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Anal. Chem. 1984, 56, 1344-1347
Matrix Effect in Determination of 2,3,7,8-Tetrachlorodibenzodioxin by Mass Spectrometry Yves Tondeur,' Phillip W. Albro, J. Ronald Hass,* Donald J. Harvan, and Joanna L. Schroeder Department of Health and Human Services, National Institute of Environmental Health Sciences, Laboratory of Molecular Biophysics, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Some valldation aspects for the determlnatlon of part-pertrillion levels of tetrachlorodibenzodioxin in environmental media with gas chromatography/mass spectrometry are examined. A specific matrix effect in the determination system has been noted while using a magnetic sector Instrument operatlng in high resolution selected ion monitoring and low resolution selected reactlon monitoring modes. The results emphasize the importance of monitoring simultaneously the response of the ion source with the measured event during the GC/MS experiment. A valid analytical methodology aiming at the accurate quantitative determination of trace quantities of contaminants in complex and partially cleaned-up samples should include such a precautionary measure.
Since the accidental release of 2,3,7,8-tetrachlorodibenzodioxin (TCDD) into the environment in 1976 at Seveso, Italy, considerable progress has been accomplished toward the development of sensitive, specific, and rapid analytical procedures for monitoring minute quantities of this toxic material (1-5). Validation (6, 7) of such procedures requires proper evaluation of the spiking technique and the determination of both the recovery and reproducibility of the extraction scheme. Moreover, it should provide a justification for the introduction of any additional cleanup step and adequately assess the determination system. This last aspect of the validation becomes particularly critical for quantitation at low partper-trillion (pptr) levels of a given chemical in a complex environmental sample. Its matrix has been known to affect the amplitude of the response characteristic of the substance when nonchromatographic inlet systems are used in mass spectrometry. Earlier Haddon (8),while developing trace analysis methods for toxins by using direct insertion probe and high-resolution mass spectrometry, noticed an enhancement of sensitivity (100 times for &atoxin B1 and even greater for alfatoxin M2) when partially purified urine samples were analyzed relative to pure standards. Hass et al. (9) conducted some experiments where TCDD was measured by selectively monitoring the loss of COCl (direct insertion probe inlet) obtained by collision activation of the mass selected molecular ion. Significant reductions (over 50% relative to pure standard) in the response for 100 pg of TCDD mixed with various amounts of methyl stearate (400 ng to 1 pg) were observed, whereas an enhancement in the signal for octachlorodibenzodioxin was measured when similar experiments were conducted (10). Others (11) described analogous observations. With the development and availability of sophisticated GC/MS instruments capable of high sensitivity and precision, it is becoming increasingly important to control the various instrumental parameters of the system while performing quantitation a t levels such as low parts per trillion in environmental samples. Tuning the instrument at its utmost Present address: NCI-FCRF, Frederick, MD 21701.
sensitivity for a better signal-to-noise response is not necessarily suitable for or compatible with acceptable quantitative work where maximum stability is preferred (12). The effects of the matrix and its consequences upon the veracity of the quantitative results obtained by using such GC/MS instrumentation are analyzed and reported herein.
EXPERlMENTAL SECTION (A) Materials. The beef liver and goat milk were purchased in a grocery store. Alumina A-540, Fischer Scientific Co. (80-200 mesh) was activated overnight at 130 "C and cooled in a desiccator before use. Sephadex Stock No. LH-20-100 lipophilic (bead particle size 25-100 pm) was obtained from Sigma, St. Louis, MO. The Sephadex beads were refluxed with MeOH and swelled in the same solvent overnight in the refrigerator before being suspended in a mixture of benzene-methanol (1:l). Sodium taurocholate (synthetic origin) was purchased from Sigma. The solvents were distilled in glass (Burdick and Jackson). 1,2,3,4-Tetrachloro-7-fluorodibenzo-p-dioxin (F-TCDD) was prepared and purified as previously described in ref 13. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was a gift from John Moore of the National Toxicology Program. Standard solutions were prepared by dissolving 1 mg of TCDD in 100 mL of benzene and the successive dilutions (down to 1pg/wL solution) were made with hexane. F-TCDD standard solutions were prepared directly in hexane. Isotopic purity of '*C6-TCDDwas 98%. (B) Preparation of the Samples. Beef liver tissue has been processed by following the recommended procedure described by Albro and Corbett (14). Lipid content of the milk was measured by using the described method (15). Development of a Procedure for Recovering TCDD from Milk. (a) Spiking procedure. Five milliliters of milk was thawed in a 15-mLculture tube. Then, while the milk was sonicated in a bath cleaner, 5 pL of a freshly prepared MezSO solution containing TCDD and its internal standard (F-TCDD) was added. After being sonicated for 5 min, the solution was transferred to a rotary shaker for 10 rnin at 200 rpm at room temperature. ( b )Enzymatic hydrolysis. To the 5 mL of spiked or control milk, 650 mg of sodium taurocholate was added and the mixture was placed in a sonic bath for 10 min allowing the formation of micelles. Then 50 mg of NazEDTA was added and the mixture incubated in a rotary shaker at 37 " C and 300 rpm. Finally, 50 mg of acetone powder of rat pancreas was added and the mixture incubated at 37 O C for 60 rnin (300 rpm). After 30 rnin of incubation, the pH was adjusted to 7 by adding approximately 500 pL of KOH 1 N. (c) Extraction. Immediately following the enzymatic hydrolysis, the pH of the hydrolysate was adjusted to 9 by adding 500 pL of 1 N KOH. To this 2.5 mL of EtOH was added and the clear mixture was extracted with hexane (1X 5 mL + 1 X 3 ml), with centrifugation at lo00 rpm for 5 min between extractions and the transfer of the organic layers into another 15-mL culture tube. The combined layers were washed with 3 mL of 5% K2C03 and centrifuged as before after adding 1 mL of EtOH. The hexane layer was percolated through a funnel containing NaZSO4(granular) over a glass wool plug directly into a graduated centrifuge tube of 15 mL. The drying agent was rinsed with 2 mL + 1 mL of hexane. Finally, the sample was concentrated to near dryness under a gentle stream of Nz. A white residue was formed and was easily dissolved in C6H6-MeOH (1:l) as required for the following step. The recoveries of the extraction step were determined by spiking the milk at the 200 pptr level using I4C6-
This article not subject to US. Copyright. Published 1984 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1984
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STANDARD
IO
PG
F-TCDD
340
MI2
eL&&' ,
PEAK 100,
I
277.8093
TIME
PEAK
PROFILE
PROFILE
1
321.8935
277.8093
339.8841
Typical results obtained by GC-SIM representing the exact mass fragmentograms of the indicated ions and the TIC which includes the lock mass ion. The peak profiles correspond to the GC/MS signals of interest (OV-1 fused sllica; 150 ' C to 250 'C at 8 'C/min). Figure 1.
TCDD; they were measured to be 99.2% f 2.2 ( N = 5). It has also been found that the use of Et20 or petroleum ether as an extracting solvent instead of hexane did not significantly affect the recoveries. (d) LH-20 Sephadex fractionation. The residue from the above extraction was dissolved in 100 pL of C6€&-MeOH(1:l) and placed in a sonic bath for 1to 2 min. An 8-g Sephadex LH-20 column (19 mm i.d., bed length 15 cm) was loaded with a 200-pL pipet, followed by two washings of 100 pL each using the same solvent, Elution was performed with the same mixture, C61-&-MeOH (l:l), and the following fractions collected fraction I, 22 mL (discarded); fraction II,18 mL. Fraction I1 was concentrated with a rotatory evaporator and a water bath kept at 40 'C. The residue was transferred with 2 X 0.5 mL of CHzC12into a graduated centrifuge tube and the solvent removed under a gentle stream of Nz. ( e ) A1203-A540fractionation. The above residue was loaded on an aluminum column (3 g) and eluted according to ref 14. (C)Instrumentation and Procedure. GC. An HP-5700 gas chromatograph with a 20-m fused silica OV-1 column coupled directly into the MS source was programmed from 150 OC to 220 "Cat 8 "C/min. The quantitative measurements, based on area measurements by computer, were performed by cold, "on-column" injection, 1 pL, of the solution to be analyzed flushed with 0.5 wL of solvent; 5-pL syringes (Hamilton) were used. MS. A VG Micromass ZAB-2F coupled to a Finnigan/INCOS 2300 Data System was operated in the E.I. mode (70eV). Computer-controlled selected ion monitoring (SIM)(16,17)and selected reaction monitoring (18)were used as previously described. The exact masses monitored by SIM were m/z 321.8935 for TCDD, m / z 339.8841 for 1,2,3,4-tetrachloro-7-fluorodibenzo-pdioxin, and m / t 335.9101 for l4CG-TCDD.The lock mass (for automatic correction of any drift of at least 5 ppm) was a fragment ion from Cz14at m / z 277.8093. Usually the dwell times were 0.5 s for analyte ions and 0.2 s for lock mass ion. The resolution was set at 10000, using the 5% cross-over definition, and the accel-
erating voltage for SIM experiments was 8 kV, allowing the transmission of the lock mass ions. The mass window sampled was 200 ppm, and peak profile data were recorded. When collisionally activated dissociation-mass analyzed kinetic energy spectrometry (CAD-MIKES) experiments were involved, the accelerating voltage corresponding to the transmission of the main beam was 7 kV,the sampling rate being 1.5 s/scan. Helium was used at a pressure so that the intensity of the main beam was reduced by 50%.
RESULTS Selected ion (SIM) and selected reaction (SRM) monitoring techniques are considered individually, and the matrix effect on these analyses for low levels of TCDD in environmental samples is described. GC Selected Reaction Monitoring (with low resolution of the primary ion beam). In this detection mode, the mass spectrometer monitors the chromatographic effluent for a specifictransition characteristic of the anal@. More explicity, [MI+. from TCDD and generated under EI is selectively transmitted into the second field free region of the instrument where it undergoes a collisionally activated dissociation process. A portion of the resulting daughter ion current is then allowed to reach the collector detector assembly by scanning the ESA over a limited energy range. In particular, the characteristic decomposition of TCDD [MI+- [M - COCl]+ is used here for the GC-SRM studies. A significant enhancement in the response factor for 90 pptr of TCDD was observed both for a solvent procedural blank (relative response 1.9 f 0.2) and for a cleaned-up beef liver extract (relative response 4.8 f 0.5). These average responses (N = 3) are given relative to the pure standard. No specific interference (Le., false positive) was detected in the control beef liver extract.
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984 SPIKED
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MILK
s i 0 - a - ,r AFTER FIRST CLEAN
- UP STEP Vi2
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,
,
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,
1
1
,
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14:OO
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TIME
21:oo
PEAK
PROFILE
SPIKED MILK
S 10 - B - LH AFTER FIRST
CLEAN - U P STEP
50
Flgure 2. GC-SIM data from the run of a partially cleaned-up milk extract. The milk was splked with a mixture of TCDD (90 pptr) and F-TCDD (50 pptr) (OV-1 fused silica; 150 O C to 250 O C at 8 OC/min).
GC Selected Ion Monitoring (high resolution). In a typical experiment, the magnet current is set to a specific value and kept constant. Variation of the accelerating voltage will allow preselected ions to be sequentially switched into focus for measurement. Figure 1 represents a GC-SIM (10000 resolution) analysis of a standard mixture containing 120 pg of TCDD and 10 pg of an internal standard compound (vide infra). Two exact mass fragmentograms for ions of TCDD and the internal calibrant as well as the total ion chromatogram (TIC) are represented on the upper part of the figure; the TIC trace represents the ion current for all the monitored channels including the one of the lock-mass ion sampled for automatic correction of system drifts. Peak profiles shown in the lower part of the figure originate from the responsible species eluting from the chromatographic interface. In this particular example, each mass window is scanned over 200 ppm, the center of which corresponds to the exact mass of the sampled ion. Measurements obtained by using this mass spectrometric procedure indicate that the response factor is nearly twice (or three times) as large for 90 pptr of TCDD in a beef liver (or a milk) extract over the pure standard. Similar increases in the mass spectrometric response factors were also observed for 50 pptr of 1,2,3,4-tetrachloro-7-fluorodibenzodioxin (F-TCDD) and 70 pptr of l4C,-TCDD when analyzed simultaneously with 90 pptr of TCDD in a cleaned-up milk extract. The obvious advantage of using an internal standard for quantitative purposes is demonstrated by the compensating effect observed when the response ratios are computed. For the ratios TCDD/F-TCDD and TCDD/14CB-TCDD,one obtains 1.1 f 0.2 (N = 3) and 0.7 f 0.2 ( N = 3), respectively. Under the chromatographic conditions used here, F-TCDD elutes earlier than TCDD and 14C6-TCDDcoelutes with the
nonlabeled material. This difference in retention time properties does not influence the magnitude of the effect caused by the matrix upon the absolute response factors. But, as illustrated below, this is not always the case. Example of a ”Quantitative Interference”. In this section, the extent of cleaning-up a residue has been probed with respect to quantitative determination of TCDD with GC-SIM. Goat milk (lipid content of 25 mg/mL; RSD = 1.3%; N = 3) was spiked with a mixture of TCDD (90 pptr) and F-TCDD (50 pptr) followed by an enzymatic hydrolysis, an extraction of the digested milk, and two cleanup steps. The first step involves removal of the bulk of the lipids and aliphatic compounds on a Sephadex LH-20 column. AS evidenced by the presence of the chemical noise (Figure 2) and the effect created by an interference on the quantitative measurements, such a minimally cleaned-up sample is not useful for analysis. Legitimate ions generated by the constituents of the sample other than the analyte are responsible for this high level of chemical noise. Another distinctive feature revealed by the same figure is the presence of a “quantitative interference”. This unidentified component in the mixture coelutes with TCDD (not with F-FCDD) but does not give rise to ions in the region monitored, yet it is apparent from the sudden change in sensitivity clearly discerned on the TIC trace. From a quantitative point of view, this effect is not desirable especially when the analyte and its internal standard do not have the same retention time. Indeed, the difference between the measured area ratios, F-TCDD/TCDD, for this partially cleaned-up sample and the one obtained from the standard mixture is 70 times the standard deviation of the latter. This value drops to 2.5 times the standard deviation when the residue is further cleaned-up. Justification of this additional
ANALYTICAL CHEMISTRY. VOL. 56, NO. 8, JULY 1984
*
.-c \b
time
Figure 3. Variations of the ion current from a PFK reference ion
monitored by GC-SIM while 1 pg of Flremaster FF-1 was eluting from the capillary columns: (a) and (b) instrument tuned for maximum sensitivity; (c) instrument tuned for maximum stability.
step (A1203chromatography), during which traces of lipids as well as the PCB’s and other potential chlorinated interferences are removed, is thus well substantiated by the observed sharp reduction of both the chemical noise and the effect resulting from the aforementioned interference.
DISCUSS ON The primary goal of the work presented here lies in uncovering a specific matrix effect found to have serious effects on the quantitative assessments and therefore compromising their validity for trace amounts of toxic substances in complex media. It is not our intention to compare the two techniques of GC-SRM and GC-SIM as described above, even though the former is more susceptible to interferences, but to emphasize the fact that this particular matrix effect is present regardless of the mass spectral analysis technique used and will be apparent only if an independent measurement is made to monitor its presence. In an attempt to rationalize these observations, one can trace this matrix effect to various causes or combination of them such as (1)reduction of the analyte ionization probability, (2) degradation of the extraction efficiency of charged particles from the ion source through space charge effects, (3) collisions between molecules and ions resulting from greater operating source pressure, and (4) Grob type effect of chromatographic band narrowing, as recently reported (19). Simultaneous admission of extraneous material and analyk with subsequent reduction or enhancement in sensitivity was already known for more than 2 decades (20). Ion source stability may be related for our purpose to the repeller curve. Ion current variation with repeller voltage generally gives rise to two maxima (21).A sharp maximum occurs at low repeller voltages, which corresponds to the highest value for the ion current (best sensitivity), and a broad extremum appears at higher repeller voltages associated with a loss in sensitivity by a factor of 2 or 3 relative to the sharp peak. Quantitatively it is preferable to have the source operated at the second maximum. Under such stable conditions, the ion current per unit sample pressure is expected to remain constant over a wider range of sample pressures. To illustrate this, variations of the ion current from a reference PFK ion introduced via the liquid inlet and recorded by using the conditions described hereafter are depicted in Figure 3. SIM with a resolution of 5000 and a mass window of 400 ppm was used to monitor the reference ion while 1wg of a complex mixture of polybrominated biphenyls (Firemaster FF-1) was eluted off a capillary column. The source was operated with a trap current of 200 FA and the experiments were carried out on 3 different days. The occurrence of both positive and negative peaks (first two traces) coincides with the elution of the major constituents in the PBB mixture, i.e.,
1347
hexa - and heptabromobiphenyl isomers. An estimated flux of 50 ng/s for hexabromobiphenyl penetrating the ion source was calculated at the time the major change in sensitivity happened. These first two traces (the first one is most commonly found) were obtained when the instrument was tuned for optimal sensitivity. It should be pointed aut that apart from one occasion, we were not able to obtain a broad maximum on our instrument without being constrained to a loss of more than 1 order of magnitude in sensitivity. The last SIM trace was obtained the day after the source has been used for negative CI work using isobutane as a reagent gas, know11 to rapidly contaminate ion sources. Without any cleaning involved, the system was brought back to E1 mode and FF-1 analyzed as usual. Inspection of the repeller curve recorded before this GC-SIM experiment (third trace) revealed the presence of two maxima as described in the preceeding paragraph. No efforts to duplicate or to document this last observation with the source operated on the broad maximum were attempted.
C0NCLUSIO N A matrix effect with potentially severe consequences on quantitative evaluations has been uncovered during the analysis of trace quantities of TCDD in environmental samples by using gas chromatography coupled to mass spectrometry, regardless of the particular methodology used. The mechanism of action of such an effect is tightly connected to source instabilities when relatively significant amounts (i.e,, 50 ng/s) of undesirable material reaches the ionization chamber. It is therefore necessary to remove the bulk of the matrix before mass spectral analysis to avoid this effect and we recommend that no analysis (even screening) be performed on crude extracts at the part-per-trillion level. Registry No. TCDD, 1746-01-6; F-TCDD, 84245-14-7. LITERATURE CITED Harless, R. L.; Oswald, E. 0.; Wilklnson, A. E.; Dupuy, A. E., Jr.; McDaniel, D. D.; Han, T. Anal. Chem. 1980, 52, 1239. Shadoff, L. A.; Hummel, R. A,; Jensen, D. J.; Mahle, N. H. Ann. Chim. (Rome) 1977, 67, 583-592. Lamparski, L. L.; Nestrick, T. J. Anal. Chem. 1980, 52, 2045-2054. Chess, E. K.; Gross, M. L. Anal. Chem. 1980, 52, 2057-2061. Hass, J. R.; Friesen, M. D.; Harvan, D. J.; Parker, C. E. Anal. Chem. 1978, 50, 1474-1479. Albro, P. W. “Environmental Health Chemistry - The Chemistry of Environmental Agents as Potential Human Hazards”; McKinney, J. D., Ed.; Ann Arbor Science Publishers: Ann Arbor, MI, 1980; Chapter 8. Albro, P. W. Ann. N . Y . Acad. Sci. 1979, 320, 19-27. Haddon, W. F. “High Performance Mass Spectrometry: Chemical Applications”; Gross, M. L., Ed.; American Chemical Society: Washington, DC, 1978; ACS Symposium, Series 70, p 105. Hass, J. R.; Harvan, D. J.; Parker, C. E. Presented at the National Meeting of the American Chemical Society, Washlngton, DC, Sept 9-14, 1979. Hass. J. R.; Harvan, D. J., unpubllshed results. Baughman, R.; Meselson, M. “Chlorodioxins-Origin and Fate”; Blair, E. H., Ed.; American Chemical Society: Washington, DC, 1973; Chapter 10. Millard, 8. J. “Quantitative Mass Spectrometry”; Heyden and Son, Ltd.: London, 1978; Chapter 3. Chae, K.; Albro, P. W. J. Envlron. Sci. Health, Part 6 1982, 817, 441-445. Albro, P. W.; Corbett, B. J. Chemosphere 1977, 381-385. Yakushiji, T.; Watanabe, I.; Kuwabara, K.; Yoshida, S. J. Chromatogr. 1978. 154. 203-210 - . Harvan, D. J.; Hass, J. R.; Wood, D. Anal. Chem. 1982, 54, 332-334. Tondeur, Y.; Hass, J. R.; Harvan, D. J.; Albro, P. W. Anal. Chem. 1984. 56. 373-376. Weis;, M.’; Karnofsky, J.; Hass, R.;Harvan, D. Presented at the 27th Annual Conference on Mass Spectrometry and Allied Topics, Seattle, WA, June 3-8, 1979: Pauer No. TAMP16 Voyksner, R. D.; Hass, J. R.; Sovocool, G. W.; Bursey, M. M. Anal. Chem. 1983, 55, 744-749. Taubert, R. ErdiilKohle 1957, IO, 516. Beynon, J. H. “Mass Spectrometry and Its Applications to Organic Chemistry”; Elsevier: New York, 1960; p 105.
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RECEIVED for review June 9,1983. Resubmitted February 16, 1984. Accepted February 16, 1984.
