Collision Cross Section (CCS) Database: An Additional Measure to

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Collision Cross Section (CCS) database: an additional measure to characterize steroids Maykel Hernández-Mesa, Bruno Le Bizec, Fabrice Monteau, Ana M. García-Campaña, and Gaud Dervilly-Pinel Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05117 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018

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Analytical Chemistry

COLLISION CROSS SECTION (CCS) DATABASE: AN ADDITIONAL MEASURE TO CHARACTERIZE STEROIDS Maykel Hernández-Mesa†, Bruno Le Bizec†, Fabrice Monteau†, Ana M. García-Campaña‡, Gaud Dervilly-Pinel†* †

Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), INRA UMR 1329, LUNAM Université, Oniris, Nantes F-44307, France ‡Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, Granada E-18071, Spain ABSTRACT: Ion mobility spectrometry enhances the performance characteristics of liquid chromatography-mass spectrometry workflows intended to steroid profiling by providing a new separation dimension and a novel characterization parameter, the socalled collision cross section (CCS). This work proposes the first CCS database for 300 steroids (i.e. endogenous, including phase I and phase II metabolites, and exogenous synthetic compounds), which involves 1080 ions and covers the CCS of 127 androgens, 84 estrogens, 50 corticosteroids and 39 progestagens. This large database provides information related to all the ionized species identified for each steroid in positive electrospray ionization mode as well as for estrogens in negative ionization mode. CCS values have been measured using nitrogen as drift gas in the ion mobility cell. Generally, direct correlation exists between mass-to-charge ratio (m/z) and CCS because both are related parameters. However, several steroids mainly steroid glucuronides and steroid esters have been characterized as more compact or elongated molecules than expected. In such cases, CCS results in additional relevant information to retention time and mass spectral data for the identification of steroids. Moreover, several isomeric steroid pairs (e.g. 5β-androstane3,17-dione and 5α-androstane-3,17-dione) have been separated based on their CCS differences. These results indicate that adding the CCS to databases workflows increases selectivity, thus improving the confidence in steroids analysis. Consequences in terms of identification and quantification are discussed. Quality criteria and a construction of an inter-laboratory reproducibility approach are also reported for the obtained CCS values. The CCS database described here is made publicly available.

Steroids are organic compounds which play important roles in biochemical and physiological processes. Despite they are endogenous substances, steroids can be exogenously administrated for therapeutic purposes, but also in order to improve sport performance in both humans and animals in competitions, or as growth promoters in livestock for increasing the economic benefit. Doping in sports is worldwide forbidden as well as such practice for growth promoting purposes in food producing animals is also banned within European Union countries 1. Hence, the detection of misuse of these substances in samples containing naturally occurring steroids is a great challenge for doping control and food safety laboratories. Moreover, and considering the importance of such a large family of compounds in human health, the development of steroid fingerprints is gaining interest in the scientific community to increase the knowledge about the metabolic mechanisms and effects of diseases, chemical exposures, etc.2 Under this context, steroid profiling, also known as steroid fingerprinting, is appealing because it represents a potential and efficient tool for the overall characterization of steroids presented under their free or conjugated forms (e.g. glucuronide, sulfate), as well as esters. Consequently, their global analysis allows insight into metabolic variations, leading to the

discover of biomarkers related to diseases, chemical exposures, steroids misuse, etc.3,4 Therefore, reliable and precise analytical methods, also offering high sensitivity and selectivity, are increasingly required for the qualitative and quantitative determination of these compounds. However, the establishment of an analytical workflow for targeted steroid analyses or for steroid profiling is not exempt of drawbacks. Traditionally, gas chromatography-mass spectrometry (GC-MS) has been selected as analytical technique to this end, whereas nowadays the analysis of steroids is also carried out by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS)5. Nevertheless, these methodologies present several disadvantages such as the requirement of derivatization procedures in GC-MS methods6 or the low sensitivity observed when ESI is employed due to the poor ionization of some steroids7. In addition, the selectivity provided by GCMS and LC-MS methods may not be sufficient for those analytes which are poorly fragmented when tandem mass spectrometry (MS/MS) analyses are performed or for those cases where isomeric compounds are present. In fact, the recognition of isomeric steroids is considered of high value as certain steroids can be related to specific metabolic disorders and diseases8.

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Consequently, their characterization can be of high importance for the identification of specific biomarkers in metabolism studies. Moreover, certain isomeric steroids can be the result of exogenous administration of these substances9,10. Therefore, detailed structural information of steroids serving as targeted analytes is highly desired in order to ensure unequivocal measurements, as a positive result involves legal penalties in the case of their fraudulent use. Under this context, additional information to retention time and mass spectral data is needed to support the full characterization of steroids and their related metabolites present in biological matrices. In the last decades, ion mobility spectrometry (IMS) has reemerge as separation technique due to its high potential for achieving the separation of isomeric compounds as well as the isolation of targeted compounds from background noise11. Although the principles of IMS are known since the beginning of the 20th century12, it has not been until the commercialization of ion mobility spectrometers coupled to MS when this technique has caught the interest and attention of scientists from a wide range of fields13,14. Ion mobility mass spectrometry (IM-MS) is currently applicable to the separation and structural elucidation of large biomolecules (e.g. proteins15, lipids16, etc.) as well as for the analysis of small molecules such as residues and contaminants17. IMS is a gas-phase technique in which ionized compounds are separated based on their mass-to-charge ratio (m/z) and shape, under an applied voltage and at atmospheric or reduced pressure18. Separation occurs in a neutral carrier/buffer gas, usually He, N2 or CO2, although other gases can also be considered 19 . In drift time ion mobility spectrometry (DTIMS) and travelling wave ion mobility spectrometry (TWIMS), ions pass through the drift cell according to their averaged cross-sectional area, also referred as collision cross section (CCS) and represented as Ω20. In both modes, CCS can be related to their mobility in the drift cell through the Mason-Schamp equation21. Under this principle, compact molecules migrate faster than elongated molecules because they present lower CCS, being less susceptible to colloid with the molecules of buffer gas. Although the integration of IMS in traditional LC-MS workflows is very useful for increasing peak capacity by the introduction of an additional separation dimension22, its applicability can be extended beyond this purpose23. CCS is, per se, an intrinsic property of a given compound and is considered a highly specific molecular descriptor. Therefore, its determination may provide additional information to retention time and mass spectra for identification purposes, improving the confidence in analytical results24. CCS gives valuable complementary information about those molecules which show low correlation between their CCS and m/z. Nevertheless, the implementation of CCS as identification parameter is still in its early stages due to the lack of CCS databases on which we can rely on. In this sense, a wide number of molecules remain uncharacterized in terms of CCS. In the field of contaminants and residues analysis, first CCS databases have been recently reported25,26,27,28. Moreover, CCS databases remain fundamental in metabolomics where a wide number of compounds from different chemical families are detected and need to be identified 29,30,31,32. Indeed, this step is currently recognized as the bottleneck of metabolomics studies. Within this framework, this work describes the first large CCS database for steroids as well as discusses the potential of this parameter for the identification of isomeric steroids. This

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database covers a total of 300 compounds (free, conjugated and steroid esters) and includes androgens, estrogens, progestagens and corticosteroids. The generated database is publicly available to support steroid characterization. Until the moment, only a few tens of steroids have been characterized in terms of CCS28,33,34. Furthermore, additional CCS studies are extremely required in order to compare CCS values since it will improve compound identification and will give more confidence to the use of CCS as identification parameter35.

EXPERIMENTAL SECTION Chemicals and reagents. Standards of steroids were acquired from Steraloids (Newport, RI, USA), Sigma Aldrich (St. Louis, MO, USA), and National Measurement Institute (NMI, Pymble, Australia), and stock standard solutions of them (100 µg/mL or 1 mg/mL) were kept in methanol at -20°C. In general, standard solutions in 85/10/5 (%, v/v/v) water/methanol/acetonitrile (10 µg/mL) were considered for CCS characterization, except for steroid esters. Steroid esters (10 µg/mL) were injected in 50/10/40 (%, v/v/v) acetonitrile/methanol/water. Sodium formate (0.5 mM) in 90/10 (%, v/v) propan-2-ol/water was employed for mass calibration. Instrument calibration in terms of CCS was accomplished using the Major Mix IMS/ToF Calibration Kit (Ref. 186008113) provided by Waters (Manchester, UK). Leucine-Enkephalin (Waters) was employed as a lock mass standard (2 ng/mL in 50/50 (%, v/v) water/acetonitrile solution containing 0.2% (v/v) of formic acid).

Sample analysis. Flow injection analysis (FIA) was considered as analysis mode for the CCS characterization of steroids. In these experiments, an ACQUITY UPLC System from Waters was employed. However, a stainless steel flexible capillary for flow restriction was considered instead of a LC column. The capillary (2 m × 0.12 mm, with 1/16 in female connector on both ends) was supplied by Agilent Technologies. Other conditions (i.e. mobile phase, gradient program) are indicated in section ‘Sample analysis’ of Supporting Information (SI). IM-MS conditions. A hybrid quadrupole/traveling wave ion mobility/orthogonal acceleration time-of-flight geometry instrument (Synapt G2-S HDMS) from Waters (Manchester, UK) was employed for IM-MS analyses. The instrument was equipped with an ESI interface. For positive mode, ESI parameters were established as follows: capillary voltage: 3 kV, cone voltage: 31 V, source temperature: 120°C, desolvation temperature: 350°C, desolvation gas flow: 800 L/h, cone gas flow: 50 L/h, and nebulizer gas pressure: 6 bar. Gas flows of ion mobility instrument were fixed to 2 mL/min for trap gas, 180 mL/min for helium cell gas and 90 mL/min for IMS gas. Velocity and height of both StepWave were adjusted to 300 m/s and 5 V, respectively. Trap DC bias and IMS DC bias were fixed to 47 and 3. IMS wave velocity and height were established at 1000 m/s and 40 V, respectively. Furthermore, 311 m/s and 4 V were considered as trap cell parameters whereas 219 m/s and 4 V were selected as transfer cell parameters. For negative mode, ESI parameters were established as follows: capillary voltage: 2.5 kV, cone voltage: 10 V, source temperature: 120°C, desolvation temperature: 350°C, desolvation gas flow: 800 L/h, cone gas flow: 50 L/h, and nebulizer gas pressure: 6 bar. Gas flows of ion mobility instrument were fixed to 0.4 mL/min for trap gas, 180 mL/min for helium cell gas and 100 mL/min for IMS gas. Velocity and height of both StepWave were adjusted to 300 m/s and 5 V, respectively. Trap DC bias and IMS DC bias were

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Analytical Chemistry

fixed to 47 and 2, respectively. IMS wave velocity and height were established at 600 m/s and 40 V, respectively. For trap and transfer cell, these parameters were established at 311 m/s and 4 V, and at 219 m/s and 4 V, respectively. Quadrupole resolution was established to 12.5 for MS/MS analyses.

CCS measurements. CCS values were measured in nitrogen as drift gas. This is an important aspect of the reported CCS database since CCS does not only depends on the molecule of interest but also on the nature of the drift gas employed19,36. CCSs were obtained by the application of CCS calibration curves which were created using the Major Mix CCS calibration solution (Figure S1, SI). TWIMS calibration procedure has been previously described 37. Calibration curves covered a m/z range between 195 and 1013, and a CCS range from 138 to 306 Å2 for analyses in positive mode. In negative mode, calibration curves covered a m/z from 318 to 1082 and a CCS range from 130 to 322 Å2. In the case of negative mode and ions with m/z lower than 318, CCS values were estimated by extrapolation. CCS calibration was carried out considering singly charged ions, so CCS measurements were only applicable to singly charged ions. All the ionized species detected for each steroid were identified with a deviation lower than 5 ppm in relation to their exact mass. Experiments were performed in triplicate and each analyte was individually studied. Software and data analysis. Mass spectra and ion mobility spectra were analyzed using MassLynx 4.2 which includes DriftScope V.2.8 (Waters; Manchester, UK) and allows to obtain the CCS values. CCS was also predicted by the machine learning tool ‘MetCCS Predictor’38,39. Molecular descriptors required for CCS prediction were obtained from DrugBank database40. RESULTS AND DISCUSSION This work reports the first large CCS database for steroids which encompasses endogenous compounds (e.g. glucuronide and sulfate metabolites) and exogenous substances (e.g. steroids esters) as well as deuterated steroids. Moreover, this study investigates compounds and derivatives from different steroid families including C21 pregnanes (n = 39), C21 corticosteroids (n = 50), C19 androgens (n = 127) and C18 estrogens (n= 84). Detailed information of the investigated steroids, the ions observed under ESI conditions as well as their m/z and CCS can be found in SI (see CCS dataset for steroids). All steroids have been characterized considering positive ionization mode. Estrogens except estrogen-related esters have also been characterized under ESI(-) conditions in order to evaluate the consequences of protonation and deprotonation processes (in positive and negative ionization modes, respectively) on the structural properties of these steroids. This database does not only provides information about the most abundant ion observed for each compound, but it also reports the CCS of all ions identified for each steroid (e.g. [M+H]+, [M+Na]+, [2M+H]+ or [2M+Na]+, and fragments resulted from the loss of glucuronide groups, sulfate groups, ester moieties or molecules of water). In short, a total of 300 compounds involving 1080 ions (i.e. protonated and deprotonated molecules, fragment ions, cationic adducts, dimers) have been identified and characterized in terms of m/z and CCS. In an attempt of describing the whole set of observations and before any further discussion, Figure 1 shows a general view of the CCS and m/z of singly charged steroid-based ions identified in positive mode (n = 296). For positively charged

C18-based species (estrogens), m/z ranges between 257 and 1063 whereas CCS ranges from 165 to 325 Å2. In the case of positively charged androgens, m/z ranges from 251 to 957 and CCS ranges from 160 to 339 Å2. Positive ions of progestagens and corticosteroids present a m/z between 241 and 941 with a CCS between 157 and 322 Å2. On the other hand, CCS and m/z of singly charged ions observed for estrogens in negative mode (n = 53) are represented in Figure S2, SI. Singly charged estrogen negative ions, including dimers, cover a m/z range between 265 and 949 and involve CCSs from 162 to 314 Å2. Initially, this work discusses an integrated approach of the correlation existing between m/z and CCS for steroids. It also presents the first observations resulted from the study of the most abundant ion observed for each steroid. Based on this discussion, the structural differences between protonated ions and sodium adducts are further evaluated. Moreover, the structural differences existing between steroid families are assessed. As a following step, it is discussed the potential of CCS as identification parameter in the analysis of steroids and which provides useful quality data for the achievement of this purpose. Finally, it is shown the applicability of IMS for the separation of steroids based on their CCS differences.

Holistic view of the correlation between CCS and m/z for steroids. CCS is a molecular characteristic significantly correlated to m/z. Thus, a correlation between both parameters is expected for a group of compounds belonging to the same chemical family due to their structural similarities28,30. In this sense, steroids and their derivatives have been classified according to their number of carbons resulted from the basic steroid skeleton (Figure S3, SI). They have been mainly divided in estrogens (derived from estrane skeleton which is a molecular skeleton based on 18 carbons), androgens (derived from androstane skeleton based on 19 carbons) and progestagens and corticosteroids (mainly derived from pregnane skeleton which is based on 21 carbons). Under this consideration, it is observed a good correlation between CCS and m/z for estrogens analyzed in positive mode (Figure 1B) as well as in negative mode (Figure S2, SI) according to the regression models proposed. A high correlation is also obtained in the case of androgens (Figure 1A) and of progestagens and corticosteroids (Figure 1C). In general, CCS and m/z are non-linearly correlated 41,42, as observed in Figure 1. However, and in the majority of the cases, we have also observed that data were better fitted to a lineal model when narrow ranges of m/z were evaluated (Δm/z ~ 300)41. Consequently, this has been the criteria followed throughout the manuscript. Despite the correlation existing between both parameters for all the ions from the same steroid family (Figure 1), two groups of ions are initially well differentiated in all cases, as shown in Figure S2, SI for the ions resulted from the negative ionization of estrogens. On one hand, singly charged estrogen monomers present high linear correlation between CCS and m/z (r = 0.9817), showing low dispersion of data points. On the contrary, singly charged estrogen-based dimers present a worse linear correlation (r = 0.8426) and a broader spread of data. This fact is even more clear between singly charged monomers and dimers of androgens, estrogens, progestagens and corticosteroids occurred under ESI positive conditions (Figure S4, SI). It is observed that the group of steroid monomers covers a m/z range from 241 to 543 (except the sodium adduct of estradiol diglucuronide, m/z 647.2310) and a CCS range from 157 to 265 Å2. On the other hand, all steroid dimers show a m/z higher than 563 and a CCS higher than 245 Å2 (Figure 1D). In summary,

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Analytical Chemistry for steroid monomers, good linear correlation is observed between CCS and m/z for compounds with the same steroid molecular skeleton (r > 0.8545), whereas the correlation between both parameters is lower for steroid-based dimers (r < 0.7228). As a consequence of the lower correlation existing between CCS and m/z for steroid-based dimers, CCS may provide useful and additional information to m/z for the identification of steroids. However, the identification of steroids through their dimers can result in an ineffective strategy for their determination in biological samples. Not all steroids tend to form dimers or, in general, they are not often detected. Despite the higher correlation observed for monomers in comparison to dimers, the data dispersion around the fitted regression curves (Figure S4, SI) shows that several steroid monomers present a lower or greater CCS than the CCS predicted from the linear regression curves (e.g. nortestosterone decanoate, [M+Na]+, m/z 451.3182, CCS = 256.1, CCS greater than expected; epitestosterone glucuronide, [M+H]+, m/z 465.2483, CCS = 206.0, CCS lower than expected). As a consequence, such unexpected CCS values could involve deviating molecular shapes (more compact or more extended) from their steroid A) 350

counterparts and potentially provide additional information for the characterization and identification of these compounds. Focusing only on the most abundant ion of each steroid, high correlation (r = 0.9441) is obtained between CCS and m/z for these ionized species (Figure 2A). Figure 2A also shows that the majority of ions are located within an interval of ±4% from the mean CCS value. In general, sodium adducts resulted from the ionization of androgens (e.g. boldenone, CCS = 195.6 Å2; dianabol, CCS = 199.9 Å2; or boldenone acetate, CCS = 207.5 Å2) present a CCS greater than the CCS predicted from the fit (CCS deviation > +4%). On the other hand, a wide number of [M+H]+ species resulted from the ionization of corticosteroids (e.g. halcinonide, CCS = 203.2 Å2; fluocinolone acetonide, CCS = 201.5 Å2; or 11-dehydrodexamethasone, CCS = 188.1 Å2) show smaller structures than those expected from the linear model (CCS deviation < -4%). Both trends suggest that the structural properties (in terms of CCS) of protonated ions and sodium adducts should not be studied together. They also indicate that steroids possess different structural properties depending on the number of carbons of their steroid skeleton, with consequences on their spatial size as evidenced by the observed CCSs.

B) 350 y = 8.0605x0.5441 R ² = 0.9463

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Figure 1. Representation of CCS vs. m/z for all singly charged ions resulted from the ionization of: A) androgens (n = 127), B) estrogens (n = 80), C) progestagens and corticosteroids (n = 89) under ESI(+) conditions. D) Steroid-based dimers resulted from steroid ionization.

Differences between the CCS of protonated molecules and sodium adducts. Structural differences between protonated molecules and their related sodium adducts are clearly shown in Figure 2B for corticosteroids and progestagens. In order to evaluate these structural differences, CCS vs.

m/z has been represented for those corticosteroids and progestagens showing both ionized species (Figure S5, SI). After statistical treatment of the data (t-test, α = 0.05), a good linear correlation between CCS and m/z is achieved in both cases (r =

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0.9402 for [M+H]+ and r = 0.9595 for [M+Na]+; n = 42). Moreover, a CCS difference of 24.6 ± 1.6 Å2 is shown between the protonated molecule and the sodium adduct of the selected steroids. This difference indicates the contribution of the sodium ion to the structure of sodium adducts in comparison to the structural properties of protonated molecules. If we consider all steroids which result in [M+H]+ and [M+Na]+ when ESI(+) conditions are applied, the structural difference between both ions is 22.8 ± 5.9 Å2 (n = 143). However, 12.6% of the considered steroids present a difference much lower than this value.

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Analytical Chemistry

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Figure 2. Representation of CCS vs. m/z for: A) the most abundant ion observed for each steroid in ESI(+) mode, B) [M+H]+ and [M+Na]+ of progestagens and corticosteroids, and C) all the [M+H]+ species observed.

In general, sodium adducts of steroid glucuronides are more compact than expected based on the structural differences in terms of CCS between their related [M+H]+ and [M+Na]+ species. The same effect is observed for the sodium adducts of other steroids with larger moieties (e.g. steroid esters possessing functional groups of alkyl chains which contain phenyl groups) as indicated in Table S1, SI. The sodium adducts of these steroids probably tend to fold into more compact structures than smaller and more rigid steroids because they can better embrace the sodium atom. However, other steroids with shorter moieties than steroid glucuronides or esters also exhibit more compact structures than expected (i.e. 18-hydroxycortisol, medroxyprogesterone acetate, flugestone acetate, megestrol acetate). For these compounds, this effect could be due to the distribution of the charge in the molecule. These steroids possess several electronegative atoms (mainly oxygen), leading to a high density of negative charge in one part of the molecule and favoring the accommodation of the sodium atom in the charged specie. This effect is highly remarkable in the case of 11-ketoestrone which possesses a very low CCS difference between both [M+H]+ and [M+Na]+ ions (ΔCCS = 8.7 Å2), but does not possess large moieties which are able to be fold. Finally, steroid esters based on hemissucinate moieties present sodium adducts with smaller CCS than their related protonated molecules. It means that the hemissucinate group is highly folded into the molecule when a sodium ion is caught during the ionization process.

Structural differences of steroid families. According to Figure 2C, similar linear trends are observed between CCS and m/z for steroids with either androstane or estrane skeletons (molecular skeletons based on 19 and 18 carbons, respectively) whereas a different trend is shown for progestagens and corticosteroids which are based on a steroid skeleton of 21 carbons (pregnane skeleton). Apparently, the presence (androstane skeleton) or the absence (estrane skeleton) of a methyl group in position C10 of the steroid skeleton does not have any influence on the structural properties of these compounds in terms of CCS. On the contrary, Figure 2C suggests that the presence of an ethyl moiety (C20 and C21) in position C17 can influence the CCS of progestagens and corticosteroids in comparison to the CCS of androgens and estrogens. Therefore, it appears that CCS values cannot be extrapolated within closely structurally related steroid families. In other words, the CCS of androgens and estrogens has to be independently evaluated of the CCS of progestagens and corticosteroids in order to identify correctly those steroids exhibiting smaller or larger structures than expected. CCS as additional identification parameter. In general, the CCS of protonated ions are within the threshold of ±4% from the fit (Figure S6, SI). However, the CCS of certain steroids is out of the established deviation range. In the case of androgens and estrogens, large molecules such as the protonated molecule of testosterone esters (i.e. testosterone decanoate, CCS = 231.3 Å2; testosterone cypionate, CCS = 217.7 Å2; and testosterone enanthate, CCS = 215.0 Å2) and of 19-nortestosterone esters (i.e. 19-nortestosterone decanoate, CCS = 229.1 Å2; and 19-nortestosterone laurate, CCS = 237.2 Å2) present higher CCS than predicted from the linear regression curves. Consequently, this provides relevant and additional information for their identification when steroid analyses are carried out. On the other hand, the loss of water molecules from the protonated molecule leads to important CCS deviations from the fit for the [M-2H2O+H]+ specie of 19-noretiocholanolone glucuronide

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Analytical Chemistry

Additionally, other protonated species, which were not the most abundant species but resulted from the ionization of their related steroids with enough abundance for being detected at trace concentration levels, also present a CCS value out of the established threshold of ±4%. This emphasizes the importance of the CCS characterization of all steroid ions since it provides more information about the molecules and facilitates their identification. In this sense, CCSs smaller than expected were observed for dichlorisone acetate ([M-H2O+H]+, CCS = 192.6 Å2), epitestosterone glucuronide ([M+H]+, CCS = 206.0 Å2) and its deuterated-d3 form ([M+H]+, CCS = 206.0 Å2), and hydrocortisone hemisuccinate ([M-H2O+H]+, CCS = 194.7 Å2). This last case represents a particular case because, as described above, its protonated molecule represents a ionized specie more elongated than expected. The case of epitestosterone glucuronide (CCS = 206.0 Å2) is especially relevant since its epimer testosterone glucuronide presents a larger CCS (219.8 Å2). Thus, the protonated molecule of both compounds can be easily distinguished by their CCS. Finally, it has also been shown that the [M-H2O+H]+ fragment of pregnadiol isomers (CCS ~ 182 Å2) tends to be specie more elongated than predicted from the regression curve .

Due to the CCS deviations from the fit presented by some sodium adducts, epimers such as etiocholanolone glucuronide (CCS = 208.8 Å2) and epiandrosterone glucuronide (CCS = 232.0 Å2) or epitestosterone sulfate (CCS = 201.0 Å2) and testosterone sulfate (CCS = 220.2 Å2) can be differentiated. Furthermore, the [M+Na]+ specie of 6β-ol-etiocholanolone (m/z 329.2087, CCS = 190.3 Å2), which has been characterized as a more compact molecule than expected, can be distinguished by its CCS from positional isomers (i.e. 11β-hydroxyetiocholanolone, CCS = 199.3 Å2; and 16α-hydroxyetiocholanolone, CCS = 195.1 Å2) and from isobaric compounds (oxandranlone, CCS = 199.5 Å2). A) 280

y = 0.2123x + 131.21 R ² = 0.6683

270 260 250 240 C C S (Å2 )

and 19-norandrosterone glucuronide (CCS = 191.2 and 194.6 Å2, respectively). Furthermore, fluoxymesterone ([M+H]+, CCS = 178.3 Å2) and oral turinabol ([M-H2O+H]+, CCS = 172.9 Å2) result in CCS deviations from the fit of around -4%. The presence of halogen atoms in these molecules (fluorine and chlorine, respectively) close to a hydroxyl and a keto group (high electronegative groups due to the oxygen) probably induces a high density of negative charge in one part of the molecule. Consequently, the proton is closely attached to the molecule and leads to more compact molecules in comparison to other steroids. Regarding corticosteroids and progestagens, molecular structures more elongated than expected (CCS deviation of +4% from the fit) can be attributed to the protonated molecules of 17-caproxyprogesterone (CCS = 215.8 Å2), hydrocortisone hemisuccinate (CCS = 216.2 Å2) and diflucortolone pivalate (CCS = 219.8 Å2).

230 220 210 200 190

expected CCS ± 4% androgens estrogens

180 170 230

B) 280

280

330

380

430

480

580

630

680 m /z

260 250 240 230 220 210

200

expected CCS ± 4%

190

corticosteroids

180

On the other hand, the sodium adduct was the most abundant ion observed for several steroids analyzed under positive ionization conditions. Lower correlation between CCS and m/z is obtained when [M+Na]+ and [M-H+2Na]+ species are analyzed (r < 0.8261; Figure 3) in comparison to the high correlation shown by protonated species (r > 0.9545; Figure S6, SI). Therefore, it seems that CCS is more promising as identification parameter for sodium adducts than for protonated molecules. A total of 133 sodium adducts have been detected for 85 androgens and 44 estrogens, including [M+Na]+ and [M-H+2Na]+ species. The linear correlation coefficient between CCS and m/z is 0.8175 for these ions, and 25% of them show a CCS deviation higher than ±4% from their expected value. A list of the compounds characterized as smaller or larger molecules than expected is included in Table S2, SI. In general, sodium adducts of steroid glucuronides are more compact ions than expected whereas steroid esters, except of estradiol diacetate epimers, are more elongated ions than predicted from the regression curve. Moreover, sodium adducts of smaller steroids which possess several electronegative atoms (i.e. O, F, Cl) but not large moieties, are also more compact than expected (i.e. 4-fluoro-19nortestosterone, 4-chlorotestosterone, estetrol and 11-ketoestrone). A possible chelation between the sodium ion and the molecules of estetrol and 11-ketoestrone is suggested.

530

y = 0.1588x + 149.90 R ² = 0.6824

270

CCS (Å2 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 10

progestagens

170 230

280

330

380

430

480

530

580

630

680 m /z

Figure 3. Representation of CCS vs. m/z for the sodium adducts observed in positive ionization mode for: A) androgens and estrogens, and B) progestagens and corticosteroids.

On the other hand, similar correlation between CCS and m/z is also observed for sodium adducts of corticosteroids and progestagens (r = 0.8261), but resulting in lower data dispersion. Consequently, very few compounds present CCS deviations equal or higher than ±4% from the fit (only 13% of 75 steroids). Three [M+Na]+ species of corticosteroids (i.e. budesonide, diflucortolone pivalate and flumethasone 21-pivalate) exhibit CCS that are significantly greater than the average. On the contrary, a molecular structure more compact than expected is attributed to one progestagen (i.e. 17-hydroxypregnenolone) and six corticosteroids (i.e. triamcinolone 16,21-diacetate, dexamethasone 21-phosphate, prednisolone 21-hemisuccinate, hydrocortisone hemisuccinate, cortisol 21-acetate and 18-hydroxycortisol). In general, all these compounds possess several electronegative atoms in the same part of the molecule. Thus, the sodium ion is closely attached and it leads to more compact molecules than in the case of other steroid adducts with similar m/z. Furthermore, CCS demonstrates to be a powerful tool for

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Analytical Chemistry

distinguishing positional isomers such as 18-hydroxycortisol (CCS = 200.4 Å2) and 6β-hydroxycortisol (CCS = 211.4 Å2). Finally, several estrogens resulted in [M+H]+ and [M-H]- species when they were ionized under positive and negative conditions, respectively. A difference lower than 2% has been mainly observed between the CCS of both [M+H]+ and [M-H]- ions resulted from the same estrogen. Consequently, both protonation and deprotonation processes have not effect on the structural properties of these molecules in terms of CCS. Figure S7, SI shows the representation of CCS vs. m/z for all estrogens exhibiting both ionized species. On the contrary, the [M+Na-2H]specie of estradiol diglucuronide (m/z 645.2154, CCS = 240.1 Å2) present a CCS much lower than its related protonated form ([M+Na]+, m/z 647.2310, CCS = 264.4 Å2) and, as a consequence, a CCS deviation from its expected CCS value. Probably, during negative ionization, a proton is lost from each glucuronide group and, as a result, both moieties are folded around a sodium ion leading to a more compact molecule.

Quality criteria for CCS measurements. In order to evaluate the accuracy and reproducibility of the reported CCSs, the CCS of testosterone ionized species were monitored within five months. Figure S8, SI shows the variability of CCS for [M+H]+ of testosterone during this time. Testosterone has been selected as representative steroid because its CCS has been already reported and accuracy can be evaluated based on these previous results. According to our experimental results, the CCS of testosterone [M+H]+ ion is 173.0 Å2 (n = 28). All CCS measurements performed within the five months involved a deviation lower than 0.6% from the average value, accomplishing with the tolerance of 2% currently accepted for CCS measurements37. Moreover, CCS was measured at different concentration levels (standard solutions from 0.5 to 10 µg/L analyzed by FIA) and wave velocities (500 to 1250 m/s) in order to demonstrate the robustness of CCS measurements. A deviation lower than 0.6% from the average CCS was achieved in both studies. These results ensure that CCS can be measured with precision through the time at different operational conditions. In this sense, it is important to reach high precision in CCS measurements if CCS pretends to be further implemented as identification parameter for instance in hormone monitoring plans. In addition, the CCS of testosterone [M+H]+ specie has been predicted by MetCCS Predictor, resulting in a CCS value of 174.4 Å2. Therefore, our measurements for the CCS of testosterone are within a tolerance threshold of 2%37 when is compared with the predicted CCS (Figure S8, SI). The experimental CCS of testosterone (173.0 Å2) has also been compared with the value previously reported by Chouinard et al.34 using DTIMS (174.5 Å2) and by Hines et al.28 using TWIMS (170.6 Å2), resulting in a CCS deviation lower than 1.4% in both cases. Moreover, MetCCS Predictor has been employed for the estimation of the CCS those steroids resulting in [M+H]+ under positive ionization conditions and whose molecular descriptors (i.e. polar surface area, rotatable bond count, etc.) have been previously described. These molecular descriptors are required for CCS prediction by the abovementioned machine learning tool. CCS has been predicted for 16 androgens, 11 estrogens, 20 corticosteroids and 7 progestagens (Table S3 and Table S4, SI). Predicted CCS values are reported for the [M+H]+, [MH2O+H]+, [M+Na]+ and [M-H]- species observed for these compounds. In general, the predicted values are similar to the experimental CCS of the [M+H]+, [M-H2O+H]+ and [M+H]- ions

identified for androgens, estrogens and progestagens. A deviation lower than 2% between both values has been obtained for 74.2% of the observed ionized species, whereas this difference is higher than 4% for only 10.6% of all cases. However, CCS prediction by MetCCS should not be applied to steroid sodium adducts. A deviation greater than 4% has been observed between the experimental CCS and the predicted value for 86.4% of the investigated steroids. It has been observed that MetCCS assigns an average difference of 6.5 ± 0.3 Å2 (n = 22) between the CCS of protonated molecules and sodium adducts whereas it was experimentally observed that this difference is 22.8 ± 5.9 Å2. On the other hand, CCS deviations greater than 2% are normally observed for corticosteroid ions, so the CCS prediction is not as accurate as in the case of other steroid families. Inter-laboratory reproducibility studies are highly required in order to evaluate if a tolerance threshold of 2% is well established for CCS measurements or can be reduced to 0.5% as recently shown by Stow et al.43. This will allow to take an important decision about the implementation of CCS as identification parameter in analytical workflows. As an approach to the evaluation of inter-laboratory reproducibility, the CCSs reported in our database have been compared with the CCS values already published (Table S5, SI). In general, CCS differences lower than 2% have been found for 82.3% of the CCSs previously measured by DTIMS34 and for 90.6% of the CCSs previously reported by TWIMS28. However, differences greater than 0.5% have been obtained for 96.1% of the CCS values evaluated. In this sense, new developments in instrumentation and the establishment of normalized CCS calibration strategies when using TWIMS are of great importance for improving the precision of CCS measurements. In this regard, when TWIMS is selected as IMS mode, the instrument has to be calibrated prior to CCS measurements44. Therefore, we have also investigated the frequency with which CCS calibration has to be performed since this information results fundamental for the implementation of this technique in routine laboratories. In our case, and within our quality procedure, both MS and CCS calibration are sequentially performed when system calibration is required. CCS deviation and mass error in relation to exact mass were evaluated for testosterone before performing CCS measurements in order to decide if system calibration was required or not. A maximum CCS deviation of 2% and a maximum mass error of 5 ppm were established as quality control thresholds. It was observed that system calibration was usually required because mass tolerance limit was overpassed rather than for not fulfilling CCS criteria. It involved to carry out system calibration each 7 to 10 days. Moreover, and as a proof of concept, CCS measurements have been carried out in urine samples from calves treated with nandrolone10. Urine samples were submitted to hydrolysis and solid phase extraction prior to LC-IM-MS analysis. Nandrolone ([M+H]+) was detected observing an average difference of 1.1 ppm and 0.2% from its exact mass and the CCS reported by the database, respectively. This preliminary study shows that CCS is not affected by the matrix. However, a more in-depth study should be carried out in order to evaluate the effects of biological matrices (e.g. urine, serum, etc.) on the CCS of a wide range of steroids.

Steroids separation based on their CCS. Steroids usually tend to result in singly charged species when they are ion-

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Analytical Chemistry

LC-MS workflows. Epitestosterone glucuronide ([M+H]+, CCS = 206.0 Å2) has been separated from its isomer testosterone glucuronide ([M+H]+, CCS = 219.8 Å2), which differ in the α and β positions of the glucuronide group at C17 (Figure S10B, SI). Indeed, it can be expected the separation of isomers with structural differences related to large moieties. Nevertheless, the separation of molecules with smaller structural differences can be also achieved. Androstenedione and its epimer (Figure 4B) as well as 19-noretiocholanolone glucuronide and 19-norandrosterone glucuronide (Figure S10C, SI) have been separated by IMS. In both cases, molecular pairs only differ in the α and β positions of the hydrogen atom at C5. In general, the separation of molecules with close CCS values greatly depends on the resolving power of the IMS instrument. Ion mobility spectrometers with high resolving power are currently available and IMS separations can be achieved for analytes with CCS differences of 0.5%45.

ized by ESI under both operational modes (positive and negative conditions). However, several doubly charged ions (i.e. [M2H]2-, [M-C6H8O6-2H]2-) have been observed for estrogens ionized under negative conditions and they are perfectly separated from singly charged ions (Figure S9, SI). In this sense, it has also been observed the separation of doubly charged dimers from their related singly charged monomers for estradiol disulfate and estradiol diglucuronide (Figure 4A), which provides crucial information for correct quantification of compound concentration. On the other hand, IMS provides a powerful tool for the separation of steroids possessing similar m/z but different CCS such as 4-fluoro-19-nortestosterone ([M+H]+) and trenbolone ([M+Na]+) (Figure S10A, SI). Moreover, steroids with smaller or larger molecular structures than expected can be separated from its isomers by IMS, enhancing the current limitations of

Signal intensity

A)

OH

%

[2 M-2H]2m /z = 623.2334

HO

[M-H]m /z = 623.2334 CCS = 255.0 Å2

50

O HO HO

O

OH OH

O O

O

HO

O OH

2

B) Signal intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4

6

%

50

[M+H]+ m /z = 289.2162 C C S = 167.0 Å2

O 2

H

H

O

O H

4

HH

[M+H]+ m /z = 289.2162 CCS = 176.9 Å2

H

6 -

Drift time (milliseconds)

O H

HH

HH

H

12

O

O H

HH O H

10

8

O

8

Drift time (milliseconds)

2-

Figure 4. Ion mobility spectra related to the separation of: A) [M-H] and [2M-2H] species of estradiol diglucuronide, IMS conditions: wave velocity, 600 m/s; wave height, 40 V; and B) 5β-androstane-3,17-dione and 5α-androstane-3,17-dione. IMS conditions: wave velocity, 1000 m/s; wave height, 40 V.

Finally, IMS has also shown its potential to distinguish between molecules of the same compound with different protonation sites, also known as protomers28,46. Similar behavior has been observed for the [2M+Na]+ ion of 1,6-didehydroprogesterone (m/z 643.3758). Up to three different sodium dimers were identified and separated for this compound. This hypothesis was confirmed by collision induced dissociation (CID) experiments in the transfer cell (Figure S11, SI).

CONCLUSIONS New trends in global and/or untargeted analyses require additional information for the identification of a wide range of compounds. Within this framework, we report the first large CSS database for supporting steroid profiling as well as for giving more confidence to the results obtained in the routine analysis of steroids. In total, 300 steroids have been characterized in terms of CCS considering nitrogen as drift gas. Despite high correlation has been found between CCS and m/z for steroid monomers, several compounds have shown to be more compact or elongated molecules than other steroids possessing equal or similar m/z. For these steroids, CCS provides useful complementary information for their identification. A higher number of molecules with CCSs deviated from the expected CCS values are observed when sodium adducts are examined in comparison

to protonated molecules. Steroids with large moieties such as steroid glucuronides and steroid esters are more likely to lead to smaller or larger structures than those predicted from the large set of steroids. However, the [M+Na]+ of smaller molecules such as 11-ketoestrone or estetrol has also resulted in a more compact molecule. On the other hand, inter-laboratory studies are highly required for the implementation of CCS as identification parameter in steroid analyses involving legal consequences. In this sense, our inter-laboratory study approach has delivered promising results, but they have to be interpreted carefully. The threshold of 2% for CCS measurements has been accomplished for the majority of the assayed steroids but not for all of them. Furthermore, these studies should be accompanied by the evaluation of the influence of a wide range of matrices on the CCS of steroids.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. CCS dataset for steroids (PDF) Steroid skeleton, theoretical CCS values, CCS comparison with CCS values previously reported, ion mobility separation of steroid pairs (DOCX)

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Analytical Chemistry All authors have given approval to the final version of the manuscript.

AUTHOR INFORMATION Corresponding Author * Fax: +33 2 40 68 78 78. E-mail address: [email protected] (G. Dervilly-Pinel) and [email protected].

ACKNOWLEDGMENT Maykel Hernández-Mesa wishes to express his appreciation to Fundación Ramón Areces (Spain) for a postdoctoral fellowship.

Author Contributions

“for TOC only” CCS (Å2 ) 350

300

[M+Na]+ m/z = 475.2307 CCS = 205.4 Å2

e s trogens

[M+Na]+ m/z = 475.2307 CCS = 223.4 Å2

250

200 40.0

60.0

80.0

Drift time (bins)

150 200

400

600

800

1000

1200 m/z

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