Simultaneous Determination of Twelve Tetrahydrocorticosteroid

Oct 29, 2009 - [9,12,12,21,21-d5]-THE (98.5 atom % D) was obtained from CDN. Isotopes Inc. ... CTC Analytics, Zwingen, Switzerland). The ionization ...
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Anal. Chem. 2009, 81, 10124–10135

Simultaneous Determination of Twelve Tetrahydrocorticosteroid Glucuronides in Human Urine by Liquid Chromatography/Electrospray Ionization-Linear Ion Trap Mass Spectrometry Shigeo Ikegawa,*,† Maki Hasegawa,† Rika Okihara,† Chikara Shimidzu,‡ Hitoshi Chiba,§ Takashi Iida,| and Kuniko Mitamura† Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan, Hokkaido University Hospital, Kita-14 Nishi-5, Kitaku, Sapporo 060-8648, Japan, Faculty of Health Sciences, Hokkaido University, Kita-12 Nishi-5, Kitaku, Sapporo 060-0812, Japan, and Department of Chemistry, College of Humanities and Sciences, Nihon University, 3-25-40 Sakurajousui, Setagaya, Tokyo 156-8550, Japan A liquid chromatography/electrospray ionization (ESI)mass spectrometry (MS) method for the direct determination of 12 tetrahydrocorticosteroid glucuronides in human urine has been developed. The analytes were 3and 21-monoglucuronides of tetrahydrocortisol, tetrahydrocortisone, tetrahydro-11-deoxycortisol, and their 5rstereoisomers. The mass spectrometric behaviors of these glucuronides in negative-ion ESI-MS/MS revealed the production of intense, structure-specific product ions within the same group of glucuronides. Regioisomeric glucuronides could be distinguished by collision-induced dissociation and tandem mass spectrometry. Using a linear ion trap instrument operating in the negative-ion mode and by monitoring the transition ions of [M - H]f [M - H - CH2O]- for 3-monoglucuronides and [M - H]- f [M - H - CH2OG]- for 21-monoglucuronides, a sensitive and specific assay was developed. Initial steps in the assay were a simple solid-phase extraction and the addition of [9,12,12,21,21-d5]tetrahydrocortisone-3-glucuronide (prepared by enzymeassisted synthesis) as an internal standard. The method was applied to determine the 12 tetrahydrocoticosteroid glucuronides in urine from healthy subjects and from patients with excessive cortisol production. The method described here appears to be useful for clinical and biochemical studies. Glucocorticoids, such as cortisol and cortisone, play an essential role in normal physiology by modulating metabolic and immune responses. Their altered secretion is involved in the pathogenesis of several endocrine diseases.1-5 In humans, glu* To whom correspondence should be addressed. Phone: +81-6-6721-2332. Fax: +81-6-6730-1394. E-mail: [email protected]. † Kinki University. ‡ Hokkaido University Hospital. § Hokkaido University. | Nihon University. (1) Bondy, P. K. In Metabolic Control and Disease, 8th ed.; Bondy, P. K., Rosenberg, L. E., Eds.; W. B. Saunders Company: Philadelphia, PA, 1980; pp 1427-1499. (2) White, P. C.; Mune, T.; Agarwal, A. K. Endocr. Rev. 1997, 18, 135–156.

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cocorticoids undergo extensive phase I and phase II biotransformations. These biotransformations makes them much more polar and enhances their urinary excretion. The main pathways of phase I metabolic reaction are reduction of the R,β-unsaturated carbonyl system to form tetrahydrocorticosteroids (3R-hydroxy products), oxidation and reduction at C-11, as well as reduction at C-20. These biotransformations not only increase the polarity of the molecules but also offer a site for phase II biotransformations such as glucuronidation.1,5-7 Such phase II metabolites may make up 90% of the excreted metabolites8-11 and are therefore an important class of conjugates for assessment of altered corticosteroid metabolism in disease conditions. The most common conjugation reaction in man is (ethereal) glucuronidation at carbon atoms C-3 or C-21. Most of the metabolites, such as tetrahydrocortisol (THF) and tetrahydrocortisone (THE) and their 5R-stereoisomers (alloTHF and allo-THE), are excreted in urine largely as 3- and 21monoglucuronides.9 Tetrahydro-11-deoxycortisol (THS) and its 5Rstereoisomer (allo-THS) are metabolites of 11-deoxycortisol that is a biosynthetic precursor of cortisol. THS and its 5R-isomer are known to be excreted in urine in healthy individuals as well as in disease states as their 3- and 21-monoglucuronides.12-16 Therefore, a suitable analytical method must measure all 12 tetrahy(3) Newell-Price, J.; Trainer, P.; Besser, M.; Grossman, A. Endocr. Rev. 1998, 19, 647–672. (4) Remer, T.; Maser-Gluth, C.; Wudy, S. A. Mini Rev. Med. Chem. 2008, 8, 153–170. (5) Stewart, P. M. In Williams Textbook of Endocrinology, 11th ed.; Kronenberg, H. M., Melmed, S., Polonsky, K. S., Larsen, P. R., Eds.; Saunders, Elsevia: Philadelphia, PA, 2008; pp 445-503. (6) Bongiovanni, A. M.; Cohn, R. M. In Chemical and Biological Aspects on Steroid Conjugation; Bernstein, S., Solomon S., Eds.; Springer-Verlag: New York, 1970; pp 410-450. (7) White, P. C. In Principles and Practice of Endocrinology and Metabolism; Becker, K. L., Ed.; Lippincott: Philadelphia, PA, 2001; pp 704-719. (8) Kornel, L.; Miyabo, S.; Takeda, R. Steroidologia 1971, 2, 197–236. (9) Kornel, L.; Saito, Z. J. Steroid Biochem. 1975, 6, 1267–1284. (10) Kornel, L.; Miyabo, S. Steroids 1975, 25, 697–706. (11) Aranoff, G.; Ro ¨sler, A. Acta Endrocrinol. 1980, 94, 371–375. (12) Pasqualini, J. R.; Jayle, M.-F. Biochem. J. 1963, 88, 315–318. (13) Vielhauer, W.; Mok, M.; Mittelstaedt, G. V.; Gless, K. H.; Vecsei, P. Acta Endocrinol. Suppl. 1976, 202, 74–75. (14) Pasqualini, J. R. Bull. Soc. Chim. Biol. 1963, 45, 277–300. (15) Kornel, L. Biochemistry 1965, 4, 444–452. (16) Pasqualini, J. R.; Jayle, M. F. C. R. Hebd. Seances Acad. Sci. 1963, 257, 2345–2347. 10.1021/ac9018632 CCC: $40.75  2009 American Chemical Society Published on Web 10/29/2009

Figure 1. Chemical structures of tetrahydrocorticosteroid glucuronides used in this study.

drocorticosteroid glucuronides in human urine in order to define corticosteroid metabolism and hypothalamic-pituitary-adrenal cortical axis activity. In the literature, measurements of such phase II metabolites have been done indirectly using gas chromatography/mass spectrometry (MS).17-21 More recently, liquid chromatography (LC)/MS has been used for the measurement of glucuronic acid conjugates of THF, allo-THF, and THE in human urine after enzymatic deconjugation22-25 followed by chemical derivatization26 of the liberated steroids. However, as documented elsewhere,27 the major problems associated with the deconjugation approaches are the incomplete hydrolysis (mainly due to urine matrix effect) and as well as the variability in enzyme preparations. In addition, this methodological approach provides no information about the type and site of conjugation. Direct measurement of tetrahydrocorticosteroid glucuronides as the intact molecule seems to be the optimal analytical method. The direct analysis using LC/MS of THE-3-glucuronide in bovine urine has been reported.28 However, no attempts have been made to develop an analytical (17) Ulick, S.; Tedde, R.; Wang, J. Z. J. Clin. Endocrinol. Metab. 1992, 74, 593– 599. (18) Shackleton, C. H. J. Steroid Biochem. Mol. Biol. 1993, 45, 127–140. (19) Kasuya, Y.; Shibasaki, H.; Furuta, T. Steroids 2000, 65, 89–97. (20) Shibasaki, H.; Tanabe, C.; Furuta, T.; Kasuya, Y. Steroids 2001, 66, 795– 801. (21) Rivero-Marabe´, J. J.; Maynar-Marin ˜o, J. I.; Garcı´a-de-Tiedra, M. P.; Gala´nMartı´n, A. M.; Caballero-Loscos, M. J.; Maynar-Marin ˜o, M. J. Chromatogr., B 2001, 761, 77–84. (22) Cho, H.-J.; Kim, J. D.; Lee, W.-Y.; Chung, B. C.; Choi, M. H. Anal. Chim. Acta 2009, 632, 101–108. (23) Hauser, B.; Deschner, T.; Boesch, C. J. Chromatogr., B 2008, 862, 100– 112. (24) Saba, A.; Raffaelli, A.; Cupisti, A.; Petri, A.; Marcocci, C.; Salvadori, P. J. Mass Spectrom. 2009, 44, 541–548. (25) Raffaelli, A.; Saba, A.; Vignali, E.; Marcocci, C.; Salvadori, P. J. Chromatogr., B 2006, 830, 278–285. (26) Yamashita, K.; Nakagawa, R.; Okuyama, M.; Honma, S.; Takahashi, M.; Numazawa, M. Steroids 2008, 73, 727–737. (27) Bowers, L. D.; Sanaullah, J. Chromatogr., B 1996, 687, 61–68. (28) Antignac, J. P.; Le Bizec, B.; Monteau, F.; Andre´, F. Steroids 2002, 67, 873–882.

method that would quantify all 12 tetrahydrocorticosteroid glucuronides in human urine. In this study, we have developed a direct method using LC/ electrospray ionization (ESI)-MS/MS that appears capable of simultaneous determination of 3- and 21-monoglucuronides of tetrahydrocorticosteroids. The structures measured by this analytic approach are shown in Figure 1. These compounds are tetrahydrocorticosteroid glucuronides having slight differences between each other in substitution at C-3 and C-21 and in A/B ring juncture (5R- and 5β-steroid). One objective of our work was to determine whether collision-induced dissociation (CID) with linear ion trap mass spectrometry would identify structure-specific ions of sufficient intensity to be useful for analytical purposes and to determine whether such ions, if present, could be used to distinguish isomeric pairs of glucuronides. In this paper, the formation of major product ions of selected tetrahydrocorticosteroid glucuronides under negative-ion ESI-MS/ MS are interpreted, and fragmentation pathways, which have not been previously reported, are proposed for those glucuronides. A method for LC separation of the individual glucuronides was developed. [9,12,12,21,21-d5]-THE-3-glucuronide was synthesized enzymatically as an internal standard (IS), and a solid-phase extraction procedure was shown to give adequate recovery. The final method was used to quantitate the 12 tetrahydrocorticosteroid glucuronides in healthy volunteers as well as patients with excessive cortisol production. MATERIALS AND METHODS Materials. All reference standard tetrahydrocorticosteroid glucuronides were chemically synthesized29-32 and were a generously gift of Dr. Toshio Nambara, emeritus professor of Tohoku (29) Hosoda, H.; Saito, K.; Ito, Y.; Yokohama, H.; Ishii, K.; Nambara, T. Chem. Pharm. Bull. 1982, 30, 2110–2118. (30) Hosoda, H.; Yokohama, H.; Ishii, K.; Ito, Y.; Nambara, T. Chem. Pharm. Bull. 1983, 31, 4001–4007. (31) Hosoda, H.; Takasaki, W.; Miura, H.; Tohkin, M.; Maruyama, Y.; Nambara, T. Chem. Pharm. Bull. 1985, 33, 4281–4287. (32) Hosoda, H.; Osanai, K.; Fukasawa, I.; Nambara, T. Chem. Pharm. Bull. 1990, 38, 1949–1952.

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Table 1. Trivial and Systematic Name of Tetrahydrocorticosteroid Glucuronides, Their Retention Times, and SRM Transitions

abbreviation

trivial name

systematic name

retention time (min)a

THF-3G THF-21G THE-3G THE-21G THS-3G THS-21G allo-THF-3G allo-THF-21G allo-THE-3G allo-THE-21G allo-THS-3G allo-THS-21G

tetrahydrocortisol 3-glucuronide tetrahydrocortisol 21-glucuronide tetrahydrocortisone 3-glucuronide tetrahydrocortisone 21-glucuronide tetrahydro-11-deoxycortisol 3-glucuronide tetrahydro-11-deoxycortisol 21-glucuronide allo-tetrahydrocortisol 3-glucuronide allo-tetrahydrocortisol 21-glucuronide allo-tetrahydrocortisone 3-glucuronide allo-tetrahydrocortisone 21-glucuronide allo-tetrahydro-11-deoxycortisol 3-glucuronide allo-tetrahydro-11-deoxycortisol 21-glucuronide

3R,11β,17, 21-tetrahydroxy-5β-pregnan-20-one 3-glucuronide 3R,11β,17, 21-tetrahydroxy-5β-pregnan-20-one 21-glucuronide 3R,17,21-trihydroxy-5β-pregnane-11,20-dione 3-glucuronide 3R,17,21-trihydroxy-5β-pregnane-11,20-dione 21-glucuronide 3R,17,21-trihydroxy-5β-pregnan-20-one 3-glucuronide 3R,17,21-trihydroxy-5β-pregnan-20-one 21-glucuronide 3R,11β,17, 21-tetrahydroxy-5R-pregnan-20-one 3-glucuronide 3R,11β,17, 21-tetrahydroxy-5R-pregnan-20-one 21-glucuronide 3R,17,21-trihydroxy-5R-pregnane-11,20-dione 3-glucuronide 3R,17,21-trihydroxy-5R-pregnane-11,20-dione 21-glucuronide 3R,17,21-trihydroxy-5R-pregnan-20-one 3-glucuronide 3R,17,21-trihydroxy-5R-pregnan-20-one 21-glucuronide

13.0 16.5 14.0 17.0 16.8 19.2 12.8 16.1 13.6 16.6 16.4 18.7

SRM transition (m/z) 541 f 511 541 f 335 539 f 509 539 f 333 525 f 495 525 f 319 541 f 511 541 f 335 539 f 509 539 f 333 525 f 495 525 f 319

a Column, TSKgel OSD-100S (150 mm × 2.0 mm i.d.); mobile phase, (A) 5 mM AcONH4 (pH 6), (B) MeCN, 5% B-50% B (0-25 min); flow rate, 200 µL/min.

University; their structures are shown in Figure 1. Trivial and systematic names of the compounds are listed in Table 1. [9,12,12,21,21-d5]-THE (98.5 atom % D) was obtained from CDN Isotopes Inc. (Quebec, Canada). Uridine-5′-diphosphoglucuronic acid (UDPGA, trisodium salts) and D-saccharic acid-1,4lactone were purchased from Sigma-Aldrich (St. Louis, MO). An Oasis HLB cartridge (10 and 60 mg of solid phase) was provided by Waters Co. Ltd. (Milford, MA). Methanol, acetonitrile, and ammonium acetate for LC/MS were HPLC grade purchased from Nacalai Teque, Inc. (Kyoto, Japan). Distilled water of HPLC grade was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). An amount of 5 mM ammonium acetate buffer (pH 4) was prepared by dissolving anhydrous ammonium acetate in HPLC grade H2O and adjusting the pH with acetic acid. All other chemicals and solvents were analytical grade and obtained from Nacalai Teque Inc. All glasswares were silanized with trimethylchlorosilane. LC/ESI-MS/MS. The LC/ESI-MS/MS analysis was performed on a Finnigan LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA) equipped with an ESI probe and coupled to a Paradigm MS4 pump (Michrom Bioresources, Inc., Auburn, CA) and an autosampler (HTC PAL, CTC Analytics, Zwingen, Switzerland). The ionization conditions for verifying the structures of glucuronides were as follows: ion source voltage, -4 kV; capillary temperature, 270 °C; capillary voltage, -20 V; sheath gas (nitrogen gas) flow rate, 50 arbitrary units (arb units); auxiliary gas (nitrogen gas) flow rate, 5 arb units; tube lens offset voltage, -100 V. For tandem MS (MS/MS) analysis, helium gas was used as the collision gas and the normalized collision energy was set at 10-60%. The LC separations were conducted on a semimicro reversed-phase column, TSKgel ODS-100S (5 µm; 150 mm × 2.0 mm i.d.) from TOSOH Co. (Tokyo, Japan). The mobile phase was 5 mM ammonium acetate (pH 6.0) and acetonitrile, used at a flow rate of 200 µL/min. Preparation of Rat Live Microsomes. Animal studies were approved by the Institutional Animal Care and Use Committee of Kinki University. Male Wistar rats (230-250 g), given a commercial pellet diet and water ad libitum, were used. Microsomes were isolated from liver using differential centrifugation33 and were (33) Luukkanen, L.; Elovaara, E.; Lautala, P.; Taskinen, J.; Vainio, H. Pharmacol. Toxicol. 1997, 80, 152–158.

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stored at -70 °C until use. Protein concentration of the microsomes was determined with a commercial BCA protein assay kit (Pierce Chemical, Rockford, IL). Enzyme-Assisted Synthesis of [9,12,12,21,21-d5]-THE3-Glucuronide. A suspension of microsomes (5 mg of protein) in 4.5 mL of 100 mM Tris-HCl buffer (pH 7.4) containing 5 µM MgCl2, 2.5 µM D-saccharic acid-1,4-lactone, 0.01% Triton-X100 was preincubated with each [9,12,12,21,21-d5]-THE (250 nmol) in methanol (50 µL) at 37 °C for 10 min. The reaction was initiated by the addition of 25 mM UDPGA in 100 mM Tris-HCl buffer (pH 7.4, 500 µL), and the mixture was incubated at 37 °C for 180 min. The reaction was terminated by the addition of 100 mM acetic acid (25 µL). After centrifugation at 3000 rpm for 5 min, the supernatant was loaded onto an Oasis HLB cartridge (3 mL, 60 mg), previously activated with methanol (1 mL) and H2O (1 mL). After washing with H2O (1 mL), the glucuronide was eluted with methanol (1.5 mL). After evaporation of the solvent under a gentle stream of N2 gas, the residue was redissolved in methanol (50 µL) and submitted to purification of the desired glucuronide by highperformance liquid chromatography (HPLC) with UV detection (215 nm) on a TSKgel ODS-100S column (5 µm, 150 mm × 2.0 mm i.d. TOSOH Co.). HPLC used a linear gradient of 20% solvent B (acetonitrile) increasing to 80% solvent B against solvent A (5 mM ammonium acetate, pH 4) over 40 min at a flow rate of 200 µL/min. The fractions containing the desired glucuronide were collected from 50 incubation mixtures and evaporated to dryness to give [9,12,12,21,21-d5]-THE-3G (4.64 mg, 68.2% yield) whose structure was confirmed by negativeion LC/ESI-MS/MS. The isotopic purity of the labeled compound as d5-form was 98.5 atom % D. Calibration Curves. Standard stock solutions of each tetrahydrocorticosteroid glucuronide were prepared gravimetrically in methanol at the concentration of 200 µg/mL. LC/MS standards were prepared by dilution of stock solution with methanol to give concentrations of 6.25, 12.5, 25, 62.5, 125, 250, 500, 1250, 2500, 5000, and 10000 ng/mL. The IS solution of [9,12,12,21,21-d5]-THE3G at a concentration of 20 µg/mL was prepared gravimetrically in methanol. All stock standard and stock solutions were stored at -4 °C or below and allowed to equilibrate at room temperature for at least 15 min before use. In the calibration

study, a 40 µL aliquot of each LC/MS standard was mixed with 5 µL of the IS solution; the mixture was evaporated to dryness under a gentle stream of N2 gas at room temperature. The residue was redissolved in 50 µL of 15% (v/v) acetonitrile, and 10 µL of this solution was injected into the LC/ESI-MS/MS system. The calibration curves were constructed by plotting the peak area ratio of each glucuronide relative to that of [9,12,12,21,21-d5]-THE-3G (IS) against the weight of the glucuronide. The limit of detection (LOD) was calculated on the most intense transition, with the criterion of a signal-to-noise ratio exceeding 3. Analysis of Tetrahydrocorticosteroid Glucuronides in Human Urine. Informed consent was obtained from all volunteers and patients, and the experimental procedures were conducted in accordance with the ethical committee of Kinki University and Hokkaido University Hospital. Urine samples were collected at 10:00 a.m. from healthy volunteers aged 22-25 years. Pooled urine samples were obtained from female patients aged 33-49 years with pituitary tumor, adrenal tumor, adrenal cancer, pituitary adenoma, and ectopic ACTH. Urine was kept frozen at -80 °C until analysis. For analysis, the urine samples were thawed and then centrifuged at 1710g at 4 °C for 5 min to eliminate solid residue. To a 50 µL aliquot of the resulting supernatant was added 5 µL of the IS solution, and the mixture was diluted with 100 µL of 5 mM ammonium acetate buffer (pH 5). The samples were thoroughly mixed and then applied to an Oasis HLB cartridge (10 mg, 1 mL). After washing with H2O (1 mL), the glucuronides were eluted with methanol (1 mL). After evaporation of the solvents under a gentle stream of N2 gas at room temperature, the residue was redissolved in 50 µL of 15% (v/v) acetonitrile, and a 10 µL aliquot was injected to the LC/ESI-MS/MS system. Preparation of Blank Urine Sample and Limit of Quantitation (LOQ). Urine (10 mL) of healthy volunteer was stirred for 2 h with activated charcoal powder (1 g) (Wako Pure Chemical Industries, Ltd.) and then centrifuged at 1710g (4 °C, 10 min) followed by filtration. The filter was a Millex-HA filter unit (Millipore, Billerica, MA) with a mixed cellulose acetate membrane (pore size, 0.45 µm). Repeated LC/ESI-MS/MS analyses have shown that the charcoal adsorption procedure removes essentially all tetrahydrocorticosteroid glucuronides. (For analytical details, see above paragraph.) The LOQ was defined as the lowest concentration of each glucuronide that could be detected, assuming a recovery of 80% or more. Precision and Accuracy. For the precision study, the concentration of each glucuronide in a urine sample was determined five times on the same day (intra-assay precision) and five times on the different days (interassay precision). The precision was obtained in terms of the relative standard deviation (RSD %). Analytical recoveries (%) were obtained with the test sample (n ) 5), in which 50 µL of the human urine was mixed with 10 µL aliquots of 0.5, 5, or 10 µg/mL stock solution. A 5 µL aliquot of IS solution was added, and sample was then acidified with the addition of 100 µL of 5 mM ammonium acetate buffer (pH 5). The concentrations of the glucuronides found in the urine were defined as F, and the analytical recoveries were calculated as [F(in added sample)/{F(in nonadded sample) + amounts of added tetrahydrocorticosteroid glucuronide}] × 100 (%).

RESULTS AND DISCUSSION Collision-Induced Fragmentation of Glucuronides. During the past decade, LC in combination with tandem mass spectrometry has been used to determine a variety glucuronic acid conjugates of biologically important substances with low molecular weight.34-37 Nonetheless, the general behavior of tetrahydrocorticosteroid glucuronides in collision-induced fragmentation has not been extensively investigated. Therefore, a study of the mass spectrometric behaviors of tetrahydrocorticosteroid glucuronides was judged to be essential in the development of direct analysis methods by LC/MS. In addition, interpretation of product ion spectra of the glucuronides and exploration of their CID pathways are of importance for mass spectrometric characterization. Such data should be helpful in understanding specificity of the product ions of tetrahydrocorticosteroid glucuronides that are, in turn, useful for their detection, identification, and confirmation of structure. Accordingly, we examined the collision-induced fragmentation of tetrahydrocorticosteroid glucuronides using ESIlinear ion trap MS. Typical negative-ion CID spectra of 3- and 21glucuronides of THF and allo-THF are shown in Figure 2. In the full-scan negative-ion mode, all of the tested glucuronides yielded the deprotonated molecule [M - H]- as the predominant base ion with no significant fragment ions. The high stability of [M - H]- ion is presumably due to the localization of the negative charge at the carboxyl group on the glucuronic acid moiety. Further MS/MS experiments on the selected precursor ion [M - H]- at the collision energy of 40% permitted the precise identification of the fragment ions produced by classical CID in the collision cell. The steroid nucleus was very stable under low-energy CID; however, the glucuronic acid residue and dihydroxyacetone side chains were readily fragmented. These fragment ions were found to be divided into two categories, depending on the site of conjugation. The first category corresponded to ions characteristic of the 3-glucuronides, which behaved similarly. Nonetheless, the relative abundances of the product ions differed significantly within the glucuronides (Table 2). Thus, the 3-glucuronides showed the loss of a water molecule [M - H - H2O]- or [M - H - CH2O]ion as the base peak with the weak [M - H - 2H2O]- and [M - H - CH2O - H2O]- ions generated by successive loss of a water molecule from [M - H - H2O]- and [M - H - CH2O]ion; the latter ion probably resulted from the loss of formaldehyde (CH2O) involving cleavage of C20-C21. In agreement with previous studies,34,35 the 17R-hydroxy function seems to be involved in the cleavage of C20-C21 with concomitant loss of CH2O, a phenomenon probably linked to stabilization at the negative charge. All 3-glucuronides generated the [M - H - CH2CO - H2O]- ion with modest abundance, attributable to fragmentation of the dihydroxyacetone side chain. With lower or modest abundance, a [M H - H2O - CH2O - CO2]- ion formed by the loss of water and carbon dioxide from [M - H - CH2O]- was also observed, although this ion species was not generated by THF- or THS(34) Volmer, D. A.; Hui, J. P. Rapid Commun. Mass Spectrom. 1997, 11, 1926– 1933. (35) Antignac, J.-P.; Le Bizec, B.; Monteau, F.; Poulain, F.; Andre´, F. Rapid Commun. Mass Spectrom. 2000, 14, 33–39. (36) Kuuranne, T.; Vahermo, M.; Leinonen, A.; Kostianen, R. J. Am. Soc. Mass Spectrom. 2000, 11, 722–730. (37) Mattox, V. R.; Vrieze, W. D. J. Org. Chem. 1972, 37, 3990–3996.

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Figure 2. Negative-ion ESI-MS and CID spectra of (A) THF-3G, (B) allo-THF-3G, (C) THF-21G, and (D) allo-THF-21G. The [M - H]- ions at m/z 541 of these glucuronides were collisionally activated at 40%. Table 2. Diagnostically Significant Ions Obtained from the CID Spectra on the [M - H]- Ions and the Ratio of [M - H - CH2O]- to [M - H - H2O]- of Tetrahydrocorticosteroid 3-Glucuronidesa m/z product ion

THF 541

THE 539

THS 525

allo-THF 541b

allo-THE 539b

allo-THS 525b

-

523 (100) 511 (51) 505 (7) 493 (7) 481 (21) 463 (3) N.D.c 423 (6) 405 (2) 393 (3) 347 (3) 335 (5) 317 (3) N.D. 301 (2) 175 (1) N.D. 0.51

521 (88) 509 (100) 503 (4) 491 (8) 479 (27) 461 (2) 447 (6) 421 (5) 403 (1) 391 (5) 345 (2) 333 (12) 315 (2) 305 (2) N.D. 175 (1) N.D. 1.14

507 (100) 495 (42) 489 (7) 477 (6) 465 (21) 447 (2) N.D. 407 (8) 389 (2) 377 (2) 331 (3) 319 (10) N.D. N.D. N.D. 175 (1) 157 (1) 0.42

523 (100) 511 (94) 505 (2) 493 (23) 481 (22) 463 (3) 449 (15) 423 (8) 405 (5) 393 (2) 347 (2) 335 (15) 317 (4) 307 (2) 301 (5) 175 (2) 157 (1) 0.94

521 (14) 509 (100) 503 (1) 491 (14) 479 (7) 461 (