Anal. Chem. 1983, 55, 762-764
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Utilization of Natural Isotopic Abundance Ratios in Tandem Mass Spectrometry Kathleen E. Slngleton and R. Graham Cooks" Department of Chemistry, Purdue Universlv, West Lafayette, Indiana 47907
Karl V. Wood Englne/Fueis Laboratory, Instltute of Interdlsclpiinary Engineerlng Studies, Chemistry Building, Purdue University, West Lafayette, Indiana 47907
Isotope abundance ratios in various types of mass spectrometry/mass spectrometry (MS/MS) spectra (parent, daughter, and neutral loss scans) are used to facliltate identification of halogenated components In simple and complex mixtures. By monitoring reactions due to the several isotopic constituents of an ion, one can use the characteristic signal Intensity ratio, with the mass data, to identify the type and number of polyisotoplc elements in the ion. Thls information is contalned Independently in the parent, neutral loss, and daughter scans. This approach is even applicable to mlxtures so complex that the characteristic isotope pattern cannot be recognized in the mass spectrum. General expressions have been derived to predict the expected Isotopic abundance ratios for MS/MS spectra.
Mass spectrometry/mass spectrometry (MS/MS) is suited to the analysis of complex mixtures as it requires little or no sample preparation (1-5). Several MS/MS scanning modes can be used in such work. Parent and neutral loss spectra both characterize sets of compounds with specified properties, whereas daughter spectra identify individual constituents (6, 7). Specificity can be further increased by using ancillary techniques (8) in conjunction with the two stages of mass analysis inherent in MS/MS. These techniques include selective methods of ionization (9),collision-induceddissociation accompanied by charge inversion (IO),chemical derivatization of the bulk sample prior to MS/MS (11),and preseparation during evaporation of the sample into the ion source (11). The value of MS/MS scanning modes is enhanced by considering isotopic abundance ratios when analyzing spectra. When ions containing polyisotopic elements are considered, not only is it necessary for particular peaks to be present in the spectra but they must exhibit appropriate intensity ratios. This additional restriction permits greater certainty in the identification and/or confirmation of components present in a mixture. Isotopic patterns in MS/MS spectra are different from those of mass spectra. This can facilitate spectral interpretation as has already been shown for the case of daughter scans (12). This paper illustrates, extends and applies these ideas in examining both simple and complex mixtures using several MS/MS scan procedures.
EXPERIMENTAL SECTION MS/MS spectra were obtained at low (20 eV) ion energy on a Finnigan triple quadrupole mass spectrometer equipped with an INCOS data system (13). Mass spectra were recorded by scanning the third quadrupole (Q3) and setting the first and second quadrupoles (Q1 and Q2, respectively) to pass all ions. MS/MS scanning utilized the second quadrupole in the rf-only mode as a combined focusing device and collision cell. Q2 passed all ions generated by collision activated dissociation (CAD) of a
specified precursor ion. Daughter spectra were obtained by setting Q1 to pass an ion of selected m/z ratio and scanning Q3 to record the fragment ions produced by CAD in Q2. Parent spectra were obtained by setting Q3 to pass a selected ion and scanning Q1, thus characterizing the set of parent ions which produce a specified daughter on CAD in Q2. Neutral loss spectra were obtained by simultaneously scanning Q1 and Q3 with a mass offset characteristic of a particular functional group, so as to screen a mixture for constituents containing this functional group. Solid samples were introduced into the mass spectrometer via a direct insertion probe. Liquid samples were introduced via the solid probe inlet, and regulated with a Granville-Phillips valve to maintain a constant sample pressure in the ion source of ca. 3 X lob torr. The axial kinetic energy of ions entering the collision quadrupole (the difference between the potential of Q2 and the grounded ion source) was 20 eV. The potential of Q3 relative to Q2 was -2 eV. The offset potential of Q1 was set to optimize ion beam intensity. Argon was used as the collision gas at a pressure torr. The ion source was operated in the electron of ca. 2.3 X impact mode, with the temperature maintained at ca. 550 K. All compounds were purchased commercially and used without further purification. The nitrogen bases extract was provided by Chuck Schmidt of the Pittsburgh Energy Technology Center.
RESULTS AND DISCUSSION The use of isotope ratios will first be illustrated with reference to neutral loss scans. Particular cases of each scan type will be considered before the general cases are described. Consider two successive neutral loss scans taken with the mass difference set to correspond to loss of a fragment which includes an atom of the stable isotope of interest. For chlorine-containing mixtures, the mass offsets chosen can be 35 amu and 37 amu, corresponding to the loss of 35Cland 37Cl, respectively, or a higher mass fragment containing one chlorine atom may be selected (vide infra). Any ion appearing in both scans with the appropriate 35C1/37C1isotope ratio can be confiimed as originating from a chlorine-containingcompound. For any monochlorinated fragment lost from any chlorinated precursor this ratio (35C1:37C1or the corresponding higher fragments) is 3:l; in the general case of a polychlorinated neutral fragment the ratio of peak heights between the several neutral loss scans will always have the normal isotopic abundance signature characteristic of the neutral fragment. This is illustrated in Figure 1 which shows the neutral loss scans obtained from mixtures containing chlorinated and brominated compounds. The two spectra associated with bromine loss give ions of approximately equal abundance, corresponding to the 79Br:81Brratio of 1.02 (1.11f 0.03) while ions in the spectrum associated with chlorine loss approximate the 35C1:37C1 ratio of 3.08 (3.16 f 0.36). The main source of error was sample pressure regulation during the course of the two successive neutral loss scans. One can also consider relative peak intensities within neutral loss spectra. In the case at hand, no characteristic multiplets are present, demonstrating that all halogenated components are
0003-2700/83/0355-0762$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983 Neutral LOSS Spectra
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Figure 1. Neutral loss (35 amu and 37 amu) spectra of a mixture containing Chlorinated compounds and spectra due to loss of 79 amu and 81 amu for a mixture containing brominated compounds. The chlorine and bromine spectra were normalized independently.
Parent Scan of 289' I
324
Figure 2. Parent scan of m l z 289 from 2,4,6,3',5'-pentachlorobiphenyl.
monochlorinated or monobrominated. In fact, the chlorine compound mixture consisted of chlorobenzene (mol wt 112), benzyl chloride (mol wt 126), and 1-(chloromethy1)naphthalene (mol wt 176). The bromine compounds were bromobenzene (mol wt 156), o-bromotoluene (mol wt 170), and @-bromostyrene (mol wt 182). An additional or alternative method for confirming the presence of components containing a stable isotope is a parent scan, which records all precursors of a given fragment ion. In contrast to the neutral loss experiments, ion abundance ratios within a scan are now of primary interest. A fragment ion resulting from the loss of an atom of the stable isotope, for example, (M - Cl)+, is selected. A parent scan of this ion should show two molecular ions correspondingto the 35Cland 37Clconstituents in the usual isotopic ratio. This is illustrated in Figure 2, which is the parent scan of m/z 289, the (M- C1)+ ion of 2,4,6,3',5'-pentachlorobiphenyl. The parent spectrum contains the parent ions, m/z 324 and m/z 326, in a 3:l ratio. This characteristic chlorine ratio is obtained in spite of the fact that the isotopic intensity ratio for the m/z 324 and m/z 326 ions of pentachlorobiphenyl is 1:1.6. The 3 1 ratio results because the fragment ion chosen, m/z 289, is produced by the loss of a single chlorine atom from the molecular ion, thus the observed isotopic abundance is 3:1, characteristic of a single chlorine atom. Selection of any other isotopic variant of the (M - C1)+ion gives the same ion abundance ratio in the parent scan. Thus, it is the isotopic distribution in the neutral fragment which determines the ratio of peak intensities re-
Figure 3. (a) Electron impact mass spectrum, (b) neutral loss 63 amu spectrum, (c) daughter spectrum, m l r 288, from a nitrogen bases extract from a solvent refined coal mixture spiked with 150 ppm 1,2,4-trichlorodibenzo-pdioxin,and (d) daughter spectrum of m l r 288 derived from authentic 1,2,4-trichiorodibenzo-pdioxin.
corded within a parent spectrum. For the third type of scan, daughter scans, it is again ion abundance ratios within individual spectra which are of primary interest. These spectra display all fragment ions derived from a chosen precursor. In these spectra the fragment ions have isotope ratios which reflect the number of atoms of the polyisotopic element in the neutral fragment, as well as the chosen isotopic version of the parent ion. An extreme case is the choice of an isotopically homogeneous ion, e.g., containing six 35Clatoms, which can only give singlets in the daughter spectrum. The general case of mixed isotopes and loss of various numbers of atoms of the polyisotopic element has been discussed by McLafferty and co-workers (12). These examples show the ease with which multiple MS/MS scans can be used to glean compositional information on simple mixtures. The same procedures are also applicable to the analysis of constituents in complex mixtures where the isotope abundance ratios are masked by interfering ions in the mass spectrum. In this case, neutral loss scans can be used to screen for the presence of specific functional groups or molecules. A neutral loss 35 amu scan may be chosen to screen for chlorine-containing molecules whereas a neutral loss 63 amu scan (loss of COC1, characteristic of chlorinated dibenzo-p-dioxins (14-18)) will screen specifically for chlorinated dibenzo-p-dioxins. An application of these scanning modes to a complex mixture is illustrated in Figure 3. The complex mixture consists of a nitrogen bases extract from solvent refined coal spiked with 1,2,4-trichlorodibenzo-p-dioxin at ca. 150 ppm. It is not possible to identify the molecular ion or the characteristic isotope cluster (mlz 286, 288, 290) of 1,2,4-trichlorodibenzo-p-dioxin in the mass spectrum, Figure 3a, due to interference from other constituents of the complex mixture. By scanning for neutral loss 63 amu (loss of COCl), Figure 3b, the precursor ions at m / z 223 and 286 are clearly identified. The isotope ratios of these ions show the characteristic signatures for one and two chlorines, respectively. In selecting the mass offset to be 63 amu, a 35Cl atom is specified in the neutral fragment, therefore the precursor ions must actually contain two and three chlorines, respectively. This information can be confirmed by taking daughter scans. When this is done for the second ion, (M + 2)+, in the isotope cluster selected from the neutral loss scan, examination of the ratio (M + 2 - 35)+/(M 2 - 37)+ provides a rapid means
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
Table I. Isotopic Abundance Ratios in MS/MS Spectra scan type parent daughter neutralloss mass spectrum
between spectra (a
+ b)n-z
( a t b)"
(a t b ) Z
within a spectrum ( a t b)'
n-ic
'
z -x' c x (a t b)n-z
(a t b)"
for determining the number of chlorine atoms present in the fragment ion. (Ratios of 0:1, 1:1,2:1, or 3:l ...,correspond to the presence of 0, 1,2, or 3... chlorine atoms.) For example, in the daughter spectrum of 288+ (Figure 3c), the 253:251 ratio is 2:1, indicating that these ions contain two chlorine atoms. This again indicates the presence of three chlorine atoms in the m/z 288 ion. This information is also evident in the 2:l ratio of abundances for 225:223. In the case where the (M + 2)+ ion selected is from the molecular ion isotope cluster, comparison of this daughter spectrum to the daughter spectrum of the same ion in a standard can be used to identify the given constituent (see Figure 3d), in spite of the massive interference evident in the mass spectrum. Generalized equations have been derived to give the variom abundance relationships for polyisotopic species examined by the several MS/MS spectra (Table I). Chlorine is chosen as the polyisotopic atom; however, the equations are applicable to any compound containing a polyisotopic element. For these equations, the terms are defined as follows: a and b represent the relative isotopic abundances of %C1and W l , respectively, n is the number of chlorine atoms in the precursor ion, i is a term number in the isotope cluster of the precursor ion, z is the number of chlorine atoms in the neutral fragment, and x ranges from 0 to z (each value of x representing a term number in the fragment ion cluster). The following descriptions of these expressions first refer to ion abundance ratios within individual scans. The relative abundances of peaks in a given isotope cluster within a daughter spectrum are described by the expression ( n - i l)!(i - l)! - n-ic2-x [ n - i 1- ( z - x ) ] ! ( z - x ) ! ( i - 1- x ) ! x !
+
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It should be noted that the expression is independent of the natural isotopic abundances and dependent only on the number of polyisotopic atoms present. For a case in which n = 6, i = 4, and z = 3, the peaks in the daughter spectrum are given by the ratio 1:991. The x = 0 term represents the loss of 35Cl. In a case such as n = 6 , i = 4, and z = 4 where it is not possible to lose four 35Cl,the value at x = 0 is undefined, and the cluster begins at x = 1with the loss of three 35Cl and one 37Cl. In contrast, the ratios of total ion abundances between daughter spectra are given simply by the binomial expansion (a b)". For ion abundances within parent spectra, the isotope cluster ratio is described by the binomial expansion (a + b)z. For example, the parent scan of (M - 70)+ (70 = 35C12)for a compound containing four chlorine atoms will show a cluster characteristic of two chlorines. The same spectrum will be generated for each choice of daughter ion. However, when comparisons are made between parent spectra, total abun-
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dances will be in a ratio given by the relative natural abundances of the various selected ions, viz., (a + b)n-Z. The isotope cluster ratios within neutral loss spectra can be described by the binomial expansion (a b)n-z. For example, the neutral loss 72 amu spectrum of a compound containing three chlorine atoms will show a cluster characteristic of one chlorine. (Note the isotope cluster would be the same, as would the masses at which it is observed, for neutral loss 70 amu and 74 amu from a three chlorine-containing compound.) Similarly, the ratio of intensities between neutral loss spectra representing the various isotopic choices can be described by the coefficients of the binomial expansion (a b)z. Summarizing, there are two sets of ion abundance ratios of interest for each type of scan: those within a particular scan and those between the various isotopically distinct scans of the same type. The former are of chief interest for parent and daughter scans, while both types are useful for neutral loss scans. The ratios of ion abundances for each of the six scan types is summarized in Table I.
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CONCLUSION MS/MS neutral loss, parent, and daughter scanning modes can be used to assist in analyzing complex mixtures through recognition of specific classes of compounds as evident from isotopic abundance ratios. The several isotopic versions of each spectrum are identical in appearance (but not intensity) for the neutral loss and for the parent scans. However, for daughter spectra the particular isotopic version of the parent ion which is selected determines the appearance of the spectrum. Isotopic abundance ratios within and between individual parent, neutral loss and daughter scans occur as given in Table I.
LITERATURE CITED Cooks, R. G.; Glish, G. L. Chem. Eng. News 1981, Nov 30, 40. Kondrat, R. W.; Cooks, R. G. Anal. Chem. 1978, 5 0 , 81A. Maugh, T. H. Science 1980, 209, 675. McLafferty, F. W. Science 1981, 274, 280. Yost, R. A.; Enke, C. G. Anal. Chem. 1979, 57, 1251A. Clupek, J. D.; Cooks, R. G.; Wood, K. V.; Ferguson. C. R. Fuel, in press. Zakett, D.; Schoen, A. E.; Kondrat, R. W.; Cooks, R. G. J. Am. Chem. SOC.1878, 107, 6781. McLuckey, S. A.; Cooks, R. G. I n "Tandem Mass Spectrometry"; McLafferty, F. W., Ed.; Wlley: New York, In press; Chapter 15. Ciupek, J. D.; Zakett, D.; Cooks, R. G.; Wood, K. V. Anal. Chem. 1882, 5 4 , 2215. Bowie, J. H.; Blumenthal, T. J. Am. Chem. SOC. 1975, 97, 2959. Zakett, D.; Cooks, R. G. I n "New Approaches in Coal Chemistry"; Blaustein, G. D., Bockrath, B. C., Friedman, S., Eds.; American Chemlcal Society: Washington, DC, 1981; ACS Symp. Ser., Chapter 16. Todd. P. J.: Barbalas. M. P.: McLaffertv. F. W. Ora. Mass Soectrom. lS82, 17 (2), 79. Slayback, J. R. B.; Story, M. S. Ind. Res./Dev. 1881, (Feb), 129. Buser, H. J. Chromatogr. 1975, 107, 295. Buser, H.; Rappe, C. Chemosphere 1978, 2, 199. Mahle. N. H.; Shadoff, L. A. Blomed. Mass Spectrom. 1982, 9 , 45. Pllmmer, J. R.; Ruth, J. M.; Woolson, E. A. J. Agrlc. Food Chem. 1973, 21, 90. Safe, S.;Jamieson, W. D.; Hutzinger, 0.; Pohland, A. E. Anal. Chem. 1975, 47, 327.
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RECEIVED for review October 27,1982. Accepted January 18, 1983. This research was supported with funds from NSF (CHE-8011425). K.E.S. acknowledges the support of CONOCO through a grant to the Purdue Coal Research Center.
