to obtain clearly separated phases, the upper aqueous phase was discarded and 1 ml of carbonate buffer solution of pH 9 was added and magnetically stirred for 5 min. The magnetic
CCC-0306-042X/78/0005-0591$02.50 @ Heyden & Son Ltd, 1978
BIOMEDICAL MASS Sf'ECTROMETRY, VOL. 5, NO. 10, 1978 591
W. D. LEHMANN, H.-R. SCHULTEN AND N. SCHRODER
stirrer was removed and the mixture centrifuged for separation of the two phases. Then 700 p l of the lower phase were transferred into a 1 ml reacti-vial, the solvent was blown off under a stream of nitrogen and the residue dried under vacuum conditions. The residue was redissolved in 50p1 absolute methanol and 5 pI aliquots were used for quantitative analysis by FDMS. The steps of the work-up procedure employed, which correspond to the first steps of an assay developed by Eksborg and P e r ~ s o n , ’ are ~ displayed schematically in Fig. 1.
10 p m tungsten wires activated at high temperature.26 The length of the carbon microneedles was 20 pm on average. The F D ion currents were recorded electrically with a secondary electron multiplier and the signals were accumulated by a type CAT-1024 Varian multi-channel analyser. The mass spectrometer multi-channel analyser coupling was achieved by triggering the time averaging computer from the cyclic magnetic scan of the mass ~ p e c t r o m e t e r . ~ ~For ’ ~ ’ all quantitative determinations, the lowest resolution available was adjusted in order to realize maximal transmission and a broad flat-topped peak shape.
Internal standards [2H2]Cholineiodide and [2H9]acetylcholineperchlorate were used as internal standards. Both compounds were prepared following a procedure given by KarlCn el af.” The degree of labelling of the quaternary ammonium ions obtained was determined by FDMS and the use of a multi-channel analyser. The following isotopic distributions were found: [’H2]choline: 1.2% -do; 1.7% d l ; 97.1% - dZ. [2H9]Acetylcholine: 4.9%- d 8 ; 95.1% -d9.
Mass spectrometry The studies were performed on a type 731 Varian MAT double focusing mass spectrometer equipped with a combined EI/FI/FD ion source. F D emitters used were
Brain tissue (about 20 mg)+ 1 ml HC1044% +internal standards at 0°C
Homogenization (30 min) Spinning (20 min)
Supernatant (350p1)+1.5 m10.5 m Na2HP04 (HzO)+dipicrylamine (CH,CI,)
RESULTS A N D DISCUSSION A direct quantitative analysis of choline and acetylcholine in a crude tissue extract appeared promising, because of (i) the ability of FDMS to produce fragment poor or even fragment free mass spectra, (ii) the outstanding sensitivity of FDMS for the detection of organic and inorganic cations” as compared with neutral organic molecules, and (iii) the encoura in results obtained in recent quantitative FD studies.2L-I In these studies it was the use of stable isotope labelled internal standards in particular which enabled quantitative data of good precision and accuracy to be obtained. Furthermore, the frequently observed strong fluctuations of the F D ion currents which hamper the measurement of relative intensities considerably could be compensated by averaging over a large number of cyclic scans. This is most convenient1 accomplished by the use of a multi-channel a n a l y ~ e r ~accumulating ~?~’ the electrically recorded signals of subsequent cyclic scans. This technique was also applied in this study and Fig. 2 shows the beneficial effect of signal accumulation for the determination of FD ion current ratios. The accumulation of such a large number of cyclic scans allows a precise determination of the intensity ratio (m/e 104)/(m/e 106), whereas this cannot be achieved using a single scan plot.
B
Stirring (20 min) Spinning (1 min)
I
Lower phase + 1 mi carbonate buffer pH 9
(b)
Stirring (5 min)
Lower phase (700 pl) dried down under N2and redissolved in 50 pI methanol
FD quantitation of 5 @I aliquots m / e 104, m / e 106for 12Holcholine/[2H21 m / e 146, m / e 155 for [2Ho]acetylcholine/[zH9] Figure 1. Schematic display of the work-up procedure used for the determination of choline and acetylcholine in rat brain tissue by FDMS.
592 BIOMEDICAL MASS SPECTROMETRY, VOL. 5,
NO. 10,
1978
Figure 2. Accumulation of the FD ion currents of choline ( m / e 104) and [2H21choline( m / e 106) with a time averaging computer. (a) Single scati; (b) 200 scans accumulated.
@ Heyden & Son Ltd, 1978
DETERMINATION OF CHOLINE AND ACETYCHOLINE IN RAT BRAIN
The linearity between the molar composition of the sample/standard mixtures and the observed peak height ratios was confirmed by establishing a calibration curve. Two solutions were prepared, containing 107.4 nmol ml-’ choline (solution A ) and 107.4 nmol ml-’ dideuterated choline (solution B) respectively. The calibration curve observed in the investigation of a number of different mixtures of these two solutions is displayed in Fig. 3. The investi ation of model mixtures of [2Ho]acetylcholine and [8H9]acetylcholine also resulted in a linear calibration curve. For the determination of choline and the dideuterated internal standard from the various tissue samples, the mass region from about m / e 100 to m / e 115 was scanned magnetically 50 times for one determination. The mass range was chosen in order to include a signal at m / e 113 corresponding to [2H9]~holine. This species had not been added to the samples but rather originated from hydrolysis of the internal standard [2H9]acetylcholine during the work-up procedure. The extent of hydrolysis observed was finally used to correct the observed intensity for [2Ho]choline for an amount that stemmed from hydrolysis of [2Ho]acetylcholine. Figure 4 shows a typical plot for [2Ho]choline/[2H2]/[2H9] obtained in the analysis of tissue samples. It is remarkable that the detected mass range was free of interfering background ions when an emitter heating current between 10 and 25 mA was used. Under these conditions intense and relatively stable emission of the ammonium ions and, most importantly, constant ion cilrrent ratios were observed. Below 10 mA emitter heating current, some interference of more volatile constituents of the sample mixture occurred and from about 25-30 mA emitter heating current upwards (depending on the diameter of the FD emitter) the observed relative intensities became more or less dependent upon the emitter temperature. In particular,
1.
7H3 H C-N+-CD~--CH~-OH 3 1
1
’i/
CH3 m / e 106
H3
It
HO-CH2-CH2-N-CH 1 CH3
3
m / e 104 I
Figure 4. Molecular ion group of [2H21choline ( m l e 104), [2H21choline( m l e 106). and [2H&holine ( m l e 113) from a rat brain sample (hippocampus superior). Fifty cyclic magnetic scans were accumulated at 1 5 rnA emitter heating current.
the signal at m / e 113 increased rapidly, indicating a thermally induced hydrolysis of the [2H9]acetylcholine standard to [2H9]cholineo n the FD emitter. Table 1 ives the relative abundances for [2Ho]choline/[’H2]/[ Hg] in the tissue samples under investiga-
B
tion all being obtained between 10 and 25 mA emitter heating current. The direct analysis of acetylcholine and its [2H9] analogue also resulted in clear-cut mass spectra. Figure 5 shows a typical plot for the analysis of a tissue extract. Between about 10 and 25 mA emitter heating current intense ion emission and constant ion current ratios could be observed and all determinations were performed in this range. Again, at heating currents greater than 20-25 mA, surface reactions could be observed. In the mass range scanned for [2Ho]acetylcholine/[2H9], an additional signal appeared at m / e 148. This probably corresponds to [2H2]acetylcholine and indicates a thermally induced exchange of the ester moiety between choline and acetylcholine. Table 2 contains the observed relative abundances for acetylcholine and its 12H9]analogue in the same tissue Table 1. Isotopic abundances obtained by FDMS and a multichannel analyser in quantitating choline using [2H,]choline as internal standard in various samples of rat brain tissue Tissue
0
10
20
30
LO
50
50
70
80
90
100
pl solution B added to 100 pI solution A
Figure 3. Calibration curve for the determination of choline by FDMS using [2H2]cholineas internal standard.
@ Heyden & Son Ltd, 1978
Number of measurements
Frontal cortex Cerebellum Colliculus Hippocampus superior Hippocampus inferior Striatum caudatum
2 3 2 3 4 2
Rel. int. m f e 104
24.41t0.9 15.21t1.4 30.21t0.9 19.7~k0.1 22.3+0.8 33.01tl.O
Rel. int. m l e 106
Rel. int. m f e 113
100 100 100 100 100 100
3.9+0.4 5.410.3 12.11t0.6 4.8*0.3 3.1 +0.2 9.61t1.2
BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 10, 1978 593
w.
D. LEHMANN, H.-R. SCHULTEN AND N. SCHRODER ~~
0 H~c-L--CH~-CH
CH
2 1
3
~
Table 2. Isotopic abundances obtained by FDMS and a multichannel analyser in quantitating acetylcholine using [*H,]acetylcholine as internal standard in various samples of rat brain tissue
!
-AH 1 3
~
I
m / e 1L6
Tissue
I
Frontal cortex Cerebellum Colliculus Hippocampus superior Hippocampus inferior Striatum caudatum
Figure5. Molecular ion group of [ZHo]acetylcholine( m / e 146) and [2H9]acetylcholine ( m / e 155) from a rat brain sample (hippocampus inferior). Fifty cyclic magnetic scans were accumulated at 22 mA emitter heating current.
4 3 3 3 5 2
Rel. int. rnle 146
Rel. int. rnle 155
5.8k0.4 1.4kO.05 9.5k 0.6 4.9 0.3 4.9k0.2 5.210.2
*
100 100 100 100 100 100
Table 3. Tissue concentrationsof choline and acetylcholine in various rat brain regions calculated from the ion abundances displayed in Tables 1 and 2 Concentration Tissue
samples as listed in Table 1 for the choline determination. The tissue concentrations for acetylcholine were calculated directiy from the peak height ratios displayed in Table 2. The observed peak heights for [*Ho]choline at m / e 104, however, had to be corrected for a certain amount of choline originating during the work-up procedure as described above. The calculated values for the choline and acetylcholine tissue concentrations are summarized in Table 3. The concentration data found in this FD study generally show good agreement with a number of previous studies using G C and EIMS.42-44For instance, a significantly low value for acetylcholine in the cerebellum was found in all of these investigations. Additionally, the data obtained show a good precision (between 0.6 and 10% on a relative basis), although the ratio sample to standard deviated considerably from unity especially for the acetylcholine determinations. The clear results obtained can be ascribed mainly to the extraordinarily high sensitivity of FDMS for the organic cations under investigation. Using a freshly prepared FD emitter an ion current lasting several hours could be obtained when a 5 pl aliquot of the extract was applied to the FD emitter. Since the accumulation of 50 scans was performed in c. 10 min it can be estimated that the signals for acetylcholine, one of which is shown in Fig. 4, represent between 2 and 10 pg of the quaternary ammonium ion. Thus, the assay shows an impressive sensitivity even when it is compared with novel GCMS procedure^.^^^' Simultaneously, high specificity is achieved by direct analysis of the quaternary ammonium
Number of measurements
Frontal cortex Cerebellum Colliculus Hippocampus superior Hippocampus inferior Striatum caudatum
choline nmol g-’ fresh weight
61.6k2.4 56.6* 5.7 110.8 k 3 . 5 100.4+0.6 97.813.7 161.4k 5.1
Concentration acetylcholine nmol Q fresh weight
14.3*1.0 5.2 i 0.2 34.7 2.2 24.8 i 1.5 21.1 10.8 24.7* 1.1
*
ions avoiding, for example, interference from endogenous tertiary amines which is a possible source of error when analysing the demethylation product of ~ h o l i n e . ~ ~ Finally, the use of two differently labelled internal standards enables the data to be corrected for the influence of hydrolysis during the work-up procedure. In conclusion, the combined use of ion pair extraction, stable isotope dilution and field desorption mass spectrometry is a highly sensitive and specific procedure for the quantitation of choline and acetylcholine in tissue samples. Since the required work-up procedure is simple and the mass spectrometric detection of the ammonium ions by F D is very sensitive, the analysis of much smaller sample sizes or considerably lower concentrations than investigated in this study appears possible.
Acknowledgements This work was financially supported by the Deutsche Forschungsgemeinschaft, the Ministerium fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen, and the Fonds der Deutschen Chemischen Industrie. We wish to thank Dr H. M. Schiebel. Technische Universitat Braunschweig, for his encouraging advice and fine collaboration.
REFERENCES 1. D. J . Jenden and L. B. Campbell, in Methods of Biochemical Analysis, ed. by D. Glick, Vol. 18, p. 183. Wiley, New York (1971). 2. I. Hanin (ed.), Choline and Acetylcholine: Handbook of Chemical Assay Methods, Raven Press, New York (1974). 3. A. P. De Leenheer and R. A. Roncucci, (eds.), Quantitative Mass Spectrometry in Life Sciences, Elsevier Scientific, Amsterdam (1977).
594 BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 10. 1978
B. J . Millard, Quantitative Mass Spectrometry, Heyden, London (1978). 5. W. D. Lehmann and H.-R. Schulten. Angew. Chern. Int. Ed. Engl. 17,221 (1978). 6. D. J . Jenden, I. Hanin and S. I. Lamb, Anal. Chern. 40, 125 (1968). 7. I. Hanin and D. J . Jenden, Biochem. Pharmacol. 18, 837 (1969).
4.
@ Heyden & Son Lid, 1978
DETERMINATION OF CHOLINE AND ACETYCHOLINE IN RAT BRAIN 8. D. J. Jenden, R. Booth and M. Roch, Anal. Chem. 44, 1879 ( 1972).
9. I. Hanin, R. Massaralli and E. Costa, J. fharmacol. Exp. Ther. 181,10 (1972). 10. H. D. Beckey, lnt. J. Mass Spectrom. Ion Phys. 2,500 (1969). 11. H. D. Beckey and H.-R. Schulten, Angew. Chem. Int. Ed. Engl. 14,403 (1975). 12. H.-R.Schulten, in Methods of BiochemicalAnalysis, ed. by D. Glick, Vol. 24, p. 313.Wiley and Sons, New York (1977). 13. H. D. Beckey, Principles of Field Ionization and Field Desorption Mass Spectrometry, Pergamon Press, Oxford (1977). 14. D. A. Brent, D. J. Rouse, M. C. Sammons and M. M. Bursey, Tetrahedron Left. 4127 (1973); M. C. Sammons, M. M. Bursey G. W. Wood, J. and C. K. White, Anal. Chem. 47,1165 (1975); M. Mclntosh and P.-Y. Lau, J. Urg. Chem. 40.636 (1975). 15. G. W. Wood and P. Y. Lau, Biomed. Mass Spectrom. 1, 154 (1974). 16. M. C. Sammons, M. M. Bursey and D. A. Brent, Biomed. Mass Spectrom. 1, 169 (1 974). 17. H.-R. Schulten and F. W. Rollgen, Org. Mass Spectrom. 10, 649 (1975). 18. H.-R. Schulten and F. W. Rollgen,Angew. Chem. Int. Ed. Engl. 14,561 (1975). 19. H. J. Veith, Org. Mass Spectrom. 11,629(1976). 20. G. W. Wood, P.-Y. Lau and G. N. Subba Rao, Biomed. Mass Spectrom. 3, 172 (1976). 21. H. Ogino, T. Matsumura, K. Satouchi and K. Saito, Biomed. Mass Spectrom. 4. 326 (1977). 22. K. Levsen and H. Schwarz, Angew. Chem. lnt. Ed. Engl. 15, 509 (1976) 23. H. H. Gierlich, F. W. Rollgen, F. Borchers and K. Levsen, Urg. Mass Spectrom. 12,387(1977). 24. S. Eksborg and B. A. Persson, Acta Pharm. Suec. 8, 205 (1971). 25. B. Karlen, G. Lundgren, 1. Nordgren and B. Holmstedt, in Choline and Acetylcholine: Handbook of Chemical Assay Methods, ed. by I. Hanin, p. 163. Raven Press, New York
(1974). 26. H.-R. Schulten and H. D. Beckey, Org. Mass Spectrom. 6,885 (1 972). 27. W. D. Lehmann and H A . Schulten, Anal. Chem. 49, 1744 (1977). 28. H.-R. Schulten, Cancer Treat. Rep. 60, 501 (1976). 29. 1. Jardine, M. N. Kan, C. C. Fenselau, R. Brundrett, M. Colvin, G. Wood, P.-Y. Lau and R. Charlton, in Proceedings of the Second International Symposium on Stable Isotopes, Oak Brook Illinois, October 1975, p. 138. National Technical Information Service, US Department of Commerce, Springfield, Virginia, ERDA CONF-751027(1976).
@ Heyden & Son Ltd, 1978
30. W. D. Lehmann, H. D. Beckey and H A . Schulten,Anal. Chem. 48, 1572 (1976). 31. W. D. Lehmann, H. D. Beckey and H.-R. Schulten, in Quantitative Mass Spectrometry in Life Sciences, p. 177.Elsevier, Amsterdam (1977). 32. H A . Schulten, W. D. Lehmann and M. Jarman, in Quantitative Mass Spectrometry in Life Sciences, p. 187.Elsevier Amsterdam (1977). 33. S.Pfeifer, H. D. Beckey and H.-R.Schulten, Z. Anal. Chem. 284,193(1 977). 34. W. D. Lehmann and H.4. Schulten, Angew. Chem. Int. Ed. Engl. 16,184(1977). 35. W. D.Lehmann and H.-R. Schulten, Biomed. MassSpectrom. 5,208 (1 978). 36. W. D. Lehrnann and H . 4 . Schulten, Angew. Chem. Int. Ed. Engl. 16,852(1977). 37. W. D. Lehmann, H A . Schulten and H. M. Schiebel, Z. Anal. Chem. 289,ll (1978). 38. H.4. Schulten, R. Ziskoven and W. D. Lehmann, Z. Naturforsch. Teil C 33,178 (1 978). 39. H . 4 . Schulten, U. Bahr and W. D. Lehmann, Biomed. Mass Spectrom. 5,536 (1 978). 40. H.-R. Schulten and W. D. Lehmann, in Quantitative Mass Spectrometry in Life Sciences 11, ed. by A. P. De Leenheer, R. Roncucci and C. van Peteghem, Elsevier Scientific, Amsterdam (1978). 41. H.-R. Schulten, R. Ziskoven and W. D. Lehmann, Z. Naturforsch. Teil C 33, 178 ( 1978). 42. L. B. Campbell and D. J. Jenden, J. Neurochem. 17, 1697 ( 1970). 43. W. B. Stavinoha, S. T. Weintraub and A. T. Modak, J. Neurochem. 23,885 (1974). 44. S.1.Weintraub, A. T. Modak and W. B. Stavinoha, Brain Res. 105,179 (1976). 45. D.J. Jenden, M. Roch and R. A. Booth.Ana1. Biochem. 55,438 (1973). 46. W. M. Einolf and C. Fenselau, Biomed. Mass. Spectrom. 1, 195 (1974). 47. J. Shabanowitz, P. Brynes, A. Maelicke, D. V. Bowen and F. H. Field, Biomed. Mass Spectrom. 2,164 (1975). 48. D. J. Jenden and I. Hanin, in Choline and Acetylcholine:
Handbookof Chemical Assay Methods, ed. by I. Hanin, p. 135.Raven Press, New York (1974).
Received 6 April 1978 @ Heyden & Son Ltd, 1978
BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 10, 1978 595
