Ion mobility-mass spectrometry separation of steroid

Int. J. Ion Mobil. Spec. DOI 10.1007/s12127-016-0213-4

ORIGINAL RESEARCH

Ion mobility-mass spectrometry separation of steroid structural isomers and epimers Christopher D. Chouinard 1 & Christopher R. Beekman 1 & Robin H. J. Kemperman 1 & Harrison M. King 1 & Richard A. Yost 1

Received: 21 November 2016 / Revised: 11 December 2016 / Accepted: 13 December 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Drift tube ion mobility spectrometry (DTIMS) coupled with mass spectrometry was evaluated for its capabilities in rapid separation of endogenous isomeric steroids. These compounds, which included eight isomer groups, were investigated as protonated and sodiated species and collision cross sections were measured for all ionization species of each steroid. Pregnenolone (CCS N 2 176.7 Å 2 ) and 5αdihydroprogesterone (CCSN2 191.4 Å2) could be separated as protonated species, and aldosterone (CCSN2 197.7 Å2) and cortisone (CCS N2 211.7 Å2) could be separated as sodiated monomers. However, the sodiated dimers of the remaining isomers yielded increased separation, resulting in baseline resolution. Specific structural differences including ring conformation and the chirality of hydroxyl groups were compared to evaluate their relative effects on collision cross section in isomers. These results indicated that C5 ring conformation isomers androsterone and etiocholanolone, which both contain a C3 α-hydroxyl group, yielded similar dimer CCS. Yet these compounds were well resolved from their respective β-hydroxyl epimers, trans-androsterone and epietiocholanolone. Alternative drift gases were evaluated, and carbon dioxide drift gas offered slight improvement in isomer resolution well, including allowing separation of testosterone (CCS CO2 330.0 Å 2), dehydroepiandrosterone (CCSCO2 312.6 Å2), and epitestosterone (CCSCO2 305.6 Å2). Finally, different metal cation adducts, including alkali, Electronic supplementary material The online version of this article (doi:10.1007/s12127-016-0213-4) contains supplementary material, which is available to authorized users. * Richard A. Yost [email protected] 1

Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611, USA

alkaline earth, and first row transition metal adducts were analyzed, and several of these species provided improved resolution between steroid epimers. Overall, this study shows that drift tube ion mobility is a promising tool for improved separation of isomeric steroids. Keywords Ion mobility spectrometry . Mass spectrometry . Steroids . Isomers

Introduction Steroid hormones comprise a class of structurally diverse small molecules, spanning a wide range of polarities, which are commonly analyzed for their role in numerous physiological processes. Early analytical methods involved radioimmunoassays (RIA) [1] and gas chromatography–mass spectrometry (GC-MS) [2, 3], with which assays were developed as early as in the 1960s. RIA methods were predominant for several decades, but within the last 10 years mass spectrometry (MS) has become the technique of choice in steroid analysis, especially when coupled with liquid chromatography (LC) [4] which does not require derivatization to increase volatility, as is needed with GC methods. However, the class of steroids contains numerous isomers and LC methods often suffer from limited resolution of structurally similar compounds, requiring extended analysis time to achieve separation. Tandem MS has allowed more rapid identification and quantification of steroids by characteristic fragment ions, but this approach often fails when dealing with similar fragmentation patterns created by some isomers, especially stereoisomers. Because of these difficulties, there has arisen a need for an orthogonal separation method capable of distinguishing isomeric steroids based on both structural and stereochemical

Int. J. Ion Mobil. Spec.

differences. One such method that has shown promise is ion mobility spectrometry (IMS) [5, 6], which allows rapid separation (milliseconds) of isomeric compounds and can easily be integrated with existing GC- and LC-MS methods. Ion mobility techniques have demonstrated capability in separation of isomers across many other biological classes including carbohydrates [7–10], lipids [11–13], peptides [14, 15], vitamin D metabolites [16], and amino acids [17]. Because of these capabilities, interest in IMS for clinical applications has risen [18], but IMS analysis of steroids has been limited. Classical drift tube ion mobility (DTIMS) has been used in tandem with gas- and supercritical fluid chromatography to measure the mobility constants of estrone, progesterone, and testosterone [19]. Since then, other mobility techniques including field asymmetric IMS (FAIMS) [20], or differential mobility spectrometry (DMS), have seen application in steroid analysis. FAIMS primarily operates as an ion filter to improve signal-to-noise ratio in targeted quantitative analysis, and examples of such improvements have included identification of androgenic anabolic steroids for anti-doping [21]; separation of endogenous isomer pairs pregnanolone/allopregnanolone [22], corticosterone/11d e o x y c o r t i s o l a n d 1 7 - h y d r o x y p r o g e s t e r o n e / 11 deoxycorticosterone [23]; and testosterone quantitation in human plasma [24]. In contrast to FAIMS, DTIMS and the more recently developed traveling wave IMS (TWIMS) [25] allow separation and identification of analytes in complex samples in a more global manner. Since early applications, DTIMS has been coupled with molecular imprinted polymers and corona discharge for testosterone quantitation in human urine [26]. Recently TWIMS has also been used to determine epimers of testosterone glucuronides following UPLC [27] and to separate steroid isomer pairs following derivatization with p-toluenesulfonyl isocyanate [28]. Although IMS techniques have shown progress in separation capabilities, isomers remain difficult to routinely separate. This is especially true when dealing with epimers that differ only in their stereochemistry at one chiral center and thus differ very little in collision cross section (CCS). As such, many strategies have been employed to introduce more variation in CCS. One such strategy, preanalysis derivatization, has been used to improve detection of methamphetamine metabolites [29] and carbohydrates [30] in conjunction with ion mobility. This strategy has also been implemented for steroids, in which derivatized epimers testosterone/epitestosterone, α-estradiol/β-estradiol, and androsterone/trans-androsterone [28] exhibited improved separation. Other strategies have included altering the drift tube gas composition [31, 32], either by changing the buffer gas or introducing chiral solvents, and forming solution-phase adducts with alkali/ transition metal ions and chiral ligands [13, 33–37]. Recently, our group has studied the mobility separation and gas-phase structure of a model steroid isomer pair,

epimers androsterone and trans-androsterone [38]. Notably, it was shown that sodiated dimers of these compounds adopt unique binding modes that allow baseline resolution. This study focuses on classical DTIMS coupled with mass spectrometry for the improved separation of underivatized endogenous steroid isomers, by investigating various ionization species and their relative mobility differences. The primary focus is the separation of steroid dimers, as previous results have indicated a significant improvement in resolution of these species [38]. Furthermore, in addition to separation of common cation adducts, those formed with other alkali, alkaline earth, and transition metal cations are also demonstrated. Finally, alternative drift gases (e.g., helium, argon, carbon dioxide) were investigated for their relative separation capabilities. Collision cross sections were measured for each ionization species identified for all steroids, to provide a comprehensive library for these compounds under various experimental conditions.

Experimental methods Materials and reagents Androsterone, trans-androsterone, α-estradiol, β-estradiol, etiocholanolone, corticosterone, aldosterone, cortisone, cholesterol, and lathosterol were purchased from SigmaAldrich (St. Louis, MO). Testosterone, epitestosterone, 17-hydroxyprogesterone, and 11-deoxycorticosterone were purchased from Cerilliant Corporation (Round Rock, TX). Dehydroepiandrosterone was purchased from Acros Organics (Geel, Belgium). Epietiocholanolone and 13 C3-testosterone were purchased from IsoSciences (King of Prussia, PA). Pregnenolone was purchased from MP Biomedicals (Santa Ana, CA). 5α-Dihydroprogesterone was purchased from Pfaltz & Bauer (Waterbury, CT). 11-Deoxycortisol was purchased from TCI Chemicals (Portland, OR). 5α-Cholestanone was purchased from Alfa Aesar (Ward Hill, MA). Solids were prepared as 10 μg/mL neat solutions in Fisher Optima LC-MS grade methanol, purchased from Fisher Scientific (Pittsburgh, PA), with no additives (i.e., formic acid or sodium salt). Lithium acetate, magnesium acetate tetrahydrate, and calcium acetate monohydrate were purchased from Acros Organics (Geel, Belgium). Potassium acetate was purchased from Alfa Aesar (Haverhill, MA). Rubidium acetate was purchased from Strem Chemicals (Newburyport, MA). Cesium acetate was purchased from MP Biomedicals (Santa Ana, CA). Strontium acetate was purchased from Sigma-Aldrich (St. Louis, MO). Barium acetate was purchased from Chem-Impex International, Inc. (Wood Dale, IL). These standards were combined


with the aforementioned steroid standards and were diluted to a final concentration of 10 μg/mL in Fisher Optima LC-MS grade methanol or a 1:1 mixture of Fisher Optima LC-MS grade water and Fisher Optima LC-MS grade water, depending on their relative solubility.

MS data processing was performed using Agilent IM-MS Browser B.07.01.

IM-MS analysis

Ion mobility separation was evaluated for eight groups of steroid isomers, chosen to investigate several structural differences. These groups comprised structural isomers with differences in presence and position of ketone/hydroxyl groups, double bonds, and A- and D-ring functional groups. The stereoisomers chosen exhibited either differences in the conformation of their four ring structure or epimers that differed in their chirality at one stereocenter (i.e., α- vs. β-hydroxyl groups). Structures for all compounds are shown in Figures S1–S8.

All analyses were performed with an Agilent 6560 IMQTOF instrument (Santa Clara, CA). Standard solutions were direct infused by syringe pump at a flow rate of 10 μL/min. All compounds were analyzed in positive mode using both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). An Agilent Jet Stream (AJS) source was used to perform electrospray ionization (ESI) with capillary voltage of 4000 V. The ESI source conditions were as follows: capillary voltage: +4000 V; nozzle voltage: +1000 V; drying gas: 325 °C at 5 L/min; sheath gas: 275 °C at 8 L/min. The APCI source conditions were as follows: capillary voltage: +3500 V; corona current 4 μA; drying gas: 325 °C at 5 L/min; vaporizer: 325 °C. The IM-QTOF instrument consists of a 78 cm uniform field drift tube maintained at approximately 4 torr nitrogen drift gas and 32 °C. These constant drift tube conditions allow direct comparison of drift time spectra, which were acquired over a 60 ms window. A modified version of the MasonSchamp equation (Eq. 1) [5, 39, 40] was used to measure collision cross section (CCS) for ions of interest: ð18π Þ1=2 ze Ω ¼ 16 ðkB TÞ1=2

1 1 þ mI mB

1=2

td E 760 T 1 L P 273:2 N

ð1Þ where ze is the charge, kB is the Boltzman constant, mI is the analyte ion mass, mB is the buffer gas molecule mass, td is the corrected drift time, E is the electric field strength in V · cm−1, L is the drift tube length in cm, P is the drift tube pressure in Torr, T is the drift tube temperature in Kelvin, and N is the drift tube number density in cm−3. To calculate the corrected drift time (td), the drift tube field was varied over eight field strength steps from 9.6 to 18.6 V/cm (drift tube voltage 750–1450 V). Total drift time, tD, was plotted versus the inverse of the drift tube voltage and the linear trend line was extrapolated to the y-intercept, which represents the non-drift tube time, t0. This value was then subtracted from the measured total drift time to yield the corrected drift time. All drift time spectra shown were acquired at 18.6 V/cm, unless otherwise noted; optimal peak resolving power was achieved at this field strength. Time of flight mass spectra were acquired in full scan high resolution mode over a range from m/z 100 to 1700. All IM-

Results and discussion

Separation of protonated species Because steroids typically suffer from low ionization efficiency with electrospray ionization (ESI), many mass spectrometry-based methods use atmospheric pressure chemical ionization (APCI) following liquid chromatography to identify protonated species. As such, all compounds were first analyzed individually using APCI, and the mobility drift time spectra for the most abundant ions (e.g., [M + H] + , [M + H-H 2 O] + , etc.) were compared. Mobility separation was achieved between the protonated species [M + H]+ of several endogenous steroids including β-estradiol, testosterone, 17-hydroxyprogesterone, 5αdihydroprogesterone, and cortisone (Fig. 1a). However, these species of different molecular weights can be easily differentiated with mass spectrometry alone. Isomers, which cannot be distinguished by their parent m/z, were also compared due to the capabilities for IMS in separation based on compound shape (i.e., structural differences). Several of these isomers, such as testosterone and dehydroepiandrosterone (DHEA) that differ in the position of their hydroxyl/ketone groups and double bond (Figure S2), showed minimal separation for their protonated species (Fig. 2b). Other pairs, such as pregnenolone and 5α-dihydroprogesterone (5α-DHP), were nearly baseline separated (Fig. 2c). In general, drift peaks for the protonated species were not Gaussian in shape, with tailing (e.g., DHEA), shoulders (e.g., pregnenolone), and/or multiple drift peaks (e.g., 5α-DHP); this is likely attributed to charge-location isomers, in which protonation can occur at any one of several locations on the molecule, altering the collision cross section and the drift time spectrum. CCS were measured for all protonated steroids and the values for the most abundant ionization species of each compound are listed in Table 1. Other ionization

Int. J. Ion Mobil. Spec. Fig. 1 a Overlay of drift spectra for the protonated monomers of five different endogenous steroids, along with their corresponding collision cross sections; and overlays for structural isomers b testosterone and dehydroepiandrosterone (DHEA), and c pregnenolone and 5α-dihydroprogesterone (5αDHP)

species produced with APCI (e.g., [M + H-2H2O]+) exhibited lower intensity and minimal separation. Separation of sodiated adducts with electrospray ionization Due to the limited separation of protonated steroid isomers, and based on previous work from our group showing IMS resolution of sodiated steroid epimers [38], ESI was also used to investigate other potential ionization species, specifically sodiated adducts. Without addition of any modifiers/salts, the major ionization species observed for each compound included the [M + Na]+ and [2 M + Na]+ species. The sodiated monomers did not yield significantly improved resolution over the protonated species, with the exception of aldosterone and cortisone which were baseline resolved (RS = 1.95). CCS Fig. 2 Overlays of drift spectra for testosterone and dehydroepiandrosterone (DHEA) as a sodiated monomers and b sodiated dimers. The inset mass spectrum is collected for the drift range from 32 to 35 ms, showing the fragmentation of sodiated dimers post-drift tube

were measured for sodiated monomers and are listed in Table S1. Although monomer separation was minimal, additional peaks in the drift spectra were identified for all compounds. For example, the sodiated monomers of testosterone and DHEA exhibited overlapping drift peaks at 25 msec (Fig. 2a), but also showed lower intensity baseline-resolved drift peaks in the 32–35 msec range; these peaks are attributed to fragmented [2 M + Na]+ dimers, and confirmed by mass spectrometry (Fig. 2b). This post-drift tube fragmentation has been reported previously [38]. Drift spectra for the intact sodiated dimers indicated that mobility separation was considerably improved for these species within each isomer group. For example, although CCS for the sodiated monomers (Fig. 2a) of testosterone and DHEA were very similar, 196.7 and 195.5 Å2, respectively (yielding RS = 0.16), the CCS for the sodiated dimers (Fig. 2b) were 269.0 and 251.3 Å2,

Int. J. Ion Mobil. Spec. Table 1 Collision cross sections for the most abundant protonated species of each steroid, collected in nitrogen drift gas, and resolution (RS) between isomers

Steroid

Ionization Species

m/z

CCS (Å2), N2

RS, N2

α-Estradiol β-Estradiol

[M + H-H2O]+

255.175

0.01

[M + H]+

289.217

162.4 ± 0.2 162.2 ± 0.2 174.5 ± 0.2 174.3 ± 0.2 174.7 ± 0.2 174.1 ± 0.2 174.3 ± 0.2 167.7 ± 0.2 167.2 ± 0.2 176.7 ± 0.2 191.4 ± 0.2 186.3 ± 0.2 188.1 ± 0.2 189.4 ± 0.2 190.4 ± 0.2 193.8 ± 0.3 194.6 ± 0.3 204.6 ± 0.3 203.9 ± 0.2 205.4 ± 0.3

0.07# 0.09

Testosterone Dehydroepiandrosterone Epitestosterone Androsterone trans-Androsterone

+

[M + H-H2O]

273.222

[M + H-H2O]+

273.222

Pregnenolone 5α-Dihydroprogesterone

[M + H]+

317.248

17-Hydroxyprogesterone 11-Deoxycorticosterone

[M + H]+

331.227

Corticosterone 11-Deoxycortisol

[M + H]+

347.222

Aldosterone Cortisone Cholesterol 5α-Cholestanone Lathosterol

[M + H]+

361.202

[M + H-H2O]+

369.352

Etiocholanolone Epietiocholanolone

0.06*

0.02 1.23 0.28 0.14 0.02 0.03* 0.01#

*Resolution between testosterone/dehydroepiandrosterone and cholesterol/5α-cholestanone. # Resolution between testosterone/epitestosterone and cholesterol/lathosterol

respectively (RS = 2.10). This pattern was observed for the other isomer pairs including structural isomers 17-hydroxyprogesterone/11-deoxycorticosterone (Figure S9a) and corticosterone/11-deoxycortisol (Figure S9b), and epimers testosterone/epitestosterone (Figure S9c) and androsterone/ trans-androsterone Figure (S9d). CCS for all sodiated dimers are listed in Table 2. Our group has previously compared experimental results with theoretical modeling for elucidation of structural differences that attribute to the mobility separation observed in the dimers of steroid epimers androsterone and trans-androsterone [38]; the results presented here indicate that similar structural differences may contribute to separation of the dimers of many other steroid isomers as well. Structural differences affecting Gas-phase structure: C3 hydroxyl epimers vs. C5 ring conformers The relative effects of two different structural variations on CCS were compared for structural isomers androsterone and etiocholanolone that differ in the conformation of their ring at the C5 position (Fig. 3a), and their respective C3 hydroxyl epimers trans-androsterone and epietiocholanolone. An overlay of the sodiated dimer drift spectra for these four compounds revealed similar mobilities for the isomers with the same chirality at their C3 hydroxyl group (Fig. 3b), regardless of the C5 ring conformation. However, baseline separation was observed between compounds with different C3 stereochemistry (i.e., androsterone and trans-androsterone, and etiocholanolone and epietiocholanolone). Androsterone and

etiocholanolone (3α-hydroxyl group) also showed the unique presence of fragmented trimer drift peaks in the 36–38 msec range; the propensity of 3α-hydroxy steroids to preferentially Table 2 Collision cross sections for the sodiated dimer of each steroid, collected in nitrogen drift gas, and resolution (RS) between isomers. Bold values for resolution indicate baseline separation of RS ≥ 1.5 Steroid

[2 M + Na]+ m/z

CCS (Å2), N2

RS, N2

Testosterone Dehydroepiandrosterone Epitestosterone* Androsterone trans-Androsterone Etiocholanolone Epietiocholanolone Pregnenolone 5α-Dihydroprogesterone 17-Hydroxyprogesterone 11-Deoxycorticosterone Corticosterone 11-Deoxycortisol Aldosterone Cortisone Cholesterol 5α-Cholestanone Lathosterol*

599.408

269.0 ± 0.5 251.3 ± 0.4 250.7 ± 0.3 242.6 ± 0.3 256.3 ± 0.3 247.4 ± 0.3 262.5 ± 0.4 264.3 ± 0.4 260.5 ± 0.4 260.1 ± 0.3 292.8 ± 0.4 289.9 ± 0.6 269.3 ± 0.4 266.1 ± 0.4 269.9 ± 0.5 303.8 ± 0.6 303.5 ± 0.6 308.1 ± 0.6

2.10*

603.439 603.439 655.470 683.429 715.416 743.377 795.700

1.89# 1.81 1.90 0.31 2.77 1.94 0.57 0.03* 0.46#


Int. J. Ion Mobil. Spec. Fig. 3 Comparison of sodiated dimer drift spectra for structural differences in (a) androsterone, trans-androsterone, etiocholanolone, and epietiocholanolone. These four isomers differ in the stereochemistry of their C3 hydroxyl group and the ring configuration at C5. The difference in C3 stereochemistry affects (b) drift spectra for these compounds more than C5 ring configuration

form trimers, in comparison with their 3β-hydroxy isomers, is a result of higher gas-phase stability for these multimers [38]. Comparison of drift spectra with alternative drift gases IMS separation was also investigated using several alternative drift gases that differ in their molecular weight, polarizability, and calculated radius. The gases chosen included helium (monoatomic, smaller molecular weight and radius than nitrogen), carbon dioxide (larger molecular weight and radius), and argon (similar in polarizability and radius, but monoatomic). Comparison of mass spectra collected with each drift gas revealed little difference for each compound. Collision cross sections were measured for the most abundant species, which Table 3 Collision cross sections for the sodiated dimer of each steroid, collected in helium, argon, and carbon dioxide drift gas. Bold values for resolution indicate baseline separation of RS ≥ 1.5

again included sodiated monomers (Table S2) and dimers (Table 3). Several trends previously reported in literature for other compound classes were seen in comparing collision cross sections and drift spectra in the different drift gases. Specifically, collision cross section and drift time were shown to be proportional to the polarizability and radius of the drift gas; collision cross sections in helium are smaller than in argon, which are smaller than in nitrogen, and so on. However, this trend appears to depend more on the polarizability and the radius of the drift gas rather than the overall mass. This is best demonstrated by comparing argon and nitrogen. Argon, although more massive, is monoatomic, less polarizable, and has a smaller calculated radius. In contrast, nitrogen, although less massive, yields longer drift times and

Steroid

[2 M + Na]+ m/z

CCS (Å2) He

RS, He

CCS (Å2) Ar

RS, Ar

CCS (Å2) CO2

RS, CO2

Testosterone DHEA Epitestosterone Androsterone trans-Androsterone Etiocholanolone Epietiocholanolone Pregnenolone 5α-DHP 17-OHP 11-DOC Corticosterone 11-Deoxycortisol Aldosterone Cortisone Cholesterol 5α-Cholestanone Lathosterol

599.408

164.2 ± 0.2 151.6 ± 0.2 148.7 ± 0.2 141.8 ± 0.1 149.1 ± 0.2 141.6 ± 0.1 149.1 ± 0.2 155.6 ± 0.2 154.5 ± 0.2 153.0 ± 0.2 169.2 ± 0.2 169.0 ± 0.2 158.0 ± 0.2 157.1 ± 0.2 159.1 ± 0.2 183.4 ± 0.2 182.5 ± 0.2 187.9 ± 1.3

1.57*

239.3 ± 0.5 224.6 ± 0.3 221.1 ± 0.3 218.3 ± 0.2 227.1 ± 0.3 218.0 ± 0.3 226.5 ± 0.3 235.2 ± 0.3 231.8 ± 0.4 230.6 ± 0.5 248.7 ± 0.5 251.4 ± 0.5 237.5 ± 0.3 236.7 ± 0.3 237.9 ± 0.4 271.4 ± 0.5 269.6 ± 0.5 273.9 ± 0.5

1.75*

269.5 ± 0.2 252.6 ± 0.2 246.8 ± 0.2 241.8 ± 0.3 254.0 ± 0.4 240.8 ± 0.3 252.1 ± 0.3 258.2 ± 0.5 258.0 ± 0.4 255.2 ± 0.4 280.7 ± 0.4 280.3 ± 0.4 263.7 ± 0.4 260.6 ± 0.4 265.5 ± 0.4 298.5 ± 0.5 300.6 ± 0.9 293.8 ± 0.5

2.08*

603.439 603.439 655.470 683.429 715.416 743.377 795.700

1.82# 1.15 1.49 0.11 2.29 1.52 0.45 0.10* 0.15#

1.71# 1.02 1.41 0.28 1.97 1.61 0.28 0.15* 0.38#

2.50# 1.53 1.54 0.31 2.77 1.90 0.76 0.43* 0.24#



Changing drift gas did yield some marked improvement in mobility separation. For example, improvement was seen for previously unresolved isomers, specifically within the testosterone group. Although the sodiated dimer of testosterone was separated from its structural isomer DHEA and epimer epitestosterone, these two compounds were not well resolved in nitrogen (Fig. 5a). Use of carbon dioxide drift gas allowed improved separation of these compounds, providing mobility resolution for all three of these endogenous steroids (Fig. 5b). In addition, resolution between protonated pregnenolone and 5α-dihydroprogesterone (RS = 1.23 in nitrogen) was increased to RS = 2.96 in carbon dioxide (Fig. 5c-d). CCS and isomer resolution for other protonated species in CO2 are listed in Table S3.

Alternative cation adducts of androsterone epimers Fig. 4 Comparison of sodiated dimer overlays and corresponding resolution between epimers androsterone and trans-androsterone in a helium, b nitrogen, and c carbon dioxide drift gas

larger collision cross sections. Furthermore, differences in cross section between isomers tended to increase further with larger drift gases as well. This pattern is illustrated in Fig. 4, which shows overlays of drift spectra and collision cross sections for androsterone and trans-androsterone in helium, nitrogen, and carbon dioxide. The overall trends of collision cross section (as a function of m/z for the various isomers) are represented in all four gases for sodiated monomers (Figure S10) and dimers (Figure S11); these plots display graphically the trends in separation between all isomers within a group (plotted as vertical sets for the same m/z) for the different experimental conditions.

Fig. 5 Overlays of drift spectra for sodiated dimers of testosterone, epitestosterone, and dehydroepiandrosterone in a nitrogen drift gas, and b carbon dioxide drift gas, demonstrating improved separation between DHEA and epitestosterone; and overlays of drift spectra for protonated monomers of pregnenolone and 5αdihydroprogesterone in c nitrogen drift gas, and d carbon dioxide drift gas

Formation of alternative cation adducts (i.e., alkali, alkaline earth, and transition metals) has been used in several ion mobility studies to augment separation by altering gas-phase ion structure. Here we chose a model isomer pair, epimers androsterone and trans-androsterone, to investigate differences in separation for these various cation adducts. These compounds were chosen because their gasphase structures and ion mobility behavior have been studied previously [38]. Cation adducts included alkali (lithium, sodium, potassium), alkaline earth (magnesium, calcium, strontium, rubidium), and first row transition metals (scandium (III), chromium (III), manganese (II), iron (II), cobalt (II), nickel (II), copper (II), and zinc (II)). The most abundant ionization species identified for monovalent cation adducts were similar to those observed with sodium, specifically singly charged monomers [M +

Int. J. Ion Mobil. Spec. Fig. 6 Comparison of dimer overlays and corresponding resolution between epimers androsterone and transandrosterone with first row transition metal cation adducts a manganese, b cobalt, c copper, and d zinc

X]+ and dimers [2 M + X]+. Adducts with the alkaline earth and transition metals also preferentially formed +1 species; because all metal ions were introduced as acetate salts, formation of the +1 species included acetate as the counter anion (i.e., [M + XII + Acetate]+ and [2 M + XII + Acetate]+ for divalent cations). The +2 species were identified in some instances at extremely low intensity, and these will not be discussed. CCS were measured and the resolution between epimers was compared with that obtained for protonated and sodiated species. In general, little improvement in separation was observed for alkali and alkaline earth metals with both monomer and dimer species, and the resulting intensity of these ions was lower. However, first row transition metal adducts did produce improvement in resolution over sodiated dimers, notably for copper (II) (RS = 2.85) and zinc (II) (RS = 2.01), albeit at slightly lower intensity. Drift spectra for these adducts are shown in Fig. 6, indicating the potential to improve overall separation for other isomeric steroids. Table 4 Optimal separation conditions for each isomer group, indicating ionization species, drift gas environment, and resolution Isomer Group

Testosterone/DHEA Testosterone/Epitestosterone Androsterone/trans-Androsterone Etiocholanolone/Epietiocholanolone Pregnenolone/5α-DHP 17-OHP/11-DOC Corticosterone/11-Deoxycortisol Aldosterone/Cortisone Cholesterol/5α-Cholestanone Cholesterol/Lathosterol

Ionization Species Drift Gas [2 M + Na]+ N2 [2 M + Na]+ CO2 [2 M + CuII + Ac]+ [2 M + Na]+ [M + H]+ [2 M + Na]+ [2 M + Na]+ [M + Na]+ [2 M + Na]+ [2 M + Na]+

N2 N2 CO2 N2 N2 CO2 CO2 N2

RS

2.10 2.50 2.85 1.90 2.96 2.77 1.94 2.00 0.43 0.46

Conclusions In summary, DTIMS-MS was utilized for rapid separation of endogenous steroid structural isomers and epimers. Although limited separation was observed for the protonated and sodiated monomers of most isomers, baseline resolution was achieved for many of the isomer pairs as sodiated dimers. Several structural differences were investigated, showing that some of these differences (i.e., C5 ring conformation) contributed less variation to sodiated dimer collision cross section than others (i.e., α- vs. β-hydroxyl groups). Separation was further augmented with use of carbon dioxide drift gas, in comparison with smaller and less polarizable gases helium, argon, and nitrogen. In addition, alternative cation adducts were investigated and in particular several of the first row transition metal adducts (copper and zinc) improved resolution between epimers. Optimal separation conditions (highest resolution achieved) for each isomer group are listed in Table 4. DT-IMS shows significant potential for improving analysis of isomeric steroids by providing rapid separation capabilities to existing MS methods. Acknowledgements The authors gratefully acknowledge financial support from Agilent Technologies, Wellspring Clinical Lab, and the University of Florida Graduate Fellowship.

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