Table VI. CI(NH3) Mass Spectra of Selected Amines Major peaksa ComTemp, pound (mol wt) O C M+1 Others: m/e (96) 4 7 8*
(183) (190) (176)
160 150 160
75 100 0
220
17
66(12), 49(100) 161(9), 95(11), 78(48) 131(58), 114(100), 69(13), 63(11), 49(11) 227(14), 131(56), 114(100),66(8), 49(154) 78(9),61(55) 95(19), 78(29) 176(4),63(3), 46(4)
(179) 160 100 (207) 125 100 (177) 150 100 a (M 1)+-NH3peaks are generally present with relative intensities 5 2 5 % as are (2M 1)+ peaks particularly at higher 10 12 14c
+
+
sample pressures. These peaks are omitted together with all those arising from the spectrum of NH3 reagent gas. Run as serotonin creatinine (mol wt 113) sulfate. This spectrum was obtained only after allowing the sample to evaporate in the direct probe for 9 min. A t shorter times, the spectrum was dominated by a peak at m / e 192, apparently resulting from a more volatile aryl methyl ether impurity present in the sample (E1and FD suggests that about 2% of this impurity is present).
the reagent gas ions. However, in this case, the samples were handled in a completely routine manner to better approximate the results which would be obtained in the case of an unknown of biological origin. Thus the characteristics of CIMS when used in these circumstances can be summarized as simple and reliable operation and high sensitivity. Ion currents due to sample are a t least as high for the same sample flow as in EIMS, and protonated molecular ion currents greater than A a t the collector are obtainable. CONCLUSIONS The present work demonstrates the potential of FDMS for analysis and structure determination of biogenic amines and
related compounds. The comparisons with spectra obtained by other techniques allow for some assessment of their relative strengths. The caveat contained in the last paragraph of the paper by Milne et al. (1)emphasizes the difficulties inherent in the application of CIMS to biological samples and it is likely that formidable problems would be associated with FDMS also. In introducing this latter alternative, one escapes the problems related to vaporization of an unstable, polar sample and thereby gains an element of flexibility in that nonvolatile salts which have improved spectral characteristics may be employed. To counter this, one may expect a loss in sensitivity and, perhaps, a new set of sample preparation difficulties. We have not explored this latter area, except to show that the presence of alkali metal salts (NaCl, CsC1) does not interfere with smooth field desorption of isoquinoline 13, even when massive amounts are added to the sample. It may also be noted that no evidence of sodium containing organic ions was found in any of the commercial samples studied, a result which our experience suggests is more likely to arise from favorable field desorption characteristics of the compounds than from scrupulous absence of sodium salts. ACKNOWLEDGMENT The authors acknowledge Maurice Hirst for generous samples of isoquinolines. We thank Robert Charlton, Pui-Yan Lau and John Olekszyk for technical assistance. LITERATURE C I T E D (1) G. W. A. Milne, H. M. Fales,andR. W. Colburn, Anal. Chem., 45, 1952(1973). (2)G.Cohen and M. Collins, Science, 167, 1749 (1970). (3)V. E. Davis, M. J. Walsh, and Y-L. Yamanaka, J. Pharmcol. Exp. mer., 174, 401 (1970). (4) H. D. Beckey, Int. J. Mass Spectrom. /on Phys., 2, 500 (1969). (5) A. M. Hogg, Anal. Chem., 44, 227 (1972). (6) E. Waser and H. Sommer, HeIv. Chim. Acta, 6, 61 (1923). (7) J. Reisch, R. Pagnucco, H. Alfes, N. Jantos, and H. Mollmann, J. Pharm. Pharmacol., 20, 81 (1968).
RECEIVEDfor review December 1 , 1 9 7 5 . Accepted March 8, 1976. We are grateful to the National Research Council of Canada for generous support.
Determination of Chlorinated Dibenzo-p-dioxins in Pentachlorophenol by Gas Chromatography-Mass Spectrometry W. W. Blaser,” R. A. Bredeweg, L. A. Shadoff, and R. H. Stehl Analytical Laboratories, Dow Chemical U.S.A., Midland, Mich. 48640
A gas chromatographic-mass spectrometric procedure has been developed for the determination of chlorinated dibenzo-pdioxins in pentachlorophenol. The chlorinated dibenzo-pdioxins are isolated from the sample matrix using ion exchange chromatography. Since the detection system is highly specific, no further sample preparation is necessary.
Several papers (1-5) have drawn attention to the chlorinated dibenzo-p-dioxin (CDD) content in technical grades of domestically manufactured pentachlorophenol. In these, the analytical method employed to measure CDD has been electron capture gas chromatography. This detection system is relatively nonspecific to the large number of structu984
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
rally similar components found in these samples (6);thus, a multistep clean-up procedure was employed to prepare the samples prior to quantitation. The purpose of this paper is to describe a simple analytical technique for the accurate determination of CDD employing ion exchange chromatography followed by gas chromatography-mass spectrometry (GUMS) determination. Since the mass spectrometer can monitor the molecular ions from the particular component of interest to the exclusion of other ions, the specificity attained allows minimal sample clean-up prior to quantitation with high sensitivity. EXPERIMENTAL Solvents. All solvents used in the procedure were “distilled in glass” quality obtained from Burdick and Jackson Company,
500mv. f.8
(A
A
460\
4 2
12
8
16
4
50mv. f . 8 .
8 456,450,460
L i
I
4
8
I
i 12
I
I
16
5 0 0 m v . f.8.
C
TIM E (min.)
I
I
1
4
0
12
TIME(min.)
Figure 1. Mass chromatogram of hexachlorodibenzo-pdioxin (A) 5
figlml hexachlorodibenzo-pioxin standard. (B)1 .O-ml eluate from
0.125 g typical pentachlorophenol
I 16
Flgure 2.
Mass chromatogram of octachlorodibenzo-pdioxin
( A ) 48 fig/ml octachlorodibenzo-pdioxin standard. (B) Chlorinated phenoxy phenols through 21 K (OH- form) ion exchange. (C)1.0 ml eluate from 5.0-g
sample DOWlClDE EC-7 Muskegon, Mich. 49442.
Ion Exchange Resin. One liter of 50-100 mesh DOWEX 21K anion exchange resin (obtained from The Dow Chemical Company, Midland, Mich. 48640) in the chloride form was placed in a beaker and allowed to equilibrate with distilled water for several hours to ensure that the beads were fully swollen. The resin was subsequently slurried and transferred to a glass column (2-inch i.d. by 30 inches) having a glass wool plug above the Teflon stopcock and the water drained to the top of the resin bed. It is extremely important that a large diameter column be used for this step in the procedure, since there is a 20% volume increase of the resin in going from the chloride to the hydroxide form. Use of small diameter columns may present an explosion hazard, since the small column may not accommodate the large volume change. The glass column used should be wrapped with tape to prevent injury should the column break. Approximately 3 1. of 5% aqueous sodium hydroxide were passed through the resin (flow rate -10 ml/min) to convert it to the hydroxide form. Sufficient distilled water was then passed through the resin to remove the excess sodium hydroxide. When the eluate was neutral, approximately 1 gallon of anhydrous methanol was passed through the resin to remove the water. If the resin was not used immediately, it was refrigerated to retard decomposition. Preparation of Gas Chromatographic Column. A column packing of 3% SE-30 on Anachrom ABS, 90-100 mesh, was prepared using conventional techniques. Several injections of Sylon BTZ (N,0-bis(trimethylsilyl)acetamide, trimethylchlorosilane, and trimethylsilylimidazole in a 3:2:3 ratio) were introduced into the column a t elevated temperatures to deactivate any remaining adsorptive sites. Standard Solutions. Standard solutions were prepared by dissolving appropriate amounts of the chlorinated dibenzo-p-dioxins in benzene. The chlorinated dibenzo-p-dioxins used for standards were those compounds previously synthesized and reported by Aniline (7). The purity of the hexachlorodibenzo-p-dioxin(a mixture of two isomers) was greater than 99%. No attempt was made in this study to separate or quantitate individual HCDD isomers. It was assumed that each isomer produced the same proportion of molecular ions under constant conditions. The octachlorodibenzo-p-
dioxin used for standardization had a purity of 99.9%. Sample Preparation. Eighty milliliters of prepared DOWEX 21K resin (OH- form) were transferred to a glass column (0.7-inch i.d. by 24 inches) equipped with a glass wool plug above the Teflon stopcock and allowed to settle. The solvent was drained until it was just above the resin bed. Two column volumes (-50 ml) of 1:l (v/v) benzene:methanol were passed through the resin. Then 5.0 f 0.1 g of sample was dissolved in 20 ml of 1:1 (v/v) benzene:methano1 and transferred to the ion exchange column with a minimum amount of the same solvent. The CDD were eluted from the column with 1:l (v/v) benzene:methanol in the 26-150 ml fraction. The solvent from the eluate was evaporated in a small vial at ambient temperature using a stream of dry air, and the remaining residue dissolved in 1.0 ml of benzene in a 5-dram vial. Gas Chromatographic-Mass Spectrometric Analysis. The GC column, prepared as previously described, was installed in the GC-MS and allowed to equilibrate under the conditions listed: Instrument LKB-9000 GC/MS; column: 3-fOOt X %-inch glass (2-mm i.d.) packed with 3% SE-30 on Anachrom ABS, 90-100 mesh; column temperature: 230 "C, isothermal; injection port temperature: 240 "C; separator temperature: 290 "C; Carrier gas: helium a t 40 ml/min; ion source temperature: 290 "C; trap current: 60 @A;accelerating voltage: 3.5 kV; ionizing electron energy: 24 eV; slits: 0.25 mm/0.25 mm; and resolution: -400. For hexachlorodibenzo-p-dioxin (HCDD), the magnetic field and accelerating voltage alternator (AVA) were adjusted such that m / e = 388, 390, and 392 were monitored. The multiplier voltage was approximately 85% of the maximum (see Figure 1). For octachlorodibenzo-p-dioxin (OCDD), the magnetic field and AVA were adjusted such that m/e = 456, 458, and 460 were simultaneously monitored. The multiplier was approximately 75% of maximum (see Figure 2). The column was pre-conditioned with several injections of a typical sample residue solution until the peak areas from injections of standard solutions were constant. Two-microliter injections of standard solutions and sample residue solutions were used. Quantitation was achieved by comparison of ion-peak areas integrated by an IBM-1800 computer system. ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
985
When analyzing for HCDD, the fragment ion at mle = 388, 390, 392 due t o the loss of two chlorine atoms may be used t o quantitate the OCDD if the sample originally contained at least 30 ppm. DISCUSSION
The GC/MS procedure described here is highly specific for the analysis of chlorinated dibenzo-p -dioxins. Limits of detection of 0.5 ppm are readily achieved using only ion exchange chromatography for matrix removal. Although not all components present are completely resolved by the chromatographic column, the specificity of the mass spectrometric detector is such that no interference is encountered. An exception to this, although rarely encountered, occurs when a relatively large component elutes simultaneously with the CDD, thereby depressing CDD ionization in the ion source. All commercial pentachlorophenol can contain chlorinated phenoxyphenols. Previous workers (8, 9) have shown that erroneous results may be obtained if these compounds are not completely removed prior to GC or GC/MS quantitation. The chlorinated phenoxyphenols are only partially soluble in basic aqueous solutions and are, therefore, not completely separated from the neutral components when subjected to liquid-liquid extraction. When subsequently subjected to the high temperatures of the gas chromatograph, these chlorinated phenoxyphenols may condense to form chlorinated dibenzo-p-dioxins in the gas chromatograph. A sample of chlorinated phenoxyphenols isolated from commercial grade pentachlorophenol was purified by several recrystallizations. The purified material was analyzed by mass spectrometry (CEC 21-11OB-resolution approximately 1500) using the direct probe technique ( I O ) . The data indicated that the material was a mixture of octachlorophenoxyphenol ( m / e = 458, with the characteristic isotope pattern for eight chlorine atoms) and nonachlorophenoxyphenol (mle = 492 with the characteristic isotope pattern for nine chlorine atoms). . No evidence was found for the presence of a significant amount of octachlorodibenzop-dioxin (M+ = 456). T o verify that the chlorinated phenoxyphenols do condense to form dioxins, a solution of this material was injected into the GC/MS operating a t the conditions of analysis for OCDD; approximately 2% of the material was converted to OCDD. The peak width was considerably broader than the peak width of an OCDD standard. An aliquot of the chlorinated phenoxyphenol solution was subjected to the ion exchange procedure. The concentrated eluate was injected as before. No peak was observed a t m / e = 456, 458, and 460 (Mf for OCDD). Likewise, when the molecular ions for nonachlorophenoxyphenol ( m / e = 492, 494, 496) were monitored, no peak was observed. Thus, the ion exchange procedure effects complete removal of the chlorinated phenoxyphenols and their potential interference in the GC-MS analysis. The procedure, as described, was developed primarily for the analysis of DOWICIDE EC-7 pentachlorophenol. This material typically contains less than 1 ppm hexachlorodibenzo-p-dioxin and less than 30 ppm octachlorodibenzop-dioxin. When the procedure is applied to other domestic technical grade pentachlorophenol, the sample size must be reduced appropriately to avoid solubility problems. Typically, the large sample size is retained to avoid difficulties with sample inhomogeneities, but the material is diluted with 1:l benzene/methanol and an .aliquot is taken. Aliquots which contain 0.1-0.3 gram of sample have been typically used. The analyses of several samples of domestically produced pentachlorophenol are summarized in Table I. Heptachlorodibenzo-p-dioxin and the chlorinated diben986
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
Table I. Analysis of Domestically Manufactured Pentachlorophenol
Sample
b
Hexachlorodibenzo-p- Octachlorodibenzo-pdioxin, ppm (wt/wt) dioxin, ppm (wtlwt)
18 1550 B 9 1250 27 575 C D 19 1980 DOWICIDE N.D." 14 EC-7 (laboratory produced) DOWICIDE N.D. 2 EC-7 (typical production) a N.D. = Not detected with a detection limit of 0.5 ppm. Samples A through D were samples of pentachlorophenol which were domestically manufactured by Dow and three other manufacturers. Ab
Table 11. Determination of Chlorinated Dibenzo-pDioxins by Gas Chromatography-Mass Spectrometry
Hexachlorodi- Octachlorodibenzo-p-dioxin benzo-p-dioxin Sample Solvents only Mixed chlorophenoxyphenols (7,8, and 9 chlorines) DOWICIDE EC-7
a
Added, Found, Added, Found, PPm PPm PPm PPm 19 1.5 17 1.3 0.0 N.D. 0.0
