Mutagen Isolation Methods - ACS Publications - American Chemical

19 Mutagen Isolation Methods Fractionation of Residue Organic Compounds from Aqueous Environmental Samples M. Wilson Tabor and John C . Loper 1

1,2

Department of Environmental Health and Department of Microbiology and Molecular Genetics , University of Cincinnati Medical Center, Cincinnati, OH 45267

Downloaded by FUDAN UNIV on January 24, 2017 | http://pubs.acs.org Publication Date: December 15, 1986 | doi: 10.1021/ba-1987-0214.ch019

1

2

A general preparative procedure, based on high-performance liquid chromatography (HPLC) and the Salmonella microsome mutagenicity assay, has been developed for the isolation of mutagenic components from samples of complex mixtures of residue organics obtained from environmental waters. This procedure features preliminary HPLC separation to characterize the sample; preparative-scale HPLC separation with mutagenic bioassay of the fractions; further HPLC separation of bioactive fractions, employing different elution techniques; and chemical-biological characterization of isolated mutagenic components. Results of the application of this approach to residue organics from drinking water, ground water, and waste water are presented along with chemical characterization, via high-resolution mass spectrometry, of the constituents of a mutagenic subfraction isolated from waste water residue organics.

T R A C E L E V E L S O F A N T H R O P O G E N I C O R G A N I C C O M P O U N D S in environmental waters have heightened concern as to the possible human health impact of chronic exposure to these contaminants via routes such as drinking water. One response to this concern in the United States has been the enactment of a series of federal acts, including the Safe Drinking Water Act of 1974, PL 93-523, and the Clean Water Act of 1977, PL 95-217. Although the implementation of this federal legislation has yielded improvements in the nation's drinking water and national waterways regarding substances such as toxic metals (J), a recent review (2) concluded that "further studies of the identities, carcinogenicity, 0065-2393/87/0214/0401$06.00/0 ® 1987 American Chemical Society

Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986.


402

ORGANIC POLLUTANTS IN WATER

mutagenicity, mode of formation, and practical methods of removal are needed for the organic contaminants". The goal of our studies has been to understand the biohazardous components of complex mixtures of organics f r o m aqueous samples in terms of their prevalence, concentration, chemical structure, origin, and mechanism of biological effects. O n e specific focus of this research effort has been the isolation, identification, and chemical-biological characterization of mutagenic components f r o m aqueous environmental samples. The first step required for this process was the application of methods for the isolation of residue organics f r o m aqueous samples, for example, drinking water, ground water, and waste water (3,4), b y using columns of X A D resins to concentrate the organics. Residue organics have been defined as those organics adsorbed b y X A D - 2 / X A D - 7 resins under the conditions described and recovered b y the solvent elution method e m p l o y e d i n the U . S . E n v i r o n m e n t a l Protection Agency ( U S E P A ) Interim Protocol for drinking water (5). In the past, these organics were referred to as nonvolatile residue organics (6-JO) on the basis of previous descriptions of organics in drinking water (11). F o l lowing isolation, the residue organics can be tested for mutagenicity and other biological end points v i a a variety of short-term bioassay procedures (12) and/or fractionated for the isolation of mutagenic components for chemical-biological characterization. The need for the fractionation of residue organics isolated from water samples to assess mutagenicity and to identify the biohazardous constituents has been discussed (8, 13). O n the basis of this need, a biological approach to the chemical fractionation and separation of residue organics is the method of choice for the isolation of biohazardous compounds f r o m complex mixtures of residue organics. W e introduced a general method based upon high-performance liquid chromatography ( H P L C ) for the fractionation of residue organics from drinking water. This method assessed the polarity distribution of constituents by using an analytical H P L C separation followed b y preparative-scale H P L C separation of the residue organics for mutagenicity testing and compound identification (6, 7). The strategy for this method is summarized in Figure 1. This approach led to the first identification of a previously unidentified mutagen from drinking water residue organics (14). The use of H P L C for the separation of residue organics from aqueous samples for mutagenicity testing has been extended to studies of many other types of water samples f r o m different parts of the w o r l d . Baird and co-workers (15, 16) have used such an approach in their studies of mutagenicity of residue organics from drinking water, river water, storm runoff, reclaimed waste waters, and other waste waters. Jolley and co-workers (27, 18) have applied H P L C to the separation


19.

TABOR AND LOPER

403

Mutagen Isolation Methods RESIDUE

ORGANICS

BIOASSAY

HPLC SEPARATION

Fi

F

2

BIOASSAY

F

F

3

N

HPLC SEPARATION


I

ETC,

Figure 1. Schematic of the strategy for the coupled bioassay-analytical fractionation of residue organics isolated from aqueous environmental samples. and waste water effluent residue organics to study the relationship of mutagenicity to disinfection methods and to fractionate residue organics f r o m other polluted waters for bioassay of separated fractions. In a study of genotoxic components of oil shale retort process water, Strniste et al. (19) first separated these samples b y a classical l i q u i d - l i q u i d extraction scheme followed b y H P L C separation of the extracts for bioassays. O n e research group (20) reported the use of H P L C to fractionate drinking water residue organics for mutagenicity assessment. Our research group has made extensive use of H P L C for the fractionation of drinking water and waste water residue organics (3, 4, 6-10, 21, 22). Residue organics f r o m samples of ground and river water and f r o m samples of various stages in the processing of both drinking waters and of diverse waste waters have been fractionated i n these studies. This approach has provided a better assessment of the mutagenic potential of such residue organics. T h e purpose of this chapter is to present the specifics of our H P L C approach as applied to residue organics f r o m a variety of aqueous samples, particularly as the method is used in the isolation of mutagenic constituents for compound identification. E x amples of the separation of residue organics f r o m a variety of aqueous environmental samples w i l l be used to illustrate the applicability of the approach.

Methods Instrumentation. H P L C separations were performed on a Waters Associates system consisting of t w o M o d e l 590 pumps, a M o d e l U 6 K injector, a M o d e l 680 automated gradient controller, and both M o d e l 440 fixed wavelength (254 nm) and M o d e l 480 variable wavelength



404


absorbance detectors. Chromatograms were displayed on a Fisher Recordall Series 5000 two-pen recorder. F o r preparative-scale (mil ligram level) separations, a Waters Associates Z-module radial compres sion column ( R C M ) unit was used. The R C M unit was fitted with an 8-mm X 10-cm column packed with 10-μπι silica particles bonded with octadecylsilane for reverse-phase separations; for normal-phase separa tions, similar R C M columns packed with 10-μπι silica particles were used. F o r analytical-scale (microgram level) separations, prepacked 3.9-mm X 30-cm columns (Waters Associates), packed with 10-μπι silica particles bonded with octadecylsilane for reverse-phase separations or with 10-μπι silica particles for normal-phase separations, were used. Mass spectral (MS) determinations were performed on a Kratos M S 80 high-resolution mass spectrometer as described previously (3,13). Sample Description. Finished drinking water I (DWI) was from a city that draws its raw water f r o m a river polluted b y chemicals f r o m numerous industrial, municipal, and agricultural sources. T o prepare finished drinking water, the raw water is treated b y a series of settling, coagulation, and flocculation steps, and the final product is chlorinated to a residual level of 1-2 m g / L . Finished drinking water II (DWII) was f r o m a city that draws its raw water f r o m a network of streams and rivers that principally drain wilderness regions. The raw water is settled in a series of reservoirs and then chlorinated to a residual level of 2-4 m g / L for distribution. Finished drinking water III (DWIII) was f r o m a city that draws raw ground water f r o m a major U . S . aquifer system. The raw water is treated b y p H adjustment to > p H 10 with lime to remove minerals, followed b y a series of settling steps. The final product is chlorinated to a free chlorine residual of 1-2 m g / L , a process that lowers the p H to 9.0. Because this aquifer is recharged in part b y stream bank infiltration f r o m a river subject to multiple points of contamination f r o m industrial, municipal, and agricultural sources, residue organics were isolated f r o m both the river water (RW) and the raw ground water (GW) for mutagenesis-separation assessment. In addition to these drinking waters and raw waters, both influent waste water (IWW) and effluent waste water ( E W W ) f r o m a municipal sewage treatment plant, heavily polluted (>803> b y volume) b y in dustrial discharges, were examined. Reagents. Organic solvents for H P L C separations—methylene chloride, methanol, isopropyl alcohol, hexane, and acetonitrile—were obtained as H P L C grade f r o m Fisher Scientific. T y p e I water for H P L C and for the preparation of other aqueous solutions was purified as described previously (7). A l l H P L C solvents were filtered through a 0.45-μπι Millipore membrane filter (Millipore Corporation) and degassed


19.

TABOR A N D LOPER

Mutagen Isolation Methods

405


by 15 m i n of sonication while under reduced pressure immediately prior to use. Sample-enrichment purification cartridges, S E P - P A K S (Waters Associates), packed with silica particles bonded with octadecylsilane, were activated and used according to methods described previously (6, 7, 9) for the processing of H P L C fractions for mutagenesis testing. A l l other chemicals were of reagent grade and were used without further purification. Isolation of Residue Organics f r o m Waters via X A D Chromatography. Residue organics were isolated f r o m the water samples via X A D chromatographic procedures developed in our laboratory. Drinking water and ground water samples were processed via the X A D procedure described in publications (3, 9, 20, 22, 22) and detailed in the Interim Protocol developed for the U S E P A (5). Waste water samples were processed via a modification of the X A D procedure (4). Biological Analysis. Tester strains T A 9 8 and TA100 for the Salmonella microsomal mutagenicity tests were provided b y B. Ames. Characteristic properties of the bacterial tester strains were verified for each fresh stock, and their mutagenicity properties were verified again by using positive and negative controls as part of each experiment, as recommended (23). Mutagenesis tests requiring metabolic activation used polychlorinated biphenyl mixture Aroclor 1254, induced rat liver 9000 X g supernatant fraction, S9 (Litton Bionetics). Mutagenesis assays, without (— S9) and with (+S9) metabolic activation, were conducted as described previously (3, 7, 20, 24). T h e detection of mutagenic activity in experimental samples was based upon a dosedependent response exceeding the zero-dose, spontaneous control value by at least twofold; that is, the ratio of total revertant colonies per plate to spontaneous colonies per plate was >2. In some situations involving H P L C subfractions in which the amount of sample was limiting, semiquantitative determinations of mutagenesis were made as described previously (3, 10). A l l recoveries of bioactivity from concentrated or fractionated residue organic samples were based upon an expression of mutagenesis per liter equivalent, representative of the original water sample. T y p i c a l mean revertant colony counts, ± standard error, obtained f r o m spontaneous plates and positive control plates from our laboratory for the time period of the experiments described herein, were reported recently (3, 21). H P L C Separations. SAMPLE PREPARATION FOR INJECTION. Isolated residue organics were dissolved in water/acetonitrile solvent mixtures for reverse-phase H P L C separations as follows: sample was dissolved in a m i n i m u m volume of acetonitrile and diluted with water until


406


a volume equal to the acetonitrile was added or until the solution became cloudy; in the latter case, additional acetonitrile was added, about 10$ of the total volume or until the solution became clear again. For normal-phase H P L C separations, residue organics were dissolved in methylene chloride/hexane solvent mixtures as follows: sample was dis solved in methylene chloride, minimum volume, then diluted with an equal volume of hexane b y using the same procedure and criteria fol lowed for the acetonitrile/water sample solutions.


ANALYTICAL-SCALE SEPARATIONS.

A p r e l i m i n a r y analytical reverse-phase H P L C separation of an aliquot of the residue organics was conducted to characterize the sample of residue organics according to the polarity of the components. The separation was accomplished by injecting 25 μ ί of a l-μg/μL· solution of the residue organics into the H P L C unit operating under the following conditions: flow rate, 2.0 m L / m i n ; initial mobile phase, water. F o l l o w i n g sample injection, the column was washed with water for 5 m i n or until no more 254-nm absorbing components were eluted; that is, there were no peaks >5% of full-scale absorbance setting, usually 0.5 or 1.0 absorbance units full scale. After the water wash, a linear mobile-phase gradient of 45-min duration was initiated f r o m 100$ water to 100$ acetonitrile. O n comple tion of the gradient, the column was washed with acetonitrile for 10 min or until no more U V - a b s o r b i n g components were eluted. In general, results f r o m this H P L C separation indicate the composition of the sample of residue organics. F o r example, if the majority, >75$, of the sample components eluted at a solvent composition of 80$ water:20$ acetonitrile or later, the sample w o u l d be ready for separation via preparative-scale reverse-phase H P L C to collect fractions for mutagenesis testing. However, if the majority, >75$, of the sample eluted before the 80$ water:20$ acetonitrile solvent composition, then the sample w o u l d be examined v i a an analytical-scale normal-phase H P L C separation prior to scale-up to the preparative level. T o run a preliminary analytical-scale normal-phase H P L C separa tion, 25 μ ί of a l-μg/μL solution of residue organics was injected into the H P L C unit fitted with a normal-phase column. The column was washed with hexane flowing at 2.0 m L / m i n for 5 m i n or until no more U V - a b s o r b i n g components were eluted. F o l l o w i n g the hexane wash, a linear gradient of 30-min duration was initiated from 100$ hexane to 100$ methylene chloride. O n completion of the gradient, the column was washed with methylene chloride until no more UV-absorbing components were eluted. F o l l o w i n g this wash, a linear mobile-phase gradient of 30-min duration was initiated from 100$ methylene chloride to 100$ isopropyl alcohol. O n completion of this second gradient, the column was washed with isopropyl alcohol for 10 m i n or until no more


19. TABOR A N D LOPER


407

UV-absorbing components were eluted. T h e results of this H P L C separation identified those samples most suited for preparative-scale normal-phase separation into subfractions for mutagenesis testing.


INITIAL PREPARATIVE-SCALE

SEPARATIONS.

H P L C O n the basis of the results of the analytical scale H P L C separation, one of the following preparative-scale H P L C separations was used for the initial fractionation of the complex mixture of residue organics for mutagenesis testing. F o r these separations, mobile-phase f l o w rates of 2.0 m L / m i n were used, and sample sizes f r o m 40 to 80 m g were loaded per fractionation run. E a c h preparative-scale run involved the collection of a series of fractions, each of w h i c h contained components eluted with a specific combination of isocratic and linear-gradient mobile-phase compositions. In each case, the respective fraction for each mobilephase composition was collected until no more U V - a b s o r b i n g components were eluted, then the mobile phase composition was changed along with the collection vessel for the collection of the next fraction.

The initial preparative-scale reverse-phase H P L C fractionation i n volved the collection of the following five fractions based on mobilephase composition: Fraction A , 100$ water; Fraction B, a 5-min linear gradient f r o m 100$ water to 75$ water:25$ acetonitrile, then isocratic until no more U V - a b s o r b i n g components are eluted; Fraction C , a 5-min linear gradient to 50$ water:50$ acetonitrile, then isocratic until no more U V - a b s o r b i n g components are eluted; Fraction D , a 5-min linear gradient to 25$ water:75$ acetonitrile, then isocratic as before; and Fraction E , a 5-min linear gradient to 100$ acetonitrile, then isocratic until no more U V - a b s o r b i n g components are eluted. T h e fractions were processed for mutagenesis testing and/or further separation according to the following procedures: T h e aqueous solution fraction, Fraction A , was concentrated via gentle evaporation under a stream of dry nitrogen according to procedures described previously (4). Fractions B - E were processed v i a our previously published procedure employing reversephase S E P - P A K cartridges (6, 7, 9). In this procedure, H P L C subfractions B - E were diluted with two volumes of water, and this solution was passed through a C18 S E P - P A K , previously activated as follows: Slowly pass 10 m L of acetonitrile through the S E P - P A K according to the instructions f r o m the manufacturer; the S E P - P A K then is washed with 20 m L of T y p e I water. Following the slow passage of the H P L C subfraction through the S E P - P A K , 5 m L of air is gently passed through the S E P - P A K to remove the residual solvent. T h e residue organics are eluted f r o m the S E P - P A K by the slow passage of 5 m L of methylene chloride through the cartridge. T h e volumes of the methylene chloride concentrates are recorded, and these samples are stored i n Teflon-capped amber vials



408


at —20 °C until mutagenic testing. These solutions were then processed as described in the following paragraphs for the preparation of dimethyl sulfoxide ( D M S O ) solutions for bioassay. F o r preparative-scale normal-phase H P L C separations, the following fractions were collected: Fraction A , isocratic hexane wash until no more U V - a b s o r b i n g components eluted; Fraction B, a 5-min linear gradient f r o m 100$ hexane to 65$ hexane:35$ methylene chloride, then isocratic until no more U V - a b s o r b i n g components are eluted; Fraction C , a 5-min linear gradient from 65$ hexane:35$ methylene chloride to 35$ hexane:65$ methylene chloride, then isocratic until no more U V - a b sorbing components are eluted; Fraction D , a 5-min linear gradient to 100$ methylene chloride, then isocratic until no more UV-absorbing components are eluted; Fraction E , a 5-min linear gradient from 100$ methylene chloride to 65$ methylene chloride:35$ isopropyl alcohol, then isocratic until no more U V - a b s o r b i n g components are eluted; Fraction F , a 5-min linear gradient from the previous solvent to 35$ methylene chloride:65$ isopropyl alcohol, then isocratic until no more U V - a b s o r b i n g components are eluted; and Fraction G , a 5-min linear gradient f r o m the previous solvent to 100$ isopropyl alcohol, then isocratic until no more U V - a b s o r b i n g components are eluted. These fractions were processed for mutagenesis testing and/or further H P L C separations b y gentle evaporation under a stream of nitrogen (4, JO), described as follows. The volume of each H P L C subfraction was reduced b y using a micro Snyder apparatus. Usually a measured aliquot of an extract is concentrated at one time, rather than concentrating the whole extract. The sample is gently heated b y using an N - E v a p bath (Organomation). As the solution was concentrated, some constituents came out of the solution. In those cases, a small volume of acetone was added to keep the components in solution until sufficient evaporation had occurred to azeotrope the remaining H P L C solvent f r o m the subfraction. Usually three to four additions of acetone were required. The final volumes of the acetone concentrates were recorded, and these samples were stored in Teflon-capped amber vials at —20 °C until mutagenesis testing. A t the time of bioassay, an aliquot of the residue solution was removed from the sample vial; typically, this aliquot was adjusted to the necessary bioassay volume with D M S O .

ADDITIONAL PREPARATIVE-SCALE

SEPARATIONS.

H P L C After mutagenesis assessment of the H P L C fractions f r o m the initial preparative-scale separation just discussed, those fractions containing mutagenic constituents are further separated on H P L C by employing the following strategy: F o r example, if the mutagenic constituents were found to be in Fraction D f r o m an initial reverse-phase H P L C preparative-scale separation, that is, a mobile-phase composition of 25$ water:75$ acetonitrile, a



19.

TABOR A N D LOPER

Mutagen Isofotion Methods

409

solution of the bioactive fraction would be injected into the H P L C unit. The separation w o u l d be accomplished on a reverse-phase column b y using an initial mobile phase of 40% water:60$ acetonitrile. Following a wash of 5 m i n , or until no more U V - a b s o r b i n g constituents are eluted (collected as Fraction D - l ) , a linear gradient of 20-min duration to a mobile-phase composition of 20$ water:80$ acetonitrile w o u l d be run. During this gradient, fractions w o u l d be collected at 5-min intervals, that is, four fractions, Fractions D - 2 through D - 5 . A t the completion of the gradient, the collection vessel w o u l d be changed for the collection of Fraction D - 6 , and the column w o u l d be washed isocratically with the latter mobile phase until no more U V - a b s o r b i n g constituents eluted or for a time period of 10 m i n . The six fractions, D - l through D - 6 , w o u l d be processed for this hypothetical case via the S E P - P A K procedure (6, 7, 9) for mutagenesis testing as described earlier. The same general approach w o u l d apply for an additional H P L C separation of mutagenic fractions from either initial normal or reverse-phase H P L C preparativescale separations. If further fractionation of mutagenic subfractions is required to isolate constituents f r o m fractions collected during the second separation, a similar H P L C strategy may be employed (7).

Results and Discussion The purpose of this chapter is to present a general approach for the isolation of mutagenic constituents f r o m residue organics of aqueous environmental samples. The isolated mutagens then can be characterized chemically as to their identity (14) or characterized biologically-biochemically as to their mechanism of mutagenesis (25, 26) or other properties. The overall strategy for this approach to isolation is outlined in Figure 1. This strategy of a coupled bioassay-chemical fractionation procedure was proposed b y Loper and Lang (27), based largely upon their mutagenicity and carcinogenicity studies of residue organics isolated b y K o p f l e r et al. (28) as part of a U S E P A five-city study (24) of drinking water organics. Originally, the procedure was developed b y using reverse-phase H P L C as a method for the chemical fractionation of the complex mixture of residue organics (7, 9). However, it was found subsequently (JO) that residue organics f r o m some drinking water samples w o u l d require a combination of reversephase and normal-phase H P L C separations to isolate mutagens for c o m p o u n d identification. Therefore, the more general approach described herein was developed. Mutagenic Activity of Residue Organics from a Variety of Aqueous Samples. Samples of residue organics from differing aqueous environmental sources have been found to contain a w i d e variation not only in



410


the total amount of mutagenic activity per liter equivalent of water but also in tester strain specificity and requirements for metabolic activation (8, 13, 28). Results of the mutagenic activities for residue organics from a variety of aqueous samples are summarized in Table I. In general, from these data and those reported b y others, mutagenic activity of residue organics on a per liter equivalent basis was found to be the greatest for waste waters f r o m industrially contaminated municipal sewage treatment plants (e.g., I W W and E W W , Table I) (4, 29, 30), whereas the lowest mutagenic activity appears to be associated with waters such as raw ground waters, drinking waters f r o m ground sources (e.g., G W , Table I) (3), settled river water as a drinking water source sampled prior to disinfection (22), and domestically contaminated municipal treatment plant waste waters prior to disinfection [(32); Tabor and Loper, unpublished]. These data suggest a w i d e variation in the chemical constituents of the residue organics f r o m different waters. Preliminary analytical-scale H P L C separations of the residue organics are required to assess the distribution of organic constituents and are useful in planning preparative-scale H P L C separations for mutagenic compound isolation. A n a l y t i c a l - S c a l e H P L C Separations. Reverse-phase H P L C chromatography favors the distribution of the semi- and nonpolar constituents of a sample of residue organics, whereas normal-phase H P L C chromatography favors the distribution of semipolar constituents (32). This approach is illustrated in Figure 2 b y the chromatograms of residue organics f r o m a waste water sample separated b y both reverse-

Table I. Mutagenic Activity for Residue Organics Isolated from a Variety of Aqueous Environmental Samples Net Revertants per Liter Equivalent

0

TA98 Sample

-S9

Drinking Water I (DWI) Drinking Water II (DWII) Drinking Water III (DWIII) Ground Water (GW) River Water (RW) Influent Waste Water (IWW) Effluent Waste Water (EWW)

93 124 56 39 53 90 2800

TA100 +S9 36 48 30

—

67 12000 3800

-S9

+S9

328 218 184

149

— — — —

— — — — — —

N O T E : — indicates the response was not equal to or greater than 2X the spontaneous rate. ° Net revertants = total revertants — spontaneous revertants, i.e., controls. Representative average spontaneous rates ± standard deviation (n > 15) over the time period for these experiments were TA98 - S9,15 ± 3; TA98 + S 9 , 2 8 ± 6; TA100 - S9,122 ± 10; TA100 + S9,126 ± 14.


TABOR AND LOPER

Mutagen Isofation Methods

411


19.

TIME(min)(
Ζ Η Ζ

G Η

r

r

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58

Ο

h—

Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986. / Plate

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Figure 5. Direct-acting mutagenic activity of HPLC fractions (1-4, Figure 4) obtained by sequential step elution of residue organics isolated from finished drinking water I. "MIX" indicates the mutagenic activity measure for an aliquot of residue organics proportionately reconstituted from the separate fractions; "SUM" is the arithmetic sum of the net revertant colonies for each dose for the four fractions.

Liter Equivalents

TA98


£ 0 1


416


one generally finds the bulk of the mutagenic activity of a given mixture of residue organics to have eluted i n one or possibly two of the broad fractional cuts. Therefore, the next and subsequent preparative-scale H P L C separations (Steps II, III, etc.) of the active fraction(s) f r o m Step I w i l l involve one of two approaches. T h e first approach uses the same chromatographic mode, that is, reverse phase or normal phase, used in Step I; the second approach involves the alternate chromatographic mode. In either case, the general strategy of H P L C fractionation followed b y bioassay of an aliquot of the collected fractions is applicable (Figure 1). The application of the same preparative chromatographic mode for Step II as employed i n Step I was described b y Tabor and L o p e r (7) for the isolation of a TA100 promutagen f r o m drinking water residue organics. T h e p r o m u t a g e n subsequently was i d e n t i f i e d as 3-(2chloroethoxy)-l,2-dichloropropene (14). In the first step, bioactivity was isolated i n the 50:50 water:acetonitrile fraction. T h e Step II H P L C fractionation was accomplished b y a shallow linear solvent gradient that started at a solvent composition 5$ less in acetonitrile, that is, the strong solvent, and went to a composition 20$ greater in strong solvent than the isocratic solvent composition used for elution of the mutagenic fraction i n Step I. (NOTE: A shallow linear gradient is defined as a 1$ solvent composition change per minute at a mobile-phase f l o w rate of approximately one column volume per minute.) This approach should be generally applicable to the separation of bioactive fractions f r o m Step I that originally eluted at solvent compositions >20$ strong solvent. The application of the second approach, use of a chromatographic mode for Step II alternate to that used in Step I, is illustrated b y the isolation of an S9-dependent mutagenic subfraction f r o m residue organics isolated f r o m an effluent waste water sample, E W W i n Table I. In this isolation, Step I involved the separation of the residue organics via normal-phase preparative H P L C . T h e resulting chromatogram for this separation is shown i n Figure 6. Bioassay of aliquots of the collected fractions showed that >80$ of the S9-dependent T A 9 8 mutagenesis had been eluted b y 100$ methylene chloride in Fraction 5, Figure 6, and no activity for T A 9 8 was detected in the later eluting components. Because this mutagenic fraction eluted in a semipolar region, preparative-scale reverse-phase H P L C was applied for the Step II separation. T h e separation involved a series of isocratic and linear gradient elutions using combinations of water and acetonitrile, as shown i n Figure 7. Bioassay of the collected fractions showed that >90$ of the S9-dependent T A 9 8 mutagenesis h a d b e e n e l u t e d v i a an i s o c r a t i c w a s h of 50:50 water:acetonitrile into Fraction 4, Figure 7. Identification of Mutagens i n Waste W a t e r — E W W . Attempts to further separate this fraction into discrete components v i a H P L C were Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986.

19.

TABOR A N D LOPER

417


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Mutagen Isolation Methods - ACS Publications - American Chemical

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