Anal. Chem. 1988, 60, 1933-1936
1933
technique. The certified value for the a-tocopherol in the cod liver oil SRM is also provided in Table IV. The multidimensional HPLC procedures provide rapid analysis time, minimal sample handling, and analyte specificity. The accuracy of the multidimensional HPLC method is M % , and the precision is within 6%. The availability of SRMs 1563 and 1588 will provide the analyst with materials for use in the validation and comparison of analytical methods for the determination of fat-soluble vitamins in lipidic foodlike matrices.
c1
E N
a
N
Y
al 0 C
ACKNOWLEDGMENT The authors thank Hoffman-La Roche, Basel, Switzerland, for the gift of tocol and Karlheinz Ballschmiter, University of Ulm, Federal Republic of Germany, for the cod liver oil sample. We also thank Gary D. Byrd and Richard G. Christensen for their technical assistance in establishing the purity of the reference compounds used in this study and Robert C. Paule for statistical analysis of the analytical data. Registry No. Retinyl acetate, 127-47-9;vitamin D2,50-14-6; dl-a-tocopheryl acetate, 52225-20-4; a-tocopherol, 59-02-9.
m
f In
n U
> 3
I
I
5
10
I
1
15
LITERATURE CITED
20
Retention time (min)
F W e 6. Chromatogram of cod liver oil SRM 1588 using normal-phase HPLC.
GPC/reversed-phase HPLC analysis. A chromatogram from the analysis of the cod liver oil using direct-injection normal-phase HPLC is shown in Figure 6.
CONCLUSIONS Table IV provides both gravimetric values and the concentrations of retinyl acetate, dl-a-tocopheryl acetate, and ergocalciferol in SRM 1563-2 as determined by the multidimensional HPLC procedures. The measured values are in agreement, in all cases, with the gravimetric amounts of each vitamin added to the coconut oil. The certified concentrations for SRM 1563-2 (fortified coconut oil) were derived from a weighted combination of the HPLC results and the gravimetric data. The uncertainty associated with these values is f 2 standard deviations of the mean values determined by each
(1) Method of Vitamin Assay, 3rd ed.; Interscience: New Ywk, 1966. (2) Margolls. S. A. Reference MaferMs for Organic Nutrienf Measurem n f ; Special Publication 635; National Bureau of Standards: Galthersburg, MD, 1982. (3) Landen, W. 0. J . Assoc. Off. Anal. Chem. 1980, 6 3 . 131-135. (4) Landen, W. 0.; Eienmuiier, R. R. J . Assoc. Off. Anal. Chem. 1979, 6 2 , 283-289. (5) Williams, R. C.; Schmidt, J. A.; Henry, R. A. J . Chromafogr. Sci. 1972, 70, 494-501. ( 6 ) Ueda, F.; Makino, T.; Kazama, A. Vitamins 1969, 39, 176-160. (7) Ueda. F.; Makino, T.; Kazama, A. Vitamins 1969, 40, 84-90. (8) Holasov6, M.; BlattnB, J. J . Chmmafogr. 1976, 723, 225-230. (9) Cerflflcafe of Analysis , SRM 1563, Chokferol and Fat-Soluble Vita mlns in Coconut oil; National Bureau of Standards: Gaithersburg, MD. 1987. (10) Cerfiffcafeof Analysis, SRM 7588, Organics in Cod Liver Oil; National Bureau of Standards: Gaiihersburg, MD, 1988.
-
RECEIVED for review February 5,1988. Accepted May 2,1988. Certain commercial products are identified to specify adequately the experimental procedure. Such identification does not imply endorsement or recommendation by the National Bureau of Standards, nor does it imply that the materials identified are necessarily the best available for the purpose.
Detection of Neutral Products of Chemical Ionization Reactions Charles Allgood and Burnaby Munson* Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 The neutral products of chemical Ionization (CI) reactions have been detected In an unmodified commercial chemical ionization mass spectrometer source. The neutral products are ionized in subsequent ion/moiecule reactions with reagent gas ions. The reiatlve abundances of these ions show a characteristic increase with increasing electron current. The neutral product of benzene has been detected at m / z 79, CsH7+, in the CH, C I spectrum of bibenzyi and 4-methylbenzophenone. Chlorobenzene, the dominant neutral product from C I reactions, was Identified in the CH, C I spectrum of l,l-bls(p-chlorophenyl)-2,2,2-trlchloroethane ( p ,p’-DDT). I n addition, thls method has been used to determine that protonated benzil, CsHsCOCOCeHs, fragments to form C,H,CO+ and neutral benzaldehyde, C,H,CHO, rather than C,H, and
co. 0003-2700/88/0360-1933$01.50/0
Table I. Relative Ion Intensities for Selected Ions in the CHd CI Mass Spectrum of Bibenzyl as a Function of Total Source Pressurea
pressureb
183c/105d
7ge/105
0.425 0.500 0.550 0.600 0.650
0.0195 0.0190 0.0190 0.0189 0.0180
0.0290
0.0370 0.0480 0.0500 0.0505
Electron current = 0.65 mA. Source pressure in Torr. mlz 183 = (M
+ H)+. d m l z 105 = C,H,C,H,+.
‘ m l z 79 = C,H,+.
The neutral products of ion/molecule reactions have been detected in radiation chemistry experiments (I,2 ) , in ultralow-pressure reactors (3), in ion cyclotron resonance (ICR) 0 1988 American Chemical Society
1934
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15. 1988
experiments (4),and under high pressure or chemical ionization (CI) conditions ( 5 ) . The ICR and CI experiments utilized unusual experimental conditions to demonstrate that the neutral products could be detected for certain carefully chosen systems. In this paper, we wish to report the detection of the neutral products of reactions in an unmodified CI source from several classes of compounds under standard high pressure or CI conditions and to illustrate analytical applications of the detection of neutral products of CI reactions for structure elucidation and the determination of mechanisms of fragmentation reactions.
EXPERIMENTAL SECTION The mass spectra were obtained with a Hewlett-Packard 5980 quadrupole mass spectrometer using the chemical ionization mode of the dual electron impact (EI)/CI source. The data were collected with an IBM XT computer interfaced to the mass spectrometer with a Technivent interface and software (Technivent, Inc., St. Louis, MO). The instrument was usually operated in the selected ion monitoring mode, recording five to ten ion channels. However, full range CI mass spectra were obtained for all compounds with this instrument and with a Du Pont 492 B mass spectrometer, which has been modified for high-pressure operation (6). The electron energy was nominally 70 eV. The source temperature was approximately 250 "C. The CI source operates with no repeller or withdrawal plate voltage in the high-pressure region. The pressure was measured with a capacitance manometer (MKS Instruments, Burlington, MA) connected directly to the source through a metal probe. The externally measured electron current was varied from approximately 0.20 to 0.70 mA. The values of the electron current within the high-pressuresource are not known, but are expected to be proportional to these values. The methane reagent gas (Matheson, UHP) was maintained at approximately 0.5 Torr for these experiments. The samples were obtained from several commercial sources and were used without further purification. GC and MS analyses showed no significant impurities in these compounds. All of the samples were introduced through a metal probe under conditions such that the sample concentration in the source remained relatively constant for the several-minute duration of a set of experiments. Approximately 30 s was allowed to elapse after each change in the electron current before taking measurements; however, this time interval did not appear to be a critical parameter in these experiments. Each ion channel was sampled for 3 ms, and approximately 15 readings were averaged to obtain each point. The total time to sample all of the ion channels at each electron current was approximately 1.2 s including settling times. During this time, no signficant variation was observed in the ion current (samplesize). The electron current was varied randomly to eliminate the possibility of changes due to changes in sample size during the course of an experiment. Similar experiments could be done on samples introduced through a gas chromatograph and were done for other compounds in experiments not reported in this paper. RESULTS AND DISCUSSION To establish whether or not the neutral products could be detected by subsequent ion/molecule reactions under CI conditions in this unmodified, standard CI source, experimenb were tried with bibenzyl, C6H5CH2CH2C6H5.The CH4 CI mass spectrum of bibenzyl is very simple: a dominant fragment ion a t m / z 105, an abundant MH+ ion at m / z 183, and traces of C6H7+at m / z 79, as shown in Figure la. One can explain this spectrum in terms of proton transfer to the benzene ring with rapid migration of H around the ring, followed by decomposition to give neutral benzene and CsHg' a t m / z 105. This C8Hg+ion is either the phenylethyl ion, C6H&HzCHz+,formed by simple cleavage of the aromaticCHz bond or C6H5CHCH3+,the methylbenzyl ion, formed by simultaneous cleavage of this bond and rearrangement of the ion to this lower energy structure. Previous experiments using a modified CI source indicated that the neutral product of benzene could be detected by the
a.
I I
EC
=
.25 mA (M+Hl '
C8,' +,-.
,
I!,
68
88
181 128 140 160 188 288
68
88
111 120 148 168
181 208
mass spectrum of bibenzyl at low (a)and high (b) electron currents: t = 250 OC;P = 0.50 Torr. Figure 1. CH, C I
,035,
1
I
.015-/
'oloi&& 107/105
,005
. ,000 0.0
O0 . . 11
0.2 0 . 2o
0.3 5
0.4
0.6 I
w0 . 5 ~
0.7
E l e c t r o n C u r r e n t . mA
Relative ion currents for selected ions in the CH, C I mass spectra of bibenzyl as functions of electron current: 791105 or C8H7+/CH and 1071105 or C8H1,+1C8HO+correspond to the lefthand scale: MH /C8H,+ or 1831105 corresponds to the right-hand scale; t = 250 OC; P = 0.50 Torr. Flgure 2.
+
increase with increasing electron current of the ratio of ion currents, C6H7+/C8Hg+,according to the following reactions (5, 7): CH5+ C ~ H ~ C H ~ C H Z CCsH,+ ~ H ~ C6H6 CH,
+
CH5+ + C6H6
-
C6H,+
+
+ CH4
+
(1)
(2) The spectrum of bibenzyl in Figure lb, obtained with a much larger electron current than the spectrum in Figure l a (0.75 mA compared with 0.25 mA) shows a low-abundance, but easily detectable, ion a t m / z 79, C6H7+,which may be compared with the traces of C6H7+in Figure la. The data of Figure 2 show the expected increase of the 1(79)/1(105) ratio with increasing electron current in agreement with the previous observations (5, 7). The observation that the 1(79)/1(105) ratio appears to extrapolate to zero a t zero electron current suggests that all of the protonated benzene at m/z 79 is formed by the two-step reaction above. Consequently, although the more complex dissociative proton-transfer reaction CH5+ + C ~ H ~ C H ~ C H ~ C G H ~ C6H6CH=CH2 + C6H7++ CHI (la) is 29 kcal exothermic, it is not involved in the formation of C&+. Figure 2 also shows the constancy of the 1(183)/1(105) or [MH+]/ [C8H9+]ratio with variations of electron current, as one would expect for two ions that were formed by com-+
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988
peting bimolecular reactions of CH5+and C2H5+ ions with the sample. The CHI CI mass spectrum of benzene has been reported to contain a low-abundance ethyl adduct ion, (M C2H5)+, CaH11+ at m / z 107, approximately 17% of the base peak (8). This ion should be formed in this mixture from ion/molecule reactions of CZH5' with benzene. The ratio of ion currents, I ( 107)/I(105), increases with increasing electron current, as shown in Figure 2. In addition, it is possible that the ethyl ion reacts with bibenzyl by the dissociative addition reaction C2H5' + C ~ H ~ C H ~ C H ~-*C CSHll+ G H ~ + C ~ H S C G H(3) ~
1935
+
to produce neutral ethylbenzene, which can be detected by subsequent proton-transfer reactions from the CH4CI reagent ions as follows: CH5+ + CzH5CGH5 C8Hll+ + CH4 (4)
-
This mechanism for the formation of protonated ethylbenzene at m/z 107 will also cause the 1(107)/1(105) ratio to increase with increasing electron current, as shown in Figure 2. The ratio 1(107)/1(79) is independent of electron current at 0.10 f 0.02 for these experiments. Under similar conditions, pure benzene introduced as a sample gives a ratio of 0.046 f 0.006 for 1(107)/1(79), an observation that suggests that reaction 3 is occurring to some extent to form protonated ethyl benzene in the CHI CI spectra of bibenzyl. The lower ratio for (M + CzHs)+/(M + H)+ in these experiments than in those reported previously (8)results from the higher temperature and lower pressure in the current experiments. Since the formation of protonated benzene at m / z 79 by reactions 1plus 2 involves two consecutive ion/molecule reactions of CH5+ and the formation of C8H9+involves only a single reaction of CH5+,the ratio 1(79)/1(105)should increase with increasing reaction time (at constant electron current). An increase in pressure in a conventional CI source increases the number of ion/molecule collisions and the reaction time; consequently, one would expect that the ratio 1(79)/1(105) should increase with increasing pressure, as is shown in Table I. The ratio, I(183)/1(105), is essentially independent of pressure. The CI mass spectra of more complex molecules also give rise to neutral fragments that are detectable by these experiments of the variation of ionic ratios with electron current. For example, the CHI CI spectrum of p,p'-DDT contains small amounts of MH+ ions (18%), the most abundant fragment ions corresponding to the loss of C6H5C1from MH+ (loo%), less abundant fragment ions corresponding to the loss of HCl from MH+ (50%),and a relatively minor species from the loss of CHC13 from MH+ (5%), in agreement with previously reported data (9). One observes in Figure 3 that the ratio of isotopic abundances that is proportional to the C&Cl+/(M + H - C6H5C1)+ ratio is proportional to the electron current. Consequently, we may attribute the formation of d l of the C6H6C1+ions to the consecutive reactions p,p'-DDT + CH5+ -* (M + H - C&Cl)+ C&&1 (5)
+
+
C&C1 + CH5+ -* C&C1+ CH4 (6) For the reasons given above for the ions from the neutral products of bibenzyl, one would expect to observe (M + CZH5)' ions for chlorobenzene; and the ratio (C6H5C1+ C2H5)+/ (C6H6Cl)+should be independent of electron current. These results are also shown in Figure 3, 0.020 0.001. When pure chlorobenzene is introduced as a sample, under identical experimental conditions, the ratio I(107)/I( 105) is 0.018 f 0.001, essentially the same as the value obtained for p,p'-DDT. The agreement between these two values confirms the formation of neutral chlorobenzene and indicates the
*
I
.01
.000.0
0.1
0.2
0.3
0.5
0.4
E I ~ c t r o n Current,
0.6
.00
0.7
mA
Figure 3. Relative ion currents for selected ions in the CH, C I mass spectra of p,p'-DDT as functions of electron current: 1411113 and 1131147 correspond to the left-hand scale and 2351247 correspond to the right-hand scale; 113 = 35CIC H +; 141 = 35CIC6H5C,H,+; 235 = 36C12isotope of (M H - CHC13)'; 147 = 37C13isotope of (M H - C6H5CI)+.
+
+
absence of a dissociative addition reaction of CzH5' with DDT analogous to (3). Other ionic ratios are independent of electron current: (M + H)+/(M + H - C&Cl)+, (M + H - HCl)+/(M + H C6H5C1)+,and (M + H - CHCl,)+/(M + H - C6HC1)+. The last ratio is shown in Figure 3 for one isotope of each species, 1(235)/1(247). These ratios reflect the ratios of products of proton-transfer reactions to DDT. The ratios of the abundances of the chlorine isotopes of each ionic species are also independent of electron current. The CHI CI mass spectrum of CHC13 consists mostly of CC13+ions. However, since the loss of chloroform is a minor pathway in the fragmentation of protonated DDT ( 5 % ) , only small amounts of these ions can be detected a t even the highest electron currents studied: 1(117)/1(113) = 0.1. The neutral product from the other major fragmentation reaction of MH+ is HCl; however, no significant amounts of H2C1+are observed under any conditions. We presume that the HC1 reacts rapidly with the walls of the source of the mass spectrometer. The dominant ionic products in the CHI CI mass spectra of aliphatic chlorides are (M + H - HC1)+ ions; however, no significant amounts of H2C1+were detected with these compounds either. In the example of bibenzyl, there was only one possible neutral product that could be formed during the fragmentation of the protonated molecule. For benzil, C6H6COCOC6H5,the CHI mass spectrum contains predominantly C6H5CO+at m/z 105 (Figure 4a). This major fragment ion can be formed by the following two equally reasonable mechanisms that cannot be differentiated from the ionic species formed: C&&OCOC6H5 C&COCOC&5
C&+
+ CH'5
-
-
C&,CO+ C&&O+
C,jHsCHO (7) C6H6
+ co
(8) The correct mechanism can be determined by detecting the neutral products, either benzaldehyde (7) or benzene and carbon monoxide (8), by subsequent reactions of these neutral products with the methane reagent ions. Figure 4b clearly shows reaction (7) to be the dominant fragmentation pathway, since the ionic ratio, C6H5CHOH+/C6H5CO+ or I(107)/1(105), increases with increasing electron current while only very small amounts of protonated benzene are detected at m / z 79 and the 1(79)/1(105) ratio does not increase with increasing electron current. Protonated CO, HCO' at m / z 29, cannot be detected in these experiments because of the large amounts of C2H5' ions at m / z 29 from CHI. Calculations show that the rate constants for reaction of CH5+ with benzene and
1936
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988 ,020
, f
20 18
4-Methylbenzophenone
C Y u C
11
180
120
148
200
188
168
228 005
t
93/79
t
0OOi.c 0.0
I 0.1
-
6
-
4
5
I-0
0.2
0.3
0.4
0.5
0.6
0.7
E l e c t r o n C u r r e n t , mn
,005
,000
j & L
0.0
/"
0.1
Flgure 5. Relative ion currents for selected ions in the CH, C I mass spectra of 4-methylbenzophenone as functions of electron current. Solid points (left-hand scale) correspond to (protonatedtol~ene)/('~C, isotope of CEH5CO+)and open points (right-hand scale) correspond to (protonated toluene)/(protonated benzene).
791105
1. .05 !
=
--.-L-., I
0.3
0.2
0.4
Electron Current.
0.5
-0.6
0.7
.oo
m4
Flgure 4. (a) CH, C I mass spectrum of benzil, CEH,COCOC6H5: P = 0.50 Torr; t = 250 OC; electron current = 0.35 mA. (b) Relative intensity of selected ions as functions of electron current: 107/105 or CEH,CHOH+/CBH5CO+ and 791105 or C&+/C&CO+ correspond to the left-hand scale; 211/105 or MH+/C6H,+ corresponds to the right-hand scale.
benzaldehyde are approximately the same: k(CH5+, C6H5CHO)/k(CH5+,C6H6)= 1.07 (10). Figure 4b also shows that the MHf/C6H5CO+ratio, Z(211)/Z(105),is independent of electron current, as expected for two ions formed by processes of the same kinetic order. In molecules with more than one major fragment ion there must also be more than one neutral product that may be detected in these type of experiments. The substituted benzophenones, for example, give major peaks in their CH, CI spectra corresponding to the substituted and unsubstituted benzoyl ions, as shown in reactions 9 and 10. (C&jCOC6H,X + H)+ XC&4CO+ C & 3 (9) (C6H5COC&X
+ H)+
+
C6HjjCO'
+ C&jX
(10)
Benzene and substituted benzene are formed as neutrals. In 4-methylbenzophenone, for example, the neutral molecules formed from dissociative proton transfer are toluene and benzene. Both species are detected as their (M + H)+ ions and have the expected dependence on electron current, as shown by the linear increase for 1(93)/1(120),[CH3C6H6+]/ [C&CO+], and the constant ratio of ion currents, 1(93)/1(79), [CH&,&+] / [ C,H,+], in Figure 5 , in agreement with expectations from the mechanisms of formation. The abundance of ions at m/z 93 is greater than the abundance of ions at m / z 79, in qualitative agreement with the fact that formation of C6H&O+ is greater than the formation of cH3C6H5CO+.The relative abundances are given by [C6H7+i / [CH3CGH6+1 = ka[C6H61 /Kb[CH3C6H51
-
ka[CH3C6H,CO+] /kb[C&I5CO+]
in which ka and kb are the rate constants for the reaction of benzene and toluene with methane ions. The Langevin theory ( I I ) , which takes into account the molecular polarizability and reduced mass, gives a theoretical ratio of 1.08 for k a / k b . The measured ion current ratio for [CH3C6H4CO+]/[C6H5CO+]is 1.15, giving a theoretical value of 1.25 for the ratio [C&+] / [CH3C6H6+],compared to the experimentally obtained value of 2.0 f 0.2 shown in Figure 5. This method of detecting neutral products of chemical ionization reactions may be helpful in structure determination because both the ionic species as well as the neutral products from CI reactions can be detected a t the same time under identical experimental conditions. These and other experiments suggest that this two-step mechanism may be occurring to a small extent in forming the CI mass spectra of many compounds. Further work in this area is being undertaken at present. h g k t r y NO.p , p '-DDT, 50-29-3;C6H,CH&H&H, 103-29-7; 4-CH3CeH,COC6H, 134-84-9;CsH,jCOCOCsH5, 2154-56-5.
LITERATURE CITED (1) Cacace, F. I n Kinetics of Ion-molecule'Reactions; Ausloos, P., Ed.; NATO Advanced Study Institutes Series B, Vol. 40; Plenum: New York, 1979: p 199. (2) Lias, S. G.: Ausloos, P. Ion-Molecule Reactions: Their Role in Redia tion Chemistry; American Chemical Society: Washlngton, DC, 1975. (3) Marinelli, W. J.; Morton, T. H. J. Am. Chem. SOC. 1978, 700, 3536. (4) Brauman, J. A.; Lieder, C. A. Int. J. Mass. Spectrom. Ion fhys. 1975, 17. 307. (5) Blom, K.; McGuire, J. M.; Hauer. C. R.; Munson B. Org. Mass Spectrom. 1982, 17, 345. (6) Spreen, R. C. Ph.D. Thesis, University of Delaware, 1983. (7) Blom, K. F. Ph.D. Thesis, University of Delaware, 1983. (8) Field, F. H.; Munson, M. S. 8. J. Am. Chem. SOC. 1967, 89, 1047. (9) Safe, S.; Huntzinger, 0. Mass Spectrometry of Pesticides and follutants; CRC: Boca Raton, FL, 1973; p 24. (IO) Su, T.; Chesnavich, W. J. Chem. Fhys. 1982, 72, 5183. (11) Harrison, A. Chemical Ionization Mass Spectrometry; CRC: Boca Raton, FL, 1983.
-
RECEIVED for review February 16, 1988. Accepted May 16, 1988. This work was supported in part by a grant from the National Science Foundation (CHE-8312954).
