Volume 6, Number 1 Finnigan Corporation February, 1976

Selective Reagents for Chemical Ionization

Mass Spectrometry

by Donald F. Hunt Department of Chemistry

University of Virginia Charlottesville, VA 22901

Much of the power of chemical ionization mass spectrom­etry stems from the finding that the characteristics of the Cl mass spectrum produced is dependent on both the nature of the reagent gas and the type of ion-molecule reaction used to ionize the sample. Different structural information can be obtained with different reagent gases. As a consequence, it is possible to control the type and quantity of structural information obtained from a mass spectrum by varying the nature of the reagent gas used in the Cl mode of operation.1

As indicated in Figure 1, the extent of fragmentation pro­duced from a particular sample can be controlled by vary­ing the exothermicity of the ionizing reaction .1 Electron bombardment of H2 , CH., and (CH3) 3CH produces H3 +, CH5 +, and (CH3)JC+ respectively and each of these ions functions as a Bronsted acid toward the neutral organic molecule.

Since the proton affinity (PA) of H2 and, therefore, the Brons ted acidity of H3 + is considerably greater than that of CH5+, CI(H2) spectra exhibit much more fragmentation

H~ + H, - H,+

CH. + W-CHs~

H = - 101 kcal PA = 101

H ~ - 127 kcai PA - 127

(CH,hC = CH• + H+ - (CH3)p• H = - 195 :ai PA = 195

than those obtained with methane. In the case of dihydro­testosterone (Figure 1) ionization with H3 + causes frag­mentation in the vicinity of both functional groups as well as on the carbocyclic ring system. With CH5 + fragmenta­tion is largely restricted to loss of water from both the ketone and alcohol functional groups. Ionization with the still weaker Bronsted acid, (CH3 )JC+ affords a spectrum where most of the ion current is carried by the M+ 1 ion. Loss of water, presumably from the alcohol moiety, gen­erates an M - 17 ion which only accounts for 10% of the total sample ion current. Because isobutane Cl spectra contain a paucity of fragment ions, this reagent gas is

ideally suited for high sensitivity , quantitative analysis by the isotopic dilution method.

As part of a continuing effort to develop CIMS into a powerful method for the identification, structure elucida­tion , and quantitation of organic compounds, we have explored the analytical utility of a number of reagent gases for both positive and negative Cl mass spectrometry.

Gases studied to date include argon-water, ammonia, deuterium oxide, and nitric oxide in the positive ion mode; oxygen and hydrogen in the negative ion mode. Examples illustrating the analytical potential of each of these Cl reagents are outlined below.

100 OH

Cl(~ M+l (a) M+l- H20

50 . O ! MW 290 •2H2 o H

w 100

I I I I I ll~ u z M+l <( Cl (ME THANE) 0 z (b) ~ 50 Ill -H 0 <( 2

-2H2~ II ..J Ill w 100 a:

Cl (ISOBUTANE) Mtl

(c) 50 -

0~--~~--~.~~--r--r--~.~~~.~~.

100 140 180 220 260 300 M/e

Figure 1: Dihydrotcstosterone Ci mass spectra recorded with (a) hydrogen, (b) methane, and (c) isobutane as the reagent.

· ~

Argon-water

We find this reagent mixture to be particularly valuable for solid probe and GC-MS analysis of biological samples where the amount of material available for examination is frequently sufficient for only one experiment.2 Cl spectra recorded with argon-water exhibit abundant M + 1 ions characteristic of sample molecular weight as well as all

the electron impact type fragmentation so useful for eluci­dating or confirming molecular structure.

Electron bombardment of argon-water (20/1) at 1 torr pro­duces abundant ions at m/e 40 (Ar+), 80 (Ar2 • ), and 19 (H30 "'"). Excited argon neutrals (Ar") and large numbers of low energy electrons are also generated under the above conditions. As expected H30 · functions as a Bronsted acid in the gas phase and protonates organic compounds containing a heteroatom. Since the proton affinity of water is quite high (PA = 167± 4 kcal/mole) , the energy trans­ferred to the sample in the proton transfer reaction is relatively small and the abundant M + 1 ions that result rarely undergo extensive fragmentation. In contrast to the situation with H30 +, electron transfer (oxidation) occurs when sample molecules encounter Ar' in the ion source. Since the recombination energy of Ar' is 15.8 eV and the ionization potentials of most organic compounds are in the range 9-12 eV, most of the radical cations produced in the oxidation reaction undergo fragmentation along pathways very similar to those observed in conventional electron impact mass spectrometry. Collisions between organic sample molecules and excited argon neutrals or low energy electrons may also result in sample ionization but here the exothermicity of the reactions is low and the molecular ions generated are usually stable toward fur­ther fragmentation .

For the purpose of comparison, conventional El and Cl (Ar-H20) spectra of 4-decanone are shown in Figure 2. Reaction of this ketone with H30 + affords a single ion corresponding to protonated sample, M+ 1. In contrast the El spectrum displays a relatively weak molecular ion, M+. and a series of fragment ions derived from a-cleavage and Mclafferty rearrangement pathways. Cl with argon­water as the reagent affords a spectrum which is essen­tially the sum of those produced by El and Cl with water. All of the fragment ions produced by El as well as the abundant M+ 1 ion found in the CI(H20 ) spectrum are displayed in Figure 2b. With respect to sensitivity, we find that the total sample ion current obtained by the Cl method with Ar-H20 is the same and about 30 times greater with N2-H20 than either El or CI(He-H20).3

100 El 43

(a)

w (.) 50 z Cl 0 z ::::) a:J 100 Cl

Cl 40 43 -' w (Ar·HzOl cr

50 (b) 19

0 40

MW 156

71

58

71

80 M/e

~ 86 1131 M+

156

157 Mil

120 160

Figure 2: Eland CI(Ar-H20) mass spectra of 4-decanone.

It should also be mentioned that the Cl argon-water (nitrogen-water) system is ideally suited for obtaining accurate mass measurement data on a double focusing magnetic sector instrument by either the peak matching technique or with the aid of an on-line automated data acquisition and processing system.2 Perfluorokerosene (PFK) is employed as the internal standard and affords a spectrum identical to that produced in the El mode of operation. Proton transfer from H3Q+ to the fluorocarbon does not occur and the on ly ions generated result from electron transfer between Ar•· and PFK.

Deuterium Oxide

Deuterium oxide is an excellent Cl reagent for the deter­mination of active hydrogen in organic compounds and for the differentiation of 1°, 2°, and 3° amines.4 GC-MS analysis can be carried out by adding deuterium oxide to the GC carrier gas as it enters the Cl source.

At a pressure of 0.4 torr electron bombardment of 0 20 affords abundant ions at m/e 22(0 30 +), 42(020 h D·, 62(D20 h D+, 82(020 )40 + and 1 02(020 )50 +. These ions in turn function as Bronsted acids and deuterate most or­ganic compounds. In addition, all hydrogens bonded to oxygen, sulfur, and nitrogen atoms are exchanged for deuterium while the sample molecule is in the Cl source. As indicated in Figure 3b, the most abundant ion in the Cl (D20) spectrum of the nucleoside, adenosine. occurs at m/e 274 and corresponds to d5-adenosine .L o~ . Since this same ion appears at m/e 268 in the CI(H20 ) spectrum (Figure 3a), the above result clearly indicates that all five acidic hydrogens in the nucleoside suffer exchange in the ion source when 020 is employed as the reagent gas. In addition, two fragment ions resulting from clevage of the glycosidic linkage are also observed. These appear at m/e 136 and 140 and correspond to a d3-sugar moiety and d2-adenine + o·. To determine the nitrogen substitution pattern in an un­known amine it is necessary to record two Cl spectra. One spectra must be taken with methane or isobutane to ascertain the sample molecular weight and one with deuterium oxide to count the number of exchangeable hydrogens and thereby determine the extent of substitu­tion on the nitrogen atom.

N~ 100 Cl <H20 l 268 MW 267 N~

w (a) (/N IN~ u 55 z Cl 50 37 HOC~ 0 19 ~133 z :::> HO OH 136 CD Cl 100

Cl (D2o) 274 ...J w a: (b)

50 62 42

0 40 80 260 M/e

Figure 3: (a) Cl water and (b) Cl deuterium oxide mass spectra of adenosine.

In addition to the above examples, we have also obtained CI(D20) spectra of a number of compounds containing one or more common organic functional groups. Our find­ings indicate that hydrogens bonded to heteroatoms in alcohols, phenols, carboxylic acids, amines, amides, and mercaptans undergo essentially complete exchange in the ion source when 0 20 is employed as the reagent gas at a pressure of 0.4 torr. Small amounts of deuterium incorporation (less than 15%) occur in ketones , alde­hydes, and esters but, in general, this does not complicate the analysis.

Ammonia

Ammon ia is an excellent Cl reagent for determining the molecular weight of polyfunctional organic molecules and for the selective ionization of basic components in com­plex organic mixtures.5 Electron bombardment of am­monia at 1 torr produces the set of ions, (NH3 }nH- (n = 1, 2, and 3) which occur at m/e 18, 35, and 52. These ions, in turn , function as weak Bronsted acids and weak electrophiles toward other organic compounds in the gas phase. Proton transfer from NH4 + to the organic sample is observed if the proton affinity of the compound is greater than that of ammonia (PA = 207 kcal/mole}. Of the com­pounds studied to date only amides,6 amines,6 •

7 and some a, ,8-unsaturated ketones8 fit into this category and, therefore , exhibit M+ 1 peaks in their ammonia Cl spec­tra. As indicated in Figure 4a almost no fragmentation accompanies the proton transfer reaction since the pro­cess is only mildly exothermic.

In addition to M+ 1 ions, spectra of compounds derived from the above categories also display peaks correspond­ing to the electrophilic attachment of NH4 + to the organic sample.5 Ketones, aldehydes, esters and acids also add NH4 + (Figure 4) but are not sufficiently basic to remove a proton from the ammonium ion. Spectra of these com­pounds, therefore, exhibit a single ion corresponding to M+ NH4 + . Neither proton transfer or electrophilic attach­ment is observed in the CI(NH3} spectra of simple ethers, alcohols , phenols, nitro compounds, hydrocarbons, or aromatics: no ions are produced from these compounds. In contrast, difunctional molecules are readily ionized if two functional groups can interact simultaneously with the ammonium ion through formation of intramolecular hydrogen bonds. These results suggest that ammonia may find utility as a reagent for probing stereochemical relationships in organic compounds.

As illustrated in Figure 4, CI{NH3} spectra contain abun­dant ions characteristic of the molecular weight of poly­functional molecules such as derivatized and underiva­tized sugars. El and methane Cl are unsatisfactory for the mol. weight analysis of this class of compounds.

Nitric Oxide Nitric oxide has shown considerable promise as a Cl reagent for organic functional group analysis,9 the qualita­tive analysis of hydrocarbons, 10·11

•12 and for the enhance­

ment of molecular ion abundance in spectra of alkaloids and TMS derivatives of biological compounds.13 Under electron impact at 1 torr, nitric oxide affords a high abun­dance of NO+ ions. Studies on the ion-molecule reactions

100 (a)

50 18

35 NHCOC H 637 M+ l 181 16 4 +

M+N~

~2 MW 163

1 00 +-~~~L-----------~~~-----(b) 35

5 0 18

r 100 1-~~~~-------------L--~L-/(\ .,. NJ.H7"'"

{ 0 (c)

50 ~I (l~ -~ 181 52 I '-.,./" ... I MW 116 ~ I 00 -t----L--~-'--------1-.L-------~ (d) 35 174 M-+ NH:

S::,,. ...J 50 w a:

191 18r i2

1 00 ~-4~~--------------L-~-----

(e) 35

50 18

Ho~o\. HO~OH

ISO

5 2 MW ISO

198

o +-~~~~i1~~·~~~-4--~~ 0 40 100 140 ISO 220

M/e

Figure 4: Cl ammonia mass spectra of (a) n-butyranilide, (b) lauric acid, (c) n-propyl propionate, (d) 4-decanone, (e) glucose.

of this reagent species indicate that NO+ functions as an electrophile, hydride abstractor, and one-electron ac­ceptor toward organic samples. As shown in Figure 5, CI(NO) spectra of simple ketones and esters exhibit a single peak at M+ 30 corresponding to the electrophilic addition of NO- to the sample molecule. Aldehyde spec­tra (Figure 5b) show two ions, M+ 30 and M- 1. The latter species is produced by NO"" abstraction of the hydrogen attached to the carbonyl group. Acids suffer electrophilic addition and also lose a hydroxyl group to give an M - 17 ion (Figure 5d).

As indicated in Figure 6a, n-amyl alcohol and primary alcohols in general afford nitric oxide spectra containing three ions, M- 1, M- 2+ 30, and M- 3. Formation of the latter two ions results from oxidation of the alcohol to the corresponding aldehyde which suffers either e lectrophilic attachment or hydride abstraction on reaction with NO- . Abstraction of hydrogen from the carbon bearing the hydroxyl group by NO- generates the M- 1 ion.

Secondary alcohols (Figure 6b) afford CI(NO) spectra con­taining ions corresponding to M- 1, M - 17 and M- 2+ 30. NO M- 3 ions are observed in spectra of secondary al­cohols. Tertiary afford spectra containing a single ion

100 • (a)

50 .

100

(b)

50 1&.1 u z <I 0 z 100 ~ Cl) <I (c)

...J 50 1&.1 a:

100

(d)

50

0 0

0 30 NO

+ ifCH3

150

M+ +

NO

MW 12 0

+ NO 113

0 M-1

~l..H +

M+NO ·

MW 114 144

I No+

16 0 0

~o)l...cH3 M+NO+

MW 130

No'., 0

~l..oH

40

MW 130

80

M/e

160 +

M ... NO

1"3 M-17

120 160

Figure 5: Cl nitric oxide spectra of (a} acetophenone, (b) n-hep­taldehyde, (c) n-amyl acetate, and (d) heptanoic acid.

100

5 0

wiOO u z <(

0 ,. ~ 50

CD <(

...1 100 w Q:

50 -

0 0

(a) NO+

30

No• (b) 30

(c) +

NO

30

116 M-2+30

~OH

MW 88 8 5

1,87

OH 116

~ M-2+ 30

MW 88 71 87

I I 71

~ OH

I I

40 80 M/ e

12 0

Figure 6 : Cl nitr ic oxide spectra of (a) n-amyl alcohol, (b) sec-amyl alcohol and (c) t-amyl alcohol.

which corresponds to M-OH (Figure 6c). Thus it is pos­sible to use the nitric oxide Cl technique to differentiate primary , secondary and tertiary alcohols.

Another exciting aspect of the CI(NO) method is the find­ing that many hydrocarbons afford spectra which contain only one or two ions.10

•1

1.12 The CI(NO) spectrum of de­

cane (Figure 7), for example, shows an M- 1 ion which carries over 96% of the total ion current. In contrast CI(CH4) and El spectra of decane exhibit an abundance of low molecular weight fragment ions.

Nitric oxide CIMS is also useful for differentiating cyclic alkanes from olefins. M - 1 is the only ion produced from cyclohexane. Olefins such as 3-decene, on the other hand, afford spectra containing two ions, M - 1 from hy­dride abstraction and M+ 30 from electrophilic addition of NO+ to the double bond (Figure 7c). Spectra of dienes show the above two ions plus a third species correspond­ing to M+. Generation of this molecular ion presumably occurs by transfer of an electron from the olefin to the NO+ ion.

141 ~ M· l

100

50 - 30 +

NO MW 142

w 100 u z <I 0

50 z ~

(a) II 220

30 NO+ -+0- M

(b) +NO

CD <t

...J 100

MW 190 [ I w a::

30 (c) 139

NO+ M- H 170

M+NO 50

3- DECENE

MW 140

0

20 60 100 140 180 220 M/e

Figure 7: Cl nitric oxide spectra of (a) n-decane, (b) p-di-t-butyl­benzene, and (c) 3-decene.

Oxygen

Studies with oxygen indicate that this reagent will be use­ful for the analysis of alcohols, 10 polycyclic aromatics, 11

sulfur compounds, and polychlorinated aromatic pesti­cides14 by negative chemical ionization (NCI) mass spec­trometry.

Electron bombardment of oxygen at 1 torr under negative ion conditions affords 0 2- . o-. and a large population of thermal (or near thermal) electrons. Alcohols react with 0 2 to form hydrogen bonded adducts, (M + 0 2t. and with 0 to form M - 1 ions. 10 These two ions account for 97-100% of the sample ion current for most simple al­cohols (Figure 8). The NCI(02) method appears ideally suited for the molecular weight analysis of alcohols since even molecules with a tertiary hydroxyl group fail to undergo fragmentation when oxygen is employed as the Cl reagent.

NCI(02) spectra (Figure 9) of polycyclic aromatics exhibit

100 32 120 ~OH .:.

w (a) • M+02 u 02 z 50 MW 88 <l: 0 !. z 0 8 7 :::>

I Q) I <l: 100 .:. 32 M -__] M•l5

w 02 00 a:: (b)

50 . -0 MW 152

0 I

I I I ,

0 40 80 12 0 16 0 2 0 0 M/ e

Figure 8: NCI(02) spectra of (a) n-amyl alcohol, (b) acenaphthylene.

MW 320 100 Cl 176 NCI ( Oz)

CI JOCOJO:CI

"' (a) Cl 0 Cl u

50 CIJOr~-z M-~02-0CI M '02 0 H < M

0 z Cl 0 301 320 335 => .. s s. CXl < 100

35 Cln Clm c12H50C~ C1t4 0CI~ ..J

"' cr0--6 a: 50 ~ I 305

(b) n m • 5 6 7 ....

0 ff, 20 40 180 220 260 300 340 380

M/e

Figure 9: NC1(02) spectra of (a) TCDD, (b) PCB mixture.

abundant M- and (M+ 15)- ions. Attachment of a thermal electron to the sample produces M- which then under­goes a reaction with molecular oxygen to form OH and a phenolic anion corresponding to (M+ 15) . Sulfur het­erocyclics are easily distinguished from other aromatics having a molecular weight at the same nominal mass. Under NCI(02 ) conditions sulfur heterocyclics afford abundant (M + 32) ions resulting from addition of oxygen to the radical anion of the aromatic molecule. Presumably the structure of these ions correspond to radical anions of sulfones. Work is in progress to evaluate the applicability of NCI(02) mass spectrometry to the analysis of high boil­ing petroleum fractions.

Oxygen is also an ideal reagent for the analysis of 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD) 14 in the presence of other chlorocarbon contaminants, such as polychloro­biphenyls, DDT, and DOE. El and CI(CH4 ) spectra are unsatisfactory for this purpose since these contaminants all afford fragment ions which occur in the molecular weight region of TCDD spectra. In contrast, when oxygen is employed as a reagent gas for analysis by NCI mass spectrometry, TCDD spectra are produced in which > 80% of the sample ion current is carried by an ion at m/e 176. Formation of this ion (whose m/e is well out of the region containing peaks from the contaminants (Figure 8a)) in­volves capture of a thermal electron to form a radical anion followed by reaction with molecular oxygen. One possible reaction pathway is shown below. Use of the

NC1(02) method on a 2 pg solid probe sample afforded a signal for m/e 176 with a SIN > 50/1.

CI~O~CI e- CI(Y0~CI CI~0~CI ----<> CI~O~CI

m/ e 320

_o· - 0

c1 r(Y0 ~CI --o;-<> CI~O~CI -----9

Cl0°+ 0~CI

Cl~o' O~CI

m/ e 176

Hydrogen

Electron bombardment of hydrogen at 1 torr affords H­in the negative ion mode. This ion is a very strong Bron­sted base in the gas phase and abstracts a proton from most organic samples. Since a large fraction of the energy liberating in these acid-base reactions remains in the molecule containing the newly formed bond (i.e. H2), very little fragmentation accompanies the ionization process (Figure 1 0). By addition of ca. 1% of various organic molecules (CH3N02 , CH3COCH3 , CH30H, PhCH3 ) to the hydrogen reagent it is possible to wipe out the H reagent ion and generate the corresponding (M - 1) ion from the organic additive (CH2N02, CH2COCH3 , CH30 , PhCH2 ) .

The significance of the above procedure is that it facili­tates generation of a wide spectrum of anionic organic bases and nucleophiles for use as Cl reactant ions. Efforts to explore the analytical utility ot several of these ions in CIMS are currently underway in our laboratory.

100

-50 H

100 w (.) z q: 0

50 z H

::> al q:

..J 100 w a::

50 H

0 0

OH 0 ~

(a)

~

OH

{b)

OH

(c)

MW 156

~OH 0

MW 130

~OCH3

40

0

MW 144

80

M/e

129

M-1

I 120

143 t.H

I 55

-I M

160

Figure 10: NCI(H2) spectra of (a) 4-decanone, (b) heptanoic acid, and (c) methyl heptanoate.

REFERENCES

1. For recent reviews see: (a) F. H. F1eld, " ton-Molecule Reactions," J. L. Franklin, Ed., Plenum Press, New York (1 972); (b ) F. H. Field,

" MTP International Review of Science, Physical Chemistry, Vol. 5, A. Maccoll , Ed., Buttersworth, (1972): (c) M.S.B. Munson, Anal. Chem. , 43, 28A (1971 ).

2. D. F. Hunt and J . F. Ryan Ill , Anal. Chem. , 44, 1306 (1972). 3. G. P. Arsenault, J. Amer. Chern. Soc., 94, 8241 (1972). 4 . D. F. Hunt, C. N. McEwen, and A. A. Upham, Anal. Chern., 44, 1292

(1972). 5. D. F. Hunt, Adv. Mass Spectrometry , 7, 517 (1974). 6. I. Dzidic, J. I . ner. Chern. Soc., 94, 8333 (1972). 7. (a) D. F. Hunt, C. N. McEwen, and A. A. Upham, Tetrahedron Lett.,

4539 (1971); (b) M. S. Wilson, I. Dzidic, and J . A. McCloskey, Bio· chim. Biophys. Acta , 240, 623 (1971 ).

8. I. Dzidic and J . A. McCloskey, Org. Mass Spectrom., 6, 939 (1972). 9. D. F. Hunt and J . F. Ryan, J.C.S. Chern. Comm., 620, (1972).

10. D. F. Hunt, C. N. McEwen, and T. M. Harvey, Anal. Chem., 1n press. 11 . D. F. Hunt and T. M. Harvey, Anal. Chern .. 10 press. 12. D. F. Hunt and T. M. Harvey, Anal. Chem., 1n press. 13. B. Jelus, B. Munson, and C. Fenselau, (a) Anal. Chern .. 46, 729

(1974): Biomed. Mass Spectrom., 1, 96 (1974). 14. D. F. Hunt, T. M. Harvey and J . W. Russell , J.C.S. Chern. Comm. ,

151 (1975).

About Our Authors: ------------.,

Dr. Donald F. Hunt is a University of Massachusetts graduate, his dissertation work dealing with organotransition metal chemis­try. Dr. Hunt spent a year postdoctoral at MIT and then joined the Chemistry Department at the University of Virginia where he is currently an associate professor.

Dr. Israel Hanln is a graduate of UCLA. his dissertation dealing with detection for acetylcholine. Dr. Hanin then spent a year at the Karolinska Institute followed w1th a postdoctoral and staff position at NIMH. He Is currently assistant professor at the Uni­versity of Pittsburgh and Director of the Western Psychiatric Institute and Clinic.

Application of Gas Chromatography-Chemical

Ionization Mass Spectrometry to the Analysis of Microquantities of Choline

and Its Esters 1

by Israel Hanin, Department of Psychiatry, WPIC, University of Pittsburgh School of Medicine,

Pittsburgh, PA 15261

Introduction

The late nineteen sixties witnessed the origin of two ex­citing and powerful chemical techniques- Chemical Ionization Mass Spectrometry (CI/MS),2 and Mass Frag­mentography (MF).3 Within less than a decade, both tech­niques gained wide acceptance, and have been applied to myriad applications in the research laboratory. Whereas Cl has been used more frequently in the domain of basic and applied chemistry, MF has gained considerable popularity in its applications within the biological sphere .• Recently CI/MS has been coupled with MF, thus com­bining these two powerful tools into one integrated ap­proach, CI/MF.

This report describes one such application of CI/MF in the analysis of microquantities of choline and its esters. Choline and acetylcholine are both essential biological

constituents. Acetylcholine is released from nerve end­ings in the peripheral and central nervous system of mammals. It has also been found in certain invertebrates, insects, and even plants. Acetylcholine plays a role in muscle contraction, and transmission of nervous impulses both in the brain, and in the peripheral nervous system. Choline is the immediate precursor of acetylcholine. It is a normal constituent of the mammalian diet, and is utilized in vivo for the biosynthesis of lipids and membranes, in addition to the formation of acetylcholine. Recently, there has been an upsurge in the study of acetylcholine func­tion and metabolism, because of the indication that an imbalance in this metabolism may be the cause of certain disease states of both peripheral and central origin .~

A variety of specific and sensitive chemical techniques have been developed for the analysis of choline and acetylcholine in tissue extracts.6 Of these, the gas chro­matographic (GC) approach 7 has proven to be most versatile . This versatility is inherent in the ability to com­bine this basic GC approach with Electron Impact MF (EI/MF),8 and, as demonstrated here, with CI/MF,' to achieve the added inherent advantages of these powerful techniques.

Chemical Analysis - GC/EI/MS

Analysis of choline and its esters by GC is achieved following the esterification of choline in the presence of acetylcholine, to an ester analog of choline, and the sub­sequent demethylation of these esters. Demethylation is implemented either by pyrolysis or by chemical reaction with sodium benzenethiolate.7 The resulting tertiary amines are volatile, and thus are amenable to analysis by GC and GC/MS.

{ AceCiylhocl~~.ne t _E...:...sle_rd_•c_al•_on-+( Choline ester l Demelh lal•on j Oemelhylaled } GC MS , ~ r Acetylchohne r y ~ Ch0t1ne 95191$

Our initial studies utilizing GC/MS provided the basic El spectrum for all demethylated choline esters. 9 This spec­trum 1s shown in Figure 1. Based upon these observa-

II lClCl

~ a: w 0....

w en a: co ~ 5Cl

t5 a: 1-z w u oc w 0....

0 ,I l I 20 5Cl

11/E

58

.I

lCICl

_J a: 1-0 I-

LL.

3Cl 0

~ 1-z w u 0:: w 0....

Figure 1: GC/EI/MS spectrum of dtmethylaminoethyl acetate.

tions, MF assays of choline esters were subsequently conducted by focusing on m/e 58, which is the most prominent fragment ion obtained by GC/EI/MS of the demethylated choline esters (see Figure 2 for GC/EI/ MF record at m/e 58) . This fragment corresponds to the di­methylmethylenimmonium ion, (CH3h .,.N = CH2.

100

50

SPECTRUI1 NUI1BER

Figure 2: GC/EI/MF record at m/e 58 for dimethylaminoethyl ace­tate, propionate and butyrate, respectively.

Chemical Analysis - GC/CI/MS

The GC/EI/MS analyses described above are sensitive and quite specific. We, nevertheless, have recently de­veloped a newer approach which utilizes GC/CI/MS to improve even further this methodology for the analysis of choline and its esters. This approach was utilized be­cause of the increased inherent specificity of CI/MS over EI/MS. Furthermore, the m/e 58 fragment, which is the base peak for the demethylated choline esters analyzed by GC/EI/MS, is also the base peak for a large number of other tertiary amines. Therefore, using only this fragment plus a retention time could provide questionable evidence as to the identification of a GC peak, and interfering com­pounds could not be easily distinguished.

Experiments were performed using a Finnigan Model 3200 GC/MS System containing a differentially pumped Cl source and an all glass interface between the GC and the MS. The GC was equipped with a U-shaped silanized glass column (150 em; 2mm 1.0.) packed with Pennwalt 223 Amine packing (SG-100 mesh).

Typical fragmentation patterns with ions at m/e 72 and m/e (M + 1) were obtained for each of the choline esters tested. Figure 3 illustrates the Cl spectrum obtained for demethylated acetylcholine, utilizing methane as the reactant gas.

Methane as the reactant gas yielded approximately equal abundances of fragments at m/e 72 and m/e 132. When isobutane was used as a reactant gas, a predominance of the m/e 132 [ = (M + 1)] fragment was observed. (See Figure 4). An example of GC/CI/MF application to the analysis of choline esters is shown in Figure 5. In this case, the in­strument was focused selectively on m/e 72 and m/e (M+ 1) (132 for demethylated acetylcholine; 146 for de­methylated propionylcholine; 160 for butyrylated and demethylated choline).

Figure 3: GC/CI/MS spectrum of d imethylaminoethyl acetate. Methane used as reactant gas. (Reproduced w ith per­mission from Analytical Biochemistry.)

~" •w .. '•·•''~!;; • J8 ·.:?. ct,.~'f.WR.. s·c e• ::... c· :SCY · ~.f

Figure 4: GC/CI/MS spectrum of dimethylami noethyl acetate. !so­butane used as reactant gas. (Reproduced with permis­sion from Analytical Biochemistry.)

'l .. .. ,, ..

iii ... ... r ""

..

Figure 5: G CICI/MF spectrum of dimethylaminoethyl acetate, pro­pionate and butyrate, respectively. Methane used as reactant gas. (Reproduced w ith permission from Analy­tical Biochemistry.)

Quantitative Capabilities Of GC/CI/MF

The quantitative nature of this approach was ascertained by determining the linearity of recovery of the m/e 72 and m/e (M + 1) fragments for both demethylated acetyl­choline, and the demethylated product of choline which had been esterified, previously, to butyrylcholine. Internal standards used in this analysis were the isotopic variants of these compounds in which both methyl groups on the nitrogen had been replaced by CD3 groups; i.e., m/e 78 and m/e [ (M + 1) + 6] , respectively. Recovery was linear, and thus quantitative whether methane or isobutane were used as reactant gas. (See Figures 6 and 7.)

Sensitivity Of GC/CI/MF

The relative sensitivity of EI/MF and CI/MF in the analysis

of choline esters was compared, this time using 5% DOTS and 5% OV1 on Gas Chrom Q as the GC packing. Comparing signal to background ratios and using 13 picograms of demethylaminoethyl acetate, we observed that GC/EI/MF and GC/CI/MF yielded the identical sen­sitivities when isobutane was used as reactant gas. Using methane, on the other hand, we were able to elicit with GC/Cl/MF double the sensitivity that was obtained using GC/EI/MF.1

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Figure 6: U nearity of recovery of deuterated versus non-deuter­ated d imethylaminoethyl acetate and butyrate using GC/CI/MF. Methane used as reactant gas. (Reproduced with permission f rom Analytical Biochemistry.)

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Figure 7: Unearity of recovery of deuterated versus non-deuter­ated dimethylaminoethyl acetate and butyrate using GC/CVMF. lsobutane used as reactant gas. (Reproduced with permission from Analytical Biochemistry.)

Discussion

GC/CI/MS application exhibits distinct advantages over GC/EI/MS in its application to the analysis of choline and its esters. It is more specific and slightly more sensitive than GC/CI/MS. Furthermore, it allows the investigator to perform the analysis at higher m/e values than with GC/EI/MS, thus enhancing the rel iability of the technique. At the same time, it exhibits all the advantages of GC/EI/MS shown earlier in the analysis of choline and its esters; namely, reproducibility, ability to fu nction in the MF mode, linearity, and quantitative recovery of the fragments being monitoried.

This has been just one example favoring the application of chemical ionization in the study of a topic of biological interest. Presently, biological applications of GC/CI/MF are in their infancy. Nevertheless, it is inevitable that GC/CI/MF will play a major role in biological research in the near future.

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REFERENCES

408-732-0940 3 12·358-C522 301-468-9333 713-n4~92

HEMEL HEMPSTEAD

1. Han1n, 1., Proc. West. Pharmacol. Soc. 18 :72-73, 1975; Han1n, 1. , and Skinner, R.F., Anal. B1ochem. 66:568-583, 1975.

2 . Munson, M.S.B., and Field, F.H., J . Am. Chem. Soc. 89:1047-1052, 1967: Beggs. D .. and Yergey, A., Industrial Research, February, 1973.

3. Hammar, C.-G., Holmstedt, B., and Ryhage, R. , Anal. Biochem . 25:532- 548, 1968 .

4. Gordon. A.E., and Fngeno, A., J Chromatography 73:401-417, 1972: Costa, E .. and Holmstedt, B. (Eds.) Gas Chromatography­Mass Spectrometry m NeurobiOlogy Raven Press, New York, 1973 Jenden, D.J .. and Cho, A K., Ann Rev Pharmacal. 13:371-385, 1975.

5. For rev1ews see chapters by: a) S1lbergeld, E., and Goldberg, A.M.; b) vanWoert, M.H., and c) Wetss, B.L. , Foster, F.G., and Kupfer, D.J . tn: Biology of CholinergiC Function . A.M. Goldberg and I. Hantn (Eds.), Raven Press, New York, 1976.

6. Han1n, I. (Ed.), Choline and Acetylcholine: Handbook of Chemical Assay Methods , Raven Press, New York, 1974.

7 . a) Jenden, D.J , and Hantn, 1. . pp. 135-150; and b) Green, J.P., and Szilagyi. P.I.A .. pp. 151- 162; in: Choline and Acetylcholine: Handbook of Chemical Assay Methods, I. Hanin (Ed.), Raven Press, New York, 1974: c) Stavinoha, W.B .. & Weintraub, S.T., Anal. Chem. 46:757- 760, 1974.

8. Hammar, C.-G., Hanin, 1. , Holmstedt, B., Kttz, R.J .. Jenden, D.J. and Karlen, B., Nature 220:915-917, 1968 ; Karlen, B., Lundgren, G., Nordgren, 1. , and Holmstedt, B., m: Choline and Acetylcholine: Handbook of Chemical Assay methods , I. Hanin (Ed.), Raven Press, New York, 1974, pp 163-179.

9. Hammar, C.-G., Hantn, 1. , Holmstedt, B .. Kttz, R.J., Jenden. D.J. and Karlen, B., Nature 220:915-917, 1968.

DATELINE

Anaheim, California-

The Sixtieth Annual Meeting of the Federated So­cieties for Experimental Biology will be held at the Anaheim Convention Center, Anaheim, California, April 12 through 15. We will be exhibiting at booths E115-116, please stop in.