Anal. Chem. 1980, 52, 191-192
Finally, the overlap between phenanthrene and pyrene (Figure 2, D and C, respectively) is slight. The main pyrene emission is overlapped by anthracene, and other conditions are required for their resolution. The choice of conditions need not be a matter of fortuity as Latz et al. Suggest ( I ) , but can be made from a knowledge of the component fixed excitation and emission spectra (4).
LITERATURE CITED (1) Latz, H. W.; Ullman, A . H.; Winefordner, J. D. Anal. Chem. 1978, 50, 2148.
Sir: Our Correspondence concerning synchronous luminescence measurements of multicomponent samples was intended not as a criticism of the technique which indeed has valuable applications, but rather as a concern against the sole use of the technique. The comment by Lloyd simply reinforces our original conclusions in that additional measurements are required to reveal difficulties with the analyte solution. Had the sample been “real” and the contents unknown, sample dilution could conceivably have reduced the concentration of a component below the limit of detection. We must also take exception to the comment that our conclusions were based “on errors of fact and interpretation” due to a massive inner filter effect because Lloyd makes no distinction between prefilter, post-filter effects; and self absorption effects; the extent of the former two depend entirely upon the cell geometry and methods of illumination and measurement and the latter one depends only upon the optical depth of the analyte a t the fluorescence wavelength(s). In our Correspondence, the concentrations of the PAH compounds used were selected on the basis of the sensitivity of the emission section of the instrument used with the exception of fluorene. All peaks in Figure 1 should be labeled x l except for the fluorene peak which was x 3 owing to the higher concentration of fluorene used to show the extent of the masking. A pre-filter effect (inner filter), absorption of the exciting radiation, was of concern because of the additive absorbance of the mixture at various wavelengths but was found not to be a problem with the instrumental system used by us. The sample compartment permitted translation of the 10 X 10 mm cell along the axis of excitation, and similar results were obtained from approximately 2- to 8-mm excitation depths (with the exception of increased scatter at each extreme). This is attributed to the combination of a focused xenon arc lamp and a high efficiency monochromator producing a high excitation flux. Certainly, ratio absorbance measurements alone are not sufficient to infer a significant pre-filter effect because increased absorbance of exciting radiation can be offset by an increase in the intensity of that radiation.
191
(2) Parker, C. A. “Photoluminescence of Solutions”; Elsevier Publishing Company: New York. 1968; pp 222-226. (3) Lloyd, J. B. F. J . Forensic Sci. SOC.1971, 11, 83. (4) Lloyd, J. B. F.; Evett, I. W. Anal. Chem. 1977, 49, 1710.
H~~~ Office ~~~~~~i~ science ~~b~~~~~~~ Priory House Gooch Street North Birmingham B5 6QQ, U.K.
J. B. F. Lloyd
RECEIVED for review March 5 , 1979. Accepted July 10, 1979.
The fact that a post-filter effect existed in our case, the absorption of fluorene fluorescence by pyrene, was the point intended. It is particularly significant in that a weak fluorophor was masking a strong one. Dilution of a sample to offset high absorbance due to a weakly or nonfluorescent compound could conceivably result in loss of qualitative luminescence information about either one or both of the compounds. If in the event that masking is not complete when the post-filter condition exists, then the problem of quantitation remains complex. Finally, when the sample is an unknown, the fixed excitation and emission spectra of the components are also unknown and the relative concentrations of species would indeed need to be fortuitous if error due to spectral overlap is to be avoided. The conclusion is still held that the application of synchronous luminescence to mixtures of complex and unknown composition, although excellent for fingerprinting, requires caution in the form of more conventional measurements. Lloyd is probably correct concerning the possibility of anthracene impurity in the phenanthrene since a commercial sample was used without purification (as stated). In Figure l b , the small peaks IV and I11 should be x1 not x 3 and in the caption AA should equal 5 nm as stated in the text.
LITERATURE CITED (1) Latz, H. W.; Ullman, A. H.; Winefordner, J. D. Anal. Chem., 1978, 5 0 , 2148.
’
Present address, Department of Chemistry, Ohio University, Athens, Ohio 45701. ‘Present address, Procter and Gamble, Industrial Chemicals Division, Sharon Woods Technical Center, Cincinnati, Ohio 45241,
H. W. L a d A. H. Ullman2 J. D. Winefordner Department of Chemistry University of Florida Gainesville, Florida 32611 RECEIVED for review June 8, 1979. Accepted July 10, 1979.
Specificity of Low Resolution Gas Chromatography-Low Resolution Mass Spectrometry for the Detection of Tetrachlorodibenzo-p-dioxin in Environmental Samples Sir: A. diDomenico et al. ( I ) make the statement in their conclusions that low resolution gas chromatography-low resolution mass spectrometry (LR GC-LR MS) is sufficient for detection of 2,3,7,&tetrachlorodibenzo-p-dioxinat the part per trillion (ppt) level in environmental samples contrary to 0003-2700/80/0352-0191$01.OO/O
criticism by one of us ( 2 ) . Although they do not specifically so state, this conclusion gives the impression that LR GC-LR MS is specific for tetrachlorodibenzo-p-dioxinITCDD) in that type of analysis. For their study, LR GC-LR MS was suitable but in general, when the cleanup is not specific, positive D 1979 American Chemical Society
192
ANALYTICAL CHEMISTRY, VOL. 52, NO. 1, JANUARY 1980
Table I. EPA Phase I Dioxin Implementation Plan Beef Fat Samples Analyzed for TCDD
grazed on treated land control fortified extracts and solutionsb
no. of apparent
analyzed bvLR
positive results by LR LR
GC-LR MS
GC-LR GC-HR MS MSa
no. of
64
19
9
20 9
9 9
2 9
a In this part of the study, only those extracts showing an apparent positive result or a limit of detection greater The than 20 ppt were analyzed by LR GC-HR MS. fortification level was 20 to 100 ppt TCDD in the beef fat,
identification of TCDD in environmental samples requires the use of high resolution mass spectrometry. There is only a small amount of published data to support this conclusion (3)but there are additional data, generated by blind analysis of sample extracts, to add support to our conclusion. As collaborators in Phase I of the United States Environmental Protection Agency Dioxin Implementation Plan, we received extracts of beef fat to examine for the presence of TCDD by GC-MS. The fat samples had been obtained by the U S . Environmental Protection Agency (EPA) from animals which had grazed on land treated with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) herbicide. Extracts of these samples, extracts of control samples, and fortified control samples were prepared by an EPA laboratory and aliquots were sent to the collaborators. The extracts were identified only by a code number. Our plan of analysis was to examine all extracts by LR GC-LR MS and to then use LR GC-high resolution mass spectrometry (HR MS) (9000 resolution) ( 4 ) to examine those extracts which showed an apparent positive result or which had a limit of detection above 20 ppt. All data generated were submitted to the EPA. When the complete set of fat samples had been analyzed, the results were decoded by the EPA and the collaborators met to interpret the results ( 5 ) . Our decoded data are shown in Table I. These data support the following conclusions. (1)LR GC-LR MS is a satisfactory screening technique for TCDD in beef fat a t levels of 20 ppt and higher. (We had no false negative results.) (2) With this particular type of sample and cleanup, it is necessary to use high resolution mass spectrometry to check apparent positive results. (3) The total analytical system used was not capable of 100% certainty in identifying only the positive samples. (Two high resolution results on control samples were positive.) The other collaborators analyzed the extracts by procedures of their own choosing. One of the other collaborators performed a few analyses on the same extracts by both LR GCLR MS and LR GC-HR MS which lend additional support to our second conclusion above. The Phase I study also yielded data which illustrate the difficulty of confirming the presence of a compound near the detection limit. After the first set of results was decoded,
samples which appeared to be positive were cleaned up again and the new extracts were submitted to the collaborators under code numbers. These new extracts were examined only by LR GC-HR MS. One sample, which we found positive a t the limit of detection in the first analysis, was negative at the same sensitivity when the second extract was analyzed. With another sample, our first analysis was negative and the second analysis positive. When the data of all the collaborators were examined, several samples showed this mixture of positive and negative results near the limit of detection. (The limit of detection was defined as 2.5 times noise.) Summarizing, our disagreement is not with the work reported by diDomenico et al. ( I ) , but with their conclusion as we have discussed. Analysis by LR GC-LR MS alone is acceptable if suitable control samples are available to show the absence of interferences. In the absence of suitable controls and when cleanup is nonspecific, positive results must be confirmed by high resolution mass spectrometry. Recently, a clean-up technique using reagent modified adsorbents and high performance liquid chromatography has been described which shows promise of being specific for TCDD (6). This cleanup could eliminate the need for a high resolution mass spectrometry confirmation of identity. Throughout our discussion, the problem of interference from isomers of TCDD in a determination of 2,3,7,8-TCDD has been ignored because the LR GC-LR MS interferences we have encountered were not due to isomers. This possible isomer interference must be considered if results are reported as 2,3,7,8-TCDD. The separation of 2,3,7,8-TCDD from its isomers is improved by use of a capillary GC column in place of the LR GC column (7) but capillary columns have not been shown to give a complete separation of 2,3,7,8-TCDD from its isomers. The 22 isomers of TCDD have been separated by high performance liquid chromatography using two different columns followed by LR GC-LR MS (8).
ACKNOWLEDGMENT The beef fat extracts were prepared at the EPA Mississippi Test Facility, Bay St. Louis, Miss., under the direction of A. E. Dupuy, Jr. LITERATURE CITED (1) diDomenico, A,; Merli, F.; Boniforti, L.; et al. Anal. Chem. 1979, 51, 735-740. (2) Hummel, R. A. J . Agric. Food Chem. 1977, 25, 1049-1053. (3) Shadoff, L. A.; Hummel, R. A,; Lamparski, L. L.; Davidson, J. H. Bull. Envlron. Contam. Toxlcol. 1977, 18, 478-485. (4) Shadoff, L. A,; Hummel, R. A. Biomed. Mass Spectrom. 1978, 5, 7-13. (5) "Pesticide Chemical News", Food Chemical News, Inc.: Washington, D.C., June 23, 1976; p 17. (6) Lamparski, L. L.; Nestrick, T. J.; Stehl, R . H. Anal. Chem. 1979, 57, 1453-1458. (7) Buser, H. R. Anal. Chem. 1977, 49, 918-922. (8) Nestrick, T. J.; Lamparski, L. L.; Stehl, R. H. Anal. Chem. 1979, 51, 2273-2281.
R. A. Hummel* L. A. Shadoff Analytical Laboratories Dow Chemical USA Midland, Michigan 48640 RECEIVED for review July 23, 1979. Accepted November 1, 1979.
