Upload
voanh
View
214
Download
1
Embed Size (px)
Citation preview
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
0 4
Ol
DZ
~ .. 0 .. .. 0'
: 0~ 41 ~ 04
Ol
oz
DWA EA
O• 71172 0• 1l8/lll
,_ OWA EI
• • 71/7l • •IU/160
0 4 06 WOLE RATIO
01 10
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.)
ZOrO•IUIIll 0•1661160
16
~
: 12 r .. . ..
00 00 01 I 0 II 20
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.
Bulk Rate U.S. Postage
PAID Sunnyvale, CA. Permit No. 328
Printed Matter
PRI N TED IN U.S .A .
George Vander Velde, Editor Finnigan Corporation
1145 W. Maude, Sunnyvale, Cahlom lal4086 800 E. Northwest Hwy .. Suite 330. Palatine, Il linois 60067 6 110 Executive Blvd., Rockville, Maryland 20852 9006 Sandstone Road, Houston, Texas n 036
BASEL MUNICH
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.