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A constant ion loss method for the untargeted detection of bissulfate metabolites Malcolm D McLeod, Christopher C. Waller, Argitxu Esquivel, Georgina Balcells, Rosa Ventura, Jordi Segura, and Oscar J Pozo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03671 • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 20, 2016

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Analytical Chemistry

A constant ion loss method for the untargeted detection of bissulfate metabolites Malcolm D. McLeoda, Christopher C. Wallera, Argitxu Esquivelb,c, Georgina Balcellsb,c, Rosa Venturab,c, Jordi Segurab,c, Óscar J. Pozob* a

Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia

b

Bioanalysis Research Group. IMIM, Hospital del Mar, Doctor Aiguader 88, 08003 Barcelona, Spain

c

Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain KEYWORDS: Constant ion loss, triple quadrupole, bis-sulfates, steroids, mass spectrometry, metabolism

ABSTRACT: The untargeted detection of phase II metabolites is a key issue for the study of drug metabolism in biological systems. Sensitive and selective mass spectrometric (MS) techniques coupled to ultrahigh performance liquid chromatographic (UHPLC) systems are the most effective for this purpose. In this study, we evaluate different MS approaches with a triple quadrupole instrument for the untargeted detection of bis-sulfate metabolites. Bis-sulfates of 23 steroid metabolites were synthesized and their MS behavior was comprehensively studied. Bis-sulfates ionized preferentially as the di-anion ([M2H]2-) with a small contribution of the mono-anion ([M-H]-). Product ion spectra generated from the [M-2H]2- precursor ions were dominated by the loss of HSO4- to generate two product ions i.e, the ion at m/z 97 (HSO4-) and the ion corresponding to the remaining mono-sulfate fragment. Other product ions were found to be specific for some structures. As an example, the loss of [CH3+SO3]- was found to be important for several compounds with unsaturation adjacent to the sulfate. Based on the common behavior of the bis-sulfate metabolites two alternatives were evaluated for the untargeted detection of bis-sulfate metabolites (i) a precursor ion scan method using the ion at m/z 97 and (ii) a constant ion loss (CIL) method using the loss of HSO4-. Both methods allowed for the untargeted detection of the model compounds. Eight steroid bis-sulfates were synthesized in high purity in order to quantitatively evaluate the developed strategies. Lower limits of detection (2-20 ng/mL) were obtained using the CIL method. Additionally, the CIL method was found to be more specific in the detection of urinary bis-sulfates. The applicability of the CIL approach was demonstrated by determining progestogens altered during pregnancy and by detecting the bis-sulfate metabolites of tibolone. 1.

INTRODUCTION

In recent years, metabolomic approaches have become a key tool in the identification and discovery of potential markers in diverse fields such as disease diagnostics, doping control or forensic analysis [1-3]. Whereas targeted metabolomics is preferred for the analysis of known metabolic pathways altered by a specific status e.g. by a disease, untargeted approaches are required for the discovery of new or unexpected markers. Universal analyzers are obviously preferred for untargeted detection. Thus, untargeted methods are commonly based on NMR or MS detection [4-6]. In the case of MS approaches, untargeted methods frequently take advantage of the analytical properties of high resolution analyzers such as TOF or Orbitrap systems [7]. The acquisition in full scan mode allows for the detection of potentially any compound present in the sample with the main limitation being the ionization of the molecule. However,

these untargeted strategies also have additional limitations. Thus, the pre-concentration of the sample is normally avoided in order to minimize potential loss of the unknown metabolites. Additionally, analytes which require a specific chromatographic design can be difficult to detect. Finally, the elucidation of the structure of the metabolite and the metabolic pathway involved remain the main bottlenecks for untargeted metabolomics [6,8,9]. The triple quadrupole analyzer is the gold-standard for targeted methods due to its high sensitivity and specificity when working in the selected reaction monitoring (SRM) mode. Additionally, triple quadrupoles also play an important role in a range of untargeted approaches. The wide variety of triple quadrupole scan modes have proven useful in several untargeted scenarios; among them the detection of unknown markers with a predicted structure. In this context, the value of precursor ion and/or neutral loss scan methods have been widely demonstrated for the

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detection of biomolecules such as phosphopeptides [10], phospholipids [11] and glycosides [12]. Additionally, these open scan methods have been used for metabolic studies [13-15]. Therefore, triple quadrupole instruments are valuable for untargeted approaches mainly if the nature of the potential markers can be, to some extent, predicted. An example of this situation is the detection of phase II metabolites. The detection of phase II metabolites is important in many areas of biology. One of the groups of substances in which phase II metabolism plays a critical role is the steroid hormones. Several phase II steroid metabolites have been reported as adequate markers for doping control analysis [16-19], or serve as markers of pathologies [20]. Additionally, some phase II metabolites are biologically active including, some steroid sulfates that have been shown to modulate brain function, [21]. In the case of bissulfates, their elevated serum concentrations have been associated with disease [22] and a pregnenediol bis-sulfate has been found among a set of metabolites showing associations with all-cause mortality [23]. Several analytical methods based on triple quadrupole analysis have been reported for the untargeted detection of both unconjugated steroids [24,25] and phase II metabolites such as glucuronides [26,27], sulfates [28-30], cysteinyl [28,31] or glutathione [32] conjugates. However, methods for the detection of other phase II metabolites such as bis-sulfates are currently unavailable. The goal of this study was to evaluate the usefulness of triple quadrupole instruments for the untargeted detection of bis-sulfate metabolites. For this purpose, 23 steroid bis-sulfates were synthesized and characterized by MS. Based on the common behavior of the model compounds, two alternative untargeted approaches were proposed. The strategy using a constant ion loss (CIL) method was found to be the most sensitive and specific one. The suitability of this approach has been evaluated in two scenarios: endogenous steroids in pregnancy and the administration of the anabolic androgenic steroid tibolone.

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2.2. Instrumentation 2.2.1 UHPLC-MS/MS The study was carried out using a triple quadrupole (XEVO TQMS) mass spectrometer equipped with electrospray ionization source (ESI) interfaced to an Acquity UPLC system for the chromatographic separation (all from Waters Associates, Milford, Massachusetts, USA). Drying gas as well as nebulising gas was nitrogen. The desolvation gas flow was set to approximately 1200 L/h, and the cone gas flow was 50 L/h. A cone voltage of 30 V, and a capillary voltage of 3.0 kV were used in negative ionization mode. The nitrogen desolvation temperature was set to 450 °C, and the source temperature was 120 °C. The UHPLC separation was performed using an Acquity UPLC CSH Phenyl-hexyl column (2.1 × 100 mm i.d., 1.7 μm) (Waters Associates), at a flow rate of 300 μL/min. Water and acetonitrile:water (90:10) both with formic acid (0.01% v/v) and ammonium formate (25 mM) were selected as mobile phase solvents. A gradient program was used; the percentage of organic solvent was linearly changed as follows: 0 min, 15%; 0.5 min, 15%; 10 min, 40%; 10.5 min, 40%; 11 min, 15%; 13 min, 15%. The total analysis time was 14 min. The study of the ionization of the steroid bis-sulfates was performed by scanning the m/z range from 100 to 900 in negative mode. The fragmentation of each model compound was studied by using product ion scan methods at three collision energies (10, 20, and 30 eV) in the m/z range 20-600. For the precursor ion scan method of m/z 97, a scan range from 199-274 was selected (scan rate 5 s-1) with a collision energy of 30 eV. For the constant ion loss (CIL) strategy, dwell times of 8 ms and collision energies of 15 eV were selected for each ion transition. 2.2.2 Additional instrumentation 1

2.

H and 13C nuclear magnetic resonance (NMR) spectra were recorded using either Bruker Ascend 400 MHz or Bruker Avance 400 MHz Spectrometers.

MATERIALS AND METHODS

2.1. Reagents and chemicals The structure, molecular weight and commercial source of steroid standards used as model compounds are listed in Figure S1 (supplementary information). Chemicals and solvents including sulfur trioxide pyridine complex (SO3.py) and 1,4-dioxane were purchased from Sigma– Aldrich (Castle Hill, Australia) and were used as supplied unless otherwise stated. MilliQ water was used in all aqueous solutions. N,N-Dimethylformamide (DMF) and aqueous ammonia solution were obtained from ChemSupply (Gillman, Australia). Formic acid was obtained from Ajax Chemicals (Auburn, Australia). Solid-phase extraction (SPE) was performed using Waters (Rydalmere, Australia) Oasis weak anion exchange (WAX) 6cc cartridges.

Melting points were determined using a SRS Optimelt MPA 100 melting point apparatus and are uncorrected. For synthesis characterization, low-resolution mass spectrometry (LRMS) and high-resolution mass spectrometry (HRMS) were performed using positive electron ionisation (+EI) on a Micromass VG Autospec mass spectrometer or negative electrospray ionization (–ESI) on a Micromass ZMD ESI-Quad, or a Waters LCT Premier XE mass spectrometer. Detailed information is given in Supporting Information (Experimental S1 and S2). 2.3. Synthesis of steroid bis-sulfates

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The synthesis steroid bis-sulfates employed a previously reported method (Figure 1) [33]. A total of 23 steroids were prepared as model compounds were subjected to comprehensive characterization by MS. To generate quantitative extraction recovery and limit of detection data eight bis-sulfate reference materials (A1, A3, A5, A15, A17, A18, A24, A25) were characterised to demonstrate identity and purity. Details of the synthesis, reaction conversion and characterization data are reported in the Supplementary information together with copies of the 400 MHz 1H NMR, 100 MHz 13C NMR, and LRMS. (See Experimental section S3 supplementary information) The identity of bis-sulfate compounds used in this study were: 5α-androstane-3α,17β-diol bis-sulfate (A1), 5βandrostane-3β,17β-diol bis-sulfate (A2), 5α-androstane3β,17β-diol bis-sulfate (A3), 5β-androstane-3α,17β-diol bissulfate (A4); androst-5-ene-3β,17β-diol bis-sulfate (A5), androst-5-ene-3α,17β-diol bis-sulfate (A6), 5α-estrane3β,17β-diol bis-sulfate (A7), 5β-estrane-3α,17β-diol bissulfate (A8), 5α-estrane-3α,17β-diol bis-sulfate (A9), 7αmethyl-19-norpregn-5(10)-en-20-yne-3β,17β-diol bissulfate (A10), 5β-pregnane-3α,20α-diol bis-sulfate (A11), pregn-5-ene-3α,20α-diol bis-sulfate (A12), 16α-hydroxydehydroepiandrosterone (DHEA) bis-sulfate (A13), 16αhydroxy-etiocholanolone bis-sulfate (A14), 16α-hydroxyandrosterone bis-sulfate (A15), 11β-hydroxyetiocholanolone bis-sulfate (A16), 11β-hydroxyandrosterone bis-sulfate (A17), 16α-hydroxy-testosterone bis-sulfate (A18), 2α-hydroxy-testosterone bis-sulfate (A19), 11β-hydroxy-testosterone bis-sulfate (A20), 6βhydroxy-testosterone bis-sulfate (A21), 6β-hydroxyetiocholanolone bis-sulfate (A22), 6β-hydroxyandrosterone bis-sulfate (A23), estradiol bis-sulfate (A24) and 11β-hydroxy-epiandrosterone bis-sulfate (A25).

For the establishment of the limit of detection (LOD), reference materials at increasing concentrations (1, 2, 5, 10, 20, 50, 100 and 200 ng/mL) were extracted as previously described and analyzed by the two approaches investigated. The first concentration at which the analyte could be detected with a signal to noise ration greater than 3 was selected as the LOD of the method. 2.6. Urine samples The applicability of the method for the detection of endogenous bis-sulfates was evaluated by analyzing ten spot urine samples collected from healthy volunteers (5 males, 5 females, ages 23-45). In the evaluation of pregnancy effect, urine samples from three healthy female volunteers (ages 34-35) were collected before being pregnant and in the middle pregnancy (weeks 15-20). In all three cases, fetus growth throughout the course of the pregnancy was within the normal ranges. For the study of tibolone metabolism, urine samples from an excretion study were used. In this study, a single dose of 2.5 mg of tibolone (Boltin, Schering-Plough, Madrid, Spain) was orally administered to a healthy human volunteer (male, age 56). Urine samples collected before and eight hours after administration were used for the study. Ethical approval for these studies has been granted by the Ethical Committee of our Institute (Comité Ètic d’Investigació Clínica CEIC-Parc de Salut Mar, Barcelona, Spain). All subjects participating in the study gave their written informed consent prior to inclusion. 3.

RESULTS AND DISCUSSION

3.1. Synthesis of steroid bis-sulfates 2.4. Sample treatment Urine treatment was based on a previously reported procedure for the separation of free, glucuronide and sulfate fractions [34]. Briefly, a 3 mL aliquot of urine was passed through a pre-conditioned Oasis WAX SPE cartridge. After a washing step consisting of 3 mL of sodium phosphate buffer (0.05 M, pH 7.5), 3 mL of water, and 3 mL of methanol : ethyl acetate : formic acid (50 : 50 : 1, v/v/v), bis-sulfates were eluted using 3 mL of methanol : ethyl acetate : diethylamine (50 : 50 : 1, v/v/v). After evaporation of the elution solvent, the extract was reconstituted in 100 µL of water and 10 µL was injected into the UHPLC-MS/MS system. 2.5. Extraction recovery and LOD Extraction recovery was calculated by comparing the responses of the eight reference materials added before extraction and after the extraction. Experiments were performed in triplicate at two different concentration levels (200 ng/mL and 1 µg/mL).

The synthesis of steroid bis-sulfates was performed by sulfation with sulfur trioxide-pyridine complex, followed by solid-phase extraction purification (Figure 1) [33]. A total of 23 model bis-sulfates with a range of stereochemistries and substitution patterns were prepared to study MS behavior (See supplementary information Figure S1). Sulfate esters that can eliminate to form stable cation intermediates such as those derived from allylic or tertiary alcohols have been reported to be unstable [35]. In our experiments, bis-sulfate synthesis was normally unsuccessful for steroid diols containing these structures. In addition, eight bis–sulfate conjugates were prepared in high purity and characterised as reference materials in order to obtain extraction recoveries and limits of detection. Among them, two bis-sulfates (A24 and A25), which were not included as model compounds, were prepared to serve as an independent check of the suitability of the developed open screening methods. For these eight examples, synthesis occurred in high conversion (≥ 98%) as determined by integration of the 400 MHz 1H NMR spec-

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tra. Interestingly the sulfation of estradiol cleanly afforded estradiol bis-sulfate in contrast to earlier reports [33, 36]. We explain the greater reactivity reported in this study to the use of freshly procured sulfur trioxidepyridine complex. 3.2. MS behavior of steroid bis-sulfates 3.2.1. Ionization As expected, bis-sulfates were predominantly ionized in negative mode. Two main species were observed in the negative ion mass spectra of all model compounds (Table 1). The negative ion mass spectra of most analytes were dominated by the di-anion [M-2H]2- whereas the monoanion [M-H]- normally ranged from 5% to 62%. As an example, Figure 2a shows the mass spectrum of bissulfate A4 showing [M-2H]2- as base peak. The main exceptions for this behavior were A18 and A20 for which the [M-H]- was found as the base peak of the spectrum. A general trend was observed when comparing the [MH]- abundance with the bis-sulfate structure. Thus, bissulfates A18, A20, A22 and A23 where the sulfate esters were separated by four or fewer carbon atoms had a relatively abundant [M-H]- (52%-100%) whereas those analytes where the sulfate esters were separated by a greater distance showed less abundant [M-H]- (5%-45%). This fact presumably arises as generation of the di-anion in analytes where the sulfate esters are close in space gives rise to significant charge repulsion. 3.2.2. Collision Induced Dissociation After selecting the [M-2H]2- as precursor ions, the product ion spectra of the model bis-sulfates were dominated by two ions (Figure 2b) coming from the same fragmentation pathway (Figure 2c). One of these ions (m/z 97) corresponded to HSO4- and is common to the fragmentation of mono-sulfates [28,37]. The other ion was generated after the loss of HSO4- and its m/z depended on the molecular mass of the bis-sulfate. The m/z of this second ion was always higher than the [M-2H]2- selected as precursor ion. These two ions were found to be the most prominent ones in most of the model analytes with abundances commonly > 25%. Remarkably, the ion at m/z 97 was the base peak for most of the model compounds. Besides these two ions, additional product ions were observed in the spectra of some analytes (Table 1). Thus, ions at m/z 80 and m/z 81 corresponding to •SO3- and HSO3- respectively were observed for all tested bissulfates with significant abundances for some members (Table 1). These ions have been previously reported for the fragmentation of some mono-sulfates [37]. Additionally, bis-sulfates A13-A15, A19 and A21 containing one of the sulfates adjacent to either a carbonyl or an α,βunsaturated carbonyl showed a prominent ion (abundances between 28% and 100%) corresponding to the

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consecutive losses of •SO3- and •CH3. An example of analytes belonging to this class is shown in Figure 3 together with a feasible fragmentation pathway for the aforementioned loss. Some compounds exhibited other ions coming from consecutive losses of HSO4- and other neutral molecules such as SO3 (m/z 285 in analyte A18) or CH4 (ions at m/z 337 and 335 in analytes A3 and A5, respectively). However, the limited number of cases precluded the establishment of a general relationship between structure and MS behavior for these ions. 3.3. Method development Based on the MS information extracted from the model compounds, two alternative approaches were developed for the untargeted detection of bis-sulfates: (i) a precursor ion scan method using precursors of m/z 97 and (ii) an approach based on the constant loss of HSO4-. The development of the first approach was straightforward. Common MS software directly allows for the development of precursor ion scan methods by selecting in the last quadrupole the constant ion and scanning with the first one. In fact, the precursor ion scan of the m/z 97 has been reported for the detection of sulfate metabolites [2830]. The use of the CIL strategy implies that the product ion m’/z’ depends on the m/z of the precursor. In a general way, based on the fragmentation pathway Precursor ion (m/z) Product ion (m’/z’) + CIL (m’’/z’’) the change in m/z (∆m/z) observed during CIL can be calculated from the formula: ∆m/z = z’’[precursor ion (m/z)-CIL (m’’/z’’)]/(z-z’’) Where z and z’’ are the charge of the precursor ion and the CIL respectively. In the current application the CIL is HSO4- (m”/z” 97), z” = 1 and z = 2 for [M-2H]2- precursor ions. Thus, theory predicts a positive ∆m/z for any CIL where the m/z of the precursor ion is greater than that of the constant ion. Despite the fact that the m’/z’ of the product ion could be directly obtained from that of the precursor using the formula m'/z’ = m/z + ∆m/z, a software tool could not be found to perform this untargeted approach using a scan mode. Instead, a SRM method was developed including the theoretical product ion for each precursor ion calculated by the formula mentioned above. Due to the molecular masses of steroid hormones and metabolites (250400 Da), the precursor ions for bis-sulfates ([M-2H]2-) were restricted to the range m/z 199 to m/z 274. A SRM method containing 75 transitions was then used for the

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untargeted detection of steroid bis-sulfates. Although the transitions were pre-selected, the coverage of the whole range of molecular masses for steroid bis-sulfates allowed us to consider this approach as untargeted. Chromatographic optimization was also a key point in the method development. Several columns and mobile phase compositions were tested (for more details see supplementary information, Figure S-34). Although the use of low amounts of ammonium formate (between 1-5 mM) is usually preferred in LC-MS based methods for the detection of steroids [38], in the case of bis-sulfates a higher amount of salts was required for a proper chromatographic behaviour in all tested columns. Mobile phases containing 25 mM of ammonium formate were found to be necessary to obtain satisfactory peak shapes. 3.4. Extraction recovery and limit of detection Extraction recoveries for tested analytes are depicted in Table 2. Due to the anionic character of the analytes, the application of a SPE strategy based on WAX columns provided recoveries in the range of 44-85%, with a relatively high precision of extraction (RSD < 25%). The extraction recoveries together with the specificity obtained by the WAX extraction makes this procedure suitable for the qualitative detection of bis-sulfates in a matrix as complex as urine. However, if an accurate quantification is required, the use of adequate internal standards will be advisable to obtain suitable values. The LOD obtained by the two tested approaches are summarized in Table 2. In general, LOD below 20 ng/mL were obtained for all tested metabolites which are in the range of previous untargeted method reported for steroids [24,25,27]. The only exception was the detection of A18 by the precursor ion scan approach which a LOD of 50 ng/mL. In this case, A18 still achieved a LOD of 10 ng/mL in the CIL method. This is despite the relatively low abundance (20%) of the precursor ion [M-2H]2shown in the ionisation of A18 (Table 1). In general, CIL strategy showed lower LOD than the precursor ion scan approach. The 2-5 fold increase in sensitivity can be explained by the higher specificity of the transitions selected. The use of transitions involving increases of m/z have been reported to be more specific in the detection of other multiply charged analytes such as peptides [39]. Additionally, the acquisition in SRM mode also played an essential role in this increase in sensitivity. The main exceptions for this increased sensitivity were A15 and A24. The presence of a sulfate ester at C16 of A15 favored the alternative loss of •SO3- and •CH3 against the constant loss of HSO4- (Figure 3). Thus, the ion used for the CIL only counts for a 2% in A18 (Table 1) reducing the sensitivity of the CIL approach. Regarding estradiol bissulfate A24, its CID behavior was different to the model compounds. Thus, in contrast to them, the ion at m/z 80 was double the abundance of m/z 97 in the precursor ion scan of A24. Additionally, the ion loss of m/z 80 (m/z 350)

was more abundant than the ion loss of m/z 97 (data not shown). These results are in agreement with the fragmentation of estrogen sulfates in which the ion at m/z 80 is more abundant than the one at m/z 97 [37]. Thus, using a precursor ion scan of m/z 80 or a CIL approach based on the loss of this ion could improve the detection of estrogen bis-sulfates. In any case, the sensitivities obtained by both CIL and the precursor ion scan (20 ng/mL) were adequate for the untargeted detection of these metabolites. Finally, it was notable that the SRM acquisition used in the CIL approach also facilitated method development and the evaluation of the results. This fact is important for the application of the CIL-MS method to large sample numbers. 3.5. Applicability of the approach The applicability of the developed approach was evaluated under three different scenarios. 3.5.1. Presence of endogenous bis-sulfates The presence of endogenous steroid bis-sulfates was evaluated by analysis of ten urines collected from healthy volunteers. In most of the samples, several peaks were detected by the CIL method. Among them, some peaks were obtained at the theoretical m/z of estrogens like estriol bis-sulfate (223 349), androgens like androstanediol bis-sulfate (225 353), progestogens like pregnanediol bis-sulfate (239 381) or corticosteroids like tetrahydrocortisol bis-sulfate (262 427). The presence of bis-sulfates belonging to all the steroid families confirmed that the formation of bis-sulfates is a common metabolic pathway for steroids. An example of CIL chromatograms obtained for a control urine is shown in Figure 4. One of the potential limitations of the developed approach is the fact that the ion at m/z 97 can also correspond to H2PO4- [40]. Therefore, other bis-anionic species containing phosphate groups could be potentially detected by this method. The presence of less abundant ions in the product ion spectra e.g. m/z 80 can differentiate between bis-sulfates and other anionic species. Among the 17 endogenous compounds detected, 11 of them were sufficiently abundant to allow for the acquisition of product ion spectra. All of these presented secondary ions common to bis-sulfates (see supplementary information, Figure S-33). Additionally, the identity of some of the detected bis-sulfates was confirmed by comparison between a real sample and the model compounds. In the case of 16α-hydroxy-DHEA, pregn-5-ene-3α,20αdiol and 5-androstenediol bis-sulfates (A13, A12 and A5), both retention time and relative abundances of the selected ion transitions were identical confirming the identification (see supplementary information, Table S2 and Figure S-32).

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3.5.2. Effect of pregnancy on bis-sulfates excretion The comparison between samples collected before and during pregnancy revealed several changes in the excretion of bis-sulfates. Among them, the most significant increases were observed in the peaks with transitions 223 349 and 239 381. These transitions corresponded to those expected for the bis-sulfates of estriol and pregnanediol, respectively. Since structurally related markers e.g. 8-dehydroestriol, are associated with diseases such as Smith-Lemli-Opitz Syndrome [41], the detection of bissulfates could open suitable alternatives for the detection of these pathologies. 3.5.3. Tibolone metabolism The applicability of the CIL approach was also tested for the study of metabolism of exogenous anabolic steroids. Tibolone was selected as model compound since a bis-sulfate metabolite of this steroid has been already reported [42]. Samples collected before and after tibolone administration were analyzed and compared. The pre-administration sample showed the peaks obtained for endogenous bis-sulfates as commented above. Additionally, the post-administration sample also showed two additional peaks (Figure 5). The one at the ion transition 236 375 corresponds to a metabolite with a molecular mass of 474 which matches with the expected mass of the 3-reduced metabolite already reported as a bissulfate [42]. The second potential metabolite was detected at the ion transition 244 391. This ion transition corresponded to a bis-sulfate metabolite produced after reduction and hydroxylation of tibolone. 4.

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multiply charged metabolites broadening the applicability of the method. 5.

ACKNOWLEDGEMENTS

The authors acknowledge the financial support of WADA (project 15A29OP) and Ministerio de Economía y Competitividad (Spain) (DEP2012-35612). Spanish Health National System is acknowledged for O. J. Pozo contract (MS10/00576). The authors declare no competing financial interests. Supporting Information Available: Structure of analytes, synthesis and characterization details, chromatographic optimization and confirmation of the identity. This material is available free of charge via the Internet at http://pubs.acs.org. 6.

REFERENCES

(1) Grahan S. F., Chevalier O. P.; Roberts D.; Holscher C.; Elliott C. T.; Green B. D.; Anal Chem. 2013, 85, 18031811. (2) Raro M.; Ibanez M.; Gil R.; Fabregat A.; Tudela E.; Deventer K.; Ventura R.; Segura J.; Marcos J.; Kotronoulas A.; Joglar J.; Farré M.; Yang S.; Xing Y.; van Eenoo P.; Pitarch E.; Hernández F.; Sancho J. V.; Pozo O. J.; Anal Chem. 2015, 87, 8373-8380. (3) Castillo-Peinado L.; Luque de Castro M. D.; Anal. Chim. Acta 2016, 925, 1-15. (4) Zhang A. H.; Sun H.; Wang P.; Han Y.; Wang X. J.; Analyst 2012, 137, 293-300

CONCLUSIONS

The suitability of UHPLC coupled to triple quadrupole analyzers for the untargeted detection of bis-sulfates has been explored. Based on the common MS behavior of these metabolites, two alternative approaches were evaluated. The CIL strategy was found to be more sensitive than the precursor ion scan approach. Additionally, CIL is more specific for bis-sulfates since the occurrence of the ion at m/z 97 is common to mono-sulfate analytes. The developed CIL approach allowed for the untargeted detection of bis-sulfates at the low ng/mL range. The results presented in this study show that these levels are adequate for the detection of both endogenous levels and metabolites of exogenous steroids. The detection of endogenous levels of several steroid bis-sulfates confirms that this metabolic pathway is common for all families of steroids. Thus, the presented CIL approach can be a useful tool in the search of steroid related markers in different fields like the doping control or clinical diagnosis. Additionally, similar to other phase II metabolites such as glucuronides or mono-sulfates, it is expected that the MS behavior described for steroid bissulfates will be common to other bis-sulfates or other

(5) Coen M.; Holmes E.; Lindon J. C.; Nicholson J. K.; Chem Res Toxicol 2008, 21, 9-27. (6) Dettmer K.; Aronov P. A.; Hammock B. D.; Mass Spectrom. Rev. 2007, 26, 51-78. (7) Junot C.; Fenaille F.; Colsch B.; Becher F.; Mass Spectrom. Rev. 2014, 33, 471-500. (8) Vuckovic D. Anal. Bioanal. Chem. 2012, 403, 15231548. (9) Vaniya A.; Fiehn O.; Trend Anal. Chem. 2015, 69, 5261. (10) Carr S. A.; Huddleston M. J.; Annan R. S.; Anal. Bioanal. Chem. 1996, 239, 180-192 (11) Xia Y. Q.; Jemal M.; Rapid Commun. Mass. Spectrom. 2009, 23, 2125-2138 (12) Qu J.; Liang Q. L.; Luo G.; Wang Y. M.; anal. Chem. 2004, 76, 2239-2247 (13) Jemal M.; Ouyang Z.; Zhao W. P.; Zhu M. S.; Wu W. W. Rapid Commun. Mass Spectrom. 2003, 17, 2732-2740.

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(14) Montesano C.; Sergi M.; Moro M.; Napoletano S.; Romolo F. S.; Del Carlo M.; Compagnone D.; Curini R.; J. Mass Spectrom. 2013, 48, 49-59 (15) Pozo O. J.; Ventura R.; Monfort N.; Segura J.; Delbeke F. T. J. Mass Spectrom. 2009, 44, 929-944. (16) Gomez C.; Pozo O. J.; Garrostas L.; Segura J.; Ventura R. Steroids 2013, 78, 1245-1253. (17) Gomez C.; Pozo O. J.; Marcos J.; Segura J.; Ventura R. Steroids 2012, 78, 44-52. (18) Schanzer W.; Guddat S.; Thomas A.; Opfermann G.; Geyer H.; Thevis M. Drug Test. Anal. 2013, 5, 810-818. (19) Kotronoulas A.; Gomez-Gomez A.; Segura J.; Ventura R.; Joglar J.; Pozo O. J. J. Steroid Biochem. Mol. Biol. 2016 doi: 10.1016/j.jsbmb.2016.06.006. (20) Marcos J.; Craig W. Y.; Palomaki G. E.; Kloza E. M.; Haddow J. E.; Roberson M.; Bradley L. A.; Shackleton C. H. L.; Prenat Diagn. 2009, 29, 771-780. (21) Rupprecht R.; Holsboer F.; Trends Neurosci.1999, 22, 410-416

(37) Yi L.; Dratter J.; Wang C.; Tunge J. A.; Desaire H. Anal Bioanal. Chem. 2006, 386, 666-674. (38) Marcos J.; Pozo O. J.; J. Steroid. Biochem. Mol. Biol. 2016, 162, 41-56. (39) Liebler D. C.; Zimmerman L. Biochemistry2013, 52, 3797-3806 (40) Edelson-Averbukh M.; Pipkorn R.; Lehmann W. D. Anal Chem 2006, 78, 1249-1256 (41) Shackleton C. H. L.; Roitman E.; Guo L. W.; Wilson W. K.; Porter F. D. J. Steroid. Biochem. Mol. Biol. 2002, 82, 225-232. (42) Vos R. M. E.; Krebbers S. F. M.; Verhoeven C. H. J.; Delbressine L. P. C.; Drug Metab. Dispos. 2002, 30, 106-112. TABLES Table 1. MS behavior of 23 model steroid bis-sulfates. Relative abundance shown in brackets. Scan 2[M-2H] [M-H]

(22) Oikarinen A.; Kaar M. L.; Ruokonen A. Acta Dermato Venereologica 1980, 60, 503-507.

Product ion scan [M-2HOther HSO4] ions (>10%) 97 (100) 353 (81) 80 (20) HSO4

-

(23) Yu B.; Heiss G.; Alexander D.; Grams M. E.; Boerwinkle E.; Am. J. Epidem. 2016, 183, 650-656

A1

225 (100)

451 (7)

(24) Thevis M.; Geyer H.; Mareck U.; Schanzer W. J. Mass Spectrom. 2005, 40, 955-962.

A2 A3

225 (100) 225 (100)

451 (5) 451 (12)

97 (96) 97 (100)

353 (100) 353 (27)

80 (20) 337 (10)

(25) Pozo O. J.; Deventer K.; Van Eenoo P.; Delbeke F. T.; Anal. Chem. 2008, 80, 1709-1720.

A4 A5 A6

225 (100) 224 (100) 224 (100)

451 (15) 449 (8) 449 (7)

97 (100) 97 (100) 97 (100)

353 (40) 351 (25) 351 (47)

335 (18) -

A7 A8

218 (100) 218 (100)

437 (12) 437 (10)

97 (100) 97 (100)

339 (13) 339 (22)

-

A9 A10 A11

218 (100) 236 (100) 239 (100)

437 (30) 473 (5) 479 (15)

97 (100) 97 (100) 97 (100)

339 (7) 375 (9) 381 (8)

80 (53) -

A12 A13

238 (100) 231 (100)

477 (17) 463 (18)

97 (100) 97 (100)

379 (10) 365 (2)

80 (33), 81 (24) 367 (28)

A14

232 (100)

465 (33)

97 (100)

367 (2)

A15

232 (100)

465 (34)

97 (100)

367 (2)

80 (44) 81 (20) 369 (54) 80 (48) 81 (25) 369 (48)

A16 A17 A18

232 (100) 232 (100) 231 (20)

465 (16) 465 (45) 463 (100)

97 (100) 97 (100) 97 (100)

367 (24) 367 (27) 365 (58)

A19

231 (100)

463 (7)

97 (53)

365 (8)

A20 A21

231 (52) 231 (100)

463 (100) 463 (18)

97 (100) 97 (70)

365 (55) 365 (6)

(26) Qu J.; Wang Y. M.; Luo G. A.; Wu Z. P. J. Chromatogr. A 2001, 928, 155-162 (27) Fabregat A.; Pozo O. J.; Marcos J.; Segura J.; Ventura R. Anal Chem. 2013, 85:10, 5005-5014. (28) Gomez C.; Fabregat A.; Pozo O. J.; Marcos J.; Segura J.; Ventura R. Trend Anal. Chem. 2014, 53, 106-116. (29) Sanchez-Guijo A.; Oji V.; Hartmann M. F.; Schuppe H. C.; Traupe H.; Wudy S. A. J. Lipid Res. 2015, 56, 406-412. (30) Anizan S.; Bichon E.; Di Nardo D.; Monteau F.; Cesbron N.; Antignac J. P.; Le Bizec B. Talanta, 2011, 86, 186-194. (31) Pozo O. J.; Gomez C.; Marcos J.; Segura J.; Ventura R. Drug Test. Anal. 2012, 4, 786-797. (32) Wen B.; Ma L.; Nelson S. D.; Zhu M.; Anal Chem. 2008, 80, 1788-1799. (33) Waller, C. C.; McLeod, M. D. Steroids 2014, 92, 74– 80. (34) McKinney, A. R.; Cawley A. T.; Young E. B.; Kerwick C. M.; Cunnington K.; Stewart R. T.; Ambrus J. I.; Willis A. C.; McLeod M. D. Bioanalysis 2013, 5(7), 769–781. (35) Bi H. G.; Masse R. J. Steroid Biochem. Mol. Biol. 1992, 42, 533-546. (36) Dusza, J. P.; Joseph, J. P.; Bernstein, S. Steroids 1985, 45, 303–315

80 (45) 285 (17) 80 (84) 367 (100) 339 (19) 80 (17) 80 (100)

7



232 (100) 232 (100)

465 (62) 465 (52)

97 (100) 97 (100)

367 (19) 367 (11)

97

100

369

80

%

A22 A23

367 (92) 339 (28) -

384

225 225 224 232 232 231 215 232

353 353 351 367 367 365 333 367

20 25 7 16 11 11 10 4

5 5 2 10 5 10 20 2

20 20 10 10 10 50 20 10

FIGURES Figure 1. Scheme for the synthesis of steroid bis-sulfates.

Figure 2. MS behaviour of androstanediol bis-sulfate (A1) as model compound for steroid bis-sulfates; (a) scan in ESI- mode, (b) product ion scan at 20 eV collision energy after selection of the [M-2H]2- as precursor ion and (c) proposed pathway for the ions resulting after the ion loss of m/z 97

450

m/z 500

m/z 384

m/z 384

Figure 4. UHPLC-CIL chromatograms for a control urine sample showing peaks at the theoretical transitions for the bis-sulfates of estriol (223 349), androstenediol (224 351), androstanediol (225 353), hydroxy-DHEA / hydroxy-testosterone (231 365), hydroxy-androsterone / hydroxy-etiocholanolone (232 367), pregnenediol (238 379), pregnanediol (239 381), 17hydroxypregnenolone (245 393), pregnenetriol (246 395) and tetrahydrocortisol (262 427). The different peaks suggest the presence of different isomers or sulfation patterns. 100

2.05

2.88

100 3.44 4.32

232 367

0 2.00

4.00

6.00

8.00

3.44

%

4.00

6.00

8.00

4.19 4.87

% 0 4.00

2.96 4.02

262 427 4.00

6.00

0 2.00

225 353

6.00

8.00

0 2.00

8.00

3.54

246 395 4.00

6.00

8.00

5.44

100

5.35

2.00

0 2.00 100

231 365

100

245 393 4.00

6.00

8.00

OSO3

100

%

+

4.92

4.17 4.87

100

H

O3SO

400

(b)

2.00

OSO3

350

m/z 369

0

(b)

300

m/z 232

100

(a)

250

%

56 53 55 56 85 84 44 83

200

%

4.8 6.1 4.3 4.9 3.4 3.8 3.9 3.5

150

HSO4

224 351

0 2.00

4.00

6.00

8.00

5.34 5.85

%

A1 A3 A5 A15 A17 A18 A24 A25

Prec (m/z)

CIL PI scan Prod LOD LOD (m/z) (ng/mL) (ng/mL)

100

(a)

%

Rt (min)

Extraction Rec. RSD (%) (%)

0 50

%

Table 2. Extraction recoveries and LODs obtained for the eight bis-sulfates reference materials (PI: precursor ion).

0 2.00

239 381 4.00

6.00

8.00

H H

m/z 353

m/z 97

5.45

100

4.08 5.04

0

(c)

2.00

Figure 3. MS behaviour of 16α-hydroxyandrosterone bissulfate (A15) as model compound for steroid bis-sulfates; (a) product ion scan at 20 eV of collision energy after selection of the [M-2H]2- as precursor ion and (b) proposed pathway for the consecutive losses of •SO3- and • CH3.

4.00

4.92

100

223 349 6.00

Time 8.00

%

m/z 225

%

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 8 of 9

0 2.00

238 379 4.00

6.00

Time 8.00

Figure 5. UHPLC-CIL chromatograms obtained in the study of tibolone metabolism showing the ion transitions 236 375 (bottom) and 244 391 (top); (a) urine collected before tibolone administration and (b) urine collected between 8 and 24 h after tibolone administration.

8


Page 9 of 9

100

100

%

%

2.56 37

0 1.00

2.00

3.00

0 1.00

4.00

2.00

3.00

4.00

3.00 502

100

%

100

% 0 1.00

Time 2.00

3.00

4.00

0 1.00

Time 2.00

(a)

3.00

4.00

(b)

*Corresponding author: Óscar J Pozo e-mail: [email protected] Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003 Barcelona, Spain Phone: 0034-933160480, Fax: 0034-933160499

TOC OSO3

8.00 O3SO

CIL m/z 224

- HSO4

9.00 8.81 440

100 %

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


OSO3

0

Time 8.00

9.00

m’/z’ 351

∆m/z = z’’[precursor ion (m/z)-CIL (m’’/z’’)]/(z-z’’)

9


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