Amlications of Mass Determinations FRED P. ABRAMSONl
E. I . du Pont de Nemours & Co. Inst ru ment Products Division Monrovia, Calif. 91016
1971 Analytical Chemistry Division’s Summer Symposium, “Analytical Chemistry: A Key to Progress in Sational Problem Areas,” discussed many analyscs of significance to the welfare of the population or the economy of the country. The purpose of this paper is to present methods for a numbrr of these analyses based on a single instrumental technique-mass spectrometry-and the rationalization of these methods over traditional approaches. Several of these analyses have been reported by use of mass spectrometry but only for a particular type of problem. For example, N-nitrosodimethylamine (1, 2 ) , chlorinated pesticides (3-8), organophosphorus pesticides ( 9 ) , polychlorinated biphenyls (10-14), and organomercury (15) hnvc>been subjected to a variety of mass sprctrometric analyses, although diHE
Present address, Departmeiits of Pharmacology and Pathology, George Washirigtori University Schooi of 3Iedicine, Ranhirigton, U.C. 20005. 28A
ethylstilbesterol has not. By use of the most current capabilities of mass spectrometry, coupled with appropriate ancillary techniques such as gas chromatography, data acquisition and processing, and specific ion detection, t h r limits for each of thew adulterants lie in the lonnanogram or even in thr picogram range. The advantages of mass spectrometry are: Relatively uniform high sensitivity for all materials which are volatilizable, as opposed to the electron capture gc technique where differences in sensitivity of IOGor greater are observed (16) Excellent selectivity from interfering materials by usc of specific ion detection (14) or high resolution (6) The ability to identify uncxpcctcd materials easily The frcedom from false positive results Sonreproduction of gc times except cxperimcnts for which the mass spectrometer is not being scanned
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
Nominal Mass Spectrometry Experiments
A D u Pont Instruments Model 21-490 single-focusing mass spectrometer was used to detect the output from an Aerograph Model 1400 gas chromatograph. The mass spectrometer was equipped with the special differential pumping system and an all-glass gc/ms interface based on the molecular beam design of Waterman and Stern (17) which permits a major fraction of the materials of interest eluting from the chromatograph to be transmitted to the mass spectrometer. I n all scanning experiments the mass spectrometcr was controlled by a D u Pont Instruments Xodel 21-094 data acquisition and processing system with a scan rate of 2 sec/ decade. The library search accessory was also implemented. The gc/ms interface temperature was 2ZOoC, and the ion source temperature was 230°C. For single-ion detection experiments, the amplifier output was registered on a potentio-
SPECIAL REPORT Mass spectrometry combined with gas chromatography and/or data acquisition and processing and specific ion detection can be used to advantage to determine various adulterants of interest, such as DES,chlorinated pesticides, PCB’s, and organomercury in the
low nanogram or even in the picogram range
ipectrometry to Trace If
Environmental Toxic Materials
metric recorder through an appropriate filter network. Chlorinated Pesticides. Of principle importance to the analysis of chlorinated pesticides is the mnintenance of the chemical integrity of these highly labile materials. Xetallic surfaces and reactive sites in the chromatographic apparatus and gc/ms interface must be eliminated. Unlike gas chromatographic methods which are not particfilarly sensitive t o decomposition following the column, the identification by mass spectrometry requires intact molecules to provide recognizable mass spectra. The experiments which follow sho\T- that the criterion is fully met. Reference spectra at the 100 ng/,ul level 11-ere taken for lindane, heptachlor, aldrin, chlordane, dieldrin, p,p’-DDE, o,p’DDD, endrin, o,p’-DDT, p,p’DDD, and p,p’-DDT. These reference spectra were added to the library already in use (18) in the search routines’ isomers Of c h h d a n e ere separated and had similar spectra.
Figure 1. Computer-reconstructed total ionization gas chromatogram of pesticide sample. Column: 3% Dexsil 300 GC on 80/100 Supelcoport, 6-ft x l/s-in. Pyrex, 24OoC, 30 ml/rnin H e ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
29A
A sample containing 33 ng/p1 of heptachlor, dieldrin, and endrin v a s submitted for analysis. A chromatogram based on the total number of ions in each scan vs. the scan number for this samplc is shown in Figure 1. The major peaks at scan numbers 12, 29, 3S, and 46 were inputted to the library search routine. I’igurc 2 s h o m the result of this oprration. Heptachlor. dieldrin, and endrin are properly identified, and chlordane is the extra peak. -4 way of cxamining the entire body of data present in a chromatogram without any decisions on tho part of thc operator XT hich lvould require some initial information about thc samplc is the use of a significant peak table (19). Thi. is an ordered listing of various masses It-ith respect to their relative abundance in each spectrum, the highest value attained throughout a chromatogram being kept. Thus, n-hatevcr mass is thtx largest pralr in each of the 36 scans acquired \vi11 be listed at 1000, even if it is thc base peak from a scan with Ion intensity. The back-
ground masses 39, 41, 43, and 44 are ignored by the computer in the presentation. Table I shons significant peaks for heptachlor at m ’e 100 and 2’72 (scans 11 and 12), for chlordane at m’e 373 and 373 (scans 2s and %9), for dieldrin at m’e ’79 (scan 36), and for endrin at m / e 6 7 , S1, 263, and 77 (scans 43-46). The other masses are from hexane solvent or instrument background. One of the most important aspects of computer-based data acquisition and procewing is that it allolts the data to be taken continuously uithout any on-line decisions by the operator as to whether a particular chromatographic peak is important or not. In addition, the data from such a system are stored on niagnetic tape and can be kept infinitely long if reexamination of the data at a future time is requirrd. Such advantageq w r e demonstrated n hen an attempt i t a h inadc to identify the coniponcnt at scan 22 scvcral months aftrr the data n ere taken. Hcrc, the contributions from column bleed and the qolvent tailing ser-
iously obscure the possible identification. However, when the same scan has the background as defined by scan 20 subtracted from it, the spectrum is identified as isodrin through satisfactory agreement with the published spectrum (5) and is present in the sample at a concentration calculated on the basis of peak areas of 0.6 ng p l . Diethylstilbesteiol-Di~nethyl Ether. Diethylstilbesterol is a synthetic estrogen which has been used for the chemical caponization of poultry and for inducing neight gains in young cattle, sheep, and pigs. I t has been found to induce vaginal cancer in the daughters of women n-ho have taken diethylstilbesterol to prevent miscarriages. There is an obvious requirement to keep food products free of such adulterants. To facilitate the chromatography, Some derivative of thc til-o phenolic groups is preferable. Methylation (20) and trichloroacetylation (21) are proven derivatization techniques. The dimethyl ether of diethylstilbesterol was examined in this study. Its mass
I
Table 1. Significant Peak Table for Sample Shown in Figure 1 SIGNFPK GClD A119 DATE 12/10/71 AQRATE 10 SCTIME 2 RESPWR 600 HIMASS 400 THRESH 1
m LIBRLRI S W C H
~
I
IGNORE
mum MD&(
EPA #2 33 NANOGRAMS OF PESTICIDES
MAX INTN
FIRST OCCUR
40 55 57 67 73 79 81 100 373 375 272 56 263 149 77
1000 1000 1000 1000 1000 1000 1000 1000 1000 943 863 805 791 763 685
56 43 1 48 49 36 45 11 28
29 12 1 47 13 48
BI
B
Be
a0
R
ie
I
29 38
aa
3
MASS
0,
SCAN
8
IGNORE 39, 41, 43, 44 MILOUT 666 SEQU 38
30A
33 N A N O W OF P U T I C I D L S
eAl8
@ACKW
BASE
SWTR
in
0 B
0
43
B 0
B 0
a7 3a
su M
IONS *2**2
7503 9607 18417 4485 8586 10662 6662 4548 1671 1556 3272 11919 3231 3186 4966
EAN
I
IDOCHESS 550 0 0 0 B
e9 BASE I D . ma. rnm CHLOnMNE
373
I I
1
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
Figure 2. Library search output from four major peaks i n Figure 1. Zeros for chlordane indicate n o other compound could pass initial screening used i n search algorithm. 1000 would b e oerfect f i t
Special Report
spectrum is shown in Figure 3. Mass chromatograms of m/e 296 indicate that the detection limit is about 1 ng in this scanning mode of analysis and that the linearity of sample transmission is satisfactory to permit quantitative analyses to about + 10%. Much lower detection limits are possible by using single-ion monitoring rather than scanning. Figure 4 shows the chromatogram resulting from a 1.7 ng injection of DES-DRIE, n-hile focusing the mass spectrometer on m/e 296 only. The strong signal which results shows that as little as 10 picograms would provide a noticeable deflection. N-Nitrosodimeth ylamine. The dangers of this category of adulterants have been recently reviewed (1). The mass spectrum of a standard sample of DJINA introduced via gc is not in especially good agreement with previous data (1, 2 ) suggesting that instrumental conditions should be carefully monitored when performing such analyses. The presence of DXINA can be seen from its characteristic masses. To obtain maximum sensitivity, singleion monitoring was again employed. Mass 74, the molecular ion, was used as the diagnostic species, and the mass spectrometer v a s operating with the ionizing voltage at 22 eV to suppress the formation of fragment ions formed from background molecules which often interfere with lon-er mass number chromatography. Icigure 3 represents the output from an injection of 0.23 ng showing a peak with 1O:l S/X at the appropriate retention time for DXISA. Polychlorinated Biphenyls. The purpose of this investigation was to demonstrate the virtues of data processing for complex mixtures instead of the alternate of elegant chromatographic separation (19, I S ) . X sample of 1 pg of Arochlor 1260 was introduced, and 124 scans were taken of the resulting chromatogram. To provide resolution of the many components from this complex mixture, a series of' mass chromatograms nas generated, each of which was indicative of a particular degree of chlorine substitution. Thew i5 considerable overlap possiblr between fragment ions from
Figure 3.
Mass spectrum of 100 ng of diethylstilbesterol-dimethyl ether
Figure 4. Specific ion detection by use of mass 296 at 70 eV with 1.7-ng sample of diethylstilbesterol-dimethyl ether. Colu m n temperature, 220°C; Dexsil 300 GC
Figure 5. Specific ion detection by use of mass 74 at 22 eV with 0.25-ng sample of DMNA. Column: 10% Carbowax 400 a n d 5% KOH on 100/120 Gas Chrom P; 6-ft X l/,.in. Pyrex; 90°C; 30 m l / m i n H e flow; interface temperature, 150°C
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1 9 7 2
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Special Report
a more highly chlorinated biphenyl species and the molecular ion from a biphenyl of lower substitution. For example, the fragment ions C12H+216+ resulting from HC1 loss from heptachlorobiphenyl will overlap all of the molecular peaks from hexachlorobiphenyl except the species containing six 37Cl atoms. This species is of low relative abundance. To circumvent this difficulty, the identification of chlorine content was made starting from the highest number of chlorines (seven) to the lowest (three) and eliminating chromatographic peaks from any future consideration once any assignment was made. The spectra of these polychlorinated biphenyls are rather similar with molecular ion species as base peaks and loss of C1, generating the major fragment ion cluster. This molecular ion screening procedure shown in Figure 6 is the superposition of the five mass chromatograms, each approximately attenuated to reflect the absolute intensities of each species, with the total ion chromatogram, all generated from the data acquisition and processing system. The only possible missed assignments occur where a less substituted biphenyl coelutes with a more heavily substituted species, and only the latter is identified. Four such areas may exist, centered at scans 57, 93, 103, and 123, where there are larger peaks for the C16 species than for the C17. This sort of fragmentation is not expected and probably represents several points of coelution. Nonetheless, this procedure has identified 31 components in this mixture without using any special chromatographic care. In this procedure, merged components with their centroids only 1 scan apart (ca. 6 sec) have been separated (Figure 6). This technique xi11 be of general significance in the screening of complex mixtures where individual components or groups of components can be categorized by their mass spectra.
mass spectrometry to exclude from observation masses of elemental composition other than that desired. I n this way, materials which would cause interference even by the use of nominal mass singlepeak monitoring may be avoided. One traditional method for performing high-resolution experiments is peak matching (22) by use of an oscilloscope to monitor the output from the mass spectrometer which is being slowly alternated (ca. 1 sec) between two masses, one a reference mass and one the unknown mass. Exact mass is determined by superimposing the position of the unknown peak with
il, m / e 256 -258.260
Figure 6. Mass ch romatogra m deconvolution of Arochlor 1260 with chlorine n u m b e r a s structural probe. Top chromatogram represents total ionization, and lower five mass chromatograms identify chlorine numbers which are indicated on total ionization trace
