A review on structural elucidation of metabolites of environmental steroid hormones via liquid chromatography–mass spectrometry

Accepted Manuscript A review on structural elucidation of metabolites of environmental steroid hormones via liquid chromatography–mass spectrometry Li Ma, Scott R. Yates PII:

S0165-9936(18)30469-2

DOI:

10.1016/j.trac.2018.10.007

Reference:

TRAC 15271

To appear in:

Trends in Analytical Chemistry

Received Date: 9 September 2018 Revised Date:

4 October 2018

Accepted Date: 6 October 2018

Please cite this article as: L. Ma, S.R. Yates, A review on structural elucidation of metabolites of environmental steroid hormones via liquid chromatography–mass spectrometry, Trends in Analytical Chemistry (2018), doi: https://doi.org/10.1016/j.trac.2018.10.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A review on structural elucidation of metabolites of environmental

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steroid hormones via liquid chromatography–mass spectrometry

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Li Maa,b* and Scott R. Yatesb

Department of Environmental Sciences, University of California, Riverside, California 92521, United

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Contaminant Fate and Transport Unit, Salinity Laboratory, Agricultural Research Service, United States

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Department of Agriculture, Riverside, California 92507, United States

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* To whom correspondence should be addressed; E-mail: [email protected]

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Abstract This review covers liquid chromatography–mass spectrometry (LC-MS) strategies for

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structural elucidation of degradation and transformation products of environmental steroid

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hormones due to their estrogenic and/or androgenic effects on environmental living life. We

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focus on identification fundamentals of unknown metabolites rather than on target analysis. We

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discuss LC-MS capacities and overall MS-based steps for structural elucidation of unknown

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metabolites. Also summarized in the review are the fragmentation reactions and fragmentation

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pathways of protonated and deprotonated molecules of unknown compounds. We pay particular

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attention to versatile orthogonal techniques (e.g., isotopic labeling, hydrogen/deuterium

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exchange and model compound utilization) associated with MS and intelligent spectral

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interpretation strategies used in literature for structural elucidation of steroid hormone

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metabolites. We provide up-to-date applications of LC-MS on structural elucidation of biotic and

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abiotic transformation products of steroid hormones in environmental samples. Most of what is

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discussed in this review also applies to other emerging environmental contaminants.

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technique; degradation and transformation product; environmental steroid hormone

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Keywords: Structural elucidation; liquid chromatography–mass spectrometry; orthogonal

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Abbreviations

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APCI, Atmospheric-pressure chemical ionization

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API, Atmospheric-pressure ionization

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APPI, Atmospheric-pressure photoionization

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CID, Collision-induced dissociation

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DFT, Density functional theory

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EI, Electron ionization

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ESI, Electrospray ionization

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E1, Estrone

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E2, 17ß-Estradiol

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EE2, 17α-Ethynylestradiol

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FED, Frontier electron density

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FT-ICR, Fourier-transform ion cyclotron resonance

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GC-MS, Gas chromatography-mass spectrometry

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HCD, Higher-energy collision-induced dissociation

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H/D, Hydrogen/deuterium

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HRAM, High-resolution accurate-mass

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IT-MS, Ion-trap mass spectrometry

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LTQ, Linear trap quadrupole

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LC-MS, Liquid chromatography-mass spectrometry

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MS, Mass spectrometry

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MRM, Multiple reaction monitoring

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MSn, Multistage fragmentation

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NLS, Neutral-loss scan

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NMR, Nuclear magnetic resonance

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PIS, Precursor-ion scan

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Q-TOF, Quadrupole time-of-flight

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RDB, Ring double-bond equivalent

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TQ, Triple-quadrupole

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TOF, Time-of-flight

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WWTPs, Wastewater treatment plants

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1. Introduction Endocrine disrupting steroid hormones, including for example sex steroid hormones (majorly estrogens, androgens and progestagens) and anabolic steroids, are among the most widely

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investigated environmental pollutants during the past few decades associated with their adverse

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effects on aquatic organisms (e.g., feminization, decreased fertility and intersexuality)[1-3].

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Exogenous natural and synthetic steroid hormones (i.e., not self-produced) were generally

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reported to exhibit the highest affinities for binding to hormone receptors and thus the highest

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disrupting potencies [4, 5]. These hormones are administrated to human and livestock for

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medication purpose and are subsequently excreted through defecation and urination, primarily in

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biologically-inert conjugated form, partially in biologically-active free forms, and then reach the

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wastewater treatment plants (WWTPs) [6]. Part of the contaminants pass WWTPs unaltered,

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leading to their omnipresence in surface water. Steroid hormones have a common parent sterane

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backbone which features four fused rings, namely three cyclohexane rings (assigned as A-, B-,

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and C-rings) and one cyclopentane ring (the D-ring). The skeleton and structures of common

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steroid hormones investigated in the environment are shown in Figure 1.

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Ever since concern was raised on endocrine disruptors, numerous degradation approaches,

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biotic and abiotic, have been conducted with the purpose to remove the contaminants from the

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environmental compartments. In this regard, most of existing studies tackled parent compounds,

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while relatively less information on their metabolites and transformation products has been

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documented. This is, at least partially, due to the identification challenges. A subtle change in the

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molecular structure of the parent compounds during the degradation and transformation process

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would lead to changes in their potential for steroid receptor binding and thus subsequent

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biological activities. For example, previous studies demonstrated that the phenolic A ring of

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estrogens was responsible for their estrogenicity [7], while modification of estrogens via

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aromatic ring hydroxylation and cleavage can reduce their estrogenicity by a factor of 200–500

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[8]. Nevertheless, some hydroxylated estrogens were reported to incur carcinogenesis [9, 10]. In

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a phototransformation study on anabolic steroids, albeit the near complete phototransformation

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of parents, biological activity of photoproduct mixtures retained, suggesting the possible

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formation of more biologically-active and environmentally-persistent photoproducts [11].

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Therefore, characterization of novel biodegradation and transformation products is of uttermost

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importance for policy makers to conduct ecological risk assessment and strategy planning.

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From an identification point of view, compounds to be identified can be classified into three

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classes: “known knowns”, “known unknowns” and “unknown unknowns” [12]. A “known

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known” refers to a compound expected to be present in certain samples whose structure is well

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known and its presence need to be confirmed. An identification of “known knowns” is better

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described as an act of identity confirmation rather than identification. For example,

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environmental analysis is mostly directed at searching for the presence of target compounds

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which are known to the investigator and quantifying them if confirmed. For relatively polar

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compounds like steroid hormones, this task can be best fulfilled using liquid chromatography

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tandem-mass spectrometry based on multiple reaction monitoring (MRM) mode by referring to

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commercial standards. So far, such target-based environmental monitoring has been extensively

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conducted, exemplified by studies on occurrence and laboratory based microcosm studies on

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degradation kinetics of parent compounds [6, 13-16]. A “known unknown” represents a

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compound that is unknown to the investigator but can be found in the literature or compound

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databases. Finally, an “unknown unknown” is a compound that is not described elsewhere [12].

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The identification of “known unknowns” and “unknown unknowns” entails an access of more

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advanced instrument, versatile mass spectral libraries or databases as well as in-depth

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interpretation of mass spectra. Generally, biotic and abiotic metabolites and transformation

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products of the parent compounds in the environment fall in between the second and third

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categories. Therefore, only “known unknowns” and “unknown unknowns” are taken into account

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in the current review.

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There are two reviews published ten years ago discussing the structural elucidation of

metabolites of environmental contaminants by hyphenated MS techniques with one focusing on

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pharmaceuticals and the other on phenols [17, 18]. A few recent reviews reported the advances

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of MS in identification of transformation products of emerging environmental contaminants [19-

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24]. However, the dissociation mechanisms of ionized molecules based on collision-induced

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dissociation (CID) and identification strategies of yet-to be identified compounds based on, for

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example, orthogonal techniques (e.g., isotopic labeling, derivatization, hydrogen/deuterium

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exchange and model compound utilization), were inadequately addressed in these reviews.

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Therefore, the purpose of this review is to summarize up-to-date progress in MS techniques in

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structural elucidation of biotic and abiotic metabolites and transformation products of steroid

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hormones in the environment. Information on fragmentation pathways of reference steroid

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hormones was also generalized.

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2. Liquid chromatography-mass spectrometry (LC-MS) capabilities

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It is well known that gas chromatography-mass spectrometry (GC-MS) equipped with an

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electron ionization (EI) source is a popular tool in structural elucidation of unknown compounds

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ever since its invention [25]. However, since GC-MS is applicable for volatile and thermostable

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compounds, determination of polar and thermo-labile compounds largely relies on LC-MS.

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Atmospheric-pressure ionization (API) is soft ionization technique used in LC-MS [26]. Three

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kinds of API are routinely used: electrospray ionization (ESI), atmospheric-pressure chemical

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ionization (APCI) and atmospheric-pressure photoionization (APPI), among which, ESI,

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followed by APCI, is the most widely used technique in LC-MS [26] and is fairly suitable for

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steroid hormones. Table 1 provides a quick view of the applicability of ESI and APCI in

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identification of degradation and transformation products of steroid hormones in the environment

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based on laboratory studies. Unlike the hard ionization technique EI under which a molecular ion

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is dissociated into multiple fragments, ESI/APCI mostly yields intact protonated/deprotonated

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molecules [26], which help identify the molecular mass of the analyte.

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In essence, ionization of a molecule in ESI obeys the acid-base theory. In positive ESI,

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molecules are protonated majorly to form [M+H]+ with m/z (M+1); while in negative ESI,

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deprotonation ions [M–H]– with m/z (M–1) are readily generated. In addition, formation of other

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adducts such as [M+Na]+, [M+K]+ and [M+NH4]+ in the positive mode and [M+Cl] – and

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[M+CH3COO] – in the negative mode, for example, could also occur which can be employed to

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pinpoint the molecular identity. In APCI, protonated and deprotonated molecules are also

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commonly observed. Information concerning the ionization mechanisms of ESI/APCI was

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described elsewhere [26, 27]. In the current review, only protonated and deprotonated molecules

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in ESI/APCI are discussed, while other ionization techniques or multiply-charged ions are

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beyond the scope of this review.

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Among the common mass analyzers used so far, quadrupole and ion-trap mass spectrometers

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(IT-MS) provide unit-mass resolution and a mass accuracy of ±0.1 for mono-charged ions over

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the entire applicable m/z range; while high-resolution accurate-mass (HRAM) mass

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spectrometers, represented by time-of-flight (TOF), orbitrap and Fourier-transform ion cyclotron

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resonance (FT-ICR) mass spectrometers, feature a mass error even less than 1ppm and a

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resolution in excess of 105 (based on full peak width at half maximum height definition) for low-

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molecular-weight analytes [26]. Therefore, with the aid of HRAM-MS, the exact mass of both

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precursor and product ions is measured which enables determination of an elemental

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composition (or molecular formula). Information including advantages, disadvantages, mass

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accuracy and mass resolution power of different analyzers were included in other reviews [18,

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28]. Protonated or deprotonated molecules in ion source are usually selected as precursors for

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CID in tandem MS. CID refers to the process whereby a precursor ion, via collision with a

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neutral gas, is activated and then fragmented to form a population of product ions [29]. CID can

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occur in a collision cell of quadruple or in ion trap at low collision energy. Low-energy CID,

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together with higher-energy collision-induced dissociation (HCD) which occurs in a HCD cell of

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an Orbitrap machine, is so far the most frequently used mass spectrometry dissociation technique

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for the analysis of small molecules [30]. A wealth of structural information is provided thereof.

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Based on the abovementioned capabilities of certain analyzers in elemental composition

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assignment and its CID function, LC-ESI/APCI-MS proved itself an indispensable tool in

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environmental analytical chemistry.

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3. Fragmentation pathways of [M+H]+ and [M–H]–

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3.1 Fragmentation reactions

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Different from EI for which an odd-electron molecular ion is generated via loss of an

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electron, ESI/APCI generally yields even-electron molecular ions with mostly the addition or

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subtraction of a hydrogen ion ([M+H]+ and [M–H]–). Fragmentation of [M+H]+ and [M–H]–

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generally obeys the even-electron rule: an even-electron is apt to form an even-electron fragment

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ion via a neutral loss rather than form an odd-electron fragment ion via a loss of a radical.

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Violation of the rule may also occur in some circumstances. For instance, a loss of an alkyl

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radical group from a protonated or deprotonated molecule is observable for steroids [31]. Prior to

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the proposal of the fragmentation pathways, it is essential to know typical fragmentation

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reactions subjected to CID-MS. Studies concerning steroid hormones in this regard are relatively

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less addressed. Yet, by analogy to a recent review [32], fragmentation reactions of protonated or

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deprotonated steroids in available literature are classified into two major categories: charge-

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remote fragmentation and charge-induced fragmentation. Charge-remote fragmentation is

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characterized by charge retention in the original place with bond cleaved at a site physically

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remote from the site of the charge [33-35]. Conversely, charge-induced fragmentation is a

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fragmentation reaction in which the cleavage occurs adjacent to the apparent charge site,

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resulting in charge migration to a new site. Examples of two reactions are portrayed in Figure 2.

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It is showed that charge-remote fragmentation reactions of deprotonated ions are similar to those

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of protonated ions because they take place in sites remote from the charge site. However, charge-

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induced fragmentation reactions are distinct between protonated and deprotonated ions with

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adjacent electrons transferred towards and away from the charge site, respectively.

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3.2 Fragmentation pathway proposal

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The proposal of fragmentation pathways of a specific compound depends not only on

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knowledge of fragmentation reactions but also other valuable approaches. Particularly, the use of

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HRAM-MS enables the calculation of the molecular formulae of both precursor and product ions.

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Afterwards, the nitrogen rule can be applied to and the ring double-bond equivalent (RDB) value

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can be calculated from the molecular formula. A positive mono-charged ion has a RDB value

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ending in +0.5 and a negative mono-charge ion has a RDB value ending in –0.5. A molecule

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losing an electron has an integer value. Besides, stable-isotope labeling, multistage fragmentation

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(MSn) and certain software are also very much helpful. Noteworthy is that the concept of ion

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stability was attenuated in a number of studies to propose the structure of the product ions. Guan

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et al. [31] elucidated the fragmentation pathways of protonated anabolic steroids (e.g., boldenone,

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trenbolone, testosterone and so on) with the aid of Mass Frontier software, accurate mass

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measurement and MSn. The results suggested firstly that charge-induced cleavage was the most

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important fragmentation process. Secondly, charge-induced cleavage most likely occurred at a

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carbon atom with the most braches or strains. Thirdly, formation of priority product ions was

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pertinent to the stability of the product ions. A product on with the charge stabilized by

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resonance for instance has high stability. In a study to propose the fragmentation pathway of

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protonated estrone (E1), Bourcier et al. [36] compared the CID spectra of two marker

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compounds, namely estrone methyl ether and E1-D4 (deuterium labeled at C2, C4 and C16),

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with that of E1 to deduce the chemical structure of product ions. The use of deuterated E1

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implied methyl rearrangement the primary step to initial fragmentation of protonated E1. Sun et

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al. [37] studied thoroughly the product ions of [M–H]– of estrogens and their analogs and

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summarized that the most abundant fragment ions were consistent with the fragmentation

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pathway of retrocyclization. The overall rule is that these product ions which formed due to the

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elimination of small neutral molecules (e.g., H2, CH4, H2O, CO and CH3OH) and/or ring

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cleavage are stabilized by conjugated double bonds and an aromatic ring. As illustrated in Figure

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3, the presence of a double bond in equilin and 17ß-equilin facilitated a H2 loss, leading to the

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aromatization of ring B, and a C17 carbonyl group in equilin favors a CH4 loss, leading to the

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conjugation of a C13-14 double bond with the ketone carbonyl group. Conversely, such

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mechanisms were void in 17ß-estradiol (E2). Similar rules were iterated by Tedmon et al. [38],

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in defining the fragmentation differences between standard isobaric steroid hormones. Notably,

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density functional theory (DFT) calculation was employed in this study to predict fragmentation

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events based on the calculated lowest energy configurations of product ions and the predicted

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results generally matched what was observed in CID spectra [38]. DFT together with frontier

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electron density (FED) are common electronic theory used to predict metabolite formation of

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estrogens in the environment [39]. Elsewhere, a few more studies [40-42] investigated the CID

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spectra of a series of standard steroids and correlated the diagnostic fragmentation pattern with

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their structural feature, which holds promise guiding the structural elucidation of their

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metabolites.

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4. Overview of structural elucidation steps and strategies

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Although specific structural elucidation steps may be case dependent due to variations in

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instrumentation and the experimental conditions, the overall steps include: 1) full scan to show

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peaks of potential metabolites and their protonated/deprotonated molecules ([M+H]+ and [M–H]–

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); 2) acquisition of product-ion spectra of [M+H]+ and [M–H]– based on CID; 3) molecular

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formula assignment of [M+H]+ and [M–H]– and product ions if applicable; and 4) structural

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elucidation based on product-ion spectral interpretation and authentic standard confirmation if

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available. Overall workflow of elucidation is schemed in Figure 4.

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Generally, real samples and controls were firstly scanned in positive and negative mode

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across an appropriate m/z range. Peaks present in treated samples but absent in controls are

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regarded as prospective peaks of the unknown metabolites. The selection of potential peaks of

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interest may rely on either preliminary visual interpretation of relevant chromatograms or data

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processing software such as XCMS [43]. Appropriate screening criteria including for example

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peak area threshold were implemented to prioritize the list of potential peaks of interest. Ion

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adducts and/or dimers/trimers were used to identify the most likely protonated/deprotonated

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molecules.

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Molecular formulae of the protonated/deprotonated molecules were assessed based on accurate mass measurement on HRAM-MS. Several calculation criteria were applied to narrow

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the number of possible molecular formulae, including an accuracy error threshold, upper limits

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on the number of carbon/hydrogen/nitrogen, the nitrogen rule, isotope pattern and RDB, until

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one molecular formula is derived. Thereafter, an option to mine the chemical structure of the

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unknown compound is to search its molecular formula against known chemical structure

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databases such as PubChem, Chemspider, and SciFinder [44]. Although each database is

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incomplete, valuable information can still be drawn for ease of structural illustration. Otherwise,

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the structure can be disclosed based on the following approaches.

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Protonated/deprotonated molecules of the unknown metabolites are subject to MSn

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(multistage fragmentation of the protonated/deprotonated molecules to acquire product-ion

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spectrum), if necessary, based on CID for further structural information. The structure of the

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metabolites can be proposed based on product-ion spectral interpretation. One common and easy

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approach to product-ion spectral interpretation is by referring to the product-ion mass

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spectrometry data of existing literature and spectral libraries such as MassBank, METLIN, NIST

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and Wiley or by comparison with fragmentation patterns of commercial standards, if available.

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Besides, knowledge of CID fragmentation reactions (Section 3.1) and use of fragmentation

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pathway predicting software help the interpretation. Two widely used commercially available

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software packages are Mass Frontier and Mass Fragmenter [44]. The formula assignment of the

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product ions based on HRAM-MS also greatly facilitates the structural elucidation. Another way

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that helps structural elucidation is via comparison of protonated/deprotonated molecules of the

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metabolites with that of a related parent compound itself. This strategy derives from the premise

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that the initial metabolites conserve to some degree the basic skeleton of its parent compound.

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Mass shift between them implies certain transformation reactions. For instance, a +16 (Da) shift

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in m/z value may indicate hydroxylation, +14 (Da) methylation, –2 (Da) dehydrogenation, +2

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(Da) saturation, +18 (Da) hydration and –18 (Da) dehydration, etc. Notably, when routine MS analysis alone do not suffice to unravel the identity of an

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unknown compound, orthogonal techniques that can be readily performed on MS are employed

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to facilitate structural elucidation. Therein, 2H or 13C isotopic labeling is a sagacious strategy

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adopted. The location of modifications during the degradation and/or transformation process

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could be inferred via comparison of the spectra between non-labeled and labeled parent

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compounds. Hydrogen/deuterium (H/D) exchange is another example. Hydrogen atoms attached

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to heteroatoms (e.g., N, O and S) are easily exchanged by deuterium. Therefore, in a H/D

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exchange event, investigators measured the m/z shift between a unknown metabolite and its

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H/D-exchange counterpart to judge the presence of hydroxy (–OH), carboxylic acid (–COOH)

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and amino (–NH2) functional groups in the metabolite structure. Other orthogonal techniques

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such as derivatization and using model compounds are also feasible. Examples to illustrate the

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structures of the degradation and transformation products are elaborated in the following part.

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5. Applications in environmental samples

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5.1 Triple-quadrupole MS (TQ-MS)

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TQ features three quadrupoles where the first and third quadrupoles permit mass analysis and

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the second allows CID. Generally, TQ is not an ideal tool for structural illustration due to its low

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sensitively, low-mass resolution and accuracy in the scan mode whereby the molecular formulae

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of the unknowns cannot be determined; rather, TQ is applied more for quantitative analysis in the

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MRM mode which features high sensitivity. Nevertheless, due to the unavailability of more

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powerful instrument under certain circumstances, TQ was still availed in a few studies to

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tentatively identify the degradation and transformation products of progestagens and estrogens in

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the environmental matrix [8, 45-49](Table 1). Full scan and product ion scan with TQ were

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conducted in these studies to obtain the protonated or deprotonated molecules of the metabolites

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and their fragmental information. The structures of the metabolites were tentatively proposed by

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means of personal interpretation of the spectra and referring to literature and further confirmed

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using authentic standards if available. The unequivocal structural elucidation, however, was

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challenging without standards or other confirmation method. One thing to be notified is that to

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manifest minor metabolites on full scan spectra which would be otherwise invisible if the

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concentration of parent compound is low arising from the low sensitivity of TQ in scan mode,

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parent compounds were usually fortified at much higher concentrations than environmentally-

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relevant concentrations. In this case, the similarity in transformation pathways of parents cannot

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be guaranteed for spiked concentrations with environmentally-relevant concentrations.

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(NLS) [50] with TQ also help structural elucidation. PIS mode is used to look for the precursors

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of a particular fragment ion. For example, Zhou et al. [51] applied PIS approach to search for

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halogen-containing estrogens with halide ions as fragments. NLS is used to monitor an ion which

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upon CID has a neutral loss with a mass matching the fixed m/z difference. NLS is especially

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useful for screening metabolites of sulfate- and glucuronide-conjugated steroids which feature

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common neutral losses of 80 (SO3) and 176 Da (C6H8O). These conjugates can also be screened

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in PIS mode by the scan over all precursors that give rise to fragment ions at m/z 80 (SO3 –) and

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m/z 97 (HSO4–) or m/z 175 (glucuronate) and m/z 113 (fragment of the glucuronide moiety) [18,

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52]. However, PIS and NLS are mostly used for identification of new steroid metabolites [53] in

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biological matrix but are rarely applied in environmental samples.

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With TQ, orthogonal techniques and other intelligent strategies were adopted to assist the identification. For example, Huber et al. [8] extrapolated the O3 oxidized products of 17α-

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ethynylestradiol (EE2) by referring to two model compounds 5,6,7,8-tetrahydro-2-naphthol and

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1-ethinyl-1-cyclohexanol, which represent, respectively, the reactive phenolic moiety and the

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ethinyl group of EE2. The derived oxidation products of EE2 were identified using TQ, and the

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characteristic neutral losses of deprotonated molecules ([M-H]–) of the oxidation products also

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supported the putative structures. For example, loss of 44 (CO2), 46 (CH2O2) and 62 (CO2 + H2O)

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likely suggested the presence of one carboxylic acid, α/ß-hydroxy acid and two carboxylic acid

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functional groups in the structure, respectively [54, 55]. Elsewhere, Ma and Yates [49] employed

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“pseudo-MS3” to tentatively identify the biodegradation products of 17ß-estradiol-3- glucuronide

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and 17ß-estradiol-3-sulfate in riverine sediment. To apply “pseudo-MS3”, an increased de-

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clustering voltage in ESI source was applied to produce in-source fragmentation of the

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deprotonated molecules. One characteristic fragment with high response was selected for further

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fragmentation in a collision cell of TQ. Tentatively proposed metabolites in the study included

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hydroxylated products, keto derivatives and dehydrogenated products, among which several

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products were confirmed using authentic standards and GC-MS after derivatization. In another

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study on ligninase-mediated E2 removal [48], E2 lost one electron to generate an E2 free radical

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which self-combined via coupling reactions to form dimers and trimers. Reaction sites in E2

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molecules were proposed based on the computed charge and spin densities of the free radical.

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5.2 IT-MS

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Similar to TQ-MS, drawbacks of IT-MS arising from low-mass accuracy and resolution do

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not allow a molecular formula calculation. Yet the high full-scan sensitivity and MSn capabilities

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render IT-MS a promising technique in structural elucidation of the degradation and

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transformation products of environmental contaminants. The high full-scan sensitivity trait of IT-

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MS may make avoidable the cumbersome steps to concentrate the analytes. In a MSn experiment,

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selection of precursor ions, CID and product-ion scan are all performed in ion-trap, whereby a

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high abundance of structurally-informative product ions are yielded. Intelligent identification

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strategies, orthogonal information and electronic theory were also used in some studies to verify

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the metabolite structures proposed based on MSn. For example, in a photodegradation study of

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EE2 [56], the structures of the intermediates were determined via comparison of obtained MS4

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data with prior knowledge of EE2 photocatalytic mechanisms. The hydroxylated products were

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proposed to be the initial metabolites, arising from ˙OH attack of EE2; whereas the reaction site,

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namely the location of the hydroxyl group, was predicted based on FED calculation. In another

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example with characterization of an EE2 biotransformation product, MSn data suggested the

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presence of a hydroxyl group, an aldehyde group, and a carboxylic group in the structure [57].

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This product derivatized with dansyl chloride in the same manner as EE2 (reaction site is located

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at phenol group), in support of the preservation of the phenolic group of the parent EE2 after

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transformation. Furthermore, the early elution of the product on reversed-phase LC

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chromatograph suggested the presence of a polar group such as a carboxylic moiety. Such

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polarity oriented judgement was also stated in other studies [11, 58].

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5.3 TOF-MS and Q-TOF-MS

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High full-scan sensitivity, high-mass resolution and accuracy provided by TOF-MS make it a

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significant asset for molecular formula definition of analytes. Single TOF-MS has been proved

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useful for structural disclosure for oligomers or polymers, which are common transformation

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products of estrogens in enzyme-mediated systems [59, 60]. Nonetheless, since TOF-MS alone is

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not able to provide CID, NLS and PIS, additional structural illustration strategies and analytical

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tools are needed for further confirmation. For example, to determine the structure of EE2

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metabolites by an advanced catalytic oxidation process [61], the molecular formulae of the

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oxidized metabolites were assigned by TOF-MS, confirmed by nuclear magnetic resonance

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(NMR) and verified by FED calculation.

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During the past few years, a Q-TOF hybrid increasingly becomes a universal tool in

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environmental analysis. The so-called hybrid instrument usually means that the two different

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analyzers are tandem in space. Q-TOF can be regarded as a modified TQ with the third

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quadrupole replaced by TOF analyzer. Each analyzer alone has disadvantages, while a hybrid-

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mass system is able to integrate the merits of the two. Q-TOF hybrid mass spectrometer inherits

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acquisition modes from TQ except MRM, allowing a molecular formula calculated for not only

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precursor ions but also product ions. Wang et al. [62] confirmed the formation of oligomers as

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the major phototransformation products of estrogens in a Mn (Ⅲ)-mediated system based on

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accurate mass measurement and CID spectral interpretation. Identified oligomers were formed

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via self-coupling reaction of E2 or E1 and cross-coupling reaction of E2 and E1. The same

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approaches were applied by Wang et al. [63] to evaluate the photodegradation products of E2 on

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silica gel and natural soil; therein hydroxylated products and similar oligomers were identified.

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In both studies, DFT calculations were carried out to provide theoretical insights into the

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potential reaction sites of the substrates (e.g., hydroxylation, oligomerization, and C-C bond

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cleavage). In a study to evaluate the photolysis by-products of E1by Q-TOF, Souissi et al. [64]

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employed two model compounds, 5,6,7,8-tetrahydro-2-naphtol and 2-methylcy-clopentanone

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which represent ring A/B and ring D of E1, respectively, to deduce the possible structure of E1

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photolysis by-products. Based on CID mass spectra and accurate mass measurement, the by-

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products were proposed to be hydroxylated products and dimers. Importantly, by measuring the

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mass shift of the protonated molecules and their fragmented product ions between E1and E1-D4

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(labeled at C2, C4 and C16) photolysis by-products, the location of reaction site (e.g.,

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hydroxylation and polymerization) was tentatively localized. In a denitrifying media incubated

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with both testosterone and 2,3,4-13C-labeled testosterone (13C labeled at C-2, C-3 and C-4), a

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13

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suggested two 13C-labeled carbons from the parent were lost during the formation of this C17

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intermediate. Eventually, the two lost 13C-labeled carbons were assumed to be C-3 and C-4 via

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plausible deduction from the structure of one upstream intermediate. Table 1 listed a few more

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studies on Q-TOF-aided metabolite identification in environmental samples.

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5.4 Linear trap quadrupole (LTQ)-Orbitrap

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C-labeled intermediate with 17 carbons was identified [65]. TOF-MS accurate mass calculation

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To perform a LTQ-Orbitrap analysis, the spectrometer was switched between Orbitrap-MS for high-resolution full scan data and LTQ-MS/MS for low-resolution fragmentation data (MSn).

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With the former they are able to measure a molecular formula of the precursor and fragment ions

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with high confidence while the later enables a structure tentatively proposed. Examples of LTQ-

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Orbitrap application are also listed in Table 1. Hom-Diaz et al. [66] built an in-house library

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based on the accurate mass of the transformation products of E2 and EE2 described in the

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literature. By comparing the LTQ-Orbitrap data with that in the library, major transformation

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products of E2 and EE2 by microalgae were determined to be hydroxylated and dialdehyde

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products. Using the similar approach, Cole et al. [67] indicated that 17α-trenbolone and

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trendione were majorly bio-transformed to dehydrogenated and hydroxylated products.

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Elsewhere, Kolodziej et al. [11] tentatively proposed the structure of some photo-transformation

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products of 17α/ß-trenbolone and trendione in the similar way. Furthermore, additional

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information, NMR and UV/Vis data for instance, were appended in this study for further

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verification and/or confirmation. NMR data confirmed three proposed structures. The blue shift

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in UV/Vis spectra of the phototransformation products from that of the parents suggested the

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disruption of the conjugated 4,9,11 -system of the parents upon photolysis, congruent with the

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proposed structures.

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Recently, LTQ-Orbitrap proved itself an able tool in the investigation of potential

biologically-active by-products generated during the water treatment processes like ozonation

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and chlorination. As an example, Gervais et al. [58] characterized eight halogenation and

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hydroxylation products of EE2 during the chlorination of drinking water. The assigned formulae

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suggested all the major by-products should have a structure similar to their parent EE2. Isotope

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ratio calculation implied that chloride and bromide were added to the structure. MSn

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fragmentation patterns indicated that halogen atoms and the additional hydroxyl group were

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located on the A-ring. The near concurrent but late elution of halogenation products relative to

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that of EE2 also supported the A-ring substituted reaction because the presence of halogenated

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atoms on the A-ring would decrease the polarity of the phenolic group and thus promote the

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retention of the by-products on reversed-phase separation column. In another study, Bourgin et

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al. [68] characterized four major ozonation by-products of esrone-3-sulfate via a combination of

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MSn and H/D exchange approaches. The calculated molecular formulae and changes in m/z

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during the H/D exchange experiment determined whether a carbonyl group or a hydroxyl group

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was attached to the parent during ozonation treatment. MSn spectral interpretation provided

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further hint on the location of the carbonyl and hydroxyl groups. Finally, in-house synthesized or

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commercially available standards further confirmed the structure.

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5.5 Orbitrap, Quadrople-Linear Ion-Trap and LTQ-FT-ICR

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Apart from the above-stated mass spectrometers, Table 1 also listed examples of application of Orbitrap [69] and LTQ-FT-ICR [70] on estrogen metabolite characterization. Here, orbitrap

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functions similarly to a sole TOF analyzer, while LTQ-FT-ICR was analogous to LTQ-Orbitrap.

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6. Concluding remarks

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Low-mass accuracy and resolution MS like TQ and IT-MS hitherto still play a role in the

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tentative identification of unknowns, while to achieve unambiguous identification the authentic

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standards are essential. The unavailability of the standards substantially compromises their

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qualitative capability. The development of HRAM-MS has greatly aided metabolite

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identification arising from their accurate mass measurement capacity. As a result, a hybrid MS

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system that offers accurate mass data and CID fragmentation data has been proven to be a

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powerful tool in structural elucidation of steroid hormone metabolites in the environmental

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samples. In the application examples present here (Table 1), orthogonal techniques and spectral

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interpretation skills manifest the effectiveness of the hybrid MS systems.

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The success of structural elucidation of unknowns relies not only on instrumentation but also on better software algorithms, especially those for screening and fragmentation pathway

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prediction of the unknowns, which are not yet fully developed. Software with sufficient selection

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and filtering parameters is necessary to ensure a priori selection of interested peaks and

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elimination of false-positive and false-negative results. Advanced software solutions for

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fragmentation pathway prediction need to be developed to consider rearrangement reactions [31].

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Additionally, since quantum mechanical approaches with DFT for example were often employed

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to provide theoretical insights into the fragmentation pathways, better computational resources

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are also necessary for optimum electron density calculation.

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Summing up, this review aims to provide the progress in application of LC-MS on structural elucidation of steroid hormone degradation and transformation products in the environment.

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Structural elucidation steps together with fragmentation pathways of pronated and deprotonated

440

molecules are also discussed. The information provided in this review is useful for future

441

identification of novel metabolites of other environmental emerging contaminants. However, one

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need to bear in mind that structural elucidation based on routine MS analysis is sometimes

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inadequate to assign the identity of the unknown; alternatively, orthogonal techniques are needed

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to assist identification. Also, gaining as much as possible knowledge on experimental parameters

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and physiochemical properties of the parent compounds is pivot for identity assignment of novel

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metabolites.

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Acknowledgement

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This work was supported by the United States Department of Agriculture (USDA) under

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Project No: 2036-12130-011-00D

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[68] M. Bourgin, G. Gervais, E. Bichon, J.-P. Antignac, F. Monteau, G. Leroy, L. Barritaud, M. Chachignon, V. Ingrand, P. Roche, B. Le Bizec, Differential chemical profiling to identify ozonation byproducts of estrone-sulfate and first characterization of estrogenicity in generated drinking water, Water

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Res, 47 (2013) 3791-3802. [69] K. Sun, Q. Luo, Y. Gao, Q. Huang, Laccase-catalyzed reactions of 17β-estradiol in the presence of humic acid: Resolved by high-resolution mass spectrometry in combination with 13C labeling,

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Chemosphere, 145 (2016) 394-401.

[70] S. Karim, S. Bae, D. Greenwood, K. Hanna, N. Singhal, Degradation of 17α-ethinylestradiol by nano

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zero valent iron under different pH and dissolved oxygen levels, Water Res, 125 (2017) 32-41. [71] N.S. Rannulu, R.B. Cole, Novel Fragmentation Pathways of Anionic Adducts of Steroids Formed by Electrospray Anion Attachment Involving Regioselective Attachment, Regiospecific Decompositions, Charge-Induced Pathways, and Ion-Dipole Complex Intermediates, J Am Soc Mass Spectr, 23 (2012) 1558-1568.

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[72] K.M. Wooding, R.M. Barkley, J.A. Hankin, C.A. Johnson, A.P. Bradford, N. Santoro, R.C. Murphy, Mechanism of Formation of the Major Estradiol Product Ions Following Collisional Activation of the Molecular Anion in a Tandem Quadrupole Mass Spectrometer, J Am Soc Mass Spectr, 24 (2013) 1451-

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1455.

[73] X.Y. Ma, C. Zhang, J. Deng, Y.L. Song, Q.S. Li, Y.P. Guo, C. Li, Simultaneous Degradation of

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Estrone, 17 beta-Estradiol and 17 alpha-Ethinyl Estradiol in an Aqueous UV/H2O2 System, Int J Env Res Pub He, 12 (2015) 12016-12029. [74] W.O. Khunjar, S.A. Mackintosh, J. Skotnicka-Pitak, S. Baik, D.S. Aga, N.G. Love, Elucidating the Relative Roles of Ammonia Oxidizing and Heterotrophic Bacteria during the Biotransformation of 17 alpha-Ethinylestradiol and Trimethoprim, Environ Sci Technol, 45 (2011) 3605-3612. [75] P. Mazellier, L. Méité, J.D. Laat, Photodegradation of the steroid hormones 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) in dilute aqueous solution, Chemosphere, 73 (2008) 1216-1223.

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[76] L. Méité, B.D. Soro, N.K. Aboua, V. Mambo, K.S. Traoré, P. Mazellier, J. De Laat, Qualitative Determination of Photodegradation Products of Progesterone and Testosterone in Aqueous Solution, Am J Anal Chem, 7 (2016) 22.

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[77] Q. Yu, J. Geng, H. Huo, K. Xu, H. Huang, H. Hu, H. Ren, Bioaugmentated activated sludge degradation of progesterone: Kinetics and mechanism, Chem Eng J, 352 (2018) 214-224.

[78] L. Barritaud, G. Leroy, M. Chachignon, V. Ingrand, P. Roche, E. Bichon, G. Gervais, M. Bourgin,

estrone sulfate, Water Sci Tech-W Sup, 13 (2013) 1302-1308.

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J.P. Antignac, F. Monteau, B. Le Bizec, Identification of treatment by-products of the ozonation of

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[79] M. Fahrbach, M. Krauss, A. Preiss, H.P.E. Kohler, J. Hollender, Anaerobic testosterone degradation in Steroidobacter denitrificans - Identification of transformation products, Environ Pollut, 158 (2010) 2572-2581.

[80] P.A. Segura, P. Kaplan, V. Yargeau, Identification and structural elucidation of ozonation transformation products of estrone, Chem Cent J, 7 (2013).

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[81] J.O. Ojoghoro, A.J. Chaudhary, P. Campo, J.P. Sumpter, M.D. Scrimshaw, Progesterone potentially degrades to potent androgens in surface waters, Sci Total Environ, 579 (2017) 1876-1884. [82] J.H. Li, Y. Zhang, Q.G. Huang, H.H. Shi, Y. Yang, S.X. Gao, L. Mao, X. Yang, Degradation of

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organic pollutants mediated by extracellular peroxidase in simulated sunlit humic waters: A case study with 17 beta-estradiol, J Hazard Mater, 331 (2017) 123-131.

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[83] Y.L. Chen, H.Y. Fu, T.H. Lee, C.J. Shih, L. Huang, Y.S. Wang, W. Ismail, Y.R. Chiang, Estrogen Degraders and Estrogen Degradation Pathway Identified in an Activated Sludge, Appl Environ Microb, 84 (2018).

[84] Y.Y. Yang, L.P. Pereyra, R.B. Young, K.F. Reardon, T. Borch, Testosterone-Mineralizing Culture Enriched from Swine Manure: Characterization of Degradation Pathways and Microbial Community Composition, Environ Sci Technol, 45 (2011) 6879-6886. [85] R.B. Young, D.E. Latch, D.B. Mawhinney, T.H. Nguyen, J.C.C. Davis, T. Borch, Direct Photodegradation of Androstenedione and Testosterone in Natural Sunlight: Inhibition by Dissolved 31

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Organic Matter and Reduction of Endocrine Disrupting Potential, Environ Sci Technol, 47 (2013) 84168424. [86] L.H. Shan, Y. Li, Y.J. Chen, M.H. Yin, J.J. Huang, Z.Z. Zhang, X.F. Shi, H.M. Liu, Microbial

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hydroxylation of 17-estradiol by Penicillium brevicompactum, Biocatal Biotransfor, 34 (2016) 137-143. [87] S. Larcher, G. Delbès, B. Robaire, V. Yargeau, Degradation of 17α-ethinylestradiol by ozonation — Identification of the by-products and assessment of their estrogenicity and toxicity, Environ Int, 39 (2012)

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66-72.

[88] Y.L. Chen, C.P. Yu, T.H. Lee, K.S. Goh, K.H. Chu, P.H. Wang, W. Ismail, C.J. Shih, Y.R. Chiang,

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Cell Chem Biol, 24 (2017) 712-+.

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Biochemical Mechanisms and Catabolic Enzymes Involved in Bacterial Estrogen Degradation Pathways,

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Figure captions: Figure 1. Steroid nomenclature and structures of major steroid hormones investigated in

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literature. Figure 2. Charge-remote (outlined by dash square) and charge-induced fragmentation pathways of deprotonated 17ß-estradiol (a), protonated 17ß-estradiol (b) and protonated boldenone (c).

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Date are adapted from and modified based on [31, 36, 71, 72].

Figure 3. CID spectra (adapted from [37]) of deprotonated equilin (a), 17ß-equilin (b) and 17ß-

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estradiol and proposed formation pathways of selected product ions.

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Figure 4. Overall workflow for structural elucidation of transformation metabolites.

33

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18 11

19 1

12

C 9

10

B

A

16

15

7

5

4

8

14

6

OH

O

H

OH

H

OH

H

Estrogen H

H

H

H

HO

Estrone

OH

OH H H

H

Progesterone

O

H

H

H

H

O

Testosterone

Androstenedione

OH

Anabolic steroids

OH

H

EP

H H

O

H

TE D

H O

H

H

O

Trenbolone

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Boldenone

H

Levonorgestrel

Norethindrone

OH

Androgen

H

H

O

O

O

H

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H

H

H

Figure 1.

34

H

Ethynylestradiol

Estriol

O

H

H

HO

HO

17 -Estradiol

Progestagen

H

OH

H

SC

H HO

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R1

17

D

2 3

R2

13

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a. -

m/z 145 O

-

H

m/z 143 O

OH

-

-H2O O

OH

m/z 271 O

-

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H

m/z 253

H

m/z 271 O

OH

OH

-

O

-

-

O

m/z 271

O

m/z 239

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m/z 271

OH

OH

OH

OH

SC

-CH3OH

13 17

-

-

H

-

-

O

O

O

O

14

14

15

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15

O

-

m/z 183

b. OH2+

+ Transfer

OH

OH

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c.

OH

OH

m/z 287

OH

m/z 255

14

16

15

O

OH

17

H

13

m/z 271

OH

H OH

+ OH

H

m/z 255

m/z 255

OH

m/z 159

OH OH

+ H OH

m/z 287

m/z 271

13

OH

OH

+

+

13

+

OH

m/z 255

m/z 273

-

H

O

+

of the methyl group

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-H2O

+

OH

17

17

m/z 271

16

15

-

H16

O

16

14

m/z 271

m/z 271

m/z 271

m/z 271

16

14 15

m/z 287

rH OH

Figure 2.

35

H -H O 2 +

m/z 287

+

m/z 153

+ +

m/z 135

m/z 135

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Figure 3.

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SC

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ACCEPTED MANUSCRIPT

36

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Sample preparation

Elemental composition assignment

Match of product ion spectrum with parallel data in database or literature

Interpretation of product ions and proposal of fragmentation pathways

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Structure searching in database

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Screening for “known unknowns” and “unknown unknowns”

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Full scan and product ion scan analysis

Using orthogonal techniques and/or electronic theory to assist identification or confirm the results

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Figure 4.

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Confirmation using authentic standards if available

37

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Table 1. Structural elucidation and identification of degradation and transformation products of steroid hormones in the environment based on laboratory studies

EE2

E2, EE2 Progesterone, testosterone EE2

Progesterone 17α-TBOH, 17ß-

TBOH, TBO

MS analyzer

One intermediate was characterized 4-OH-EE2, sulfo-EE2

Biotransformation in the UV/H2O2 system Biotransformation by ammonia oxidizing bacteria and heterotrophic bacteria Photodegradation

ESI

IT

ESI

IT

APCI

IT

Photodegradation

APCI

IT

Transformation by nano zero valent iron in buffered solution

ESI

LTQ-FT ICR

Biodegradation by activated sludge from WWTP Direct photolysis

ESI

LTQ-Orbitrap

Photodegradation

Hydroxylated phenolic- or quinone-type compounds Isomerization, enolization, oxidation and hydration Hydrogenation, hydroxylation, radical coupling Dehydrogenation, saturation, ring-cleavage Hydroxylated and dialdehyde products

Comments on identification strategies

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Ion source ESI

IT

The structures of the intermediates were deducted on the basis of LC-MS data and the prior knowledge of photocatalytic mechanisms, with further identification 2 3 4 and verification by LC-MS , MS , and MS Referring to prior data of parallel studies

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E1, E2, EE2

Degradation/transformation type

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EE2

Major transformation products/reaction ·OH or ·OOH induced hydroxylation and decarboxylation reaction

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Parent compound

ESI

LTQ-Orbitrap

E1, hydroxylated and dialdehyde products

Degradation by cultivated algae

17α-TBOH, 17ß-

Dehydrogenation, hydroxylation

Biotransformation

ESI

LTQ-Orbitrap

TBOH, TBO E1-3S

Hydroxylation

Ozonation in river water

ESI

LTQ-Orbitrap

EE2

Halogenation and hydroxylation on the A-ring Carbonylation, hydroxylation

Chlorination in drinking water

ESI

LTQ-Orbitrap

Ozonation

ESI

LTQ-Orbitrap

Dehydrogenation, hydrogenation Presence of carboxylic acid and hydroxyl group Oligomers

Anaerobic biodegradation

APCI

LTQ-Orbitrap

Ozonation transformation

APCI

LTQ-Orbitrap

Laccase-catalyzed transformation in

ESI

Orbitrap

E1-3S Testosterone E1 E2

EP

E2, EE2

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1 2

38

LTQ-Orbitrap

Reference [56]

[73]

Structurally-informative fragmentation were made based n on MS ; NMR confirmation

[74]

Using a model compound 5,6,7,8-tetrahydro-2-naphthol

[75]

Identify assignment based on personal interpretation of spectra and referral to literature Accurate mass data were used for identification. MSn data were used for confirmation

[76]

Part of degradation products were confirmed using authentic standards Analogy to previously confirmed products; Part of the compounds were confirmed by NMR analysis

[77]

Degradation products of E2 and EE2 from the literature was compiled to build an in-house library which was used for identification By analogy to published data

[66]

[70]

[11]

[67]

Structure was postulated based on characteristic neutral losses Halogenated isotopic patterns helped the identification

[78]

Based on H/D exchange, MSn and accurate mass measurement strategies [4-14C]-Testosterone and NMR were used to assist identification Structural illustration based on MSn and deuteriumlabeled E1 (E1-D4) 13 C labeled E2 was used to identify cross-coupling

[68]

[58]

[79] [80] [69]

ACCEPTED MANUSCRIPT

EE2

Hydroxylation, hydrogenation, dehydrogenation, side-chain breakdown Oxidized to carboxylic acid,

products ESI

TQ

Confirmation using authentic standards if available

Ozonation

ESI

TQ

Dehydrogenation, hydrogenation, hydroxylation Hydroxylated, ring-cleavage products Oligomers

Biodegradation by bacteria from activated sludge from WWTP TiO2 photocatalysis in water matrix

ESI

TQ

ESI

TQ

Ligninase-mediated transformation

ESI

TQ

Desaturation, hydroxylation, dehydrogenation A-ring dehydrogenation product Hydroxylation, polymerization

Biodegradation by riverine sediment

ESI

TQ

Degradation by river water

ESI

TQ and Q-TOF

ESI

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Progesterone, norgestrel

darkness in humic acid-containing solution Biotransformation by microalgae isolated from lake water

Full scan to determine the precursor ions; precursor ion scan and product ion scan to determine the structure of major oxidation products; oxidation products of EE2 were derived from model compounds and were confirmed by either GC-MS or authentic standards if applicable Confirmation using authentic standards if available

[8]

[47]

Q-TOF

Byproducts were tentatively identified by referring to previous literature Molecular modeling was applied to postulate coupling reaction pathways Confirmation by GC/MS and using authentic standards if available EAWAG BBD prediction alongside literature references; confirmation by Q-TOF Confirmation using authentic standards if available

ESI

Q-TOF

Confirmation using standards

[83]

ESI

Q-TOF

[64]

ESI

Q-TOF

The use of CID, accurate mass measurement and the use of model compounds and 2H-labled E1 permitted the structure proposed Structures were proposed based on molecular accurate mass, chromatographic retention times, product ion spectra

Quantum chemical calculations were performed based on the density functional theory method to identify the potential reaction sites Three products were confirmed using authentic standards

[62]

α/ß-hydroxy acid

E2 E1 E1

E2, EE2

E1, E2, E3, EE2

Testosterone Androstenedione, testosterone

Ring-cleavage products, pyridinestrone acid Hydroxylation

Enzymatic catalyzed photolysis in humic acid-containing solution Biodegradation by degraders in activated sludge from STP Direct photolysis

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Progesterone

EP

E2-3S, E2-3G

Formation of hydroxyl, carbonyl, and carboxyl groups on A ring of parent compounds, ethinyl group intact Dimer, trimers, and tetramers

Peroxymonosulfate oxidation of steroid estrogens

Mn (Ⅲ) mediated phototransformation

ESI

Q-TOF

Dehydrogenation, hydroxylation Rearrangement, hydration, and more highly oxidized

Biodegradation by swine manure

ESI

Q-TOF

Direct photodegradation

ESI

Q-TOF

AC C

E2

SC

containing products

Progesterone, norgestrel EE2

[45]

39

[46]

[48] [49] [81] [82]

[51]

[84] [85]

ACCEPTED MANUSCRIPT

E1, testosterone

E1, E2, EE2 EE2

Dimer, trimer Two epimers

E1, E2, EE2

Dimer, trimer

Q-TOF

13

Photodegradation

ESI

Q-TOF

Biodegradation Ozonation

ESI APCI

Q-TOF Q-TOF

Quantum chemical calculations were performed based on the density functional theory method to identify the potential reaction sites Confirmation by NMR analysis Identification by referring to deuterated EE2.

[86] [87]

Biodegradation by bacterial strains and activated sludge from STP and river water Laccase catalysis Degradation by iron tetra-amido macro-cyclic ligand complex Transformation by peroxidase

ESI

HDMS-QTOF

Two metabolites were confirmed by NMR

[88]

ESI ESI

TOF TOF

Confirmation by NMR analysis

[59] [61]

ESI

TOF

C labeled-testosterone

[65] [63]

[60]

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E1, estrone; E2, 17ß-estradiol; EE2,17α-ethynylestradiol; 17α-TBOH, 17α-trenbolone; 17ß-TBOH,17ß-trenbolone; TBO, trendione; E1-3S, estrone-3-sulfate; E2-3G, 17ß-estradiol3-glucuronide; E2-3S, 17ß-estradiol-3-sulfate; STP: sewage treatment plant; WWTP, wastewater treatment plant; NMR: nuclear magnetic resonance; HDMS, high definition mass spectrometry (from Waters); IT: ion trap; LTQ-FT ICR, linear trap quadruple-Fourier-transform ion cyclotron resonance; LTQ-Orbitrap, linear trap quadruple-Orbitrap; TQ, triple quadruple; Q-TOF, quadruple time-of-flight; TOF, time-of-flight; GC-MS, gas chromatography-mass spectrometry; LC-MS; liquid chromatography-mass spectrometry; H/D exchange, hydrogen/deuterium exchange; CID, collision-induced dissociation; ESI, electrospray ionization; APCI, atmospheric-pressure chemical ionization

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3 4 5 6 7

Hydroxylation Two muconic acid derivatives with open phenolic ring structures Hydroxylated products, metacleavage product

ESI

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E2 EE2

Biotransformation

SC

E2

photoproducts A-ring cleavage product with 17 carbons E1, hydroxylated products, oligomers

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Testosterone

40

ACCEPTED MANUSCRIPT

Highlights:

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•

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•

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•

LC-MS capacities and overall MS-based steps for structural elucidation of novel metabolites of environmental steroid hormones were generalized. Fragmentation reactions and fragmentation pathways of protonated and deprotonated molecules of the metabolites were summarized. Versatile orthogonal techniques and intelligent spectral interpretation strategies used in literature for structural elucidation were specified. Up-to-date applications of LC-MS on structural elucidation of biotic and abiotic transformation products of environmental steroid hormones were provided.

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•