Screening Botanical Extracts for Quinoid Metabolites - ACS Publications

Benjamin M. Johnson, Judy L. Bolton, and Richard B. van Breemen*. Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,. Universit...
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Screening Botanical Extracts for Quinoid Metabolites Benjamin M. Johnson, Judy L. Bolton, and Richard B. van Breemen* Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231 Received June 25, 2001

Botanical dietary supplements represent a significant share of the growing market for alternative medicine in the USA, where current regulations do not require assessment of their safety. To help ensure the safety of such products, an in vitro assay using pulsed ultrafiltration and LC-MS-MS has been developed to screen botanical extracts for the formation of electrophilic and potentially toxic quinoid species upon bioactivation by hepatic cytochromes P450. Rat liver microsomes were trapped in a flow-through chamber by an ultrafiltration membrane, and samples containing botanical extracts, GSH and NADP(H), were flow-injected into the chamber. Botanical compounds that were metabolized to reactive intermediates formed stable GSH adducts mimicking a common in vivo detoxification pathway. If present in the ultrafiltrate, GSH conjugates were detected using LC-MS-MS with precursor ion scanning followed by additional characterization using product ion scanning and comparison to standard compounds. As expected, no GSH adducts of reactive metabolites were found in extracts of Trifolium pratense L. (red clover), which are under investigation as botanical dietary supplements for the management of menopause. However, extracts of Sassafras albidum (Nutt.) Nees (sassafras), Symphytum officinale L. (comfrey), and Rosmarinus officinalis L. (rosemary), all of which are known to contain compounds that are either carcinogenic or toxic to mammals, produced GSH adducts during this screening assay. Several compounds that formed GSH conjugates including novel metabolites of rosmarinic acid were identified using database searching and additional LC-MS-MS studies. This assay should be useful as a preliminary toxicity screen during the development of botanical dietary supplements. A positive test suggests that additional toxicological studies are warranted before human consumption of a botanical product.

Introduction Cytochromes P450 constitute an important family of metabolic enzymes that carry out a variety of Phase I reactions with broad substrate specificity. Xenobiotic substrates for these enzymes are usually converted to metabolites that are more hydrophilic than their precursors, allowing them to distribute preferentially in the blood and be excreted in the urine. However, some xenobiotics including botanical compounds might be activated by P450 to short-lived electrophilic intermediates capable of alkylating cellular biomolecules or participating in redox cycling reactions and causing cellular damage. For example, a botanical electrophile might contain a para-alkoxyl- or ortho-hydroxy-substituted phenol that could be metabolized to a quinone methide or ortho-quinone, respectively. An important human defense against such reactive intermediates is the nucleophilic tripeptide GSH, which binds covalently to electrophiles to form stable hydrophilic conjugates. Enzymatic and nonenzymatic conjugation between electrophilic metabolites and GSH is common in the liver, where the concentration of glutathione is approximately 10 mM (1). However, liver cells are often slow to respond to depletions of the GSH supply, and a large quantity of electrophilic material can overwhelm defense mechanisms and cause damage (2). * To whom correspondence should be addressed. Phone: (312) 996-9353. Fax: (312) 996-7107. E-mail: [email protected].

Consumer demand for dietary supplements is growing in the United States of America, where the market for botanical dietary supplements (herbal medicines) in particular now exceeds $5 billion/year (3). Herbal supplements are often perceived to be safer than drugs because of their “natural” origin, long-standing ethnomedical use, and over-the-counter availability. However, crude plant extracts typically consist of a complex mixture of compounds and might contain high concentrations of specific compounds recovered from a plant during processing. Given the molecular diversity of botanicals, it is possible that toxic compounds might be extracted and concentrated during the preparation of botanical dietary supplements. Another cause for concern is the possibility that human metabolism might activate some of these compounds to form reactive and potentially toxic products. Few botanical dietary supplements have been screened for toxicity because safety testing is not required in the United States and animal and human safety studies are time-consuming and expensive. Pulsed ultrafiltration-mass spectrometry is a drug discovery technique invented in our laboratory for the affinity selection and mass spectrometric identification of small molecules that interact with a macromolecular receptor (4). We demonstrated the use of this technology in the early stages of drug development by rapidly screening for metabolites and metabolic stability (5) and by screening drugs for metabolic activation to electrophilic intermediates (6). Here we report the use of pulsed

10.1021/tx010106n CCC: $20.00 © 2001 American Chemical Society Published on Web 10/18/2001

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Figure 2. Scheme for metabolism and toxicity screening using pulsed ultrafiltration and LC-MS-MS.

Figure 1. Structures of precursor compounds in botanical extracts that formed electrophilic metabolites during pulsed ultrafiltration LC-MS-MS screening.

ultrafiltration-mass spectrometry to screen botanical extracts for electrophilic compounds and activated metabolites. This assay complements the Ames test by screening complex botanical extracts for the formation of reactive mammalian metabolites and characterizing them using HPLC and tandem mass spectrometry. Also, this new assay provides a rapid in vitro toxicity screen to determine whether botanical dietary supplements should be tested for safety in animals and humans. The Ames test is a widely used in vitro assay to assess the possible mutagenicity of chemicals (7), in which a defective strain of Salmonella typhimurium will proliferate only if exposed to mutagenic material that helps reactivate a gene necessary for histidine synthesis. This assay can be modified to screen for mutagenic metabolites by including liver enzymes in the nutrient broth, allowing positive test results to be obtained for metabolically activated natural products such as aflatoxin and safrole (this and other relevant compounds are shown in Figure 1). However, the Ames test is an imperfect model for human toxicity because it uses prokaryotes instead of mammalian cells and tests for mutagenicity instead of cytotoxicity. Additionally, the Ames test provides no information regarding the identity of mutagenic compounds when it is used to screen mixtures such as metabolite preparations or botanical extracts. Therefore, a more selective method to predict the toxicity of such mixtures might be a useful compliment to the Ames test.

Materials and Methods Caution. The essential oil of Sassafras albidum (Nutt.) Nees (sassafras) contains a high concentration of safrole, a known carcinogen. Symphytum officinale L. (comfrey) contains toxic pyrrolizidine alkaloids. Gloves should be worn while handling these materials, and sample preparation should be carried out in a fume hood. Solutions containing rat liver microsomes are considered biohazards and should be disposed of according to appropriate guidelines. Extraction of Plant Material. Symphytum officinale L. (comfrey) was grown at the Field Station of the UIC Program for Collaborative Research in the Pharmaceutical Sciences in Downers Grove, IL. The entire plant was harvested in early October and was dried and ground. Leaves of Rosmarinus officinalis L. (rosemary) were purchased at a local supermarket. The extract of Trifolium pratense L. (red clover) was produced by PureWorld Botanicals, Inc. (South Hackensack, NJ) and standardized to a total isoflavone content of 17%.

Symphytum officinale and R. officinalis were extracted as follows. Dry, ground plant material (15 g) was suspended in 150 mL of methanol and sonicated for 30 min. After filtration, the supernatant was evaporated to near-dryness on a rotary evaporator. The extracts were stored under nitrogen at -20 °C and analyzed within 1 week. Pulsed Ultrafiltration and LC-MS-MS. A schematic of the pulsed ultrafiltration process is shown in Figure 2. The ultrafiltration chamber (1.1 mL internal volume) was constructed in-house as described previously (5), and the methylcellulose ultrafiltration membrane had a molecular weight cutoff of 30 000 Da. Dexamethasone-induced male Sprague-Dawley rat liver microsomes were diluted in 100 mM phosphate buffer at pH 7.4 prior to injection into the chamber. Fresh 100 µM phosphate buffer (pH 7.4) was pumped at 100 µL/min through the chamber fitted with the ultrafiltration membrane and stir bar. Rat liver microsomes were injected into the chamber to obtain a concentration of 1 mg/mL microsomal protein and washed for 30 min at room temperature. Fresh 10 mM stock solutions of GSH and NADP(H) in degassed water were prepared ahead of time and stored at -20 °C until immediately prior to use. Each stock solution of S. officinale, R. officinalis, and T. pratense extract was prepared by dissolving 20 mg of the extract in 500 µL of water. For each assay, 50 µL of a botanical extract stock solution was mixed with 50 µL of 10 mM GSH and 100 µL of 10 mM NADP(H) and flow injected into the ultrafiltration chamber. The ultrafiltrate was then collected over 30 min while the chamber was washed with buffer at 100 µL/min. The quantities of the samples in the ultrafiltrates were sufficient for multiple LC-MS-MS experiments. All samples were analyzed within 24 h. Chromatographic separations were carried out on 50 µL aliquots of each ultrafiltrate using a Waters (Milford, MA) Alliance 2690 HPLC system and XTerra MS C18 column (3.5 µm, 2.1 × 100 mm). The solvent system consisted of water containing 0.5% formic acid (solvent A) and methanol (solvent B) at 180 µL/min and a linear gradient as follows: 95% A for 5 min, during which the eluent containing buffer salts and unconjugated GSH were diverted to waste, and then 5-95% B over 25 min. Positive ion electrospray mass spectra were acquired using a Micromass (Manchester, U.K.) Quattro II triple quadrupole mass spectrometer. A precursor ion MS-MS scan was used to select HPLC peaks corresponding to GSH adducts. Specifically, collision induced dissociation (CID) was used to form the b1-type fragment ion of m/z 130 from the GSH moiety of conjugates, and precursor ion scanning was used during LCMS-MS to record signals indicating the m/z value of intact GSH adducts. When a GSH adduct was identified during precursor ion scanning, product ion MS-MS was used with CID to obtain structurally significant fragment ions of the metabolite for identification. The collision energy for all MS-MS experiments was 24 eV and the argon collision gas pressure was 1-3 µbar. The NAPRALERT database (8) was searched to obtain a list of all compounds previously isolated from each plant under study. Compounds with the appropriate molecular weights were

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Figure 3. Pulsed ultrafiltration LC-MS-MS screening of the essential oil of sassafras for electrophilic metabolites following activation by rat liver microsomes and trapping with GSH. Positive ion electrospray ionization, CID, and precursor ion scanning MS-MS were used for the selective detection of GSH adducts. (A) Control with the essential oil of sassafras and NADP(H) but without GSH. (B) Control with the essential oil of sassafras, GSH and NADP(H) but without rat liver microsomes. (C) Experiment containing the essential oil of sassafras, GSH, NADP(H), and rat liver microsomes. (D) Safrole standard with GSH, NADP(H), and microsomes. evaluated for structural moieties consistent with metabolic activation and conjugation with GSH. For structural confirmation, the retention times and tandem mass spectra of GSH adducts of standards of these compounds were then compared to those of unknowns obtained during screening.

Results and Discussion Extracts of four botanicals were investigated that have been used as dietary supplements, herbal remedies, flavoring agents, or spices. These included extracts of comfrey, rosemary, red clover, and the essential oil of sassafras. In addition to a history of human use, some of these botanicals were selected because they contain compounds known to be hepatotoxic or carcinogenic (comfrey, rosemary, and sassafras) or are believed to be nontoxic (red clover). Therefore, these botanicals provided both positive and negative controls to validate the screening assay. The leaves of the North American sassafras tree are dried and used to make tea and file powder, and the root bark was used formerly to flavor root beer. The essential oil is marketed as an aromatic and contains a high concentration of safrole (9). Two possible bioactivation pathways have been suggested for safrole. The first involves hydroxylation and sulfate conjugation at the benzyl carbon, followed by displacement of the sulfate group to produce a carbocation (10). The second entails O-dealkylation to yield hydroxychavicol, followed by oxidation to form an ortho-quinone or a quinone methide (11). Therefore, the essential oil of sassafras provided a suitable test sample for this assay. The pulsed ultrafiltration LC-MS-MS assays of the essential oil of sassafras and a safrole standard are shown in Figure 3. LC-MS-MS with precursor ion scanning of the ultrafiltrate following pulsed ultrafiltration with microsomes, GSH, and the essential oil of sassafras shows a peak eluting at 18.6 min with an apparent precursor ion of m/z 456 (Figure 3C). This peak corresponds to a GSH adduct of hydroxychavicol (see structure in Figure 1), which was confirmed using LC-MS-MS with

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product ion scanning of m/z 456 and by comparison to the GSH adduct of hydroxychavicol formed by microsomal activation of safrole (Figure 3D). The hydroxychavicolGSH conjugate was formed by O-dealkylation of safrole to hydroxychavicol, oxidation to the ortho-quinone, and then nucleophilic attack by GSH on the electrophilic sixmembered ring (6). For each experiment that produced positive results, two control analyses were carried out for additional characterization and validation. In one control, GSH was omitted from the incubation mixture in order to detect endogenous compounds or Phase I metabolites that coincidentally fragmented to form ions of m/z 130. The LC-MS-MS chromatogram of this control incubation for sassafras is shown in Figure 3A and shows that neither the essential oil of sassafras or its Phase I metabolites contained compounds that produced fragment ions of m/z 130. In the second control, microsomes were omitted to help determine whether GSH adducts were activated metabolically, via autoxidation, or through another nonenzymatic pathway. The LC-MS-MS chromatogram for this sassafras control is shown in Figure 3B. A peak of low intensity was detected eluting at 18.6 min. Since this analyte is much more abundant in the presence of microsomes (Figure 3C), the small amount of reactive compound detected in the control shown in Figure 3B was probably formed by autoxidation. The set of pulsed ultrafiltration-mass spectrometric assays shown in Figure 3 indicates that the essential oil of sassafras can be metabolically activated to form electrophilic intermediates that may be trapped as GSH adducts. In particular, hydroxychavicol-o-quinone was identified using this assay as a reactive metabolite of sassafras. Since conjugation with sulfate was not a part of the assay design, hydroxylated safrole did not form an unstable sulfate conjugate and then decompose to form a reactive carbocation (10). Therefore, the chromatograms shown in Figure 3 help to validate this pulsed ultrafiltration assay as an effective toxicity screen for the detection of reactive quinones but not carbocations. Symphytum officinale (comfrey) is a perennial plant that has been used ethnomedicinally to treat painful menstruation and bronchial problems (12) and to stimulate healing of external wounds (13) and broken bones (14). However, comfrey is known to contain pyrrolizidine alkaloids and is hepatotoxic (15) and carcinogenic in rats (16). The pulsed ultrafiltration LC-MS-MS assay for metabolic activation of a comfrey extract and formation of GSH adducts is shown in Figure 4. The precursor ion LC-MS-MS analysis of the ultrafiltrate showed a protonated GSH adduct of m/z 504 eluting at 11.8 min and two GSH adducts of m/z 666 eluting at 17.5 and 19.5 min (Figure 4C). The peaks corresponding to these ions were not detected in the control experiment omitting GSH (Figure 4A). However, the peaks eluting at 11.8, 17.5, and 19.5 min were detected in the control omitting microsomal protein (Figure 4B), but these peaks were of low intensity compared to the experiment containing GSH and microsomes (Figure 4C). A search of the NAPRALERT database suggested rosmarinic acid (see structure in Figure 1) as a possible match for the compound of mass 360 Da and producing the GSH adducts detected at m/z 666. Pulsed ultrafiltration LCMS-MS analysis of the authentic standard confirmed that the peaks eluting at 17.5 and 19.5 min are rosmarinic acid-GSH adduct regioisomers, and identified the analyte

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Figure 4. Pulsed ultrafiltration LC-MS-MS screening of a methanol extract of comfrey following metabolic activation by rat liver microsomes and trapping with GSH. Precursors of the GSH fragment ion of m/z 130 were detected using positive ion electrospray and MS-MS with CID. (A) Control without GSH. (B) Control without microsomes showing trace amounts of GSH adducts that were formed without metabolic activation. (C) Analysis of the activated comfrey extract indicating GSH conjugation to rosmarinic acid (m/z 666, 17.5, and 19.5 min), and to de-esterified rosmarinic acid (m/z 504, 11.7 min). Unidentified GSH adducts were detected eluting at 16.8 and 18.5 min.

Figure 5. (A) Positive ion product ion tandem mass spectrum with CID of the rosmarinic acid-GSH adduct of m/z 666 detected eluting at 19.6 min during LC-MS-MS. (B) Proposed fragmentation scheme for a GSH conjugate of rosmarinic acid.

of m/z 504 eluting at 11.8 min as a GSH adduct of deesterified rosmarinic acid. During subsequent LC-MS-MS product ion scanning of the GSH adducts detected in the comfrey ultrafiltrate, characteristic GSH peptide fragment ions were detected such as b1, b2, y2, and z2 (see fragmentation scheme in Figure 5). Although most fragmentation occurred at the amide linkages of the GSH peptide, product ion scanning also helped identify electrophilic metabolites in which the botanical components contained labile bonds. For example, the LC-MS-MS product ion analysis of m/z 666 from the ultrafiltrate of comfrey is shown in Figure 5A.

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Cleavage of the ester linkage of this rosmarinic acidGSH adduct with the positive charge remaining on the peptide moiety produced a product ion of m/z 504. The abundant fragment ion of m/z 375 was formed by ester cleavage of the y2 ion (Figure 5b). The base peak of m/z 163 resulted from ester cleavage with the charge remaining on the smaller fragment, which was probably stabilized by cyclization. These preliminary studies indicate that a total of at least three rosmarinic acid-GSH regioisomers might be formed following the bioactivation of rosmarinic acid by rat liver microsomes. MS-MS with CID of these adducts suggest that both catechol moieties of rosmarinic acid are capable of undergoing oxidation to ortho-quinones and then conjugation to GSH (not shown). The exact structures of these conjugates and the specific effects of rosmarinic acid activation in mammalian tissues are under investigation. Rosmarinic acid has been shown to have activity as an antioxidant and an inhibitor of cyclooxygenase (17, 18), but no electrophilic metabolites of this compound have been identified until now. Nakazawa and Ohsawa did not report GSH adducts or mercapturic acids in the urine of rats following administration of rosmarinic acid (19). However, it is possible that these species were overlooked since only 32% of the administered dose of rosmarinic acid was recovered in the form of other metabolites. This may have been due to the inability of the photodiode array to detect these metabolites at low concentrations. This example emphasizes the importance of careful sample handling and the use of a sensitive and selective detector in metabolism studies. In the present studies, GSH conjugates of rosmarinic acid metabolites were detected in the comfrey extract, and these adducts were probably the result of reaction of GSH with rosmarinic acid ortho-quinones. However, this pulsed ultrafiltration LC-MS-MS assay did not detect the pyrolizidine alkaloids that are probably the cause of the acute human toxicity of this plant (20). This result illustrates the selectivity of our screening assay since pyrrolizidine alkaloid toxicity does not result from metabolic activation to quinoid or epoxide intermediates. Rosmarinus officinalis (rosemary) is grown widely throughout Europe and the United States where its leaves are used as a flavoring agent. This plant was selected for study because it contains rosmarinic acid (discussed above). Rosemary also contains carnosic acid, carnosol (21), and eugenol (22) (see structures in Figure 1). Eugenol can be metabolized by P450 to a quinone methide (23) and has been shown to be toxic to rat liver hepatocytes with an LC50 of 200-300 µM (24). The results of the pulsed ultrafiltration LC-MS-MS screening for activated metabolites from the rosemary extract are shown in Figure 6. Several GSH adducts were detected including the expected derivatives of rosmarinic acid that eluted at 12.4, 17.7, 19.6, and 20.2 min, with signals corresponding to precursor ions of m/z 504, 666, 666, and 666, respectively. The peak eluting at 18.4 (m/z 456) corresponded to the hydroxychavicol-GSH adducts, formed by O-dealkylation of eugenol to give hydroxychavicol and subsequent oxidation to an ortho-quinone. This analyte was also observed in the ultrafiltration LC-MS-MS chromatogram of a eugenol standard (not shown). The chromatographic peak at 17.0 min (m/z 486) might have resulted from metabolism of either rosmarinic acid or eugenol, since this peak was observed in the ultrafiltra-

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Figure 6. Pulsed ultrafiltration LC-MS-MS screening for GSH adducts of reactive metabolites in a methanol extract of rosemary following activation by rat liver microsomes. (A) Control without GSH. (B) Control without microsomes. (C) Analysis of the ultrafiltrate of the activated rosemary extract indicating that GSH forms conjugates with the following compounds: eugenol (m/z 486, 17.0 min; m/z 456, 18.4 min), rosmarinic acid (m/z 504, 12.4 min; m/z 486, 18.5, and 19.4 min; m/z 666, 17.7, and 19.6 min), carnosol (m/z 636, 26.0 min), and carnosic acid (m/z 638, 26.7 min). Unidentified GSH adducts were detected eluting at 21.9 and 22.9 min.

tion LC-MS-MS chromatograms of both standards. One possible metabolic pathway involves GSH conjugation to the 1′-hydroxyeugenol metabolite observed previously (25). An additional metabolite of rosmarinic acid (m/z 486) was observed eluting at 19.4 min. Again, this metabolite was observed during analysis of a rosmarinic acid standard but is not a caffeic acid-GSH adduct. It is interesting to note that this screening assay did not detect the eugenol-GSH metabolite that formed following oxidation of eugenol to a quinone methide. This unexpected outcome occurred because this GSH conjugate did not form an abundant fragment ion of m/z 130 under the conditions used in this experiment (not shown). One explanation might be the possible presence of stabilizing hydrogen bonds between the hydroxyl groups on the aromatic ring and hydrogen bond acceptors on the GSH moiety. This phenomenon is not expected to occur for most GSH adducts, but it serves as a reminder that this screening assay will not detect all quinones. Furthermore, this assay is not intended to replace more rigorous toxicological studies. The peaks eluting at 21.9 min (m/z 652) and 22.9 min (m/z 471) in Figure 6 could not be identified based on the LC-MS-MS data and the NAPRALERT database. However, the species eluting at 26.0 min (m/z 636) and 26.7 min (m/z 638) were consistent with a carnosol-GSH adduct and a carnosic acid-GSH adduct, respectively. These compounds are similar in structure and have been reported to interconvert during sample preparation depending on the conditions used (26). It is interesting to note that the GSH adduct of carnosic acid was also detected in the control incubation with GSH but without microsomes (Figure 6B). It is unclear whether such compounds that undergo autoxidation are likely to be toxic, although they might be analogous to stable intermediates that are not likely to cause significant damage (27). As expected, no GSH adducts were detected in the control with no GSH (Figure 6A). As a food product, the leaves rosemary should pose no threat to human health if consumed in moderation, since small amounts of electrophilic quinones, if formed, could be deactivated by reaction with endogenous GSH. However, certain extraction methods might concentrate com-

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pounds such as rosmarinic acid or eugenol and increase the risk of toxicity of dietary supplements containing rosemary. Since our data indicate that a methanol extract of rosemary contains compounds that may be activated metabolically to redox active compounds or electrophilic species, we recommend that additional toxicological studies be carried out to establish whether dietary supplements containing extracts of rosemary are safe for human consumption. Extracts of T. pratense (red clover) are under investigation for their estrogenicity (28) and as alternatives for hormone replacement therapy in menopausal women (29). Several extracts of red clover were screened for metabolic activation using pulsed ultrafiltration LC-MSMS. Since these extracts are being evaluated in clinical trials for use as dietary supplements to help manage menopause in women, precursor ion scans were carried out over two different scan ranges for extra rigor. As in the analyses discussed above, the scan range m/z 406706 (for the first quadrupole) was used, but then a second LC-MS-MS analysis was carried out over the range m/z 706-1006. While it would have been possible to scan the entire mass range in a single experiment, doing so using a triple quadrupole mass spectrometer would have resulted in a 2-fold reduction in sensitivity. Since the two chromatograms showed no evidence of the presence of GSH adducts (not shown), there was no concern about the safety of these red clover extracts. These results demonstrate the feasibility of using pulsed ultrafiltration LC-MS-MS for the screening of botanical extracts and dietary supplements for compounds that might be activated by hepatic cytochromes P450 to electrophilic quinoid intermediates and reactive epoxides. The chemical constituents of the sample do not need to be known, and chemical diversity in the extract does not interfere with the identification of reactive metabolites since only GSH adducts are detected during the selective LC-MS-MS analysis using precursor ion scanning. After adducts are detected, LC-MS-MS with product ion scanning may be used for additional characterization to facilitate structure elucidation, and searching databases such as the NAPRALERT database facilitates the identification of the reactive metabolites and their botanical precursors. Therefore, with a minimum time and effort, botanical extracts may be screened using this approach for the formation of activated metabolites. If GSH adducts are detected in a particular preparation or extract, additional studies would be warranted to determine whether these metabolites are of concern to human health. Alternatively, additional pulsed ultrafiltration LC-MS-MS assays might be carried out to help guide the preparation of extracts containing either none or negligible amounts of the compound(s) of concern. Pulsed ultrafiltration LC-MS-MS screening for activated metabolites as described here has certain strengths and limitations regarding the types of reactive metabolites that are formed and trapped as GSH adducts. For example, quinones, quinone methides, quinone imines and reactive epoxides are readily detected using this assay (6). However, relatively unreactive epoxides such as styrene oxide might not form GSH adducts in a microsomal preparation without the addition of GSH S-transferases, which are more abundant in cytosol than in microsomes. Also, electrophilic metabolites formed by Phase I oxidation followed by Phase II conjugation and then decomposition to carbocations or nitrenium ions

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would not be identified using this assay. However, additional methods are under development in our laboratory to expand the range of reactive metabolites that can be detected. We anticipate that an array of in vitro screening assays such as this will soon be implemented, which will facilitate rapid and inexpensive prediction of the potential toxicity of prospective drugs and botanical dietary supplements.

Acknowledgment. This project was funded in part by Grant P50 AT00155 provided to the UIC/NIH Center for Botanical Dietary Supplements Research jointly by the Office of Dietary Supplements, the National Institute of General Medicine, the Office for Research on Women’s Health, and the National Center for Complementary and Alternative Medicine. The authors thank Dr. Norman R. Farnsworth, Nancy L. Booth, Daniel S. Fabricant, Spiro D. Garbis, and Aleksej Krunic for their helpful discussions and suggestions, Steven Totura for growing and harvesting S. officinale, and Dr. Dejan Nikolic and Dr. Chungang Gu for assistance with mass spectrometry.

References (1) Klassen, C. D. (1996) Casarett & Doull’s toxicology, the basic science of poisons, 5th ed., pp 177-183, McGraw-Hill, New York. (2) Thompson, D. C., Barhoumi, R., and Burghardt, R. C. (1998) Comparative toxicity of eugenol and its quinone methide metabolite in cultured liver cells using kinetic fluorescence bioassays. Toxicol. Appl. Pharmacol. 149, 55-63. (3) Eisenberg, D. M., Davis, R. B., Ettner, S. L., Appel, S., Wilkey, S., Van Rompay, M., and Kessler, R. C. (1998) Trends in alternative medicine use in the United States, 1990-1997: results of a follow-up national survey. J. Am. Med. Assoc. 280, 1569-1575. (4) van Breemen, R. B., Huang, C., Nikolic, D., Woodbury, C. P., and Venton, D. L. (1997) Pulsed ultrafiltration mass spectrometry: a new method for screening combinatorial libraries. Anal. Chem. 69, 2159-2164. (5) van Breemen, R. B., Nikolic, D., and Bolton, J. L. (1998) Metabolic screening using on-line ultrafiltration mass spectrometry. Drug Metab. Dispos. 26, 85-90. (6) Nikolic, D., Fan, P. W., Bolton, J. L., and van Breemen, R. B. (1999) Screening for xenobiotic electrophilic metabolites using pulsed ultrafiltration-mass spectrometry. Comb. Chem. High Throughput Screening 2, 165-175. (7) McCann, J., Spingarn, N., Kobori, J., and Ames, B. Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 979-983. (8) NAPRALERT, A database on natural products, Chemical Abstracts Services, Columbus, OH. (9) Kadeem, D. P., and Gage, D. A. (1995) Chemical composition of essential oil from the root bark of Sassafras albidum. Planta Med. 61, 574-575. (10) Boberg, E. W., Miller, E. C., Miller, J. A., Poland, A., and Liem, A. (1983) Strong evidence from studies with brachymorphic mice and pentachlorophenol that 1′-sulfoxysafrole is the major ultimate

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