Steroids 80 (2014) 80–91

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Tandem mass spectrometric characterization of bile acids and steroid conjugates based on low-energy collision-induced dissociation Masamitsu Maekawa a, Miki Shimada a, Takashi Iida b, Junichi Goto a, Nariyasu Mano a,⇑ a b

Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya-ku, Tokyo 156-8550, Japan

a r t i c l e

i n f o

Article history: Received 22 May 2013 Received in revised form 14 November 2013 Accepted 20 November 2013 Available online 1 December 2013 Keywords: Bile acid conjugate Low-energy collision-induced dissociation Neutral loss Product ion Steroid conjugate

a b s t r a c t We examined the characteristics of several bile acids and some steroid conjugates under low-energycollision-induced dissociation conditions using a triple quadrupole tandem mass spectrometer. According to conjugation types, we observed characteristic product ions and/or neutral losses in the product ion spectra. Amino acid conjugates afforded specific product ions. For example, glycine-conjugated metabolites routinely produced a product ion at m/z 74, and taurine-conjugated metabolites produced product ions at m/z 124, 107, and 80. When a strong peak appeared at m/z 97, the molecule contained a sulfate group. In contrast to amino acid conjugates, carbohydrate conjugates required a combination of product ions and neutral losses for identification. We could discriminate a glucoside from an acyl galactoside according to the presence or absence of a product ion at m/z 161 and a neutral loss of 180 Da. Discrimination among esters, aliphatic ethers, and phenolic ether types of glucuronides was based upon differences in the intensities of a product ion at m/z 175 and a neutral loss of 176 Da. Furthermore, N-acetylglucosamine conjugates showed a characteristic product ion at m/z 202 and a neutral loss of 203 Da, and the appearance of a product ion at m/z 202 revealed the existence of N-acetylglucosamine conjugated to an aliphatic hydroxyl group without a double bond in the immediate vicinity. Together, the data presented here will help to enable the identification of unknown conjugated cholesterol metabolites by using low-energy collision-induced dissociation. Ó 2013 Published by Elsevier Inc.

1. Introduction Cholesterol is involved in the stabilization of biomembranes, and is a precursor for the biosynthesis of small biomolecules, including bile acids, steroid hormones, and vitamin D. Humans produce cholesterol from acetyl-CoA via mevalonic acid, and also ingest cholesterol in the diet. It has a simple chemical structure possessing only a hydroxyl group at the C-3 position as a characteristic functional group. Natural cholesterol metabolites, including bile acids, steroid hormones, and vitamin D, possess hydroxyl, carbonyl, and carboxyl groups, which are often conjugated to other small molecules, particularly when detected in the

Abbreviations: CA, cholic acid; CDCA, chenodeoxycholic acid; CID, collisioninduced dissociation; DCA, deoxycholic acid; ESI, electrospray ionization; GlcNAc, N-acetylglucosamine; LC, liquid chromatography; LCA, lithocholic acid; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NPC, Niemann–Pick disease type C1; SNAG-D5-CA, 3b-sulfo-7b-N-acetylglucosaminyl-5-cholen-24-oic acid; SNAG-D5-CG, glycine-conjugated 3b-sulfo-7b-N-acetylglucosaminyl-5-cholen-24oic acid; SNAG-D5-CT, taurine-conjugated 3b-sulfo-7b-N-acetylglucosaminyl-5cholen-24-oic acid; UDCA, ursodeoxycholic acid. ⇑ Corresponding author. Tel.: +81 22 717 7525; fax: +81 22 717 7545. E-mail address: [email protected] (N. Mano). 0039-128X/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.steroids.2013.11.016

urine [1–4]. For example, the reactive chemical handles may be conjugated to amino acids [5], sulfuric acid [6], glucuronic acid [7], glucose [8], galactose [9], or N-acetylglucosamine (GlcNAc) [10]. These conjugations result in the addition of very polar functional groups onto the cholesterol metabolites; therefore, electrospray ionization (ESI) is a suitable ionization method for their mass spectrometric analysis [11–14]. Bile acids are quantitatively major metabolites of cholesterol, and all of the above-mentioned conjugations have been found in the human body. Sulfation is the most commonly observed modification made during the body’s elimination of bile acids; sulfated bile acids constitute approximately 40% of total urinary bile acids [15]. Glucuronidation is known as an important detoxification route for low molecular weight compounds including drugs [16–19]. Since Back et al. [7,20] found bile acid glucuronides in the urine of cholestasis patients, the glucuronidation of bile acids had been thought to occur almost exclusively as an ether modifying a hydroxyl group at the C-3 position. However, it is now known that the main component of bile acid glucuronides are esters linked through a carboxyl group at the C-24 position [21]. In the urine of ursodeoxycholic acid (UDCA)-treated patients with primary biliary cirrhosis, a hydroxyl group at the C-7b position

M. Maekawa et al. / Steroids 80 (2014) 80–91

was specifically conjugated with GlcNAc [22], as was also found to be the case with other bile acid-related substances [23]. Similarly, bile acids modified with glucoside at the C-3 position [23,24], and galactoside at the C-24 position [9] have been found in human urine. Some of these conjugations are also found on steroid hormones [25]. In addition, Alvelius et al. [26] found the highly concentrated abnormal cholesterol metabolites, 3b-sulfo-7b-Nacetylglucosaminyl-5-cholen-24-oic acid (SNAG-D5-CA) and its amino acid conjugates, in urine from a Niemann–Pick disease type C1 (NPC) patient. Recently, we pointed out the possibility of their metabolites as new diagnostic biomarkers of the disease [27]. Importantly, we can provide information relevant to modifications on all conjugated cholesterol metabolites by investigating the characteristics of bile acid conjugates and some steroid conjugates (Fig. 1). Accordingly, we investigated the mass spectrometric characteristics of all known conjugations using ESI-mass spectrometry (MS) and ESI-tandem mass spectrometry (MS/MS) with low-energy collision-induced dissociation (CID). The knowledge obtained here will help to enhance databases used for the identification of known conjugated cholesterol metabolites in human urine.

2. Experimental 2.1. Chemicals Cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and UDCA were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The following compounds were synthesized in our laboratories using previously reported methods: glycine- and taurine-conjugated bile acids [28], bile acid 3-sulfates [29], bile acid 3-glucuronides [30], bile acid 24-glucuronides [31], bile acid 7b-N-acetylglucosamine conjugates [32], bile acid 3-glucosides [33], bile acid 24-galactosides [34], SNAGD5-CA and its glycine and taurine conjugates (SNAG-D5-CG, SNAG-D5-CT) [35]. 17b-Estradiol 3-sulfate, 3-glucuronide, and 17-glucuronide were purchased from Steraloids, Inc. (Newport, RI, USA). All conjugated standard specimens were dissolved at 2.0 lmol/L in water/ethanol (1:1, v/v), and unconjugated standard specimens were dissolved at 20 lmol/L in water/ethanol (1:1, v/v). Ultrapure water was prepared using a PURELAB ultra apparatus (Organo, Tokyo, Japan). All other chemicals and solvents were analytical grade. 2.2. Measurement of product ion spectra ESI-MS/MS was performed using an API 5000 mass spectrometer (AB SCIEX, Framingham, MA, USA). The ion spray voltage, declustering potential, and Turbo V source heaters were set at 4500, 80 V, and 700 °°C, respectively. Nitrogen was used for Curtain Gas, Gas1, Gas2, and Collision Gas, with flow rates of 25, 40, 60, and 6 units, respectively. The spray solvent was a mixture of 20 mmol/L ammonium acetate solution (pH 7.0) and methanol (3:7, v/v). Analytes were introduced into the ESI probe through an inline filter and a short ODS column (Shim pack MAYI-ODS [36], 2.0 mm i.d.  10 mm, Shimadzu Corp., Kyoto, Japan) at a flow rate of 0.2 mL/min. Product ion spectra were measured with a scan range of m/z 5 –900 and a scan time of 1 s. The collision voltage was set from 5 to 130 V. Unit mass resolution was selected in both Q1 and Q3. Doubly charged deprotonated molecules ([M 2H]2 ) were selected as precursor ions for glycine- and taurine-conjugated bile acid 3-sulfates, SNAG-D5-CG, and SNAG-D5CT. Singly charged deprotonated molecules ([M H] ) were used for all other compounds. Data were collected and processed using Analyst 1.4.1 data collection and integration software.

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3. Results and discussion 3.1. Differences in low-energy CID patterns among bile acids and their amino acid conjugates We have already analyzed unconjugated, glycine-, and taurineconjugated bile acids using a Q-TOF instrument [37]; here, using a triple quadrupole instrument, we obtained similar product ion spectra for these conjugates, as shown in Fig. 2B, D, and F. The similarity of the spectra is not surprising because of a similarity of the ionization conditions. We performed product ion scan analysis of unconjugated, glycine-, and taurine-conjugated CDCA (Figs. 2B, D, and F). Only [M H H2O] was detected at m/z 373 using collision voltages from 30 to 60 V (Table 1). The intensity of that product ion was very low (Fig. 2A), indicating that the unconjugated bile acid was very stable under low-energy CID. Although we did not show a mass spectrum of CA, product ions with neutral losses consistent with formic acid, e.g., [M H (H2O+HCOOH)] at m/z 343 and [M H (2H2O+HCOOH)] at m/z 325, were detected at relatively high intensity using a collision voltage of 60 V (Table 1S). By contrast, we never observed product ions of LCA. Qiao et al. [38] investigated the fragmentation pathway of 18 species of bile acids predominantly comprising dihydroxylated bile acids and including amino acid conjugates, by tandem mass spectrometry using an ion trap/time-of-flight instrument operated in negative ion mode. They show very similar results, and in the product ion spectrum of CDCA, a dehydroxylated product ion appeared only at m/z 373. In addition, DCA possessing a 12a-hydroxyl group produced neutral loss of 44 Da (CO2) and 46 Da (HCOOH) as with our data. These suggested that different instruments shared similar fragment pattern under low-energy CID conditions. For glycine-conjugated CDCA (GCDCA), three product ions appeared at m/z 74, m/z 384, and m/z 386, as shown in Fig. 2D. The product ion observed at m/z 74 was NH2CH2COO , and the peaks at m/z 384 and m/z 386 were estimated to be [M H (H2O+HCOOH)] and [M H (H2O+CO2)] , respectively, which may be produced by the elimination of water and formic acid or carbon dioxide. Those three product ions were most intense at a collision voltage of 50 V. Although dehydrated product ions were found in the spectra of unconjugated bile acids, when analyzing glycine conjugates we found little dehydrated product ion (Table 1S). Instead, we observed that water was eliminated with CO2 or HCOOH; this same fragmentation was also found in CA and DCA. Both CA and DCA have a 12a-hydroxyl group, which is known to bind with a carboxyl group at the end of the side chain through intra-molecular hydrogen bonding [39]. Similarly, the carboxyl group in glycine conjugates can bind to the 12a-hydroxyl and to the 7a- and 7b-hydroxyl groups. Therefore, glycine conjugates produced [M H (H2O+CO2)] and [M H (H2O+HCOOH)] , except for GLCA, which possesses only a 3a-hydroxyl group. NH2CH2COO at m/z 74 was a common product ion in all glycine conjugates (Tables 1, 1S–3S, and 5S). The product ion spectrum of taurine-conjugated CDCA (TCDCA) is shown in Fig. 2F. Specifically, the three product ions observed at m/z 80, 107, and 124 were all derived from the taurine moiety, and were identified as SO3 , CH2CHSO3 , and NH2CH2CH2SO3 , respectively. These three product ions were common in all taurine conjugates (Tables 1, 1S–3S, and 5S). Although all product ions appeared in the product ion spectrum at a collision voltage of 80 V, the use of higher collision voltages resulted in the convergence of product ions to SO3 , at m/z 80. Since taurine conjugates have no carboxyl group, they produced [M H H2O] , at m/z 480, similar to unconjugated bile acids, but did not give rise to [M H (H2O+CO2)] or [M H (H2O+HCOOH)] . These facts also confirm the effect of intra-molecular hydrogen bonding between a hydroxyl group and a

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(A) Bile acid conjugates R3 COR4

R1

R2

H

Compound

R1

R2

R3

R4

CDCA GCDCA

OH OH

α-OH α-OH

H H

OH NHCH 2COOH NHCH 2CH 2SO3H

TCDCA

OH

α-OH

H

CDCA 3-sulfate

OSO3H

α-OH

H

OH

GCDCA 3-sulfate

OSO3H

α-OH

H

NHCH 2COOH

OSO3H

α-OH

H

NHCH 2CH 2SO3H

O

α-OH

H

OH

O

α-OH

H

NHCH 2COOH

O

α-OH

H

NHCH 2CH 2SO3H

α-OH

H

TCDCA 3-sulfate

HOH2C O

HO HO

CDCA 3-glucoside

OH HOH2C O

HO HO

GCDCA 3-glucoside

OH HOH2C

TCDCA 3-glucoside

O

HO HO

OH

CDCA 24-galactoside

OH

OH OH HO O

O

CH2OH

HOOC O

HO HO

DCA 3-glucuronide

O

H

OH

H

OH

OH

OH

OH

DCA 24-glucuronide

OH

HO O

O

OH COOH

OH AcHN O

O

AcHN O

O

UDCA 7β-N-GlcNAc

OH

β

GUDCA 7β-N-GlcNAc

OH

β

OH CH2OH

H

OH

OH CH2OH

H

NHCH 2COOH

OH CH2OH

H

NHCH 2CH 2SO3H

OH

OH

TUDCA 7β-N-GlcNAc

AcHN O

OH

β

O

(B) Other steroids R2 COR

OH AcHN O

HO3SO

Compound

O

OH CH2OH

R1

R

Compound

OH

17β-Estradiol 3-glucuronide

R1

R2

HOOC

5

SNAG-Δ -CA

O

HO HO

OH

O OH

SNAG-Δ 5-CG

SNAG-Δ 5-CT

OH

NHCH 2COOH

17β-Estradiol 17-glucuronide

NHCH 2CH 2SO3H

17β-Estradiol 3-sulfate

OH

OSO3H

HO O

O

OH COOH

OH

Fig. 1. Structures of the compounds tested in Figs. 2–7. (A) The structures of the bile acid conjugates. (B) The structures of other steroids conjugates.

carboxyl group on the fragmentation of bile acids and their glycine conjugates. 3.2. Characteristics of sulfates in low-energy CID Bile acid 3-sulfates have two acidic moieties. One is a sulfate group at the C-3 position, and the other is a carboxyl group or a sulfonic group at the side-chain terminus. In ESI mass spectra,

unconjugated bile acid 3-sulfates produced a singly charged deprotonated molecule, [M H] , as a base peak (data not shown). By contrast, glycine- and taurine-conjugated bile acid 3-sulfates yielded doubly charged deprotonated molecules, [M 2H]2 , as base peaks when we included ammonium acetate in the spray solvent (data not shown). These phenomena were consistent with previous observations [40]. CDCA 3-sulfate mainly generated HSO4 , at m/z 97, as a product ion at collision voltages of 40 V

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(A)

(B)

ᅜ104 6.0

ᅜ106 2.5

ᅜ105 7.0

COO-

391

4.0

6.0 HO

0.0

1.5

OH H

2.0

-30

-40

-50

-60

1.0

Intensity (cps)

Intensity (cps)

2.0 5.0 COO-

4.0 3.0

HO H

2.0

0.5 1.0 0.0

373

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

0

-90 -100 -110 -120 -130

100

200

300

400

(C)

700

800

900

ᅜ105 2.0

NH2CH2COO74

4.0 2.0

1.6

0.0 -40

0.6

-50

-60

-70

0.4

Intensity (cps)

0.8

Intensity (cps)

600

(D)

ᅜ104 6.0

ᅜ106 1.0

500

m/z

Collision voltage/V

CONHCH2-

CONCH -

1.2

HO

HO

CONHCH 2COO-

H

H

HO

0.8

OH

H

448 386

0.2

0.4

0.0

0.0

384

-5

-10

-20

-30

-40

-50

-60

-70

-80

0

-90 -100 -110 -120 -130

100

200

300

400

Collision voltage/V

500

m/z

600

700

800

900

ᅜ105 1.2

(E)

Intensity (cps)

80

0.3

1.4

CONHCH2CH2SO 3-

ᅜ105 1.0

0.6

0.8 0.0

1.2

-60

-70

-80

-90

1.0 0.8 0.6 0.4

Intensity (cps)

1.6

(F)

0.9 ᅜ106

CONHCH2CH2SO 3-

SO3-

HO

H HO

CH2CHSO3-

0.6

H

OH

498 NH2CH2CH2SO3107 124

0.4

0.2

0.2

480

0.0

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

Collision voltage/V

0

100

200

300

400

500

600

700

800

900

m/z

Fig. 2. Typical product ion spectra and graphs of the product ion intensities versus the applied collision voltages for unconjugated (A, B), glycine-conjugated (C, D), and taurine-conjugated CDCA (E, F). The collision energies were 50, 50, and 80 V for (B), (D), and (F), respectively. Other mass spectrometric parameters were described in the experimental section.

or higher (Fig. 3A and B). In addition, SO3 at m/z 80 was also detected and increased in intensity as the collision voltage was raised (Table 1 and Fig. 3A). Metzger et al. [41] investigated the fragmentation pattern of cholesterol 3-sulfate and reported the production of HSO4 , at m/z 97, and SO3 , at m/z 80, results that accord with the present data. Interestingly, only the LCA 3-sulfate produced [M H SO3] (with a neutral loss of 80 Da) as shown in Table 2S. Specifically, the dihydroxy and trihydroxy bile acids, with hydroxyl groups at the C-7 and/or the C-12 positions, did not show this neutral loss of 80 Da. In contrast to unconjugated bile acids, bile acid 3-sulfates lacking amino acids produced few dehydrated product ions. The doubly charged, deprotonated, glycine-conjugated bile acid 3-sulfates revealed complex fragmentation patterns upon CID. GCDCA 3-sulfate produced [M H NH2CH2COOH] , at m/z 453, [M H (H2SO4+NH2CH2COOH)] , at m/z 355, [M H (H2SO4+ CONCH2COOH)] , at m/z 329, HSO4 , at m/z 97, SO3 , at m/z 80, and NH2CH2COO , at m/z 74 (Table 1, Fig. 3C, and D). [M H NH2

CH2COOH] , at m/z 453, is a steroid skeleton-containing product ion produced by the loss of a glycine moiety that yields fragment ions at m/z 355 and m/z 329. Cleavage between C-23 and C-24 leading to a neutral loss of 101 Da may be typical of double conjugates of amino acids and sulfuric acid, because the corresponding cleavages appeared in the case of taurine-conjugated bile acid 3-sulfates as we described later. These three product ions were observed at collision voltages of around 30 V (Fig. 3C), but were nearly undetectable when the collision voltage was raised. These results indicated the relative instability of the steroid skeleton-containing product ions. On the other hand, HSO4 , at m/z 97, and SO3 , at m/z 80, were detected under a broad range of collision voltages. NH2CH2COO was detected at m/z 74 for every glycine-conjugated bile acid 3-sulfate, a result shared with nonsulfated glycine-conjugated bile acids. Therefore, the simultaneous detection of m/z 74 and m/z 97 indicated that the compound was a double conjugate bearing both a glycine and a sulfuric acid.

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Table 1 The characteristic product ions of bile acids and some steroid conjugates. Product ions Unconjugated bile acids Glycine-conjugated bile acids Taurine-conjugated bile acids Unconjugated bile acid 3-sulfates Glycine-conjugated bile acids 3-sulfates Taurine-conjugated bile acids 3-sulfates 17b-Estradiol 3-sulfate Unconjugated bile acid 3-glucosides Glycine-conjugated bile acid 3-glucosides Taurine-conjugated bile acid 3-glucosides Bile acid 24-galactosides Bile acid 3-glucuronides

Loss of 18 Da m/z 74 Loss of 18 Da m/z 97 Loss of 75 Da Loss of 98 Da

m/z 124 m/z 80 Loss of 173 (75 + 98) Da Loss of 152 Da

Loss of 80 Da Loss of 18 Da

m/z 80 Loss of 162 Da

Loss of 18 Da

Loss of 162 Da

Loss of 180 Da

Loss of 162 Da

Loss of 180 Da

m/z 124

Loss of 206 (162 + 44) Da m/z 107

Loss of 162 Da Loss of 134 Da

m/z 161 Loss of 176 Da

m/z 113 m/z 113

Bile acid 24-glucuronides 17b-Estradiol 3-glucuronide 17b-Estradiol 17-glucuronide UDCA 7b-GlcNAc GUDCA 7b-GlcNAc TUDCA 7b-GlcNAc SNAG-D5-CA SNAG-D5-CG

Loss of 18 Da Loss of 62 (18 + 44) Da Loss of 176 Da Loss of 176 Da Loss of 122 Da Loss of 203 Da Loss of 75 Da Loss of 203 Da Loss of 221 Da Loss of 75 Da

m/z 175 m/z 175 Loss of 176 Da Loss of 221 Da Loss of 203 Da Loss of 221 Da m/z 97 Loss of 98 Da

m/z m/z m/z m/z m/z m/z

m/z m/z m/z m/z m/z

SNAG-D5-CT

Loss of 98 Da

Loss of 221 Da

Loss of 301 (221 + 80) Da

Taurine-conjugated bile acid 3-sulfates, which also produced doubly charged deprotonated molecules by ESI-MS, formed sulfate group-derived product ions, taurine moiety-derived product ions, and steroid skeleton-containing product ions (Fig. 3F). As we observed with glycine-conjugated bile acid 3-sulfates, steroid skeleton-containing product ions, [M H H2SO4] (with a neutral loss of 98 Da), at m/z 480, [M H CONHCH2CH2SO3H] , at m/z 426, and [M H (CONCH2CH2SO3H+H2SO4)] , at m/z 329, were observed under lower collision voltages. HSO4 , at m/z 97, was observed most strongly at a collision voltage of 40 V, although it was produced under a broad collision voltage range. Product ions derived from the taurine moiety, SO3 , CH2CHSO3 , and NH2CH2CH2SO3 , at m/z 80, m/z 107, and m/z 124, respectively, were also detected as products of nonsulfated taurine-conjugated bile acids (Table 1). In addition, SO3 , at m/z 80, which was derived from either a sulfate group or a taurine moiety, increased with increasing collision voltage (Fig. 3E). In contrast to nonsulfated taurine-conjugated bile acids, several steroid skeleton-containing product ions were found. [M H H2SO4] , with a neutral loss of 98 Da, was detected with relatively high intensity at a collision voltage around 30 V. Estrogen, which is produced from androgen by a sequence of oxidation reactions, has a phenolic hydroxyl group at the C-3 position, and it is often conjugated with a sulfuric acid. Although bile acid 3-sulfates produced product ions of both HSO4 and SO3 , 17b-estradiol 3-sulfate gave only SO3 , at m/z 80. Similarly, a neutral loss of 98 Da, corresponding to H2SO4, was completely absent from the product ion spectra, whereas a product ion at m/z 271, which is produced by a neutral loss of 80 Da, gave a strong signal (Table 1, Fig. 3G, and H). Galuska et al., used LC–MS/MS in SRM mode to quantitate steroid sulfates containing estradiol 3-sulfate. Their transition ions were [M H] in Q1 and [M H 80] in Q3 [42]. These facts show a significant differing chemical characteristic in the phenolic and the aliphatic sulfate esters.

m/z 107

m/z 80

Loss of 199 (101 + 98) Da Loss of 249 (151 + 98) Da

m/z 97

m/z 80

m/z 74

m/z 124

m/z 107

m/z 97

Loss of 224 (180 + 44) Da m/z 80

m/z 74

m/z 85

m/z 75

m/z 75 m/z 85

m/z 75

113 113 175 202 202 124

Loss of 221 Da

85 113 100 100 107

Loss of 296 (221 + 75) Da Loss of 319 (221 + 98)Da

m/z 80

m/z 74 m/z 80 m/z 97

m/z 74

m/z 124

m/z 107

m/z 97

3.3. Low-energy CID patterns of neutral sugar conjugates Bile acid 3-glucosides have only one ionic functional group at the side-chain terminus, and therefore, they are converted to singly charged deprotonated molecules. As shown in Fig. 4B, when the deprotonated molecule was selected as a precursor ion, two product ions were formed, one, at m/z 391, corresponded to the CDCA carboxylate anion (with a neutral loss of 162 Da), and the other, at m/z 373, corresponded to an anion that had lost glucose (with a neutral loss of 180 Da). These two product ions were most intense at a collision voltage of 60 V (Fig. 4A). Although both product ions appeared in the product ion spectra of CA 3-glucoside, CDCA 3-glucoside, and DCA 3-glucoside, we did not find an anion that had lost glucose in the product ion spectra of either UDCA 3-glucoside or LCA 3-glucoside (Table 3S). The production of the intact glucose-eliminated anion may be affected by a 7a- or 12ahydroxyl group. The product ions derived from the neutral loss of carbon dioxide and formic acid were not detected. The result suggests that the elimination of the glucose moiety is more facile than the neutral loss of carboxylic acid or formic acid. Glycine-conjugated bile acid 3-glucosides produced NH2CH2COO as the most intense peak at m/z 74 (Fig. 4D). A product ion representing the loss of glucose, [M H 162] , at m/z 448, was weakly detected by comparison with the unconjugated bile acid 3-glucosides, but the related product ion [M H 180] , at m/z 430, did not appear in the product ion spectra of the glycineconjugated forms (Tables 1 and 3S). In addition, a product ion resulting from the elimination of a glucose moiety (corresponding to 162 Da) and a carbon dioxide, at m/z 404, and a product ion resulting from the elimination of an intact glucose molecule (corresponding to 180 Da) and a carbon dioxide, at m/z 386, were also detected (Fig. 4D and Table 3S). Also in taurine-conjugated forms, both [M H 162] and [M H 180] were weakly detected along with the taurine moiety-derived product ions at m/z 124, m/z 107, and m/z 80 (Table 1, Fig. 4E, and F). These data suggest that the

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(A)

(B)

ᅜ106

ᅜ105 4.0

HSO497

0.9

Intensity (cps)

Intensity (cps)

1.2

0.6

3.0

2.0 COOH

SO3-

1.0

0.3

- O SO 3

OH

H

471 80 0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

0.0 0

100

200

300

400

Collision voltage/V

500

m/z

600

700

ᅜ105 5.0

(C)

(D) 3.5ᅜ10

4.0

ᅜ106

3.0

3.0

1.2 1.0 1.0 0.8 0.0 -10

0.6

-20

-30

-40

0.4

Intensity (cps)

Intensity (cps)

NH2CH2COO74 HSO497 -

2.0

O3SO

2.5

CO

OH H OH H

CH2-

C O

2.0

453

OH

-

H

O 3SO

OH H

1.5 SO380

1.0

263 0.0

-H

CONHCH2COO-

0.5

0.2

900

-

5

1.4

800

329 355

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

0

-90 -100 -110 -120 -130

100

200

300

400

500

600

700

800

900

m/z

Collision voltage/V ᅜ105 1.0

(E)

(F)

0.8

ᅜ106 1.6

ᅜ105 8.0

0.6

HSO497

0.2

1.2

CH2-O SO 3

Intensity (cps)

Intensity (cps)

0.4

CONHCH2CH2SO3-

0.0 -20

-30

-40

-50

0.8

0.4

OH H

6.0

CH2

OH H

SO3-

4.0

CH2CHSO3-

2.0

OH H

OH

480

H

80 107 124 289

0.0

CONHCH 2CH2SO3-

-O SO 3

NH2CH2CH2SO3-

329

426

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

0

100

200

300

400

500

600

700

800

900

m/z

Collision voltage/V

OH

(G) 5.0

(H) 3.0

ᅜ106

ᅜ106 271

-O

Intensity (cps)

Intensity (cps)

2.4

OH

4.0 3.0 2.0

1.8 -O SO 3

351

1.2

SO380

0.6

1.0 0.0

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

Collision voltage/V

0

100

200

300

400

500

600

700

800

900

m/z

Fig. 3. Typical product ion spectra and graphsof the product ion intensities versus the applied collision voltages for unconjugated CDCA 3-sulfate (A, B), glycine-conjugated CDCA 3-sulfate (C, D), taurine-conjugated CDCA 3-sulfate (E, F), and 17b-estradiol 3-sulfate (G, H). The collision voltages were 80, 30, 40, and 40 V for (B), (D), (F), and (H), respectively. Other mass spectrometric parameters were described in the experimental section.

elimination of a carbon dioxide molecule may depend on an intramolecular interaction with a hydroxyl group, and that a glucose moiety and a carboxyl group in glycine-conjugated bile acid 3-glucosides can interact with each other. Bile acid acyl galactosides bear only one ionic functional group, a carboxyl group at the C-24 position, modified with a galactose through an ester bond [10]. However, these molecules

were deprotonated under ESI conditions, giving rise to [M H] ions of relatively low intensities (data not shown). The product ion spectrum of a CDCA 24-galactoside obtained at with a collision voltage of 20 V is shown in Fig. 4H. Many product ions derived from the fragmentation of a galactose moiety appeared at m/z 535, m/z 523, m/z 493, m/z 481, m/z 463, m/z 445, m/z 433, and m/z 415 with a carboxylate anion at m/z 391 and two product

86

M. Maekawa et al. / Steroids 80 (2014) 80–91

(A)

(B)

ᅜ105 1.6

ᅜ106 1.2

HO

1.2

0.4 0.0 -40 -50 -60 -70 -80

Intensity (cps)

0.9

0.6

COO COO -

1.2 HOH2C

OH

0.0

O

HO HO

H

O

OH

H

OH

0.8 553 0.4

0.3

373

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

0

100

200

300

400

500

600

700

800

900

m/z

Collision voltage/V ᅜ103 6.0

(C)

(D)

ᅜ105 3.5

ᅜ104

4.0 3.0 2.0

2.5

0.0 -40 -50 -60 -70 -80

2.0 1.5 1.0

-30

-40

-50

-60

-70

-80

O

HO HO

O

OH H

OH

CONHCH2COO-

H HOH2C O

HO HO

CONHCH2-

O

610

1.0

OH H

OH

OH H

0

-90 -100 -110 -120 -130

-H2O

HOH2C

OH

1.5

0.0

-

OH H CONHCH2COO-

HO

0.0 -20

HO OH H

2.0

0.5

-10

COO-

CONHCH2-

0.5

-5

CONHCH2COO-

CONHCH2COO-

NH2CH2 74

2.5

Intensity (cps)

3.0

Intensity (cps)

OH H

391

0.8

Intensity (cps)

COO -

ᅜ105 1.6

100

386 430 404 448

200

300

400

592

500

600

700

800

900

m/z

Collision voltage/V ᅜ104 2.0

1.0

ᅜ105 3.5

3.5

HOH2C

HO

O

OH H

OH

CONHCH2CH2SO3

3.0

O

HO HO

OH H

660

-

-60 -70 -80 -90 -100

2.0 1.5 1.0

Intensity (cps)

0.0

2.5

2.5

OH H

CH2CHSO3SO3-

2.0

0.0

480 498

1.5 NH2CH2CH2SO3124

1.0

80 107

0.5

0.5

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

0

100

200

300

400

m/z

Collision voltage/V

(G) 3.5

CONHCH2CH2SO 3-

CONHCH2CH2SO3-

ᅜ104

0.5

3.0

Intensity (cps)

(F)

1.5

(E)

600

700

900

ᅜ104 1.4

1.5

3.0

1.0

1.2

2.5

0.5

1.0

2.0

0.0

1.5 1.0

Intensity (cps)

-10 -20 -30 -40 -50

C

OH

HO

30

OHOH

0.8 HO

0.6

O

30

30

CH2OH

-H

OH

30

18

43318

CH2O -

30

553

161

0.4

463

OHO

O

H

H

493

113 O

415

445

0.2

0.5

HO O

O

HO

-

OH OH

391 COO-

Intensity (cps)

800

(H)

ᅜ104 2.0

ᅜ104

500

535

523

0.0

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

Collision voltage/V

0

100

200

300

400

500

600

700

800

900

m/z

Fig. 4. Typical product ion spectra and graphs of the product ion intensities versus the applied collision voltages for CDCA 3-glucoside (A, B), glycine-conjugated CDCA 3glucoside (C, D), taurine-conjugated CDCA 3-glucoside (E, F), and CDCA 24-galactoside (G, H). The collision voltages were 60, 70, 90, and 20 V for (B), (D), (F), and (H), respectively. Other mass spectrometric parameters were described in the experimental section.

ions of the galactose moiety at m/z 161 and at m/z 113 (Tables 1 and 3S). Product ions in the range of m/z 415–535 may be produced through the repeated elimination of water and CH2O from

the galactose moiety. Although a CDCA 3-glucoside fragmented only at the conjugation site, (Fig. 4B), CDCA 24-galactosides revealed complicated fragmentation of the sugar moiety, and

87

M. Maekawa et al. / Steroids 80 (2014) 80–91

-

OH OH

ᅜ104 6.0

(A) ᅜ105 8.0

COO-

(B)

COOH

-H

4.0

3.2

-30 -40 -50 -60 -70

Intensity (cps)

Intensity (cps)

0.0

1.6

O

OH

H

COOH

391

567

-

OOC O

HO HO

O

HO

H

OH

H

O-

HO

1.0

HO OH

COO -

4.0

2.0 4.8

H

OH

HOCH2COO-

3.0

6.4

O

O

HO

ᅜ104 5.0

5.0

505 H2C

CH2

3.0

OHO

85 2.0 75

O

113

433

1.0

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

0.0

-90 -100 -110 -120 -130

0

100

200

300

400

Collision voltage/V

(C)

500

600

m/z

700

(D)

ᅜ105 4.0

OH

ᅜ104 8.0

8.0

HO

900

OH

OH HO O

C

ᅜ104

800

OH COO-

O

O

OH COO-

O

HO H

175

567

3.0 4.0 2.0

2.0 0.0

1.0

-20 -30 -40 -50 -60

Intensity (cps)

Intensity (cps)

6.0 6.0

OHO

O

113 4.0

OH COO -

HO H

2.0

391 0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

0.0

-90 -100 -110 -120 -130

0

Collision voltage/V

ᅜ106 3.0

(F)

3.0 2.0

0.0 -10 -20 -30 -40 -50

1.0

m/z

HO

O -

OH

2.0

OH COO-

O

-

85

-50

-60

-70

-80

100

447

200

300

400

(H)

HOCH2COO-

1.0 0.5

2.0 0.0 -20 -30 -40 -50 -60

Intensity (cps)

Intensity (cps)

700

800

900

OH

HO H2C

CH2

OH

O

HO O O

O

OH COO -

O

O

75

3.0 1.5

1.5

600

O-

O-

HO

ᅜ105 3.5

-

2.5

500

m/z

2.0

3.0

O

75

0

2.5

3.5

O OH

-90 -100 -110 -120 -130

ᅜ105 3.0

ᅜ106 4.0

OOC

HO HO

Collision voltage/V

(G)

O

271

175 -40

900

OH

0.0 -30

800

CH2

HO

0.0 -20

700

113

1.0

-10

600

3.0

0.5

-5

500

4.0

1.0 1.5

400

O-

HO H2C

Intensity (cps)

Intensity (cps)

4.0

2.0

300

O-

HOCH2COO-

ᅜ105 5.0

5.0

2.5

200

OH

ᅜ105 6.0

(E)

100

85 113

2.5

HO -O

OH HO

OH COO-

O

2.0

271

447

1.5 325 1.0

1.0 0.5

0.5

0.0

0.0

175 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

Collision voltage/V

0

100

200

300

400

500

600

700

800

900

m/z

Fig. 5. Typical product ion spectra and graphs of the product ion intensities versus the applied collision voltages for DCA 3-glucuronide (A, B), DCA 24-glucuronide (C, D), 17bestradiol 3-glucuronide (E, F), and 17b-estradiol 17-glucuronide (G, H). The collision voltages were 60, 30, 40, and 40 V for (B), (D), (F), and (H), respectively. Other mass spectrometric parameters were described in the experimental section.

produced carbohydrate-derived signals, including product ions at m/z 161 and at m/z 113 as shown in Fig. 4H. As described above, bile acid 24-galactosides do not have an ionic group, and therefore charge may distribute to the galactose moiety. This is in contrast to what is observed in bile acid 3-glucosides, which possess an anionic charge at the end of the side chain. The charge distribution in the galactose moiety may complicate the fragmentation of this molecule.

3.4. Differences in low-energy CID patterns among conjugation types of glucuronides Bile acid glucuronides exist in the urine from healthy subjects as both ether type 3- and ester type 24-glucuronides [43]. Because of the differences in chemical stability between the two types of bonds that mediate these conjugations, we expected that each glucuronide would provide a characteristic fragmentation pattern.

88

M. Maekawa et al. / Steroids 80 (2014) 80–91

Product ion spectra from DCA 3-glucuronide and 24-glucuronide are shown in Fig. 5B and D. Indeed, the ether type glucuronide produced [M H (H2O+CO2)] , at m/z 505, and a product ion based on the fragmentation of a glucuronic acid moiety at m/z 433, along with an aglycone-derived product ion at m/z 391 (Table 4S). Those product ions were present at a relatively intense level along with a very weak peak at m/z 175 corresponding to a glucuronic acid moiety. Product ions at, m/z 113, m/z 85, and m/z 75 in spectra that include m/z 175 are common product ions obtained from most glucuronides, with the exception of bile acid acyl glucuronides (Table 1). By contrast, the ester type glucuronide yielded a simple product ion spectrum, with a base peak product ion derived from a glucuronic acid moiety at m/z 175, at a collision voltage of 30 V (Fig. 5C). Two other product ions, at m/z 391 and m/z 113, were produced in common with the ether type glucuronide. They were also detected in the case of the 24-galactoside. Although both types of glucuronides produced a common aglycone-derived product ion, at m/z 391, the critical difference between the two types of

(A)

(B)

ᅜ105 9.0

ᅜ104 4.5

ᅜ105 1.8

COO-

3.6

7.5

COO -

6.0

1.8

4.5

0.9 0.0

3.0

391

HO

OH H

1.5

2.7

Intensity (cps)

Intensity (cps)

glucuronides was the relative intensity of a product ion derived from a glucuronic acid moiety, at m/z 175. Although in the case of the 24-glucuronides the charge may converge at a carboxyl group on the glucuronic acid moiety, in the case of the ether type of 3-glucuronides the charge may disperse at the two carboxyl groups. This charge distribution in the glucuronic acid moiety may complicate the fragmentation of the glucuronic acid moiety in a fashion similar to what we observed with the 24-galactosides. The phenolic hydroxyl group at the C-3 position of 17b-estradiol is also conjugated with a glucuronic acid. In order to investigate the characteristic differences between glucuronides modified via phenolic hydroxyl groups as compared to the ether and ester types of glucuronides modified through aliphatic alcohols and carboxylic acids, the fragmentation patterns of 17b-estradiol 3-glucuronide and 17-glucuronide were analyzed by low-energy CID. A typical product ion spectrum from a deprotonated 17b-estradiol 3-glucuronide precursor ion is shown in Fig. 5F. A base peak was observed at m/z 271, which is an

-30 -40 -50 -60 -70

1.5

HO H

1.2

COO-

OH AcHN

OH CH2O-

O

OH AcHN O

HO

0.9

OH CH2OH

O

H

AcHN

O-

0.6

594

0.3

100

202

100

200

373 0.0

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

0

300

400

Collision voltage/V

ᅜ105 2.5

(D)

4.0

800

900

C

NH2CH2COO-

1.5 0.0 -40 -50 -60 -70 -80

Intensity (cps)

2.0

O

OH

OH CH2OH

AcHN O

HO

H

OH CH2OH

O

H

COO-

74 AcHN

651

O-

1.0

CONHCH2COO-

HO

OH H

HO

OH

0.8

AcHN

O

OH H

OH CH2O-

0.6 100 0.4

391

202

0.5

576

0.2 0.0

CONHCH2COO-

-H OH

AcHN O

HO

1.2

2.0

Intensity (cps)

700

O

ᅜ104 1.4

6.0

1.0

600

-

ᅜ103 8.0

(C)

500

m/z

448

0.0 -5

-10

-20

-30

-40

-50

-60

-70

-80

-90 -100 -110 -120 -130

0

100

200

300

400

500

600

700

800

900

m/z

Collision voltage/V ᅜ104 8.0 6.0

(F)

4.0

ᅜ105

3.5

2.0

3.0

0.0

Intensity (cps)

-70 -80 -90 -100 -110 2.5 2.0 1.5

1.0 0.0 -40

-50

-60

-70

-80

Collision voltage/V

-90 -100 -110 -120 -130

O

OH CH2OH

H

480

701

HO H

SO3-

498

NH2CH2CH2SO3

3.0

0.0 -30

HO

OH H

CH2CHSO34.0

2.0

-20

HO

AcHN O

5.0

0.5

-10

OH CONHCH2CH2SO 3-

6.0

1.0

-5

CONHCH2CH2SO3-

CONHCH2CH2SO 3-

ᅜ104 7.0

Intensity (cps)

(E)

-

107124 80 0

100

200

300

400

500

600

700

800

900

m/z

Fig. 6. Typical product ion spectra and graphs of the product ion intensities versus the applied collision voltages for unconjugated (A, B), glycine-conjugated (C, D), and taurine-conjugated UDCA 7b-GlcNAc (E, F). The collision voltages were 50, 70, and 80 V for (B), (D), and (F), respectively. Other mass spectrometric parameters were described in the experimental section.

89

M. Maekawa et al. / Steroids 80 (2014) 80–91

(A)

(B) COOH

ᅜ104 7.0

ᅜ104 2.5

Intensity (cps)

Intensity (cps)

6.0 5.0 4.0 3.0 2.0

OH -

2.0

0.0

OH CH2OH

97 COOH

1.5 -

O 3SO

1.0

451

0.0 -5

-20

-30

-40

-50

-60

-70

-80

-90

-100 -110 -120 -130

0

100

200

300

400

Collision voltage/V

ᅜ104 NH2CH2COO-

2.0

2.0

0.5

5.0

0.0 -20 -30 -40 -50 -60

0.8

Intensity (cps)

1.2

800

900

C

HSO4 97

1.5 1.0

700

CONHCH2COO-

6.0

1.6

600

(D)

2.5 ᅜ105

500

m/z

ᅜ104

(C)

Intensity (cps)

O

672

0.5

1.0

OH

-

AcHN O

-O SO 3

OH

-

O3SO

4.0

AcHN O

OH CH2OH

O

CONHCH2COO-

74

C O OH

3.0

AcHN O

-

-O SO 3

O 3SO

O

OH CH2OH

254 2.0

433

654 631

1.0

0.0

CONHCH2COO-

O OH CH2OH

O

364

0.4

0.0 -5

-20

-30

-40

-50

-60

-70

-80

-90

0

-100 -110 -120 -130

100

200

300

400

(E)

(F) ᅜ105

2.5

0.8 0.6

2.0 0.4 1.5

0.2

1.0

700

CONHCH2CH2SO3-

CONHCH2CH2SO 3-

ᅜ104 8.0

Intensity (cps)

1.0

600

800

900

CONHCH2CH2SO3-

OH

ᅜ104

3.0

500

m/z

Collision voltage/V

Intensity (cps)

AcHN O

O3SO

HSO4-

0.0

-

-O SO 3

CONHCH2CH2SO 3-

OH AcHN O

460

4.8

O

OH CH2OH

681

389

3.2

478

1.6

0.0

OH

CONHCH2CH2SO 3-

-20 -30 -40 -50 -60 0.5

OH CH2OH

O

278

HSO497

6.4

AcHN O

O 3SO

0.0 -5

-20

-30

-40

-50

-60

-70

-80

-90

-100 -110 -120 -130

Collision voltage/V

0

100

200

300

400

500

600

700

800

900

m/z

Fig. 7. Typical product ion spectra and graphs of the product ion intensities versus the applied collision voltages for SNAG-D5-CA (A, B), SNAG-D5-CG (C, D), and SNAG-D5-CT (E, F). The collision voltages were 50, 30, and 30 V for (B), (D), and (F), respectively. Other mass spectrometric parameters were described in the experimental section.

aglycone-derived ion, with several minor product ions at m/z 113, m/z 85, and m/z 75 (Table 1). Those minor product ions were probably derived from the glucuronic acid moiety, and the observed product ions were similar to those of the bile acid 3-glucuronide. By contrast, 17b-estradiol 17-glucuronide is an ether type of glucuronide similar to the bile acid 3-glucuronide. Accordingly, the overall pattern of its product ion spectrum was similar to that of bile acid 3-glucuronide, as shown in Fig. 5H. Although an aglycone-derived ion was strongly detected at m/z 271as a product ion of 17b-estradiol 3-glucuronide, the same ion was of average intensity in the product ion spectrum from 17b-estradiol 17-glucuronide. Furthermore, the glucuronic acid moiety-derived product ions observed at m/z 113, m/z 85, and m/z 75 were detected at almost the same intensities from both precursors. The product ion observed at m/z 325 may be produced by the internal cleavage of the glucuronic acid moiety. Since ether type glucuronides linked through an aliphatic hydroxyl group, e.g., bile acid 3-glucuronides and 17b-estradiol 17-glucuronide, are relatively more stable than acyl glucuronides and glucuronides linked through a phenolic hydroxyl group, product ions derived from the internal fragmentation of a glucuronic acid moiety may be found in the product ion spectra of 17b-estradiol 17-glucuronide.

3.5. Low-energy CID patterns of N-acetylglucosamine conjugates UDCA 7b-GlcNAc and its glycine and taurine conjugates were transformed to singly charged deprotonated molecules during the ESI process, and we investigated their characteristics by lowenergy CID. A typical product ion spectrum for UDCA 7b-GlcNAc is shown in Fig. 6B. An aglycone-derived product ion was clearly observed at m/z 391 as a base peak with its dehydrated ion at m/z 373. The spectrum pattern detected above m/z 300 was similar to that seen with the CDCA 3-glucoside. No steroid skeleton-containing product ions with internal cleavage of GlcNAc moiety were noted. Similar to glucose but in contrast to glucuronic acid, GlcNAc has no anionic functional group, which fragmented with internal cleavage. However, a GlcNAc moiety-derived product ion was clearly detected at m/z 202, along with its fragment ion at m/z 100. Although GUDCA 7b-GlcNAc also provided GlcNAc moiety-derived product ions at m/z 202 and m/z 100, it also strongly produced NH2CH2COO , at m/z 74, derived from the glycine moiety (Fig. 6D). In addition, two steroid skeleton-containing product ions appeared at m/z 576 (with a neutral loss of 75 Da) and m/z 448 (with a neutral loss of 203 Da), which were produced by the cleavage of an amide bond at the C-24 position or an ether bond at the

90

M. Maekawa et al. / Steroids 80 (2014) 80–91

Table 2 Estimation of conjugation type by product ions and neutral losses observed in low-energy CID. Product ion m/z m/z m/z m/z m/z m/z m/z

74 124, m/z 107, m/z 80 97, m/z 80 97, m/z 74 124, m/z 97, m/z 80 80 161, m/z 113

m/z m/z m/z m/z

175 (strong), m/z 113 175, m/z 113 113, m/z 175 (weak) 202, m/z 100

Neutral loss

Conjugation type

98 Da, 80 Da 173 Da, 75 Da 152 Da, 98 Da 80 Da 162 Da 162 Da 176 Da 176 Da (strong) 176 Da 221 Da, 203 Da 221 Da

Glycine conjugate Taurine conjugate Sulfate (aliphatic alcohol) Glycine-conjugated sulfate (aliphatic alcohol) Taurine-conjugated sulfate (aliphatic alcohol) Sulfate (phenolic alcohol) Acyl galactoside Glucoside Acyl glucuronide Glucuronide (phenolic ether) Glucuronide (aliphatic ether) N-Acethylglucosamine conjugate N-Acethylglucosamine conjugate (D5 structure)

C-7 position. In the product ion spectrum of TUDCA 7b-GlcNAc, the visibility of steroid skeleton-containing product ions was similar to that observed for the unconjugated UDCA 7b-GlcNAc (Fig. 6F). The product ions included a species lacking the GlcNAc moiety, at m/z 498, produced through a neutral loss of 203 Da; the dehydrated version of this ion also clearly appeared. Although TUDCA 7b-GlcNAc is an amino acid conjugate of UDCA 7b-GlcNAc, similar to GUDCA 7b-GlcNAc, only taurine moiety-derived product ions appeared at m/z 80, m/z 107, and m/z 124, rather than GlcNAc moiety-derived product ions at m/z 202 and m/z 100. Although SNAG-D5-CA bears two ionic moieties, a sulfate group at the C-3 position and a carboxyl group at the C-24 position, the carboxyl group is unlikely to be present as an anion under our ESI conditions because its pKa is greater than the pH of acetic acid [40]. On the other hand, the pKa values of the carboxyl group on SNAG-D5-CG and the sulfonic acid group on SNAG-D5CT are both lower than the pH of acetic acid. Therefore, SNAGD5-CA is transformed to a singly charged deprotonated molecule, [M H] , and SNAG-D5-CG and SNAG-D5-CT are converted to doubly charged deprotonated molecules, [M 2H]2 [28]. The product ion spectra resulting from the fragmentation of [M H] for SNAG-D5-CA and [M 2H]2 for SNAG-D5-CG and SNAG-D5-CT are shown in Fig. 7B, D, and F. HSO4 , at m/z 97, was noted as a product of all three amino acid conjugate types, as was the case with bile acid 3-sulfates. Furthermore, NH2CH2COO at m/z 74 for SNAG-D5-CG and taurine moiety-derived product ions at m/z 80, 107, and 124 for SNAG-D5-CT were also detected in common with other glycine and taurine conjugates (Tables 1 and 5S). By contrast, we could not detect a neutral loss of 203 Da, which is produced by the cleavage of the ether bond at the C-7 position and is commonly seen in UDCA 7b-GlcNAc and its amino acid conjugates, even though SNAG-D5-CA and its amino acid conjugates have a GlcNAc moiety at the C-7b position. Instead, we observed a typical neutral loss of [M H 221] , derived from the elimination of an intact GlcNAc molecule, in SNAG-D5-CA. A similar neutral loss was observed in the case of glycine and taurine conjugates, although in the latter cases the observed product ions [M 2H 221]2 were doubly charged, at m/z 254 for SNAG-D5-CG and m/z 278 for SNAG-D5-CT. The neutral losses corresponding to a GlcNAc molecule were different among UDCA 7b-GlcNAc and SNAG-D5-CA and its amino acid conjugates, and the presence of a double bond between C-5 and C-6 may have contributed to this different trend. In SNAG-D5-CG, since the precursor ion is a doubly charged one, the product ions that lost NH2CH2COOH and H2SO4 appeared at m/z 654 and m/z 631. A steroid skeleton-containing product ion that had eliminated both the glycine and GlcNAc moieties also appeared at m/z 433 (Fig. 7D). In SNAG-D5-CT, a similar product ion that had lost H2SO4 appeared at m/z 681, along with its fragment ion, at m/z 460, produced by an additional neutral loss of 221 Da.

4. Conclusions In this study, we investigated the CID patterns of various bile acids and steroid conjugates, and summarized the results in two simple tables (Tables 1 and 2). In the product ion analysis of conjugated metabolites, the detection of a product ion at m/z 74 strongly suggested the existence of a glycine moiety at the sidechain terminus. In the case of taurine conjugates, three product ions at m/z 80, m/z 107, and m/z 124 invariably appeared in the product ion spectrum. The detection of a strong peak at m/z 97 and another at m/z 80 indicated that the target molecule contained a sulfate group. For amino acid conjugates possessing a sulfate group, a neutral loss of 98 Da corresponded to the loss of H2SO4 and/or the neutral losses of side chain amino acids with side chain-derived product ions. The characterization of sugar conjugates was slightly more complicated as compared to amino acid conjugates and sulfates. Therefore, it was necessary to consider both product ions and neutral losses. A product ion at m/z 113 was common to all types of glucuronides (ether, ester, and phenolic ether types) and acyl galactosides. By contrast, a product ion at m/z 161 appeared only in the case of acyl galactoside, although a neutral loss of 162 Da was observed for both glucosides and acyl galactosides. However, glucosides also gave a neutral loss of 180 Da, and we could discriminate glucosides from acyl galactosides through the presence or absence of a product ion at m/z 161 and a neutral loss of 180 Da. A neutral loss of 176 Da and a product ion at m/z 175 were detected as universal fragmentations in all types of glucuronides. However, the intensity ratios of observed product ions varied greatly according to bonding patterns. In the product ion spectra of acyl glucuronides, a product ion at m/z 175 was very clearly and strongly detected in contrast to other conjugation patterns. On the other hand, a neutral loss of 176 Da gave a stronger signal than did a product ion at m/z 175 with respect to both ether and phenolic ether types of glucuronides. In particular, the intensity of a product ion derived from a neutral loss of 176 Da was overwhelmingly stronger than others in the case of phenolic ether. This information should help aid the discrimination between glucuronidation of aliphatic and phenolic alcohols. With respect to N-acetylglucosamine conjugates, neutral losses of 203 Da and 221 Da appeared in the product ion spectra. Although GlcNAc conjugates to a hydroxyl group at the C-7b position, the presence or absence of a D5-structure seemed to affect the stability of the ether bond. Two product ions, at m/z 202 and m/z 100, were detected in the case of the saturated C5-C6 bond. However, SNAG-D5-CA, SNAG-D5-CG, and SNAG-D5-CT, which possess a D5-structure, did not provide such product ions. The information obtained in this study will provide useful benchmarks to aid in the structural identification of unknown conjugated cholesterol metabolites using low-energy CID.

M. Maekawa et al. / Steroids 80 (2014) 80–91

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