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Analysis of polychlorinated dibenzofurans and dibenzo-p-dioxins in human milk by tandem hybrid mass spectrometry
Chemosphere, Vol.19, Nos.l-6, Printed in Great Britain
pp 59-66,
1989
0045-6535/89 $3.00 + .00 Pergamon Press plc
ANALYSIS OF POLYCHLORINATED DIBENZOFURANS AND DIBENZO-P-DIOXINS IN HUMAN MILK BY TANDEM HYBRID MASS SPECTROMETRY
A.P.J.M. DE JONG, A.K.D. LIEM, A.C. DEN BOER, E. VAN DER HEEFT, J.A. MARSMAN, G. VAN DE WERKEN and R.C.C. WEGMAN Laboratory of Organic-Analytical Chemistry, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bihhoven, The Netherlands.
ABSTRACT This paper describes a new method for isolation of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans from biological samples using Carbopack C chromatography and their subsequent analysis by GCMS/MS. The MS/MS technique showed to be extremely selective and sufficiently sensitive for the simultaneous measurement of all 2,3,7,8-chlorine substituted congeners of PCDD/F present at the low ppt-level in human milk. KEYWORDS MS/MS, clean-up, specificity, sensitivity, PCDDs, PCDFs, human milk. INTRODUCTION In recent years, numerous methods have been developed for the determination of polychlorinated dibenzo-pdioxines and polychlorinated dibenzofurans (PCDDs and PCDFs). Most of these methods use gas chromatography combined with mass spectrometry (GCMS) for separation and detection. At present, GCMS is the only technique able to provide the required sensitivity and specificity. The retention parameters from the chromatography provide isomer specificity while the mass spectral parameters provide class and homologue specificity. Despite the great analytical power of combined techniques, the low level of PCDD/Fs in environmental and biological samples (Smith et al., 1984; Rappe et al., 1984) together with the normally much higher levels of potentially interfering chemicals, GCMS methods require extensive clean-up procedures. Even then, analysis using low resolution mass spectrometry (LRMS) may fail because of high background levels. In such cases, GC high-resolution mass spectrometry (HRMS) has been usually recommended as the technique of choice (Cairns et al., 1980) Recently, the relatively new technique of tandem mass spectrometry (MS/MS) has shown to be an attractive alternative to HRMS. Its excellent selectivity is due to the fact that specific daughter ions are generated from the selected parent ions. When structurally unrelated ions coincide in MS-l, the different reaction that such ions may undergo during collision with gas molecules in the collision cell provide the opportunity to distinguish between the isobaric parent ions. A limitation of the technique is the relatively low abundance of the daughter ions. This is due to normally low transmission efficiencies of ions through the collision region and low fragmentation yields of stable parent ions by collision with small gaseous molecules or atoms. Nevertheless, Tondeur et al. (1987) showed that MS/MS was able to detect a few picograms of 2,3,7,8-TCDD present in a sample containing excessive amounts of polychlorobiphenyls (PCBs) and other contaminants. The aim of this study was to simplify clean-up procedures for biological samples yielding extracts free from lipids but not necessarily free from wen-known interfering chemicals like PCBs and organochloropesticides (OCs) and the subsequent analysis of PCDD/Fs by GC-MS/MS. We found that adsorption chromatography using Carbopack C can replace Amoco PX-21 in conjunction with multilayer silica and alumina chromatography resulting in a substantial time reduction for clean-up. In addition, we show that all PCDD/F congeners of interest in human milk samples, i.e. the toxic 2,3,7,8 isomers, can be determined using GC-MS/MS. 59
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Fig. 1. Normal electron impact (EI) mass spectrum of 2,3,7,8-tetrachloro-p-dioxin (upper pannel) and the coUisionally activated dissociation (CAD) mass spectrum of the molecular ion of rn/z 320 (lower pannel).
MATERIALS AND METHODS Chemicals Graphitized carbon Carbopack C, 80/100 mesh was purchased from Supelco Inc., Bellefonte, PA, U.S.A.. Unlabelled and 13C12-1abelledPCDDs and PCDFs were obtained from Cambridge Isotope Laboratories, Woburn, MA, U.S.A.. 4,4'-Dibromobiphenyl was obtained from Foxboro Analabs Inc., Schiedam, The Netherlands. All reagents used were of analytical grade.
61
Table 1. Recovery (mean + S.D.) of PCDD/Fs after clean-up using Carbopack C. dibenzo-p-dioxins
recovery (%)
2,3,7,8-TCDD 83 1,2,3,7,8-PCDD 77 1,2,3,4,7,8-HxCDD 66 1,2,3,6,7,8-HxCDD 75 1,2,3,7,8,9-HxCDD 68 1,2,3,4,6,7,8-HpCDD 81 octa-CDD 63
_+ + -+ -+ -+ -+ +
16 18 9 12 15 25 16
(7) a (7) (4) (7) (7) (7) (7)
dibenzofurans 2,3,7,8-TCDF 1,2,3,7,8-PCDF 2,3,4,7,8-PCDF 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF 1,2,3,7,8,9-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF 1,2,3,4,7,8,9-HpCDF octa-CDF
recovery (%) 72 80 82 76 67 68 60 61 53 50
+_ + _+ + + + _+ + ++
13 15 11 11 11 11 12 17 4 6
(7) (7) (4) (7) (4) (4) (4) (7) (4) (4)
a: number of determinations
Samples Fresh cow milk samples were obtained directly from a farmer. Human milk samples used were pooled aliquots of ten individual samples each, which had been collected in two different areas of the Netherlands. Sample treatment and clean-up PCDDs and PCDFs were isolated from milk according to a method described by Liem et al. (1988). In brief, sodiumoxalate was added to 150 g of milk and the fat fraction together with the PCDDs and PCDFs were isolated by repeated liquid-liquid extractions using methanol, diethyl ether and petroleum ether, respectively. To the combined extracts, appropriate amounts of t3ctz-labelled standards were added (1 pg of each compound per g of milk). Next, the extract was evaporated to dryness and the residue was dissolved in 25 ml of dichloromethane/hexane (1:1, v/v) and applied to the column containing 7 g of Carbopack C. The column was rinsed with 50 ml of dichloromethane and then refluxed with 50 ml of dichloromethane for 1 h to remove the lipids. Next, PCDD/Fs were recovered by refluxing with toluene for 20 h. After concentration of the extract to a volume of about 5 ml, the extract was further purified using alumina as described by Liem et aL (1988). The final extract was evaporated to dryness and dissolved in 50 p.1of toluene, containing 5 ng/ml of 4,4'-dibromobiphenyl as a syringe standard. An aliquot of one to six ~tl was normally used for GC/MS analysis. Instrumental Mass spectrometry. Experiments were performed on a VG 70 SQ tandem hybrid mass spectrometer with EBQQ configuration (VG Analytical, Manchester U.K.). The instrument was connected to a HP 5890 gas chromatograph (Hewlett Packard, Palo Alto, CA, U.S.A.). Instrument contro!, data acquisition and data processing was done by a VG 11/250 data system (VG, Manchester, U.K.). Ionization of samples was performed under electron impact (El) with 70 eV electrons and a f'dament emission current of 0.5 mA. An EI-only source has been installed. The source temperature was set at 250 °C. The resolving power of MS-1 (EB) was set at either 3000:1 (MS analysis) or 1000:1 (MS]MS analysis). The resolving power of the quadrupole mass analyzer was approximately unit resolution. Collisionally activated dissociation (CAD) of parent ions was performed using argon as collision gas. The collision energy was optimized for maximal response of the daughter ion at m/z 257 of 2,3,7,8-TCDD, normally between 10-15 eV. Monitoring of the argon pressure in the collision cell was not possible. Its pressure was adjusted at the high vacuum gauge of the analyzer pump line to a value of 10-6 mbarr. Gas chromato~aphy. Chromatographic separation of the samples was performed using a CP Sil 88 fused silica capillary column, 50 m x 0,22 mm I.D., film thickness 0.12 I.tm (Chrompack, Middelburg, The Netherlands). Samples were introduced by an all-glass falling needle injector (solid injector, Fa. Koppens, Best, The Netherlands). Normally, 6 p.1 aliquots were placed onto the tip of the needle and the solvent (toluene) was allowed to evaporate before the needle was dropped into the heated zone (280 °C) of the injector. The t e x t u r e of the GC oven was programmed from its initial temperature of 200 °C, one min after injection at a rate of 4 ° C/min to the final temperature of 240 °C and maintained at this temperature for 50 rain. Hehum was used as the carrier gas at a mean
62
linear velocity of approx. 32 cm/s. Multiple Reaction Monitoring (MRM). MRM measurements were performed on the two most abundant ions of the molecular ion cluster of both labelled and unlabelled PCDD/Fs, giving 40 parent ions in total. Each ion was allowed to enter the collision cell for 50 ms. This number of ions was divided into five groups of 8 ions each and measured during time intervals that match the retention time of the corresponding compounds to be analysed. The quadmpole was controlled so that synchronous measurement of the loss of 63 amu (loss of COC1 for unlabelled) or 64 ainu (loss of 13COC1 for labelled) from the indicated parent ions, respectively (i.e. 5 groups of 8 daughter ions each). Parent ions within a group were selected by high voltage switching with a fixed magnetic field. Switching between groups was achieved by magnetic field switching. The proper magnetic setting for each group was automatically adjusted by the computer system using a lock mass from perfluorokerosine.
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Fig. 2. Selected ion monitoring (SIM) profiles (upper two lraces) and selected reaction monitoring profiles (SRM) (lower pannels) following the injection of a mixture of 5 pg of 2,3,7,87TCDD (left), 5 pg of 2,3,7,8-TCDF (right) and Aroclor 1260 (245 ng), I~-heptachloroepoxide (230 ng), o,p-DDE (285 ng), o,p-DDT (471 ng) and p,p'-DDT (340 ng) onto a capillary GC-column. Note that sensitivity setting of the preamplifier during SIM measurements was 5 orders of malznitude less than durin~ SRM measurements.
63
Table 2. Levels ( in pg/g of milk fat) of 2,3,7,8-chlorine substituted PCDDs and PCDFs found in a pooled human milk sample, as determined by GC-medium resolution MS (SIR) and GC-MS/MS (MRM). Dioxins
SIR
2,3,7,8-TCDD 1,2,3,7,8-PCDD 1,2,3,4,7,8-HxCDD 1,2,3,6,7,8-HxCDD 1,2,3,7,8,9-I-IxCDD 1,2,3,4,6,7,8-HpCDD octa-CDD
MR
10 11 15 16 20 17 72 91 14 20 100 133 940 1115
Furans 2,3,7,8-TCDF 1,2,3,7,8-PCDF 2,3,4,7,8-PCDF 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF 1,2,3,7,8,9-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF 1,2,3,4,7,8,9-HpCDF octa-CDF
SIR 3 2.5 30 13 13 6 10 25 9 6
MRM 3 3 53 17 13 6 14 41 12 n.d
n.d. = not detected
12 A
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dioxins [] furans
8 6
"0
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122
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Fig. 3. Obtained detection limits (in pg injected) of PCDD/F in human milk by GC-MS/MS.
RESULTS AND DISCUSSION S..ampleclean-uo For the purpose of this study a major merit of the clean-up procedure was that the final extract will contain a high yield of the compounds of interest and that fipids will be fully removed. The presence of residual amounts of the latter in the extract will rapidly disturb the performance of the GC column. Other likely features of the procedure are that it is reliable and rapid, and excesses of other chemicals are removed. We found that Carbopack C is a useful type of active carbon. It effectively adsorbs PCDD/Fs whereas lipids are
64
easily removed by rinsing and refluxing with dichloromethane. The capacity of the column was about 5 g of fat per gram of carbon. The recovery of PCDD/F was satisfactory. It ranged from 85 to 50% for tetra to octachlorinated dioxins and furans at the level of 30 pg/g of milk fat (see Table 1). The observed gradual decrease in recovery for tetra to octa PCDD/F may indicate that adsorption of these compounds onto Carbopack C increases with increasing number of chlorine atoms in the molecule. In that case, prolonged refluxing with toluene may increase the recovery
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of these compounds. When compared to the conventional Amoco PX-21/multilayer silica/alumina clean-up method CLiem et al., 1988), the present procedure is less time-consuming. The recovery of added 13C12 labelled standards was comparable with better day-to-day reproducibility. Further study is in progress and results will be published elsewhere. M a ~ Spectra Figure 1 shows a normal EI (inset) and a CAD mass spectrum of 2,3,7,8-TCDD. The CAD spectrum of the molecular ion contains two major daughter ions at m/z 285 (M-C1) and m/z 257 (M-COC1). The abundance of these ions relative to the parent ion tends to increase both with increasing collision gas pressure and increasing collision
65
energy. However, intensities of daughter ions and the unaffected parent ion are adversely affected by increasing collision gas pressure due to a decrease in transmission efficiency. The maximal response of the M-COC1 daughter ions in MRM analysis has been established by varying the collision energy at constant collision gas pressure between repeated injections of 10 pg of 2,3,7,8-TCDD. These optimal parameter settings varied only slightly over several days of experimentation. Figure 2 shows the dramatic increase in selectivity obtained by monitoring daughter ions when compared to the monitoring of corresponding parent ions. The signal obtained from 5 pg injections of 2,3,7,8-TCDD in the presence of large (105) excesses of PCBs and OCs was fully lost in the background during selected ion monitoring (SIM) of parent ions, whereas signals of daughter ions by MRM were clearly visible. The amount of added PCBs and OCs (see legend to fig. 2) corresponds to their levels normally found in human milk (Rappe et al., 1984). This result indicates that by using GC-MS/MS, human milk samples need no further clean-up than the removal of lipids. Sensitivity As discussed above, sensitivity of MS/MS is influenced by several factors. Apart from instrumental parameters like collision gas pressure and collision energy, also the stability of the parent ion may influence the yield of daughter ions. In our experiments, optimal yields of the daughter ions were typically 1-2% of the corresponding parent ion beam entering the collision cell. When compared to HRMS, the sensitivity of the MS/MS mode was about 5-fold less. However, because of the low chemical background in MRM analysis, detection limits for tetra to octa CDD/F were attainable in the range of 1 to 10 ppt on fat basis, respectively (see Fig. 3). These limits correspond to the level of 2,3,7,8-chlorinated PCDD/Fs which are normally found in human milk, fat adipose and other biological tissue. The observed lower sensitivity with increasing number of chlorine atoms (Fig.3) can be explained on the one hand by the broader distribution of the molecular ion isotope cluster and, on the other hand by the broader gas chromatographic peak in which the higher chlorinated compounds elute, In addition, in the present work optimization of collision conditions has been performed on 2,3,7,8-TCDD. It might be that higher sensitivity is obtained for other congeners when optimization is focussed on those particular compounds. Application to human milk sa.m..,ples The method described here has been applied to the analysis of the so-called 'dirty seventeen' compounds (PCDD/F which contain chlorine atoms at positions 2, 3, 7 and 8) in a pooled human milk sample. Figure 4 shows, as an example, MRM chromatograms of tetraCDD and octaCDD. In addition, in this figure results from MRM and medium resolution SIM (R = 3000:1) are compared. In Table 2, results obtained by both determinations are shown. It follows that good agreement was obtained for both techniques. CONCLUSION This study demonstrates that using GC-MS/MS it is possible to determine PCDD and PCDF congeners at low ppt levels in human milk samples. The proposed clean-up procedure using Carbopack C appeared to be an improvement of existing methods as it allows the handling of large amounts of fat, it provided satisfactory recoveries of dioxins and furans and was rapid. Contxary to medium resolution SIR, MRM analysis were capable of the specific determination of PCDDs and PCDFs in contaminated samples without a need of extensive and time-consuming cleanup. Other features of the method are currently under investigation.
REFERENCES Cairns, T., L. Fishbein and R.K. Mitchum (1980). Review of the dioxin problem. Mass spectrometric analysis of tetrachlorodioxins in environmental samples. Biomed. Mass. Spectrom., 7,484-492. Liem, A.K.D., G.S. Groenemeijer, G.A.L. De Korte, A. Van Laar, J.A. Marsman, A.P.J.M. De Jong and R.C.C. Wegman (1988). Sample preparation for determination of PCDDs and PCDFs in eel and human milk by GC-MS. In: Sampling and Sample Treatment for the Analysis of Organic Micropollutants in the Aquatic Environment, Water Pollution Research Report Series no S , E UR 11355, Commission of the European Communities, 7689. Rappe, C., P.A. Bergquist, M. Hansson, L.O. Kjeller, G. Lindstrom, S. Marklund and M. Nygren (1984). Banbury Report 18: Biological Mechanism of Dioxin Action. C'~Id Spring Harber, New York.
66
Ryan, J.J., R. Lizotte and B.P.Y. Liau (1985). Chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans in Canadian human adipose tissue. Chemosphere, 14, 697-706. Smith, L.M., D.L. Stalling and J.L. Johnson (1984). Determination of parts-per-trillion levels of polychlorinated dibenzofurans and dioxins in environmental samples. Anal.Chem., 56, 1830-1842. Tondeur, Y., W.N. Niederhut, J.E. Campana and S.R. Missler (1987). A hybrid HRGC/MS/MS method for the characterization of tetrachlorinated-p-dioxins in environmental samples. Biomed. Mass. Spectrom., 14, 449456.
pp 59-66,
1989
0045-6535/89 $3.00 + .00 Pergamon Press plc
ANALYSIS OF POLYCHLORINATED DIBENZOFURANS AND DIBENZO-P-DIOXINS IN HUMAN MILK BY TANDEM HYBRID MASS SPECTROMETRY
A.P.J.M. DE JONG, A.K.D. LIEM, A.C. DEN BOER, E. VAN DER HEEFT, J.A. MARSMAN, G. VAN DE WERKEN and R.C.C. WEGMAN Laboratory of Organic-Analytical Chemistry, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bihhoven, The Netherlands.
ABSTRACT This paper describes a new method for isolation of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans from biological samples using Carbopack C chromatography and their subsequent analysis by GCMS/MS. The MS/MS technique showed to be extremely selective and sufficiently sensitive for the simultaneous measurement of all 2,3,7,8-chlorine substituted congeners of PCDD/F present at the low ppt-level in human milk. KEYWORDS MS/MS, clean-up, specificity, sensitivity, PCDDs, PCDFs, human milk. INTRODUCTION In recent years, numerous methods have been developed for the determination of polychlorinated dibenzo-pdioxines and polychlorinated dibenzofurans (PCDDs and PCDFs). Most of these methods use gas chromatography combined with mass spectrometry (GCMS) for separation and detection. At present, GCMS is the only technique able to provide the required sensitivity and specificity. The retention parameters from the chromatography provide isomer specificity while the mass spectral parameters provide class and homologue specificity. Despite the great analytical power of combined techniques, the low level of PCDD/Fs in environmental and biological samples (Smith et al., 1984; Rappe et al., 1984) together with the normally much higher levels of potentially interfering chemicals, GCMS methods require extensive clean-up procedures. Even then, analysis using low resolution mass spectrometry (LRMS) may fail because of high background levels. In such cases, GC high-resolution mass spectrometry (HRMS) has been usually recommended as the technique of choice (Cairns et al., 1980) Recently, the relatively new technique of tandem mass spectrometry (MS/MS) has shown to be an attractive alternative to HRMS. Its excellent selectivity is due to the fact that specific daughter ions are generated from the selected parent ions. When structurally unrelated ions coincide in MS-l, the different reaction that such ions may undergo during collision with gas molecules in the collision cell provide the opportunity to distinguish between the isobaric parent ions. A limitation of the technique is the relatively low abundance of the daughter ions. This is due to normally low transmission efficiencies of ions through the collision region and low fragmentation yields of stable parent ions by collision with small gaseous molecules or atoms. Nevertheless, Tondeur et al. (1987) showed that MS/MS was able to detect a few picograms of 2,3,7,8-TCDD present in a sample containing excessive amounts of polychlorobiphenyls (PCBs) and other contaminants. The aim of this study was to simplify clean-up procedures for biological samples yielding extracts free from lipids but not necessarily free from wen-known interfering chemicals like PCBs and organochloropesticides (OCs) and the subsequent analysis of PCDD/Fs by GC-MS/MS. We found that adsorption chromatography using Carbopack C can replace Amoco PX-21 in conjunction with multilayer silica and alumina chromatography resulting in a substantial time reduction for clean-up. In addition, we show that all PCDD/F congeners of interest in human milk samples, i.e. the toxic 2,3,7,8 isomers, can be determined using GC-MS/MS. 59
60
322
100
°'t
320
80
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324
504030-
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0
j
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Fig. 1. Normal electron impact (EI) mass spectrum of 2,3,7,8-tetrachloro-p-dioxin (upper pannel) and the coUisionally activated dissociation (CAD) mass spectrum of the molecular ion of rn/z 320 (lower pannel).
MATERIALS AND METHODS Chemicals Graphitized carbon Carbopack C, 80/100 mesh was purchased from Supelco Inc., Bellefonte, PA, U.S.A.. Unlabelled and 13C12-1abelledPCDDs and PCDFs were obtained from Cambridge Isotope Laboratories, Woburn, MA, U.S.A.. 4,4'-Dibromobiphenyl was obtained from Foxboro Analabs Inc., Schiedam, The Netherlands. All reagents used were of analytical grade.
61
Table 1. Recovery (mean + S.D.) of PCDD/Fs after clean-up using Carbopack C. dibenzo-p-dioxins
recovery (%)
2,3,7,8-TCDD 83 1,2,3,7,8-PCDD 77 1,2,3,4,7,8-HxCDD 66 1,2,3,6,7,8-HxCDD 75 1,2,3,7,8,9-HxCDD 68 1,2,3,4,6,7,8-HpCDD 81 octa-CDD 63
_+ + -+ -+ -+ -+ +
16 18 9 12 15 25 16
(7) a (7) (4) (7) (7) (7) (7)
dibenzofurans 2,3,7,8-TCDF 1,2,3,7,8-PCDF 2,3,4,7,8-PCDF 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF 1,2,3,7,8,9-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF 1,2,3,4,7,8,9-HpCDF octa-CDF
recovery (%) 72 80 82 76 67 68 60 61 53 50
+_ + _+ + + + _+ + ++
13 15 11 11 11 11 12 17 4 6
(7) (7) (4) (7) (4) (4) (4) (7) (4) (4)
a: number of determinations
Samples Fresh cow milk samples were obtained directly from a farmer. Human milk samples used were pooled aliquots of ten individual samples each, which had been collected in two different areas of the Netherlands. Sample treatment and clean-up PCDDs and PCDFs were isolated from milk according to a method described by Liem et al. (1988). In brief, sodiumoxalate was added to 150 g of milk and the fat fraction together with the PCDDs and PCDFs were isolated by repeated liquid-liquid extractions using methanol, diethyl ether and petroleum ether, respectively. To the combined extracts, appropriate amounts of t3ctz-labelled standards were added (1 pg of each compound per g of milk). Next, the extract was evaporated to dryness and the residue was dissolved in 25 ml of dichloromethane/hexane (1:1, v/v) and applied to the column containing 7 g of Carbopack C. The column was rinsed with 50 ml of dichloromethane and then refluxed with 50 ml of dichloromethane for 1 h to remove the lipids. Next, PCDD/Fs were recovered by refluxing with toluene for 20 h. After concentration of the extract to a volume of about 5 ml, the extract was further purified using alumina as described by Liem et aL (1988). The final extract was evaporated to dryness and dissolved in 50 p.1of toluene, containing 5 ng/ml of 4,4'-dibromobiphenyl as a syringe standard. An aliquot of one to six ~tl was normally used for GC/MS analysis. Instrumental Mass spectrometry. Experiments were performed on a VG 70 SQ tandem hybrid mass spectrometer with EBQQ configuration (VG Analytical, Manchester U.K.). The instrument was connected to a HP 5890 gas chromatograph (Hewlett Packard, Palo Alto, CA, U.S.A.). Instrument contro!, data acquisition and data processing was done by a VG 11/250 data system (VG, Manchester, U.K.). Ionization of samples was performed under electron impact (El) with 70 eV electrons and a f'dament emission current of 0.5 mA. An EI-only source has been installed. The source temperature was set at 250 °C. The resolving power of MS-1 (EB) was set at either 3000:1 (MS analysis) or 1000:1 (MS]MS analysis). The resolving power of the quadrupole mass analyzer was approximately unit resolution. Collisionally activated dissociation (CAD) of parent ions was performed using argon as collision gas. The collision energy was optimized for maximal response of the daughter ion at m/z 257 of 2,3,7,8-TCDD, normally between 10-15 eV. Monitoring of the argon pressure in the collision cell was not possible. Its pressure was adjusted at the high vacuum gauge of the analyzer pump line to a value of 10-6 mbarr. Gas chromato~aphy. Chromatographic separation of the samples was performed using a CP Sil 88 fused silica capillary column, 50 m x 0,22 mm I.D., film thickness 0.12 I.tm (Chrompack, Middelburg, The Netherlands). Samples were introduced by an all-glass falling needle injector (solid injector, Fa. Koppens, Best, The Netherlands). Normally, 6 p.1 aliquots were placed onto the tip of the needle and the solvent (toluene) was allowed to evaporate before the needle was dropped into the heated zone (280 °C) of the injector. The t e x t u r e of the GC oven was programmed from its initial temperature of 200 °C, one min after injection at a rate of 4 ° C/min to the final temperature of 240 °C and maintained at this temperature for 50 rain. Hehum was used as the carrier gas at a mean
62
linear velocity of approx. 32 cm/s. Multiple Reaction Monitoring (MRM). MRM measurements were performed on the two most abundant ions of the molecular ion cluster of both labelled and unlabelled PCDD/Fs, giving 40 parent ions in total. Each ion was allowed to enter the collision cell for 50 ms. This number of ions was divided into five groups of 8 ions each and measured during time intervals that match the retention time of the corresponding compounds to be analysed. The quadmpole was controlled so that synchronous measurement of the loss of 63 amu (loss of COC1 for unlabelled) or 64 ainu (loss of 13COC1 for labelled) from the indicated parent ions, respectively (i.e. 5 groups of 8 daughter ions each). Parent ions within a group were selected by high voltage switching with a fixed magnetic field. Switching between groups was achieved by magnetic field switching. The proper magnetic setting for each group was automatically adjusted by the computer system using a lock mass from perfluorokerosine.
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Fig. 2. Selected ion monitoring (SIM) profiles (upper two lraces) and selected reaction monitoring profiles (SRM) (lower pannels) following the injection of a mixture of 5 pg of 2,3,7,87TCDD (left), 5 pg of 2,3,7,8-TCDF (right) and Aroclor 1260 (245 ng), I~-heptachloroepoxide (230 ng), o,p-DDE (285 ng), o,p-DDT (471 ng) and p,p'-DDT (340 ng) onto a capillary GC-column. Note that sensitivity setting of the preamplifier during SIM measurements was 5 orders of malznitude less than durin~ SRM measurements.
63
Table 2. Levels ( in pg/g of milk fat) of 2,3,7,8-chlorine substituted PCDDs and PCDFs found in a pooled human milk sample, as determined by GC-medium resolution MS (SIR) and GC-MS/MS (MRM). Dioxins
SIR
2,3,7,8-TCDD 1,2,3,7,8-PCDD 1,2,3,4,7,8-HxCDD 1,2,3,6,7,8-HxCDD 1,2,3,7,8,9-I-IxCDD 1,2,3,4,6,7,8-HpCDD octa-CDD
MR
10 11 15 16 20 17 72 91 14 20 100 133 940 1115
Furans 2,3,7,8-TCDF 1,2,3,7,8-PCDF 2,3,4,7,8-PCDF 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF 1,2,3,7,8,9-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF 1,2,3,4,7,8,9-HpCDF octa-CDF
SIR 3 2.5 30 13 13 6 10 25 9 6
MRM 3 3 53 17 13 6 14 41 12 n.d
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Fig. 3. Obtained detection limits (in pg injected) of PCDD/F in human milk by GC-MS/MS.
RESULTS AND DISCUSSION S..ampleclean-uo For the purpose of this study a major merit of the clean-up procedure was that the final extract will contain a high yield of the compounds of interest and that fipids will be fully removed. The presence of residual amounts of the latter in the extract will rapidly disturb the performance of the GC column. Other likely features of the procedure are that it is reliable and rapid, and excesses of other chemicals are removed. We found that Carbopack C is a useful type of active carbon. It effectively adsorbs PCDD/Fs whereas lipids are
64
easily removed by rinsing and refluxing with dichloromethane. The capacity of the column was about 5 g of fat per gram of carbon. The recovery of PCDD/F was satisfactory. It ranged from 85 to 50% for tetra to octachlorinated dioxins and furans at the level of 30 pg/g of milk fat (see Table 1). The observed gradual decrease in recovery for tetra to octa PCDD/F may indicate that adsorption of these compounds onto Carbopack C increases with increasing number of chlorine atoms in the molecule. In that case, prolonged refluxing with toluene may increase the recovery
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of these compounds. When compared to the conventional Amoco PX-21/multilayer silica/alumina clean-up method CLiem et al., 1988), the present procedure is less time-consuming. The recovery of added 13C12 labelled standards was comparable with better day-to-day reproducibility. Further study is in progress and results will be published elsewhere. M a ~ Spectra Figure 1 shows a normal EI (inset) and a CAD mass spectrum of 2,3,7,8-TCDD. The CAD spectrum of the molecular ion contains two major daughter ions at m/z 285 (M-C1) and m/z 257 (M-COC1). The abundance of these ions relative to the parent ion tends to increase both with increasing collision gas pressure and increasing collision
65
energy. However, intensities of daughter ions and the unaffected parent ion are adversely affected by increasing collision gas pressure due to a decrease in transmission efficiency. The maximal response of the M-COC1 daughter ions in MRM analysis has been established by varying the collision energy at constant collision gas pressure between repeated injections of 10 pg of 2,3,7,8-TCDD. These optimal parameter settings varied only slightly over several days of experimentation. Figure 2 shows the dramatic increase in selectivity obtained by monitoring daughter ions when compared to the monitoring of corresponding parent ions. The signal obtained from 5 pg injections of 2,3,7,8-TCDD in the presence of large (105) excesses of PCBs and OCs was fully lost in the background during selected ion monitoring (SIM) of parent ions, whereas signals of daughter ions by MRM were clearly visible. The amount of added PCBs and OCs (see legend to fig. 2) corresponds to their levels normally found in human milk (Rappe et al., 1984). This result indicates that by using GC-MS/MS, human milk samples need no further clean-up than the removal of lipids. Sensitivity As discussed above, sensitivity of MS/MS is influenced by several factors. Apart from instrumental parameters like collision gas pressure and collision energy, also the stability of the parent ion may influence the yield of daughter ions. In our experiments, optimal yields of the daughter ions were typically 1-2% of the corresponding parent ion beam entering the collision cell. When compared to HRMS, the sensitivity of the MS/MS mode was about 5-fold less. However, because of the low chemical background in MRM analysis, detection limits for tetra to octa CDD/F were attainable in the range of 1 to 10 ppt on fat basis, respectively (see Fig. 3). These limits correspond to the level of 2,3,7,8-chlorinated PCDD/Fs which are normally found in human milk, fat adipose and other biological tissue. The observed lower sensitivity with increasing number of chlorine atoms (Fig.3) can be explained on the one hand by the broader distribution of the molecular ion isotope cluster and, on the other hand by the broader gas chromatographic peak in which the higher chlorinated compounds elute, In addition, in the present work optimization of collision conditions has been performed on 2,3,7,8-TCDD. It might be that higher sensitivity is obtained for other congeners when optimization is focussed on those particular compounds. Application to human milk sa.m..,ples The method described here has been applied to the analysis of the so-called 'dirty seventeen' compounds (PCDD/F which contain chlorine atoms at positions 2, 3, 7 and 8) in a pooled human milk sample. Figure 4 shows, as an example, MRM chromatograms of tetraCDD and octaCDD. In addition, in this figure results from MRM and medium resolution SIM (R = 3000:1) are compared. In Table 2, results obtained by both determinations are shown. It follows that good agreement was obtained for both techniques. CONCLUSION This study demonstrates that using GC-MS/MS it is possible to determine PCDD and PCDF congeners at low ppt levels in human milk samples. The proposed clean-up procedure using Carbopack C appeared to be an improvement of existing methods as it allows the handling of large amounts of fat, it provided satisfactory recoveries of dioxins and furans and was rapid. Contxary to medium resolution SIR, MRM analysis were capable of the specific determination of PCDDs and PCDFs in contaminated samples without a need of extensive and time-consuming cleanup. Other features of the method are currently under investigation.
REFERENCES Cairns, T., L. Fishbein and R.K. Mitchum (1980). Review of the dioxin problem. Mass spectrometric analysis of tetrachlorodioxins in environmental samples. Biomed. Mass. Spectrom., 7,484-492. Liem, A.K.D., G.S. Groenemeijer, G.A.L. De Korte, A. Van Laar, J.A. Marsman, A.P.J.M. De Jong and R.C.C. Wegman (1988). Sample preparation for determination of PCDDs and PCDFs in eel and human milk by GC-MS. In: Sampling and Sample Treatment for the Analysis of Organic Micropollutants in the Aquatic Environment, Water Pollution Research Report Series no S , E UR 11355, Commission of the European Communities, 7689. Rappe, C., P.A. Bergquist, M. Hansson, L.O. Kjeller, G. Lindstrom, S. Marklund and M. Nygren (1984). Banbury Report 18: Biological Mechanism of Dioxin Action. C'~Id Spring Harber, New York.
66
Ryan, J.J., R. Lizotte and B.P.Y. Liau (1985). Chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans in Canadian human adipose tissue. Chemosphere, 14, 697-706. Smith, L.M., D.L. Stalling and J.L. Johnson (1984). Determination of parts-per-trillion levels of polychlorinated dibenzofurans and dioxins in environmental samples. Anal.Chem., 56, 1830-1842. Tondeur, Y., W.N. Niederhut, J.E. Campana and S.R. Missler (1987). A hybrid HRGC/MS/MS method for the characterization of tetrachlorinated-p-dioxins in environmental samples. Biomed. Mass. Spectrom., 14, 449456.