Bioassay-Directed Identification of Estrogen Residues in Urine by

Urine Testing for Designer Steroids by Liquid Chromatography with Androgen ... and Electrospray Quadrupole Time-of-Flight Mass Spectrometry Identifica...
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Anal. Chem. 2004, 76, 6600-6608

Bioassay-Directed Identification of Estrogen Residues in Urine by Liquid Chromatography Electrospray Quadrupole Time-of-Flight Mass Spectrometry Michel W. F. Nielen,* Eric O. van Bennekom, Henri H. Heskamp, J. (Hans) A. van Rhijn, Toine F. H. Bovee, and L. (Ron) A. P. Hoogenboom

RIKILT Institute of Food Safety, P.O. Box 230, 6700 AE Wageningen, The Netherlands

A new approach to the search for residues of known and unknown estrogens in calf urine is presented. Following enzymatic deconjugation and solid-phase extraction, a minor part of the samples is screened for estrogen activity using a recently developed rapid reporter gene bioassay. The remainder of the bioactive extracts is analyzed by gradient liquid chromatography (LC) with, in parallel, bioactivity and mass spectrometric detection via effluent splitting toward a 96-well fraction collector and an electrospray quadrupole time-of-flight mass spectrometer (QTOFMS). The LC fractions in the 96-well plate are used for the detection of estrogen activity using the bioassay. The biogram obtained features a 20-s time resolution, and the suspect well numbers can be easily correlated with the LC/QTOFMS retention time. The mass spectral data from the thus assigned relevant parts of the chromatograms are background subtracted, followed by accurate mass measurement, element composition calculation, and identification. The method allows estrogen activity detection and identification of (un)known estrogens in urine at the 1-2 ng/L level, in compliance with current residue analysis performance for hormone abuse in cattle. The applicability of this LC/bioassay/QTOFMS approach for the identification of estrogens in real-life samples is demonstrated by the analysis of several calf urine samples, and preliminary data from a pig feed sample. The use of growth promoters for fattening purposes in cattle has been banned in the European Union (EU) since 1988.1 Interestingly, the ban prohibits all substances having hormonal action, rather then providing a black list of hormones. In contrast, residue analysis in urine, while aiming at consumer protection, fair trade, and enforcement of the ban, is still carried out on specific target compounds.2 So far, the control within the EU for illegal growth promoters in cattle and pigs revealed only a limited number of positives. Findings and analysis of illegal preparations, however, showed that steroids, natural hormones, and β-agonists * To whom correspondence should be addressed. (e-mail) michel.nielen@ wur.nl; (phone) +31 317 475615; (fax) +31 317 417717. (1) EC Council directive 96/22 (replacement of 88/146/EC). Off. J. Eur. Commun. 1996, L125, 3-9. (2) EC Council Directive 96/23. Off. J. Eur. Commun. 1996, L125, 10-32.

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are still being used.3 The targeted analysis methods for residues of steroids and β-agonists as used in the control programs are unable to detect very new or outdated compounds, which might be one of the possible explanations for the limited findings so far. The multianalyte screening ability of radioimmunoassay and enzyme immunoassay screening tools is dependent on the (limited) degree of cross-reactivity of the antibody used.4,5 The screening and confirmatory gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods are set up to the monitoring of a few selected ions or MS/MS transitions only.6,7 Even multiresidue LC/MS/MS methods will be at the end limited to a short list of a priori known hormone residues.8,9 Alternatively, receptor assays can be used to detect all compounds having affinity for a given receptor.10 This is particularly relevant in screening methods for compounds for which no detailed information is present with regard to the identity. Jungbauer and Beck described a yeast reporter bioassay for the rapid determination of estrogenic activity in environmental samples.11 In such a transcriptional activation assay, the estrogens bind to the human estrogen R-receptor, which dimerizes, travels into the nucleus, binds to the hormone response element, and initiates transcription of the lacZ reporter gene. Finally, the β-galactosidase activity is a measure of estrogenic activity. In contrast to competitive binding receptor assays, such functional activity bioassays do not show false negatives caused by signal attenuation from anti-estrogens. Recently we developed an even simpler yeast-based reporter gene bioassay for estrogens, featuring direct measurement of yeast enhanced green fluorescent protein (3) Courtheyn, D.; Le Bizec, B.; Brambilla, G.; De Brabander, H. F.; Cobbaert, E.; Van de Wiele, M.; Vercammen, J.; De Wasch, K. Anal. Chim. Acta 2002, 473, 71-82. (4) Haasnoot, W.; Kemmers-Voncken, A.; Samson, D. Analyst 2002, 127, 8792. (5) Vanoosthuyze, K. E. I.; Arts, C. J. M.; Van Peteghem, C. H. J. Agric. Food Chem. 1997, 45, 3129-3137. (6) O’Keeffe, M., Ed. Residue analysis in food, principles and applications; Harwood Academic Publishers: Amsterdam, 2000. (7) Cai, J.; Henion, J. J. Chromatogr., B 1997, 691, 357-370. (8) Van Poucke, C.; Van Peteghem, C. J. Chromatogr., B 2002, 772, 211-217. (9) Hewitt, S. A.; Kearney, M.; Currie, J. W.; Young, P. B.; Kennedy, D. G. Anal. Chim. Acta 2002, 473, 99-109. (10) Mueller, S. O. J. Chromatogr., B 2002, 777, 155-165. (11) Jungbauer, A.; Beck, V J. Chromatogr., B 2002, 777, 167-178. 10.1021/ac0490705 CCC: $27.50

© 2004 American Chemical Society Published on Web 10/21/2004

(yEGFP) for detection of estrogen activity.12,13 The only handling involved is the addition of the yeast suspension to the sample extract; after a 4- or 24-h waiting time, the fluorescence intensity of yEGFP is directly measured in a plate reader. Unfortunately, bioassay signals do not provide a clue for the chemical identity of the bioactive substance and consequently bioassay- or toxicity-directed concepts were developed in which mass spectrometric identification is combined with bioactivity screening. Particularly, in water analysis and drug discovery, experimental setups have been presented for off-line and on-line coupling of bioassays and MS.14-21 Although very elegant, the main disadvantage of on-line continuous-flow approaches is, apart from their inherent limitation to affinity principles in solution, the incompatibility of organic solvents used in flow injection analysis and LC with bioaffinity detection, nonspecific binding of receptor molecules to reaction coil surfaces, and some additional bandbroadening effects (LC). The solvent-incompatibility drawback can be partly compensated by postcolumn addition of solvent gradients, but at the cost of sensitivity and additional equipment. Offline approaches of combined bioactivity assessment and chemical identification have been successfully applied in water analysis at low and subnanogram per liter levels, for both toxic and estrogenic substances. The feasibility of chemical identification of unknowns in water has been demonstrated using quadrupole time-of-flight mass spectrometry (QTOFMS), providing high resolution and mass accuracy, which yield exact mass measurements and elemental composition calculations thereof.15-16 Very recently, the concept of combined bioassay detection and QTOFMS identification was highlighted by the successful identification of a formerly unknown illegal β-agonist in feed.22 Now we present a new concept for the identification of residues of known and unknown estrogens in calf urine, based on combined bioactivity screening of LC column effluents by a reporter gene bioassay and sensitive identification based upon LC/QTOFMS with exact mass measurement. This approach is unique in the field of hormone residue analysis and in our view the only way to comply with the ban on hormonal activity as laid down in current EU legislation.1 EXPERIMENTAL DETAILS Chemicals and Samples. Chemicals and solutions used were of analytical-reagent grade. Acetonitrile HPLC far-UV grade was (12) Bovee, T. F. H.; Helsdingen, J. R.; Koks, P. D.; Kuiper, H. A.; Hoogenboom, L. A. P.; Keijer, J. Gene 2004, 325, 187-200. (13) Bovee, T. F. H.; Helsdingen, J. R.; Rietjens, I. M. C. M.; Keijer, J.; Hoogenboom, L. A. P. J. Steroids Biochem. Mol. Biol., 2004, 91, 99-109. (14) Brenner-Weiss, G.; Obst, U. Anal. Bioanal. Chem. 2003, 377, 408-416. (15) Bobeldijk, I.; Vissers, J. P. C.; Kearney, G.; Major, H.; van Leerdam, J. A. J. Chromatogr., A 2001, 929, 63-74. (16) Bobeldijk, I.; Stoks, P. G. M.; Vissers, J. P. C.; Emke, E.; van Leerdam, J. A.; Muilwijk, B.; Berbee, R.; Noij, Th. H. M. J. Chromatogr., A 2002, 970, 167-181. (17) Farre´, M.; Klo ¨ter, G.; Petrovic, M.; Alonso, M. C.; Lopez de Alda, M. J.; Barcelo, D. Anal. Chim. Acta 2002, 456, 19-30. (18) Noij, Th. H. M.; Bobeldijk, I. Water Sci. Technol. 2003, 47, 181-188. (19) Hogenboom, A. C.; de Boer, A. R.; Derks, R. J. E.; Irth, H. Anal. Chem. 2001, 73, 3816-3823. (20) Schobel, U.; Frenay, M.; van Elswijk, D. A.; McAndrews, J. M.; Long, K. R.; Olson, L. M.; Bobzin, S. C.; Irth, H. J. Biomol. Screening 2001, 6, 291-303. (21) Derks, R. J. E.; Hogenboom, A. C.; van der Zwan, G.; Irth, H Anal. Chem. 2003, 75, 3376-3384. (22) Nielen, M. W. F.; Elliott, C. T.; Boyd, S. A.; Courtheyn, D.; Essers, M. L.; Hooijerink, H.; van Bennekom, E. O.; Fuchs, R. E. M Rapid Commun. Mass Spectrom. 2003, 17, 1633-1641.

Figure 1. Generic setup for the fractionation and identification of unknown bioactive substances using LC/bioassay/QTOFMS(/MS).

from Lab-Scan Ltd. (Dublin, Ireland). Water was purified using a Milli-Q Gradient A10 system (Millipore, Bedford, MA). 2-Hydroxyestradiol and 2-hydroxyestrone were obtained from Steraloids (Newport, RI) and taleranol and zeranol, from the Community Reference Laboratory RIVM (Bilthoven, The Netherlands). Estrone-3-sulfate was from Research Plus (Bayonne, NJ) and transresveratrol, cis-resveratrol, phloretin, trans-diethylstilbestrol, hexestrol, cis-diethylstilbestrol, and estradiol-17-valerate were from ICN (Irvine, CA) Estradiol-17-glucuronide, estradiol-3-sulfate, estriol, daidzein, genistein, naringenin, β-zearalenol, 17β-estradiol, R-zearalenol, 17R-estradiol, ethinylestradiol, zearalanone, zearalenone, dienestrol, estradiol-17-enanthate, and estradiol-17-cypionate were obtained from Sigma (St. Louis, MO). Enterodiol, coumestrol, equol, enterolactone, biochanin B, and 2,6-di-tert-butyl-p-cresol were from Fluka (Buchs, Switzerland). Bisphenol A was from Aldrich (Milwaukee, WI) and equilenin, equilin, and estradiol-17propionate were from Serva (Heidelberg, Germany). A technical mixture of 4-nonylphenol, and bisphenol B were from laboratory stock. Biochanin A was from Brunschwig (Amsterdam, The Netherlands). β-Glucuronidase/arylsulfatase (from suc Helix pomatia) was from Merck (Darmstadt, Germany). Isolute NH2 (100 mg) extraction columns were from IST (Hengoed, U.K.), and Bond Elut C18 (500 mg) solid-phase extraction columns were from Varian (Harbor City, CA). Over 100 calf urine samples were sampled from the bladder at four different slaugtherhouses. In addition, suspect urines and a pig feed sample from a control program were analyzed. Apparatus. The experimental setup for the search for (un)known estrogenic substances consisted of a gradient LC, an autosampler, a 96-well fraction collection system, a yeast-based reporter gene assay, and an electrospray ionization (ESI) QTOF tandem mass spectrometer, cf. Figure 1. The LC system consisted of two Knauer (Berlin, Germany) model WellChrom K-1001 pumps, a Knauer high-pressure dynamic mixing chamber, a GasTorr model 154 membrane degasser, and a Spark Holland (Emmen, The Netherlands) model Endurance autosampler. Liquid chromatography was performed using a Waters (Milford, MA) 150 × 3.0 mm i.d. Symmetry column packed with 5-µm C18 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

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material and a mobile phase consisting of (A) water/acetonitrile (90:10) and (B) water/acetonitrile (10:90). Gradient elution was performed at a flow rate of 0.4 mL/min, starting at 35% B and linearly programmed to 100% B in 20 min. The column effluent was split 3:1 toward a Gilson (Villiers-le-Bel, France) model FC203B 96-well fraction collector and a Micromass (Manchester, U.K.) model QTOFmicro MS system equipped with a dual ESI probe and operated in the negative ion mode at a resolution of 4500 (fwhm), source temperature 120 °C, desolvation temperature 250 °C, and cone voltage of 30 V. The second LockSpray ESI probe provided an independent flow of the lock mass calibrant 17β-estradiol glucuronide diluted in acetonitrile/water, at 5 µL/min. Data were acquired in the continuum mode from 80 to 500 Da with a scan time of 1 s and processed using Masslynx v. 4.0 software. The ionization was supported via pH adjustment to pH 10.5 with a solution of 100 mM ammonium hydroxide in acetonitrile/water (1:1), which was delivered via a second T-piece, between the column effluent splitter and the ESI probe, by a KD Scientific (New Hope, PA) model 200 syringe pump at 5 µL/min. LC/QTOFMS/MS acquisitions were performed at different collision energies, to allow both identification of small neutral losses and the observation of diagnostic fragment ions. Reporter Gene Bioassay. The reporter gene bioassay for estrogens was developed in-house and has been described elsewhere.12,13 In short, the yeast cytosensor expresses the human estrogen receptor R (hERR) and yEGFP in response to estrogens. An agar plate containing the selective MM/L medium was inoculated with the yeast ERR cytosensor from a frozen -80 °C stock (20% glycerol v/v). The plate was incubated at 30 °C for 24-48 h and then stored at 4 °C. The day before running the assay, a single colony of the yeast cytosensor was used to inoculate 10 mL of selective MM/L medium. This culture was grown overnight at 30 °C with vigorous orbital shaking at 225 rpm. At the late log phase, the yeast ERR cytosensor was diluted (1:10) in MM/L. For exposure in 96-well plates, aliquots of 200 µL of this diluted yeast culture were pipetted into each well, already containing the extracts of the urine samples. Exposure was performed for 0, 4, or 24 h. Fluorescence at these time intervals was measured directly in a CytoFluor Multi-Well Plate Reader (Series 4000, PerSeptive Biosystems) using excitation at 485 nm and measuring emission at 530 nm. The t24 - t0 (or t4 - t0) fluorescence intensity values were corrected for the corresponding values from exposed reagent blanks. Also the densities of the yeast culture were determined at these time intervals by measuring the OD at 630 nm. This was only done to check whether a urine sample was toxic for yeast. Procedures. Stock solutions of estrogens (1 mg/mL) were prepared in methanol and diluted with acetonitrile and water prior to use. The 2-mL aliquots of bovine urine samples, urine blanks, and control urines spiked with 17β-estradiol at 1 ng/mL were analyzed. The samples were adjusted to pH 4.8, 20 µL of H. pomatia was added, and enzymatic deconjugation was carried out overnight in a water bath at 37 °C. Next, 2 mL of 0.25 M sodium acetate buffer, pH 4.8, was added, and the hydrolyzed sample was subjected to solid-phase extraction (SPE) on a C18 column, previously conditioned by methanol and sodium acetate buffer. The column was washed subsequently with 10% sodium carbonate solution, water, sodium acetate buffer, water and finally 2 mL of 6602

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methanol/water (50:50). The dried C18 column was eluted with 4 mL of acetonitrile, and the eluate was applied to an NH2 column, previously conditioned with acetonitrile. The acetonitrile eluate thus obtained was evaporated to 2 mL under a stream of nitrogen gas at 40 °C. A 300-µL portion of this extract was subjected to bioassay screenings for estrogenic activity, while the remaining 1700 µL was stored in the dark at -20 °C for identification by LC/bioassay/QTOFMS, when appropriate. In the bioassay plate, samples were analyzed in triplicate as follows: three portions of 100 µL of acetonitrile extract were evaporated and redissolved in 2 µL of DMSO, and 50 µL of water was added to each well prior to addition of the yeast suspension. For bioassay-directed identification using LC/bioassay/QTOFMS, the remaining 1700 µL of acetonitrile extract was evaporated and redissolved in 80 µL of acetonitrile/water (15:65); 50 µL was finally injected into the LC/bioassay/QTOFMS system. The 20-s LC fractions thus collected in the 96-well plate (cf. Figure 1) previously filled with 2 µL of DMSO and 50 µL of water as keeper solvent were evaporated overnight in a fume hood and used for the bioassay without any additional treatment. Safety Considerations. Apart from normal laboratory safety considerations regarding the handling and use of toxic chemicals such as acetonitrile and DMSO, it should be emphasized that the use of recombinant yeast bioassays is restricted by specific regulations. However, the yeast we used is a common host strain of Saccharomyces cerevisiae that is not able to form spores (CEN.PK 102-5B, K20, URA3-, HIS3-, LEU-) and is only transformed with nonhazardous genes. This estrogen bioassay therefore requires an ML1 category biohazard laboratory only. RESULTS AND DISCUSSION General Characteristics. The methodology presented in this work relies on the performance of the bioactivity screening of estrogens, as summarized in the flowchart given in Figure 2. Many whole-cell biosensors have been developed in the past, but typically, only a few successful applications to real-life samples have been reported due to nonspecific effects caused by sample matrix components that might be toxic and influence cell growth. A very elegant way to correct for nonspecific interferences is the engineering of a dual reporter gene assay in which one reporter is expressed in response to the analyte, while a second reporter acts as an internal reference in response to the addition of a constant amount of a reference substance.23 We developed a rapid yeast estrogen bioassay based on the expression of yEGFP, which allows fluorescence measurement in a plate reader without cell wall disruption or the addition of any substrate.12,13 This yeast cell bioassay is inherently robust and thanks to the SPE cleanup step, not vulnerable toward sample matrix interferences. Although not further investigated, it is envisioned that automated SPE in a 96well plate format will further speed up the bioassay screening in routine laboratories. The application to urine samples ensures that even metabolically activated pro-estrogens will be detected, while estrogens that are metabolically deactivated via phase 2 metabolic processes such as glucuronidation and sulfation are converted back into active estrogens during the enzymatic deconjugation step. In this study, the assay is applied as a qualitative screening (23) Mirasoli, M.; Feliciano, J.; Michelini, E.; Daunert S.; Roda, A. Anal. Chem. 2002, 74, 5948-5953.

tool: above a certain fluorescence cutoff value as determined in the validation study, the urine sample under investigation is declared suspect for the presence of estrogens. This approach has been validated according to the latest EU guidelines24 and using chemically very different model estrogens such as steroids, 17β-estradiol, ethinylestradiol, mestranol; a stilbene, diethylstilbestrol; and a resorcylic acid lactone, zeranol. On each bioassay well plate, negative and positive control urine samples containing 1 ng/mL 17β-estradiol are coanalyzed for QA/QC purposes. The validation of this assay for estrogen screening in calf urine samples is discussed in detail elsewhere.25 According to Figure 2, the remaining portions of the sample extracts found suspect for bioactivity are reanalyzed by LC/bioassay/QTOFMS for bioassay-directed chemical identification using the setup of Figure 1. A minor flow part of the LC effluent is on-line adjusted to pH 10.5 and analyzed by QTOFMS. The QTOFMS provides full-scan MS data, good sensitivity, but is not as selective as a triple quadrupole MS system in MRM mode. The total ion current (TIC) chromatogram of real sample extracts will show many main components, and relevant traces of estrogens will be typically hidden in the TIC. For known estrogens, reconstructed mass chromatograms from 0.05-Da mass windows can easily solve this problem but the detection of unknown estrogenic compounds requires bioassay assistance. The parallel fractionation into the 96-well plate with subsequent bioassay screening at 20-s time resolution is crucial for the detection of unknowns and assigment of the relevant part of the TICsas indicated by the well position in the bioassaysafterward. Summing of mass spectra at the indicated retention time, background

subtraction, accurate mass measurement, and element composition calculation thereof provides the final chemical identification of unknown estrogenic compounds. As an option one might apply MS/MS acquisitions on the [M - H]- ion of interest in order to support structure elucidation of unknowns, as shown previously in the discovery of an entirely new β-agonist.22 Fractionation of LC effluents in a well plate containing DMSO/water was found to yield good recoveries over a wide polarity range from estriol to diethylstilbestrol, in accordance with LC/genotoxicity detection previously reported in the contaminants field.26 Acetonitrile/water gradient LC is compatible with the bioassay procedure for LC fractions described in the Experimental Section. Methanol/water gradients on the other hand failed, probably because of methanol residues, which cannot be removed quantitatively from methanol/ water mixtures via the evaporation procedure applied. Successful correlation of well plate numbers with LC/MS retention time demands stable operation of the postcolumn flow-split system shown in Figure 1. The correlation between LC/MS retention time and well plate number was determined by analysis of six replicates of an estrogen standard mixture containing estriol, 17β-estradiol, 17R-estradiol, estrone, and diethylstilbestrol, and the results obtained are given in Table 1. Note that the low value for the relative estrogenic potency of estriol did not allow detection by the bioassay. Some analytes of interest might show up in two adjacent wells, depending on their chromatographic peak shape and relative sensitivity in the bioassay. Nevertheless, from the data in Table 1, it can be concluded that a robust setup has been developed providing the quality of MS and bioassay correlation as required for successful bioassay-directed identification. Even when the LC/MS retention time shifted due to long-term changes in temperature conditions, a corresponding shift in well plate number was observed in the bioactivity measurement, i.e., without causing any bias on the correlation principle. As an alternative setup, one might decouple the chemical identification from the bioactivity detection by using two identical well plate fraction collectors and reinject the positive fraction from the duplicate well into a dedicated LC/QTOFMS identification system, having a shorter column and buffers or additives in the mobile phase, which would otherwise negatively effect the bioassay.22 Even LC at high pH values using an ammonia-containing mobile phase would be feasible in that alternative.27 Compound Library of Known Estrogens. It is to be expected that, depending on age, gender, and feeding regime, a significant number of calf urine samples will contain well-known estrogens originating from natural hormones such as 17R-estradiol and its metabolites estrone and estriol, phytoestrogens, and metabolites. Hence, a user library of known natural and synthetic estrogens containing relative retention times versus 17β-estradiol, negative ion MS, and MS/MS spectra was created by injection of 100 µL of standard mixtures of estrogens (100-1000 ng/mL) into the LC/QTOFMS setup of Figure 1. Data-dependent MS/MS acquisitions were triggered by an intensity threshold for the [M - H]ions in TOFMS mode and performed at three different collision energies (30, 35, and 40 eV). The results given in Table 2 include representative analytes from different chemical classes such as

(24) Commission Decision 2002/657/EC. Off. J. Eur. Commun. 2002, L221, 8-36. (25) Bovee, T. F. H.; Heskamp, H. H.; Hamers, A. R. M.; Hoogenboom, L. A. P.; Nielen, M. W. F. Anal. Chim. Acta, 2004, in press.

(26) Bobeldijk, I.; Brandt, A.; Wullings, B.; Noij, Th. J. Chromatogr., A 2001, 918, 277-291. (27) Draisci, R.; Palleschi, L.; Ferretti, E.; Marchiafava, C.; Lucentini, L.; Cammarata, P. Analyst 1998, 123, 2605-2609.

Figure 2. Analysis procedure for estrogens in calf urine using a rapid reporter gene bioassay for screening and LC/bioassay/QTOFMS for identification.

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Table 1. Validation of the Correlation between LC/QTOFMS Retention Time tR and Bioassay Well Plate Numbera

a

analyte

estriol

17β-estradiol

17R-estradiol

estrone

diethylstilbestrol

mean tR (min) mean well no.

3.78 ( 0.10 nd

8.27 ( 0.07 23/24 ( 0

9.03 ( 0.04 25/26 ( 0

10.17 ( 0.09 29 ( 0

10.97 ( 0.12 31/32 ( 0

Mean tR value of six replicates ( SD; mean well number ( well number; nd, not detected

Table 2. LC/QTOFMS/MS Data of Known Estrogensa compound

element composn

rel.RTb

QTOFMS [M - H]ion theor

QTOFMS/MS element compositionsc exp

estrone-3-sulfate 17β-estradiol-17β-glucuronide estradiol-3-sulfate estriol trans-resveratrol daidzein enterodiol cis-resveratrol phloretin coumestrol 2-hydroxyestradiol genistein equol (()-naringenin hesperetin enterolactone taleranol β-zearalenol 2-hydroxyestrone biochanin B (formononetin) R-zearalanol (zeranol) bisphenol A 17β-estradiol R-zearalenol 17R-estradiol equilenin ethinylestradiol bisphenol-B equilin estrone biochanin A zearalanone trans-diethylstilbestrol zearalenone dienestrol hexestrol cis-diethylstilbestrol 4-nonylphenol (tech mixt) BHT

C18H22O5S C24H32O8 C18H24O5S C18H24O3 C14H12O3 C15H10O4 C18H22O4 C14H12O3 C15H14O5 C15H8O5 C18H24O3 C15H10O5 C15H14O3 C15H12O5 C16H14O6 C18H18O4 C18H26O5 C18H24O5 C18H22O3 C16H12O4 C18H26O5 C15H16O2 C18H24O2 C18H24O5 C18H24O2 C18H18O2 C20H24O2 C16H18O2 C18H20O2 C18H22O2 C16H12O5 C18H24O5 C18H20O2 C18H22O5 C18H18O2 C18H22O2 C18H20O2 C15H24O C15H24O

0.36 0.39 0.41 0.46 0.51 0.54 0.57 0.63 0.71 0.71 0.76 0.76 0.77 0.78 0.81 0.82 0.83 0.86 0.94 0.95 0.99 0.99 1.00 1.03 1.09 1.14 1.15 1.16 1.19 1.23 1.29 1.31 1.34 1.34 1.36 1.37 1.70 2.47 2.71

349.1110 447.2019 351.1266 287.1647 227.0708 253.0501 301.1440 227.0708 273.0763 267.0294 287.1647 269.0450 241.0865 271.0607 301.0712 297.1127 321.1702 319.1546 285.1491 267.0658 321.1702 227.1072 271.1698 319.1546 271.1698 265.1229 295.1698 241.1229 267.1385 269.1542 283.0607 319.1546 267.1385 317.1389 265.1229 269.1542 267.1385 219.1749 219.1749

SO3-; C10H9O-; C18H21O2C4H5O2-; C5H5O3-; C18H24O2SO3-; C10H9O-; C18H23O2C10H9O-; C12H11O-; C17H19O2C10H7OC6H3O-; C8H4O2-; C14H7O3C7H6O-; C9H9O-; C17H17O2C10H7OC5H5O- ; C8H7O-; C7H7O2C15H6O5C9H7O2-; C10H9O2C8H5O2-; C10H7O2-; C9H8O4C6H5O-; C8H7O-; C7H5O2C6H3O2-; C8H7O-; C11H7O3C6H4O2-; C7H4O3-; C8H4O4C7H7O-; C8H7O-; C10H9OC17H25O3-; C9H9O-; C6H3OC17H23O3-; C17H21O2C10H9O2-; C11H11O2C13H7O2-; C14H7O3-; C15H7O4C17H25O3-; C9H9O-; C6H3OC9H9O-; C14H11O2C10H9O-; C13H11O-; C17H19OC17H23O3-; C17H21O2C10H9O-; C13H11O-; C17H19OC14H9O-; C16H13O-; C17H13O2C10H9O-; C11H11O-; C18H21O2C6H5O-; C14H11O2C10H7O-; C16H13O-; C17H13O2C10H9O-; C11H11O-; C13H11OC13H7O3-; C14H7O4-; C15H7O5C17H23O3-; C12H13O3-; C9H9OC14H9O2-; C15H10O2-; C16H13O2C10H7O3-; C9H7O-; C9H4O3C6H5O-; C8H5O-; C16H11O2C8H7O-; C9H9OC14H9O2-; C15H10O2-; C16H13O2C9H9OC14H19O-

a Conditions: negative electrospray ionization, collision energy 30-40 eV. b LC/QTOFMS relative retention time versus 17β-estradiol. c Element compositions of fragment ions calculated from QTOFMS/MS with accurate mass measurement.

stilbenes, steroids, isoflavones, isoflavanes, flavanones, coumestanes, lignanes, chalcones, and resorcylic acid lactones, having relative estrogenic potencies versus 17β-estradiol, ranging from negligible to rather strong. From this table it can be seen that some of the known estrogens have identical exact mass and element compositions. However, relative LC/MS retention time, characteristic MS/MS fragment ions, or both allow discrimination between the critical pairs 17β- and 17R-estradiol, zeranol and taleranol, equilenin and dienestrol, equilin and diethylstilbestrol, and estrone and hexestrol. In general, the MS/MS fragmentation behavior observed is dominated by small neutral losses such as loss of water, methanol, formaldehyde, carbon dioxide, methane, or ethyne. At the relatively high collision energies applied, the estrogen steroids tend to undergo fragmentation of the C-ring 6604 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

yielding, a.o., ions having their negative charge on the phenolate substructure. Some of the stilbenes and phytoestrogens show a tendency of radical formation (odd-electron ions) upon highenergy CID. The element compositions of some major MS/MS fragment ions calculated from the accurate mass determinations are included in Table 2. Bioassay-Directed Identification of Estrogens in Urine. A typical example of a reconstructed LC/QTOFMS chromatogram (summed masses at 0.05-Da mass window) together with the corresponding estrogenicity biogram as obtained from a standard mixture of estrogens (estriol, 17β-estradiol, 17R-estradiol, estrone, and DES, 1 ng each) is shown in Figure 3. The time delay between the reconstructed LC/QTOFMS chromatogram and the biogram is in the order of 0.8 min. From this figure it is evident that the

Figure 3. Reconstructed LC/QTOFMS chromatogram and reconstructed estrogenicity biogram of a standard mixture of estrogens (E3, estriol; bE2, 17β-estradiol; aE2, 17R-estradiol; E1, estrone and DES, diethylstilbestrol; 1 ng each). Conditions, see text.

Figure 4. Reconstructed LC/QTOFMS chromatogram of a calf urine sample spiked with 2 ppb of an estrogen mixture. The estrogens are hidden in the TIC background (upper panel), but their retention times were disclosed successfully by corresponding noncompliant fractions in the bioassay (indicated by arrows in the upper panel), thus allowing background subtraction and reconstruction of the mass chromatogram in the lower panel.

estrogenicity of 1 ng of estriol is negligible versus 1 ng of 17βestradiol, in accordance with its relative estrogenic potency of 5 × 10-3, cf. ref 13. The DES standard used is a mixture of transand cis-stilbene as confirmed by H NMR, and consequently, DES appears at two positions in both the chromatogram and the biogram. The same mixture was used to spike a blank calf urine sample at the 2 ng/mL level. The LC/QTOFMS TIC chromatogram shown in Figure 4 is ∼1400 times more abundant than the reconstructed mass chromatogram of the spiked estrogens.

However, the latter still allows confirmation of the presence of known estrogens in such a complex matrix, since narrow mass windows of 0.05 Da can be applied in order to reconstruct the corresponding mass chromatogram. Despite the high TIC background, element compositions of these estrogen molecules could be calculated from the accurate mass measurements of the [M - H]- ions. Generally, the following restrictions were applied: mass accuracy window (5 mDa, window of ring and double bond equivalent number (DBE) +0.5 to 20, and element options Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

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C0-30, H0-60, O1-8, F0-3, and N0-2. The presence of at least one oxygen atom was considered as a necessity for significant ion formation by deprotonation. Other halogen elements and sulfur were only considered when indicated by the isotope cluster of the [M - H]- ion. In addition, the nitrogen rule was applied to the [M - H]- ion. By doing so, C18H24O2, C18H22O2, and C18H20O2 were found for 17β/17R-estradiol, estrone, and diethylstilbestrol, respectively. From this spiked urine example, it is obvious that generic sample preparation and full-scan QTOFMS analysis yield unselective results; i.e., real samples containing unknown estrogens do require bioassay assistance in order to assign the relevant parts of the chromatogram. As an example, both the LC/QTOFMS total ion current chromatograms and the corresponding biograms are given for three urine samples found suspect for estrogenicity in the bioassay (Figure 5). In Figure 5a, a total ion current with many high-intensity signals is seen. The TIC region as indicated by the biogram was background subtracted yielding three reconstructed mass chromatograms of m/z 297, 271, and 241. Only the latter corresponded precisely with the retention time indicated by the biogram, the former ions originated from inactive substances. (Actually, the huge m/z 297 signal matches with the exact mass, element composition, and LC retention time of enterolactone, which is a known phytoestrogen but having an almost negligible estrogenic potency.13) The accurate m/z value obtained from the bioassay-directed background-subtracted mass spectrum of m/z 241 and the calculated element compositions are given in Table 3. The element compositions thus obtained were searched against electronic databases such as the Merck Index on CD-ROM and the Sigma-Aldrich search engine on the Internet. The first six calculated compositions in Table 3 were not very satisfactory: either molecules with such element compositions did not exist in electronic databases or were considered unrealistic (a molecule with only one double bond must be a carboxylic acid in order to comply with negative ESI, but it is rather unrealistic to expect estrogenic activity from a saturated carboxylic acid). Within the (5-mDa mass accuracy window, only the [M - H]- compositions C14H10N2OF and C10H13N2O5 gave entries in the database search. However, the fluorobenzylidenebenzohydrazide and thymidinelike molecules thus found are not expected to show very efficient ionization under negative ESI conditions, nor expected to have estrogenic properties at forehand. According to Figure 5a, the bioactive m/z 241 is sandwiched between huge signals from interfering analytes. Therefore, it might be argued that the accurate mass 241.0797 is less accurate than expected. Widening the mass accuracy window to (7 mDa yielded C15H13O3 for the [M - H]- ion. This element composition and the relative retention time (rel.RT) of a reference substance indicate the presence of the phytoestrogen equol, a metabolite of isoflavones which, on their turn, are well-known feed ingredients. A coeluting fragment ion having the element composition C7H5O2 supports the assignment. The bioactivity of equol in ERR transactivation assays as observed in this study was in agreement with previous findings.10 The second example given in Figure 5b highlights the use of TOFMS with accurate mass measurement. Again the backgroundsubtracted mass spectrum from the TIC region as indicated by the bioassay signal pointed to m/z 241 as an estrogenic compound. However, the LC retention time, the exact mass, and the element compositions calculated were much different from the equol case 6606

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(note that the urine sample in Figure 5b does contain some equol at ∼6.0 min retention time, but not enough to trigger the bioassay). The five element composition options calculated from the accurate mass measurement are given in Table 3. The first option C16H17O2 of the [M - H]- ion yielded the endocrine disrupter bisphenol B in electronic database searching. Bisphenol B was subsequently tested for both rel.RT and response in the bioassay and found to have a relative estrogenic potency similar to equol.13 However, the rel.RT in Figure 5b did not fully comply, so possibly the estrogenicity is caused by an isomer of bisphenol B. From the remaining element composition options, two represent saturated molecules that cannot comply with the acidity (carboxylic acid, phenol) requirement for negative ESI. The double bond equivalent numbers of the remaining options for the [M H]- ion in Table 3, C13H18O3F and C11H17N2O4, cannot yield steroidal molecules, but in theory phenolic structures would be possible. Electronic database searching did not provide any alternative molecule options for these element compositions, thereby supporting the proposal of a bisphenol B isomer. The third example shown in Figure 5c represents a calf urine sample that had been spiked with 5 ng/mL concentrations of the estrogens 17β-estradiol (m/z 271) and diethylstilboestrol (m/z 267) prior to analysis but which showed two additional estrogenic regions in the biogram. The background-subtracted mass spectra from the TIC regions as indicated by the bioassay signals yielded m/z 219 in both cases. From the accurate mass of the last eluting bioactive compound, two element compositions could be calculated. The first option given in Table 3 would be a saturated molecule, which cannot comply with the acidity (carboxylic acid, phenol) requirement for negative ESI. Electronic database searching for the second option, i.e., for the [M - H]- ion C15H23O, yielded mainly BHT and nonylphenol isomers. A technical mixture of nonylphenols and BHT were tested as references, and the former was found to comply with both the bioactivity and rel.RT observations in Figure 5c. The accurate mass measurement of the second bioactive substance in the m/z 219 trace also allowed two element compositions. The first option, C11H20O3F, would imply a molecule with only one double bond equivalent and must be a carboxylic acid. However, it is rather unrealistic to expect estrogenic activity from a saturated carboxylic acid. The second option yielded C14H19O2 for the [M - H]- ion, having a mass error of 1.2 mDa only. Electronic database searching for this composition showed many irrelevant substances that do not ionize under negative ESI conditions. However, pentamethylchromanol was also listed, being a urinary metabolite of vitamin E. A reference substance was ordered and tested both for estrogenicity and for rel.RT. LC/TOFMS of this reference provided a perfect match with the rel.RT observations in Figure 5c. However, standard solutions of this vitamin E metabolite did not yield any estrogenicity response in the bioassay, and although this substance was successfully identified, it cannot explain the signal in the biogram. Probably the relatively high signal of pentamethylchromanol either suppresses the ionization of the bioactive substance or the bioactive substance of interest itself does not ionize efficiently under negative ESI conditions. Finally, the bioassay was applied to 106 calf urine samples obtained from different slaughterhouses, according to the procedure summarized in Figure 2. One-third (35 samples) were found

Figure 5. Reconstructed LC/QTOFMS chromatograms and estrogenicity biograms of three suspect calf urines: (a) suspect urine with high levels of inactive interfering substances (m/z 271 and 297); (b) suspect urine with a bioactive substance having the same nominal mass (m/z 241) but different retention time; (c) suspect urine spiked with 17β-estradiol (m/z 271) and diethylstilboestrol (m/z 267) prior to analysis but showing two additional bioactive substances (m/z 219) in the biogram. Other conditions, see text.

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Table 3. Bioassay-Directed Identification of Estrogens in Suspect Urine Samples Given in Figure 5a bioactive no.

[M-H](m/z)

element compositions

DBE

error (Da)

Figure 5a

241.0797

Figure 5b

241.1217

Figure 5c

219.1777

C8H12N2O3F3 C11H11N2O2F2 C14H10N2OF C10H13N2O5 C7H14N2O6F C13H12OF3 C15H13O3 C16H17O2 C8H18N2O5F C13H18O3F C11H17N2O4 C10H19O4F2 C12H24O2F C15H23O

2.5 6.5 10.5 5.5 1.5 6.5 9.5 8.5 0.5 4.5 4.5 0.5 0.5 4.5

-0.0003 +0.0008 +0.0020 -0.0027 -0.0039 -0.0043 -0.0068 -0.0012 +0.0017 -0.0023 +0.0029 -0.0034 +0.0017 +0.0028

rel.RTb

proposal/comments

Y N

no entries in electronic databases no entries in electronic databases two entries, see text two entries, see text unrealistic: carboxylic acid no entries in electronic databases equol bisphenol B-like impossible: saturated molecule no entries in electronic databases no entries in electronic databases impossible: saturated molecule impossible: saturated molecule nonylphenol

Y

a Conditions: reversed-phase gradient LC with negative ESI QTOFMS. Element compositions were calculated using the following restrictions: mass accuracy window (5 mDa, ring and double bond equivalent number (DBE) range +0.5 to 20, element options C0-30, H0-60, O1-8, F0-3, and N0-2; other halogens and sulfur were only considered when indicated by the isotope cluster of the [M - H]- ion. b Relative retention time compliant versus reference compound (yes/no).

suspect for estrogenicity in the direct bioassay screening and subjected to bioassay-directed identification using LC/bioassay/ QTOFMS. In 80% of these suspect samples, the estrogenicity could, at least partly, be explained by the presence of 17R-estradiol, equol, or both. In the remaining suspect samples, no unknown synthetic estrogens were discovered, except for substances as discussed in Figure 5. Preliminary Data from Application to Feed. A wet feed sample for pigs was found suspect in the direct bioassay screening and subsequently analyzed by LC/bioassay/QTOFMS. The bioassay-directed identification approach proposed the presence of equol. The presence of such an isoflavone metabolite was unexpected in feed, but it turned out that isoflavones having weak relative estrogenic potencies might be converted to the more estrogenic metabolite equol by fermentation processes in specific feed mixtures.28 CONCLUSION A validated estrogenicity screening system has been complemented with LC/QTOFMS for bioassay-directed identification of known and unknown estrogens in calf urine samples. The system developed allows identification of bioactive estrogens at low ppb levels in both urine and feed samples. Although in the present (28) Kallela, K. Nord. Vet.-Med. 1975, 27, 562-569.

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study no newly abused synthetic estrogens have been discovered, the presence of endocrine-disrupting substances was indicated for the first time in calf urine samples. It should be emphasized that the applicability of estrogenicity screening and bioassay-directed identification of unknowns using LC/bioassay/QTOFMS is currently limited to urine from calves (