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Isolation and Identification of Diglucuronides of Some Endogenous Steroids in Dogs

Drug Metabolism and Pharmacokinetics Research Laboratories (T.M., N.Y., T.N., M.T., H.I.) and Medicinal Safety Research Laboratories (K.T., K.M.), Daiichi Sankyo Co., Ltd., Tokyo, Japan; Research Department II, Daiichi Sankyo RD Associe Co., Ltd., Tokyo, Japan (N.S.); and Association for Promoting Drug Development, Tokyo, Japan (T.I.)

(Received October 12, 2007; Accepted November 12, 2007)

	Abstract

Top Abstract Materials and Methods Results and Discussion References

 
Diglucuronidation is a novel glucuronidation reaction where the second glucuronosyl moiety is attached at the C2' position of the first glucuronosyl moiety. To examine whether diglucuronidation takes place in endogenous substrates in vivo, control urine and bile samples were collected from male Crl:CD(SD) IGS rats, beagle dogs, and cynomolgus monkeys and analyzed by liquid chromatography-mass spectrometry (LC-MS) after solid phase extraction. Several diglucuronides of C₁₉ steroids, including M1 (C₃₁H₄₆O₁₄) and M2 (C₃₁H₄₄O₁₄), were detected in the urine and bile of the dogs but not in the excreta of the rats and monkeys. A milligram quantity of M1 was successfully isolated from the pooled dog urine and analyzed by nuclear magnetic resonance (NMR) spectroscopy. M1 was unambiguously identified as epiandrosterone 3-O-diglucuronide by comparing the LC-MS and two-dimensional NMR data of M1 with those of the biosynthesized epiandrosterone 3-O-diglucuronide. M2 was identified as dehydroepiandrosterone 3-O-diglucuronide. According to these findings, the diglucuronidation reaction was proven to be occurring on steroid hormones in vivo in dogs.

As a result of a hydrophilic glucuronosyl moiety being introduced, glucuronides are generally hydrophilic enough to be excreted readily in either the bile or the urine. However, when the glucuronidation does not supply enough hydrophilicity to a molecule or when the glucuronide is still recognized as a good substrate by UGTs, subsequent glucuronidation may occasionally take place on the glucuronides. Glucuronides carrying two glucuronosyl moieties are classified into two groups, namely bisglucuronides and diglucuronides (Murai et al., 2005). Bisglucuronides are defined as glucuronides where two separate functional groups on the parent molecule are conjugated at the same time. Bisglucuronides are relatively common phase II metabolites of both endogenous and exogenous compounds (Gordon et al., 1976; Bock et al., 1992). On the other hand, diglucuronides are defined as particular glucuronides where a single functional group on the parent molecule is repeatedly conjugated by two glucuronosyl groups in tandem (Murai et al., 2005).

The first diglucuronide identified was nalmefene 3-O-diglucuronide, which was found in dog urine as a chromatographically more polar glucuronide than nalmefene glucuronide (Dixon et al., 1989; Murthy et al., 1996). After the discovery of nalmefene diglucuronide, we reported that diglucuronides could be produced in vitro from 4-hydroxybiphenyl, a phenolic compound used commonly as a model substrate for glucuronidation (Murai et al., 2002). Moreover, we found that endogenous sex steroids, namely androsterone, dihydrotestosterone (DHT), estradiol, estriol, estrone, and testosterone, also undergo diglucuronidation in vitro (Murai et al., 2005). Significant species differences were observed in the production of diglucuronides in vitro. Dog liver microsomes exhibited the highest activity for diglucuronidation, whereas monkey and human liver microsomes exhibited detectable diglucuronidation activity for particular steroids (Murai et al., 2005). Activity producing DHT diglucuronide from the corresponding monoglucuronide in recombinant human UGT isoforms was found in UGT1A8, UGT1A1, and UGT1A9, in descending order (Murai et al., 2006).

Steroids are included in a class of lipophilic small molecules that act as ligands of their nuclear receptors, and their circulating levels are regulated by a balance of steroidogenesis and metabolic inactivation by cytochrome P450, UGT, and sulfotransferase (You, 2004). The fact that androgens and estrogens are good substrates for diglucuronidation in vitro using dog liver microsomes (Murai et al., 2005) does not necessarily indicate that the diglucuronidation reaction is occurring under in vivo conditions. To date, no diglucuronides produced from endogenous compounds have been identified in vivo.

The objective of the present study was to determine whether the diglucuronidation of endogenous substances takes place in vivo. In this study, control bile and urine samples obtained from male rats, dogs, and monkeys were cleaned up using mixed mode anion exchange solid phase extraction cartridges, and the extracts were analyzed using liquid chromatography-mass spectrometry (LC-MS) to detect any endogenous diglucuronides. We were successful in detecting the [M-H]^– ions of C₁₉ steroid diglucuronides in the bile and urine of dogs but not in those of rats and monkeys. To identify the endogenous diglucuronides detected in dog bile and urine, several diglucuronides were biosynthesized using dog liver microsomes and commercially available androgens (C₁₉ steroids). The present report also describes in detail the isolation and structural elucidation of an endogenous steroid diglucuronide detected in dog urine, which has been unambiguously identified as epiandrosterone 3-O-diglucuronide, in which the second glucuronosyl moiety is attached at the C2' position of the first glucuronosyl moiety, leading to the diglucuronosyl conjugation of a single hydroxyl group of epiandrosterone at the C3 position.

	Materials and Methods

Top Abstract Materials and Methods Results and Discussion References

 
Materials. Androsterone, epiandrosterone, dehydroepiandrosterone (DHEA), DHT, and testosterone were purchased from Sigma-Aldrich Co. (St. Louis, MO). Alamethicin, D-saccharic acid 1,4-lactone, and UDPGA were also from Sigma. Deuterated dimethyl sulfoxide (DMSO-d₆, 99.9% D) was purchased from Isotec, Inc. (Miamisburg, OH). Other reagents and solvents used were of the highest grade available. Pooled rat, dog, monkey, and human liver microsomes were obtained from BD Gentest (Woburn, MA) and were stored frozen at approximately –80°C until use.

Urine and Bile Samples for Analysis of Diglucuronides. Male Crl: CD(SD) IGS rats (N = 3; body weight: approximately 200 g; age: 8 weeks old) were housed in a cage with free access to food (FR-2; Funabashi Farms Co., Ltd., Chiba, Japan) and water. Male beagle dogs (N = 3; body weight: 11–13 kg; age: 2 and 3 years old) were individually housed in cages with free access to water and were given 250 g of solid food daily (DS-A; Oriental Yeast Co., Ltd., Tokyo, Japan). Male cynomolgus monkeys (N = 3; body weight: 2.8–4.3 kg; age: 3 and 5 years old) were individually housed in cages with free access to water and were given 80 g of solid food daily (PS; Oriental Yeast Co., Ltd.). Urine samples were collected overnight prior to collecting bile samples. The rat bile samples were collected for 6 h via a polyethylene tube cannulated into the bile duct. The dog and monkey bile samples were collected from the gallbladder with syringes under anesthesia by pentobarbital for dogs and ketamine hydrochloride and xylazine hydrochloride for monkeys. The samples collected were stored frozen at or below –20°C until analysis. The protocols for all of the animal experiments were approved by the Institutional Ethical Committee for Animal Experiments.

Solid Phase Extraction of Bile and Urine Samples. The bile and urine samples were subjected to solid phase extraction using Waters Oasis MAX cartridges (6 cc, 150 mg; Waters Corp., Milford, MA). Urine samples (1 ml) were diluted with water (4 ml), and bile samples (0.1 ml) were diluted with water (4.9 ml). Each diluted sample (5 ml) was loaded onto the cartridge, which had been preconditioned by washing successively with 5 ml of methanol and 5 ml of water. The cartridge was washed with 10 ml of methanol in 50 mM sodium acetate (5 in 95) followed by 10 ml of methanol. Then, the substances retained by the column were eluted with 10 ml of formic acid in methanol (2 in 98). The eluate was evaporated to dryness under a nitrogen gas stream and reconstituted in 500 µl of acetonitrile in water (20 in 80). The samples were filtered by centrifugation using 0.22-µm centrifugal filters (Millipore, Billerica, MA), and 10-µl aliquots were subjected to LC-MS analysis.

In Vitro Glucuronidation of Steroid Hormones. Androsterone, DHEA, DHT, epiandrosterone, and testosterone were incubated with liver microsomes from rats, dogs, monkeys, and humans in the presence of UDPGA to generate steroid diglucuronides. The incubation mixture contained Tris-HCl buffer (100 mM, pH 7.5), magnesium chloride (10 mM), D-saccharic acid 1,4-lactone (5 mM), steroid substrate (200 µM), liver microsomes (1 mg protein/ml), and UDPGA (5 mM) in a final volume of 200 µl. The substrates were added as a solution in DMSO (final: 0.5% DMSO). Before starting the incubation, the microsomes were treated with alamethicin (50 µg/mg protein) for 15 min (Fisher et al., 2000). A reaction was started by the addition of UDPGA, allowed to proceed for 2 h at 37°C on a shaking incubator, and terminated by the addition of 200 µl of acetonitrile. After centrifugation, a 100-µl aliquot of the supernatant was collected and evaporated to dryness using a centrifugal evaporator. The residue was reconstituted in 100 µl of acetonitrile in water (20 in 80), and the 10-µl aliquot was subjected to LC-MS analysis.

LC-MS Analysis for Detecting the Steroid Diglucuronides Produced in Vivo and in Vitro. The mass spectrometry was performed on a LTQ Orbitrap (Thermo Fisher Scientific, Inc., Waltham, MA). Because electrospray ionization can generate intense and stable [M-H]^– ions from the steroid glucuronides (Kuuranne et al., 2000) and diglucuronides (Murai et al., 2005), the instrument was operated in the negative ion electrospray ionization mode. The capillary temperature was set at 275°C. Full scan LC-MS data were acquired using an Orbitrap detector at a resolution of 30,000 in a mass range from m/z 100 to 1000. The collision-induced dissociation (CID) was performed with a relative collision energy of 35%, and the fragment ions generated were detected with the Orbitrap detector. The LC system used was a Waters Alliance 2695. Chromatographic separations were carried out on a Capcell Pak C₁₈ MGII column (5-µm particle, 2.0 x 150 mm; Shiseido Co., Ltd, Tokyo, Japan) maintained at 30°C in a column oven. The mobile phase, which was used at a flow rate of 0.2 ml/min, was a mixture of formic acid in water (0.1 in 100, solvent A) and formic acid in acetonitrile (0.1 in 100, solvent B), and the initial mobile phase consisted of 20% of solvent B in 80% of solvent A. After the sample injection, the proportion of solvent B was increased linearly from 20 to 60% over 20 min and 60 to 80% over 0.1 min, maintained at 80% for 4.9 min, and decreased linearly from 80 to 20% over 1 min. Thereafter, the column was re-equilibrated with the initial mobile phase for 15 min. To minimize contamination of the ion source, the flow of the first 2 min and that after 20 min of each run was diverted to a waste line using a switching valve. Taking advantage of the high mass accuracy, the [M-H]^– ions of steroid glucuronides were searched for using reconstructed ion chromatograms with a mass chromatogram window of 4 mDa (theoretical exact mass ± 2 mDa). In this study, we concentrated on detecting the diglucuronides of androgens and estrogens having the chemical formulas C₃₀H₃₈O₁₄, C₃₀H₄₀O₁₄, C₃₀H₄₀O₁₅, C₃₁H₄₄O₁₄, and C₃₁H₄₆O₁₄.

Isolation of M1 from Dog Urine. Dog urine was collected for 2 days from 16 male beagle dogs, each of which received a normal diet and water, as described previously. The isolation procedures of M1 from the urine are summarized in Fig. 1. The urine samples were combined as a pool (4.0 liters), acidified (pH 4) by formic acid, and loaded onto an open column of 500 g of Amberlite XAD-2 (Rohm and Haas Co., Philadelphia, PA), which was packed in a size of 8 cm in internal diameter and approximately 20 cm in length and preconditioned with methanol followed with formic acid in water (0.1 in 100). After the sample loading, the column was washed with 3 liters of formic acid in water (0.1 in 100), and the substances adsorbed to the column were eluted with 2 liters of methanol. The methanol extracts were concentrated using a rotary evaporator to remove the methanol, and the resulting aqueous solution was supplemented with water to 500 ml and adjusted to pH 9 with ammonium hydroxide. The solution was extracted twice with 500 ml of ethyl acetate to remove any nonpolar impurities. The aqueous phase was evaporated in vacuo to a dark brown oil and was dissolved in 100 ml of formic acid in water (0.5 in 100). The solution was loaded onto an open column of 40 g of Cosmosil 75C₁₈ (75-µm particle; Nacalai Tesque, Inc., Kyoto, Japan), which was packed in a size of 3.7 cm in internal diameter and approximately 7 cm in length and preconditioned with acetonitrile followed with formic acid in water (0.1 in 100). After the sample loading, the column was washed with 100 ml of formic acid in water (0.1 in 100) and successively eluted with 100 ml each of a mixture of acetonitrile and water containing 0.1% formic acid, where a proportion of acetonitrile was increased stepwise from 10, 20, 30, 40, 50, 60 to 90%. Fractions (each 50 ml) were collected and analyzed by LC-MS on a Waters Alliance 2695 HPLC system equipped with a Waters Q-Tof Ultima mass spectrometer and a Waters 2996 photodiode array detector. The fractions containing M1 were combined and lyophilized. The residue was redissolved in 100 ml of water and adjusted to pH 9 with ammonium hydroxide. The solution was then applied onto an open column of 30 g of Bondesil SAX (40-µm particle; Varian, Inc., Palo Alto, CA), which was packed in a size of 3.7 cm in internal diameter and approximately 5 cm in length and preconditioned successively with methanol and water. The column was washed successively with 100 ml of methanol in water (5 in 95) and 100 ml of methanol. M1 was eluted with 400 ml of formic acid in methanol (2 in 98), and each fraction (40 ml) was collected. The fractions containing M1 were combined and evaporated to dryness using a rotary evaporator, and the residue was dissolved in 10 ml of acetonitrile in water (20 in 80).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Isolation procedure for M1 from control dog urine. a) Mobile phase: mixture of formic acid in water (0.1 in 100, solvent A) and formic acid in acetonitrile (0.1 in 100, solvent B); linear gradient, 25 to 65% solvent B (0–20 min); flow rate, 1 ml/min. b) Mobile phase: mixture of water in acetonitrile (45 in 55) containing 10 mM ammonium formate (solvent C) and water in acetonitrile (15 in 85) containing 10 mM ammonium formate (solvent D); linear gradient, 100 to 0% (0–15 min) solvent D; flow rate, 1 ml/min. c) Mobile phase: mixture of solvent A and solvent B; linear gradient, 25 to 65% (0–15 min) solvent B; flow rate, 1 ml/min.

M1 was further purified using an LC-10A_VP HPLC system (Shimadzu Corporation, Kyoto, Japan). First, separations were carried out on an Inertsil ODS-3 column (5-µm particle, 7.6 x 50 mm; GL Sciences, Inc., Tokyo, Japan). The mobile phase, which was used at a flow rate of 3 ml/min, was a mixture of formic acid in water (0.1 in 100, solvent A) and formic acid in acetonitrile (0.1 in 100, solvent B). After the sample injection, the proportion of solvent B was linearly increased from 20 to 40% over 6 min. The column effluent was collected in 15-s intervals using a BioFrac fraction collector (Bio-Rad Laboratories, Inc., Hercules, CA). The fractions containing M1 as assessed by LC-MS were pooled and concentrated using a rotary evaporator. In a manner similar to that described above, further HPLC purification (Fig. 1) was carried out on following columns: Inertsil ODS-3 (5-µm particle; 4.6 x 250 mm), Atlantis HILIC silica (3-µm particle, 4.6 x 150 mm; Waters Corp.), Inertsil ODS-3 (5-µm particle, 4.6 x 250 mm), and Capcell Pak C₁₈ MGII (5-µm particle, 4.6 x 150 mm; Shiseido Co., Ltd). Finally, M1 was obtained as a lyophilized, white amorphous powder (approximately 1.6 mg).

In Vitro Biosynthesis of DHEA 3-O-Diglucuronide and Epiandrosterone 3-O-Diglucuronide for NMR Analysis. DHEA 3-O-diglucuronide and epiandrosterone 3-O-diglucuronide were biosynthesized using dog liver microsomes for structural determination by NMR. In this study, androsterone 3-O-diglucuronide was not biosynthesized because the preliminary study indicated that an amount of androsterone 3-O-diglucuronide sufficient for NMR analysis would not be obtained due to the low formation rate. The incubation mixture for the biosynthesis was the same as that described previously in In Vitro Glucuronidation of Steroid Hormones except that the substrate concentration was 1 mM and the total incubation volume was 30 ml. The glucuronidation reaction was allowed to proceed for 24 h at 37°C before termination by adding 1.5 ml of formic acid. After the addition of the formic acid, the mixture was placed on ice for 15 min, and the supernatant obtained by centrifugation was applied to a Varian Mega Bond Elut C₁₈ (10 g, 60 ml), which had been preconditioned successively with formic acid in acetonitrile (0.1 in 100) and formic acid in water (0.1 in 100). After the application of the sample, the column was washed with 100 ml of a mixture of acetonitrile, water, and formic acid (5:95:0.1), and the diglucuronides were eluted with 100 ml of a mixture of acetonitrile, water, and formic acid (90:10:0.1). The column eluate was concentrated using a rotary evaporator and then further concentrated by a nitrogen gas stream, and the residue was supplemented with acetonitrile in water (20 in 80) to approximately 3 ml. The extract was separated on an Inertsil ODS-3 column (5-µm particle, 7.6 x 50 mm) as described previously. After lyophilization, DHEA 3-O-diglucuronide (approximately 7.9 mg) and epiandrosterone 3-O-diglucuronide (approximately 6.0 mg) were obtained as white amorphous powders.

NMR Analysis. ¹H-1D, correlated spectroscopy, heteronuclear single quantum correlation spectroscopy (HSQC), and heteronuclear multiple bond correlation spectroscopy (HMBC) spectra were obtained using a Unity Inova 500 NMR spectrometer (Varian, Inc.) with a Varian Nanoprobe. NMR samples were dissolved in 40 µl of DMSO-d₆ and contained 500 to 600 µg of the diglucuronides. All measurements were carried out at 25°C, and the chemical shifts were referenced to the solvent signals (2.50 ppm for ¹H and 39.5 ppm for ¹³C).

	Results and Discussion

Top Abstract Materials and Methods Results and Discussion References

 
Bile and urine from rats, dogs, and monkeys were extracted by a mixed mode anion exchange solid-phase extraction cartridge, and the extracts were subjected to the LC-MS analysis. The clean-up process using the solid phase extraction cartridge was necessary for the successful detection of the diglucuronides present in biological fluids at low concentrations because it effectively concentrated acidic compounds, such as diglucuronides carrying two carboxylic acids in the molecule, while eliminating neutral and basic compounds.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Representative reconstructed ion chromatograms of m/z 639.2633 to 639.2673 plus m/z 641.2789 to 641.2829 obtained from LC-MS analysis of dog bile (a) and dog urine (b) after solid phase extraction. The bile and urine samples obtained from the other two dogs exhibited essentially the same chromatograms.

M1, M2, and other small peaks in the dog bile and urine (Fig. 2) were characterized as diglucuronides because they all exhibited the typical fragment ions for diglucuronides at m/z 351 in their CID spectra. Based on the observed accurate mass and mass fragmentation schemes (Table 1), M1 was proposed to be a diglucuronide of C₁₉ steroid and to have the molecular formula of C₃₁H₄₆O₁₄. In a similar manner, M2 was proposed to be a diglucuronide of C₁₉ steroid and to have the molecular formula of C₃₁H₄₄O₁₄. The structure of the aglycone of these diglucuronides was difficult to identify using only LC-MS because the mass fragmentation provided only limited structural information.

For the identification of diglucuronides of C₁₉ steroids detected in dog bile and urine, androsterone, DHEA, DHT, epiandrosterone, and testosterone were incubated with liver microsomes to generate steroid diglucuronides, and the samples were analyzed by LC-MS. The dog liver microsomes generated diglucuronides in varying degrees for all the steroid hormones used in this study; however, rat, monkey, and human liver microsomes produced no detectable diglucuronides under the incubation conditions and analytical methods used in this study (data not shown). The HPLC retention times of the steroid diglucuronides biosynthesized by dog liver microsomes were as follows: androsterone 3-O-diglucuronide, 11.5 min; epiandrosterone 3-O-diglucuronide, 11.5 min; DHEA 3-O-diglucuronide, 11.0 min; DHT 17-O-diglucuronide, 12.3 min; and testosterone 17-O-diglucuronide, 10.5 min.

View larger version (9K): [in this window] [in a new window]   FIG. 3. HSQC spectrum of M1 that was isolated from dog urine. The HSQC spectrum of epiandrosterone 3-O-diglucuronide, which was biosynthesized using dog liver microsomes, exhibited exactly the same spectrum.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Chemical structure of epiandrosterone 3-O-diglucuronide (a) and DHEA 3-O-diglucuronide (b). Key HMBC correlations are shown by arrows.

M2 was identified as DHEA 3-O-diglucuronide, since M2 exhibited the same HPLC retention time and CID spectrum as those of DHEA 3-O-diglucuronide obtained in vitro (Table 1). To assess the chemical structure of DHEA 3-O-diglucuronide produced by dog liver microsomes, biosynthesized DHEA 3-O-diglucuronide was subjected to NMR analysis. Two-dimensional NMR data enabled us to completely characterize the chemical structure of the biosynthesized DHEA 3-O-diglucuronide (Fig. 4; Table 2). The first glucuronosyl moiety was attached to the C3 position of DHEA, as confirmed by the key HMBC correlation peak from the H1' proton (4.53 ppm) to the C3 carbon (78.1 ppm). In the same manner as epiandrosterone 3-O-diglucuronide, the second glucuronidation in DHEA 3-O-diglucuronide occurred at the C2' position of the first glucuronosyl moiety, which was determined by two key HMBC correlation peaks from the H2' proton (3.17 ppm) to the C1'' carbon (104.7 ppm) and from the H1'' proton (4.39 ppm) to the C2' carbon (83.4 ppm).

The regiochemistry of the diglucuronosyl moiety in both epiandrosterone 3-O-diglucuronide and DHEA 3-O-diglucuronide is attached at the C2' position of the first glucuronosyl moiety (Fig. 4), which is common among the diglucuronides characterized previously (Dixon et al., 1989; Murai et al., 2002, 2005, and 2007). The hydroxyl group at the C2' position of the glucuronosyl group in a specific monoglucuronide was recognized as a functional group for further glucuronidation by unique dog UGT isoforms, which would have similar enzymatic properties to human UGT1A8, as demonstrated in our previous study in vitro (Murai et al., 2006).

Epiandrosterone is a metabolite of testosterone that possesses less androgenic activity (Dorfman, 1948). DHEA is the principal androgen secreted by the adrenal gland and circulates in plasma mainly as the sulfate form. DHEA plays an important role as the primary precursor of many important sex steroids (Regelson et al., 1994). In humans, these androgens are excreted in the urine as sulfates or glucuronides after metabolism in the liver (Bongiovanni and Cohn, 1970). Our finding of epiandrosterone, DHEA, and other C₁₉ steroids being excreted as diglucuronides in the bile and urine of dogs suggests that the levels of circulating steroid hormones in dogs are regulated differently from other species. However, the physiological significance of the diglucuronidation pathways for steroid sex hormones in comparison to monoglucuronidation, sulfation, and the other metabolic pathways remains to be clarified. The age, sex, and pathological conditions would be possible factors that affect the level of diglucuronides in dogs, just as 17-ketosteroid sulfates and glucuronides do in humans (Kroboth et al., 1999; Jia et al., 2001).

In conclusion, we first identified the endogenous diglucuronides of steroid hormones excreted in the bile and urine of male dogs as novel metabolic pathways for the disposition of steroids in dogs. Further in vivo studies using human bile and urine should be conducted in the future to investigate whether the diglucuronidation pathway also exists in humans and whether this has any physiological significance.

	Acknowledgments

 
We thank Fumio Okoshi and Hiroyoshi Namiki for technical assistance in the animal experiments and Philip Snider for checking the English of the manuscript.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.107.019059.

ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; UDPGA, uridine 5'-diphosphoglucuronic acid; NMR, nuclear magnetic resonance; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; CID, collision-induced dissociation; HSQC, heteronuclear single quantum correlation spectroscopy; HMBC, heteronuclear multiple bond correlation spectroscopy; DMSO, dimethyl sulfoxide.

Address correspondence to: Takahiro Murai, Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. E-mail: murai.takahiro.a4{at}daiichisankyo.co.jp

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