588
Anal. Chem. 1986, 58,568-572
LITERATURE CITED (1) Druckrey, H.; Preussman, R.; Ivankovic, S.; Schmu, D. 2. Krebsforsch. 1987, 69, 103. Toth, 9.;Shubik. P. Cancer Res. 1987, 27, 43 Griswold, D. P., Jr.; Casery, A. E.; Weisburger, E. K.; Weisburger, J H.; Schnabei, F. M., Jr. Cancer Res. 1988, 619. Hecht, S. S.;Chen, C. 6.; Hoffmann, D. Acc. Chem. Res. 1979, 12,
92. Hoffmann, D.; Rathkamp, G.; Liu, Y. Y. IARC Sci. Pubi. 1974, 9 ,159. Klus, H.; Kuhn, H. fachliche Mltt. Oesterr. Tabakregle 1973, 14, 251. Hecht, S. S.;Ornaf, R. M.; Hoffmann, D. J . Natl. Cancer Inst. 1975, 54, 1237. Bharadwa], V P.; Takayama, S.; Yamada, T.; Tanimura, A. A . Gann 1976, 66, 585. Munson, J. W.; Abdine, H. Anal. Lett. 1977, IO, 777. Klus, H.; Kuhn, H. Fachliche Mltt. Oesterr. Tabakregie 1975, 16, 307. Hecht, S. S.;Ornaf, R. M.; Hoffmann, D. Anal. Chem. 1975, 4 7 , 2046. Hoffmann, D.; Dong, M.; Hecht, S. S. J . Natl. Cancer Inst. 1977, 58, 1841.
Brunnemann, K. D.; Adams, J. D.; Ho, D. P. S.; Hoffmann, D. “Proceedings of the 4th Joint Conference on Sensing of Environmental Pollutants”, American Chemical Society: Washington, DC, 1978; p 876. Chamberlain, W. J.; Arrendale, R. F. J . Agrlc. Food Chem. 1983, 31, 909. Hoffmann, D.; Ornaf, R. M.; Hecht, S. S. Science 1974, 186, 265. Henneberg, D. 2. Anal. Chem. 1981, 183,12. Watson, J. T. “Introduction to Mass Spectrometry, Biomedical, Environmental and Forensic Applicatlons”; Raven Press: New York, 1976; p 199. Millard, B. J. “Quantitative Mass Spectrometry”; Heyden, London, 1978.
(19) (20) (21) (22) (23) (24) (25) (46)
Matthews, D. E.; Hayes, J. M. Anal. Chem. 1978, 4 8 , 1375. Grob, K.; Grob, K., dr. J . Chromatogr. 1978, 151,311. Grob, K., Jr.; Neuborn, H. P. J . Chromatogr. 1980, 189, 109. Grob, K., Jr. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1978, 1 , 263. Grob, K., Jr. J . Chromatogr. 1981, 213,3 . Grob, K., Jr. J . Chromatogr. 1982, 237, i 5 . Grob, K., Jr.; Muller, R. J . Chromatogr. 1982, 244, 185. denkins, R.; Jannings, W. HRC CC, J . High Resolut. Chromatogr. dhromatogr . Comhun . 1983, 6 228. Grob, K., Jr. HRC CC J . High Resolut. Chromatogr. Chromatogr. Cammpn. 1983, 6 , 581. Jenkins, R.; Jennings, W. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1983, 6 , 582. Arrendaie, R.‘F.;Chortyk, 0. T. HRC CC, J . High Resolut. Chromafogr. Chromatogr. Commun. 1985, 8 , 62. Arrendale, R. F.; Severson, R. F.; Chortyk, 0. T. Anal. Chem. 1984, 56, 1533. Hoffmann, D.; Adams, J. D.; Brunnemann, K. D.; Hecht, S. S.Cancer R e s . 1979, 39, 2505. Chamberlain, W. 4.; Arrendale, R. F. J . Chromatogr. 1982, 234,478. Arrendale, R. F.; Severson, R. F.; Chortyk, 0. T. J . Chromatogr. 1983. I
(27) (26) (29) (30) (31) (32) (33)
254,63.
(34) Hecht, s. S.;Chen, C. 9.;Young, R.; Hoffmann, D. Beitr. Tabakforsch. Int. 1981, 1 1 , 57. (35) Hecht, S.S.;Chen, C. 8.; Dong, M.; Ornaf, R. M.; Hoffmann, D.; Tso, T. 8. Beitr. Tabakforsch. 1977, 9 ,1.
RECEIVED for review April 12, 1985. Accepted October 23, 1985. Names of products are included for the benefit of the reader qnd do not imply endorsement or preferential treatment by the USDA.
Preserving Toxicologic Activity during Chromatographic Fractionation of Bioactive Complex Mixtures Arthur L. Lafleur,* Andrew G. Braun, Peter A. Monchamp, and Elaine F. Plummer Department of Applied Biological Sciences, Energy Laboratory a n d Center for Health Effects of Fossil Fuels Utilization, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Four types of chromatographic materials were evaluated for degree of recovery of mutagenlc components during column chromatography. Materlals tested were silica, alumina, Fiorlsll, and cyanopropyl-bonded silica. A combustlon-generated complex Fixture that vas highly characlerlred and known to contaln mutagenlc components was used gs the reference sample. The cyanopropyl materlal was found to be the most efflclent material far mutagen recovery and alumlna proved to be the least efficlent.
Samples obtained from the combustion of fossil fuels are highly complex mixtures containing combustion products, unburned fuel, and insolublg particulates. They are so complex that even very powerful techniques of chemical analysis are severely challenged by them unless the samples are first fractionated into simpler sets of mixtures prior to analysis. Many fractionation schemes for complex combustion-related mixtures have been developed during the past 2 decades (1-5) and a large number of them have been reviewed (1,5). A list of more recently develoRed methods has also been reported (4) and it would appear that the most widely used methods for preliminary fractionation are column adsorption chromatography using silica gel or alumina as sorbents (1). For samples containing components of widely varying polarity such as fossil fuel combustion products and coal-derived synthetic fuels, separations based whole or in part on alumina frac-
tionation have been shown to be simple and efficient (2-4, 6). After implementation of an alumina fractionation method in our laboratory for a number of combustion samples, it became apparent that although the fractionation was efficient in terms of separation of covpound classes, the biological data indicated that much of the applied mutagenic activity of the samples could not be recovered in the fractions. Therefore, the study that is the subject of this report was initiated to determine the effect of sorbent composition on recovery of mutagenic activity using standardized, commercially available adsarption columns.
EXPERIMENTAL SECTION Apparatus. The liquid chromatographic fractionation columns were Baker-IO solid phase extraction (SPE) columns obtained from J. T. Baker Chemical Co. The columns were obtained Rrepacked with sorbent except for the alumina column, which was prepared by dry packing an SPE filter column with Woelm neutral alumina (Brockman activity I). The alumina used was obtained from ICN Pharmaceuticals,Inc., and had a particle size distribution. of 50-200 pm (70-290 mesh). The silica gel sorbent had a particle size of 40 pm and a pore size of 60 A, as did the cyanopropyl-bonded silica material used in the cyano column. The Florisil sorbent had a 100-pm particle size. All columns were packed with 1.0 g of sorbent and had a 6.0-mL solvent reservoir. The columns were constructed of polypropylene and the sorbent material was held in place by a two polyethylene fritted disks of 20-pm porosity. Chemicals and Reagents. The hexane, dichloromethme, and methanol used for fractionation were Ultrapure glass distilled
0003-2700/86/0358-0588$01.50/00 1986 American Chemlcai Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
Table I. Large-Scale Alumina Fractionation weight, mg (2) (1)
mutagenicit? PMS
(1)
(2)
238 >300
>300
125
109
>300
1
1
11
20
30
30 16
30
34
18
18
190
181
13 23
250
250
115 18
16
72
13 35 124 >300
28
>300 138
Calculated Sumsb
+
-
12
11
174
123
Applied Material
+
-
13 23 % Recovery
+
-
102 13
Mutagenicity expressed as minimum detectable mutagen concentration, in pg/mL. *Derived by adding the MDMC contributions of each fraction according to its weight to a hypothetical mixture of the fractions. solvents obtaiued from Caledon Laboi.atories, Ltd. The toluene was Analytical Reagent grade obtained from Mallinkrodt. The reference mixture was obtained from the combustion of No. 2 fuel oil as previously described (5). One milliliter of the combustion mixture contained 10.1 mg of exfractable material. Determinatidn of Total Mutagen Recovery. Approximakly 2 g of sand was packed in a 6-mL SPE filtration Column. Next, a 1.0-mL portion of the reference s b p l e was slowly added to the top of the sand and care was taken so that no material eluted through the bottom. The prepared filtration column was then coupled to the appropriate chromatographic column through an SPE adapter. For the purpose of testing for transmission of mutagens, material placed on the colbmn was eluted with 10 mL of dichloromethane followed by 6 mL of mbthanol. It was determined that this procedure would ensure complete elution with any of the columns. The two fractions were each concentrated to 1.0 mL by evaporation under nitrogen. A 100-pL aliquot of each was taken and weighed. Fractionation Procedure. The procedure used was based on fractionation schemes developed by Schiller and Mathiason (2) and by Later et al. (31, which was mddified for use in the chemical axialysis of No. 2 fuel oil (7) and its combubtion products (5). Biological data obtained from the fractionation of the reference sample using the scheme of ref 7 are f o h d in Table I. For the present study it was desired to optimize the fractionation for much smaller samples (10 mg vs. 250 mg) and reduced elution volumes in order to reduce consumption of the reference material and to accommodate more typical samples. Although the sorbent quantity and configuration are different in the present scheme, the goal was to retain the simplicity of the earlier schemes and to obtain four fractions whose composition wduld be similar to those obtained in the fractionation of No. 2 fuel oil (7). The solvents chosen for the present scheme are hexane, toluene, dichloromethane, and methanol. Methods using model mixtures were used to determine the optimum volume of each solvent required to obtain four fractions of increasing polarity. Thus, it was determined that 3.5 mL of hexane would result in a fraction containing principally aliphatic hydrocarbons with some alkyl aromatics but not polycyclic aromatic compounds (PAC). Similarly, 3.0 mL of toluene resulted in a fraction containing PAC’s and also included some moderately polar PAC’s having heteroatoms. The dichloromethane volume of 2.0 mL caused the elution of polar aromatics and PAC’s but not compounds as polar as 2-naphthoic acid, while the 5.0 mL of methanol was determined
569
to be sufficient to elute all co,mpounds in the test mixture as polar or more polar than 2-naphthoic acid. For the complex mixtuke used as a reference standard in this work, it was fodnd that the procedure deskribed here resulted in efficient separation and complete elution; however, for other samples a different procedure might be pequired to ensure elution of all components. Additional work is in progress on the application of the cyano column fractionation method with a number of other combustion-related samples. The complete procedure is as follows: The filtration column was charged with 2 g of sand as before and then a stream of nitrogen was allowed to enter the bottom of the column through its Luer fitting resulting in the conversion of the sand column into a fluidized bed. Next, the dichloromethane extract was slowly added to the fluidized sand a t such a rate that the liquid did not pass through the bed. Nitrogen was allowed to flow for a short period of time until the dichloromethane was completely evaporated. The loaded filtration column was then coupled to the top of the cyano column with an SPE adapter. The first fraction was eluted with 3.5 mL of hexane, the secohd fraction with 3.0 mL*of toluene, the third with 2.0 mL of dichloromethane, and the fourth with 5.0 mL of methanol. Each fraction was evaporated to 1.0 mL under nitrogen and three lOO-kL aliauots were measured and weighed. Bioassay. The mutagenicity of the samples was determined in S. typhimurium utilizing 8-azaguanine resistance as the genetic end point (8, 9): Frozen aliquots of strain.TM677 were grown in minimal media supplemented with 2% brain heart infusion and treated for 2 h at 37 “C in a liquid suspension with the samples a t concentrations of 0-300 gg/mL. *his was done in both the presence and absence of a 5% (v/v) Arochlor-i254 induced rat liver postmitrochondrial supernatant (PMS). Cultures treated with PMS contained an NADPH generating system. Stock solutions of the reference extract were prepared in dimethyl sulfoxide. The concentration of stock solutions ranged from 0.1 mg/mL to 30.0 mgjmL, so when diluted by a factor of 100 by the bacterial medium, it resulted in reference mixture conceni trations ranging from 1.0 mg/mL to 300 pg/mL and a Me2S8 concentration of 1.0% by volume in all cases. A 10-pL aliquot was added to 0.9g mL of bacteria with or without PMS and the NADPH generating system. Bacteria were plated in triplicate on ininimal agm, plates in the presence or absence of 50 pg/*mLof &maguanine (8AG),incubated at 37 “C and counted 2 days later. The number of mutant colonies observed divided by the plating efficiency of the culture times the dilution factor yielded an estimate of the mutant fraction (MF). The operational definition of a bacterial mhtagen in this type of assay is a sample that induces a response greater than the 99% upper cbnfidence limit for the background. The dose of mutagen yielding a mutant fraction equal to the 99% upper confidence limit-is referred to as the “minimum detectable mutaken concentration” or the MDMC. The net mutant fraction (NMF) was computed by subtracting the number of background mutant colonies dxpected from spontaneous mutation from the observed mutant fraction. A positive and negative control is also tested in the same way with every sample. Mean values for net mutant fraction and their standard errors (SEM) were computed for concentrations of 1, 10, 30, 100, and 300 pg/mL. Another measure of biological activity in complex mixtures is their ability to interfere with the attachment of cells to lectincoated plastic surfaces. Many teratogens and tumor promoters interfere with attachment (10, 11). The methods used have been described previously (12).
RESULTS AND DISCUSSION Initial Findings. Large-scale samples of the reference mixture were fractionated on alumina a number of times into several fractions according t o the procedure referred t o in the Experimental Section. On two occasions, the fractions underwent bioassay for mutagenicity. The results are shown in Table I. The estimate of recovered mutagenic activty is essentially 100% for the PMS-dependent mutagens; however, that for PMS-independent mutagens is 13% and 18% for the two fractionations. Although only a limited number of assays were performed and no rigorous statistical calculations could be performed, the data give a strong indication that PMS-
570
ANALYTICAL CHEMISTRY, VOL.
58,NO. 3, MARCH 1986 250r
Table 11. Gravimetric Recovery Data
vol, pL
mean wt, mg Nu
re1 std dev,' %
ub
concn, mg/mL
%
recovery
-
Reference Sample 100
1.013
3 0.051
+ Applied
Eluted through Sand Column 100 100
0.993
50
3 0.179 18.0 9.93 Sand + Cyanopropyl Column
0.844
3 0.057
6.7
Material Alumina Column Cyano Column -0- SFlorloil i l i c a Column Column
100-
10.1
5.0
-X-
+ -+
-
98 '0
8.44
84
7.68
76
8.40
84
20
40
60
00
40
60
00
100
250r
Sand + Silica Column 100
0.768
3 0.014
1.8
Sand + Alumina Column 100
0.840
3 0.082 Sand
100
0.835
9.8
100
50
+ Florisil Column
3 0.055
6.5
8.35
83
' 0
"N, number of samples. * u , standard deviation of the mean. 're1 std. dev, relative standard deviation.
independent (-PMS) mutagens may be lost upon fractionation. In order to test this hypothesis and to investigate a number of other sorbents that might prove more efficient, the present study was initiated. The tested sorbents included commonly used types such as alumina, Florisil, and silica as well as a bonded-phase sorbent of the cyanopropyl type. The sorbent size was reduced to 1.0 g and the elution solvents to a few milliliters in order to minimize losses during sample handling and concentration and to reduce consumption of reference material. T o further minimize sample losses not related to fractionation, the sorbents were first evaluated by using the simplest mobile phase combination that would ensure complete elution. Recovery of Applied Material. The first step in the study was to determine the amount of reference material that can be recovered from each sorbent system using this optimized mobile phase. The results can be found in Table 11. The amount of material eluted from sand was also determined. Although sand is regarded as inert, it was tested again in this study in order to confirm this supposition since elution from sand would be an ingegral part of the final fractionation procedure. As is clear from Table 11, there is no statistically significant difference in the weight of material recovered from the different column types so that it is clear that gravimetric
20
100
pg/ml
Figure 1. Performance evaluation of chromatographic materials, mutagenicity as a function of concentration for a reference mixture before and after elution from four sorbents.
recovery data would not be a practical indicator for predicting mutagenic recovery behavior. Recovery of Applied Mutagenicity. Next, the total eluent from each of the columns was tested for mutagenic activity and compared with the results obtained for the same amount of reference material. The results, shown in Figure 1, indicate that for the sorbents tested, the recovery of PMS-dependent mutagenicity was good for all columns. For PMS-independent mutagens, only the cyanopropyl column appears to give satisfactory recoveries. A statistical analysis of a portion of these data is found in Table 111. Two indicators of mutagenic potency were evaluated: One, the minimum detectable mutagen concentration or MDMC, which in some form or another is universally used as an indicator of mutagenic potency. The other parameter, the net mutant fraction a t 100 pg/mL (NMF/100), gives a direct indication of the ability of the test mixture to induce mutations a t concentrations high enough to give good precision but low enough to ensure good survival of the bacteria. The optimum NMF concentration is unlikely to be the same for a large number of compounds or mixtures, so the NMF/100 value reported here is not as widely applicable as MDMC, but in this study where the reference mixture is the same for all sorbents, it
Table 111. Recovery of Mutagenic Activity from Sorbents
control sorbent alumina cyanopropyl
PMS
Nd
MDMC" av
(+)
5 5
17 13
3 3 4
7.2 30
5
13 16 41
(-)
(+)
(-1 (+) (-)
Florisil
(+I (-1
silica
(+)
(-1
6 6 5 6
17
12
16
SEM'
N
NMF/ 100b av SEM
1.1
5
3.9
5
208 204
60 62
1.0
3 3 4 5 4 4 4
213 59 225 247 177 108 218 120
45
2.6 4.9 4.8 1.3 15 1.3 3.0
4
MDMC
recovery' NMF/100
5
75 49 17 11
85 52
OMutagenic activity is shown as the minimum detectable mutagen concentration in pg/mL. *NMF/100 = net mutant fraction at 100 pg/mL. 'Recovery: yes, mean value range shows statistical overlap with reference value range at 98% probability level (0.01 level); No, mean value range is lower than reference value and difference is significant at 80% probability level (0.10 level); mrg (marginal) if value range falls between "yes" and "non test criteria; xs, mean value range for MDMC is smaller than reference value and difference is significant at 98% probability level thus indicating recovery in excess of reference value. N , number of samples. e SEM, standard error of the mean.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
Table IV. Fractionation with Cyanopropyl Column
mean wt, mg total applied hexane toluene dichloromethane methanol recovered
10.1
0.99 3.65 1.07 3.59 9.30
u:
mg N b
re1 std dev; %
% of total
0.51
3
5
0.40 0.43 0.15 0.89
3 3 3 3
40 14 25
9.8 36.0 10.6 35.6
0.12
3
12
92.0
100
12
u, standard deviation. N , number of samples. crel std dev, relative standard deviation.
is an excellent indicator for relative mutagenic potency. From the data, i t can be seen that for both +PMS and -PMS mutagens and for both criteria of mutagenic potency, only the cyanopropyl column preserved the original mutagenic activity. For PMS-dependent (+PMS) mutagens, the data in Figure 1 indicate good recovery for all sorbents, but on inspecting the data in Table 111,it is apparent that the MDMC values for alumina and silica are much lower than for the reference sample itself thus indicating much higher activity at low concentrations. In both cases, the difference between the MDMC obtained and the reference value is significant a t the 98% probability level. The interpretation of this finding is not clear but in any case it adds some argument against the utilization of these sorbents. The marked difference in sorbent performance that is found when comparing +PMS and -PMS mutagenicity is illustrated in Figure 2. This figure shows the mean values and error bars for NMF/100 plotted for the reference material and the four sorbents. For +PMS mutagenicity, the mean values for all sorbents are very close t o the mean value for the reference sample, whereas for -PMS mutagenicity, only the value recorded for the cyanopropyl column falls within the reference mean value range. The difference between the two kinds of mutagenicity can be better understood by looking a t what is meant by P M S dependence or independence as applied t o mutagenic components. PMS-dependence means that the compounds that ultimately cause mutagenesis must first be converted to the active form by enzymes contained in the PMS mixture. The activation step almost always involves one or more oxidation steps, depending on the structure of the original molecule, that
571
result in a marked increase in the polarity and reactivity of the molecule (13,14). The compounds that require enzymatic activation for the expression of their activity are generally nonpolar or moderately polar species such as polynuclear aromatic compounds (PNA's) containing only carbon and hydrogen or sometimes containing a heteroatom. Therefore, it can be predicted that such compounds would be eluted from many types of sorbents in high yield. Species that are P M S independent do not require activation and this implies that they must be much more polar and/or reactive than the PMS-dependent types. Thus, it follows that these compounds would be the ones most likely to be bound irreversibly to active sorbents. In all the discussions concerning mutagenicity of the reference mixture, it is assumed that the mutagenicity of the whole mixture is the sum of the mutagenicities of the individual components. In other words, mutagenic additivity is being assumed for the interpretation of the data. This has never been proved rigorously for this particular mixture but for a number of other combustion generated bioactive mixtures additivity has been established. These include extracts obtained from kerosene soot and automotive diesel soot (15). I t is clear from these data that the cyanopropyl material is superior t o the others with regard to recovery of mutagenicity; therefore, it was singled out for further study to determine its efficiency in separating the reference mixture into compound classes and to determine if mutagenic activity would be preserved in this procedure as well. Fractionation of Reference Extract. The reference sample was fractionated in triplicate according to the procedure mentioned earlier into four fractions of increasing polarity. Gravimetric determinations were performed on the fractions and the results are shown in Table IV. The data from Table IV show that the sum of the fractions is very nearly equal to the weight of the applied material when the reference sample is fractionated on the cyanopropyl column. However, as we have determined earlier, the gravimetric recovery data cannot necessarily predict recovery of biological activity even though, in this case, the column sorbent has been shown to be satisfactory but under different elution conditions. Therefore, the fractions were assayed for biological activity as outlined in the Experimental Section. These results are shown in Figure 3 and Table V. Figure 3 shows a plot of net mutant fraction vs. concentration for the reference mixture and for the calculated sum
Table V. Cyanopropyl Column Recovery of Biological Activity after Fractionation
MDMC" hexane toluene DCM methanol calcd sumf applied materialg recoveryh
PMS
Nd
av
+
3 3 3 3 3 3 3 3
none none 24
-
+ + + -
+
46
74 47 none none
NMF/lOOb av
SEMe
N
0.9 3.4 15
3 3 3 3 3 3 3
9 9 152 44 26 35
3
9
11
10
SEM
AIAc
2 1 10 7 10
64 81
32
5 1 1
193
18 15 197 8 93 23 12 91 6 + 5 17 1.1 5 208 60 75 5 13 3.9 5 204 62 + 97 % 20 70 yes Yes yes 48 % 20 70 Minimum detectable mutagen concentration (MDMC) in pg/mL. *Net mutant fraction at 100 pg/mL. Attachment inhibition assay: Values in units of ID(50), which is the concentration of material in pg/mL required to reduce attachment to 50% of the control level. d N , number of samples tested. 'SEM, standard error of the mean. 'The calculated sum for MDMC takes the weight of each fraction into account. For MF/100 it is a simple sum. 8This is the value obtained for the same amount of reference material as that applied to the column. hRecovery for MDMC is yes if mean value range shows overlap with reference value at 98% probability level. -
572
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
+ PMS
T
300
T
400-PMS
300i L
2 e
2oo---x
al
z
i- - _ _ _ _ _ _ _
I
1
$ T1
IOOX
handling in the earlier experiments (Figures 1and 2 and Table 111) results in complete recovery of -PMS mutagenicity with the cyano column suggests that the losses may have been incurred in the additional sample preparation steps. For the attachment inhibition assay, the calculakd sum of the fraction is in close agreement with the activity of the whole sample prior to fractionation. The data obtained in this study suggest that in the fractionation of bioactive mixtures, although PMS-dependent activity seems relatively unaffected by fractionation, PMSindependent activity is likely to be poorly recovered with most common sorbents. Although as stated earlier, for some other combustion-derived complex mixtures, good recovery of mutagenic activity after fractionation has been reported ( I @ , but only for PMS-dependent mutagenicity. The present study confirms that +PMS mutagenicity is preserved during fractionation on a number of different sorbents but also reveals that -PMS mutagenicity is not so easily recovered. In the light of the results obtained with the cyanopropyl sorbent, it appears that this material holds promise for the fractionation of complex mixtures when preservation of biological activity is a major consideration.
ACKNOWLEDGMENT We with to thank Alexandra Hawiger and Leslie Melcer for carrying out the bioassays described here. Registry No. Silica, 7631-86-9; alumina, 1344-28-1;Florisil, 1343-88-0.
LITERATURE CITED
-x-
n "
o
20
40
60
sum
eo
io0
300 2501
T
-PMS
Pg/mi
Figure 3. Comparison of mutagenic activity of reference mixture before and after four-part fractionation on cyanopropyl column. of the activities of the fractions taken from the cyano column. There is very good agreement for +PMS mutagenicity, but for -PMS mutagenicity, it is evident that complete recovery was not achieved. A closer look a t some of the data in Table V confirms the good results for PMS-dependent mutagenicity with both the MDMC and NMF/100 values indicating complete fecovery. However, for PMS-independent mutagenicity, although the MDMC parameter indicates good recovery, the NMF/100 value indicates a 50% loss of activity. It is not clear where the losses occur, but the fact that minimizing sample
Lee, Milton, L.; Novotny, Milos, V.; Bartle, Keith D. "Analytical Chemistry of Polycycllc Aromatic Compounds"; Academic Press: New York, 1961; pp 123-125. Schiller, Joseph E.; Mathiason, Dennls R. Anal. Chern. 1977, 4 9 , 1225-1 228. Later, Douglas W.; Lee, Milton, L.; Bartle, Keith D.; Kong, Robert C.; Vassilaros, Daniel L. Anal. Chem. 1981, 53, 1612-1620. Lucke, Richard B.; Later, Douglas W.; Wright, Cherylyn W.; Chess, Edward K.; Welmer, Walter C. Anal. Chern. 1985, 5 7 , 633-639. Leary, J. A.; Lafleur, A. L.; Longwell, J. P.; Peters, W. A,; Kruzel, E. L.; Biemann, K. I n "Polynuclear Aromatic Hydrocarbons: Formation, Metabolism and Measurement"; Cook, M., Dennis, A. J., Eds.; Battelle Press: Columbus, OH, 1983; pp 799-608. Bartle, K. D.; Collin, G.; Stadelhofer, J. W.; Zander, M. J . Chem. Techno/. Biotechnol. 1979, 2 9 , 531-551. Leary, Julie A. M.S. Thesis, Massachusetts Institute of Technology, Cambridge, MA, 1982. Skopek, T. R.; Llber, H. L.; Krolewski, J. J.; Thilly, W. G. Proc. Natl. Acad. Sci. U . S . A . 1978, 75, 410-414. Skopek, T. R.; Liber, H. L.; Kaden, D. A,; Thilly, W. G. R o c . Natl. Acad. Sci. U . S . A . 1978, 7 5 , 4465-4469. Braun, A. G.; Buckner, C.; Nlchinson, B. B. Teratog. Carcinog., Mutagen. 1981, 1 , 417-427. Braun, A. G.; Emerson, D. J.; Nichlnson, B. B.; Buckner, C. A. R o c . Natl. Acad. Sci. U . S . A . 1982, 79, 2056-2060. Braun, A. G.; Nlchinson, B. B.; Horowitz, P. B. Teratog., Carcinog., Mutagen. 1982, 2 , 343-354. Gerarde, H. W. "Toxicology and Biochemistry of Aromatic Hydrocarbons"; Elsevier: New York, 1960. Williams, R. Tecwyn "Detoxication Mechanisms: The Metabolism and Detoxication of Drugs, Toxic Substances and Other Organic Compounds", 2nd ed.; Chapman & Hall Ltd.: London, 1959: pp 188-232. Thilly, W. G.; Longwell, J. P.; Andon, B. A. EHP, Environ. Health Perspect. 1983, 48, 129-136.
RECEIVED for review June 10,1985. Accepted October 1,1985. This investigation was supported by National Institute of Environmental Health Sciences Center Grant NIH-2P30ES02109-06A1, National Institute of Environmental Health Sciences Program Grant NIH-2P01-ES01640-06, and Department of Energy Contract AC02-83ERG0174.
