Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry

Abstract

Enzyme-assisted derivatization for sterol analysis (EADSA) is a technology designed to enhance sensitivity and specificity for sterol analysis using electrospray ionization⁻mass spectrometry. To date it has only been exploited on sterols with a 3β-hydroxy-5-ene or 3β-hydroxy-5α-hydrogen structure, using bacterial cholesterol oxidase enzyme to convert the 3β-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for cholesterol oxidase, making the method applicable to sterols with a 3α-hydroxy-5β-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C₂₇ acids give an intense molecular ion ([M]⁺) upon electrospray ionization and informative fragmentation spectra. The method shows promise for analysis of bile alcohols and 3α-hydroxy-5β-C₂₇-acids, enhancing the range of sterols that can be analyzed at high sensitivity in sterolomic studies.

Keywords: Girard reagent; bile alcohol; cholestanoic acid; electrospray ionization-mass spectrometry; enzyme-assisted derivatization; oxysterol; sterolomics.

Scheme 1

Enzyme-assisted derivatization for sterol analysis (EASDA): (a) EADSA of the 3β-hydroxy-5-ene function using cholesterol oxidase and [²H₅] Girard P reagent ([²H₅]GP); (b) derivatization of the 3-oxo-4-ene function with [²H₀]GP; (c) EADSA of the 3α-hydroxy-5β-hydrogen function with 3α-hydroxysteroid dehydrogenase (3α-HSD) and [²H₀]GP. Derivatives with the [²H₅]GP and [²H₀]GP reagents can be combined and analyzed in a single LC-MS run. Other isotope-coded GP reagents have been synthesized to allow triplex analysis if required [45].

Scheme 1

Enzyme-assisted derivatization for sterol analysis (EASDA): (a) EADSA of the 3β-hydroxy-5-ene function using cholesterol oxidase and [²H₅] Girard P reagent ([²H₅]GP); (b) derivatization of the 3-oxo-4-ene function with [²H₀]GP; (c) EADSA of the 3α-hydroxy-5β-hydrogen function with 3α-hydroxysteroid dehydrogenase (3α-HSD) and [²H₀]GP. Derivatives with the [²H₅]GP and [²H₀]GP reagents can be combined and analyzed in a single LC-MS run. Other isotope-coded GP reagents have been synthesized to allow triplex analysis if required [45].

Figure 1

Reconstructed-ion chromatograms (RICs) and multistage fragmentation (MS³) ([M]⁺→[M-Py]⁺→) spectra of oxidized/GP-derivatized 3α-hydroxy-5β-bile alcohols and acids: (a,b) C-3α,7α,12α-triol; (c,d) C-3α,7α,12α,26-tetrol; (e,f) CA-3α,7α,12α-triol; (g,h) cholic acid. The RICs were generated from mass spectra recorded in the Orbitrap mass analyzer at a resolution of 120,000 (FWHM definition at m/z 400), with an m/z window of ± 5 ppm. MS³ spectra were generated in the linear ion-trap and recorded at the ion-trap detector of the Orbitrap-Elite mass spectrometer. Mass accuracy for fragment ion measurements made with the linear ion-trap is typically ± 0.3 Da. Postulated compositions of fragment ions are listed in Table 1. Note that the data for cholic acid was generated on an earlier version of instrument (i.e., Orbitrap-LTQ) at lower resolution and with reduced mass accuracy.

Figure 1

Reconstructed-ion chromatograms (RICs) and multistage fragmentation (MS³) ([M]⁺→[M-Py]⁺→) spectra of oxidized/GP-derivatized 3α-hydroxy-5β-bile alcohols and acids: (a,b) C-3α,7α,12α-triol; (c,d) C-3α,7α,12α,26-tetrol; (e,f) CA-3α,7α,12α-triol; (g,h) cholic acid. The RICs were generated from mass spectra recorded in the Orbitrap mass analyzer at a resolution of 120,000 (FWHM definition at m/z 400), with an m/z window of ± 5 ppm. MS³ spectra were generated in the linear ion-trap and recorded at the ion-trap detector of the Orbitrap-Elite mass spectrometer. Mass accuracy for fragment ion measurements made with the linear ion-trap is typically ± 0.3 Da. Postulated compositions of fragment ions are listed in Table 1. Note that the data for cholic acid was generated on an earlier version of instrument (i.e., Orbitrap-LTQ) at lower resolution and with reduced mass accuracy.

Scheme 2

Structures of bile alcohols and acids, and their products of 3α-HSD oxidation and GP-derivatization.

Scheme 2

Structures of bile alcohols and acids, and their products of 3α-HSD oxidation and GP-derivatization.

Scheme 3

MS² and MS³ fragmentation of oxidized/GP derivatized bile alcohols as illustrated by C-3α,7α,12α-triol. For simplicity the fragmented Girard derivatizing group is shown in its linear isomeric form. The inset shows fragmentation route A₃ leading to the [A₃-H-(H₂O)₂]⁺ fragment ion. Cyclic isomers are depicted in Scheme 1. See Supplemental Schemes S1–S4 for fragmentation schemes of other trihydroxy- and tetrahydroxy-bile alcohols and trihydroxy-bile acids. Table 1 correlates m/z with fragment ion composition.

Figure 2

(a) RIC and (b) MS² ([M]⁺→), (c) MS³ ([M]⁺→[M-Py]⁺→), (d) ([M]⁺→[M-Py-18]⁺→) spectra of oxidized/GP-derivatized C-3α,7α,12α,25-tetrol. For comparison, MS² ([M]⁺→) spectra of C-3α,7α,12α-triol and C-3α,7α,12α,26-tetrol and the MS³ ([M]⁺→[M-Py-18]⁺→) spectrum of C-3α,7α,12α,26-tetrol are shown in Supplemental Figure S1a–c respectively. Mass spectra recorded at the peak of the RIC for these and other sterols analyzed are shown in Supplemental Figure S2. Data was generated on the Orbitrap-Elite mass spectrometer as in Figure 1. See Table 1 to correlate m/z with fragment ion composition.

Figure 3

RICs and MS³ ([M]⁺→[M-Py]⁺→) spectra of cholesterol oxidase-oxidized/GP-derivatized 3β,5α,6β-triols: (a,b) [25,26,26,26,27,27,27-²H₇]cholestane-3β,5α,6β-triol ([²H₇]C-3β,5α,6β-triol); (c,d) 3β,5α,6β-trihydroxycholanoic acid (BA-3β,5α,6β-triol). See reference [8] for a description of fragmentation pathways. GP-derivatized sterols can give syn and anti conformers resulting in twin chromatographic peaks which may or may not be resolved. Data was generated on the Orbitrap-Elite mass spectrometer as in Figure 1.

Figure 4

LC–(MS)MSⁿ analysis of oxidized/GP-derivatized C-3α,7α,12α,24R,25-pentol and C-3α,7α,12α,24S,25-pentol: (a) RIC, (b) MS² ([M]⁺→), (c) MS³ ([M]⁺→[M-Py]⁺→), and (d) ([M]⁺→[M-Py-18]⁺→) from the analysis of C-3α,7α,12α,24R,25-pentol. (e) RIC, (f) MS² ([M]⁺→), (g) MS³ ([M]⁺→[M-Py]⁺→), and (h) ([M]⁺→[M-Py-18]⁺→) from the analysis of C-3α,7α,12α,24S,25-pentol. Data was generated on the Orbitrap-Elite mass spectrometer as in Figure 1. Fragment ions are described in Table 1 and postulated structures are shown in Scheme 4.

Scheme 4

MS² and MS³ fragmentation of oxidized/GP-derivatized pentahydroxy-bile alcohols as illustrated by C-3α,7α,12α,24,25-pentol. Fragment ions with a 24-hydroxy-24-ene structure are likely to rearrange to the 24-ketone. For simplicity the fragmented Girard derivatizing group is shown in its linear isomeric form. Cyclic isomers are similar to those depicted in Scheme 1. See Supplemental Schemes S5–S7 for fragmentation schemes of other pentahydroxy-bile alcohols.

Figure 5

LC–(MS)MSⁿ analysis of oxidized/GP-derivatized C-3α,7α,12α,25,26-pentol and C-3α,7α,12α,26,27-pentol: (a) RIC, (b) MS² ([M]⁺→), (c) MS³ ([M]⁺→[M-Py]⁺→), and (d) ([M]⁺→[M-Py-18]⁺→) from the analysis of C-3α,7α,12α,25,26-pentol. (e) RIC, (f) MS² ([M]⁺→), (g) MS³ ([M]⁺→[M-Py]⁺→), and (h) ([M]⁺→[M-Py-18]⁺→) from the analysis of C-3α,7α,12α,26,27-pentol. Data was generated on the Orbitrap-Elite mass spectrometer as in Figure 1. Fragment ions are described in Table 1 and postulated structures are shown in Supplemental Schemes S5 and S6.

Figure 5

LC–(MS)MSⁿ analysis of oxidized/GP-derivatized C-3α,7α,12α,25,26-pentol and C-3α,7α,12α,26,27-pentol: (a) RIC, (b) MS² ([M]⁺→), (c) MS³ ([M]⁺→[M-Py]⁺→), and (d) ([M]⁺→[M-Py-18]⁺→) from the analysis of C-3α,7α,12α,25,26-pentol. (e) RIC, (f) MS² ([M]⁺→), (g) MS³ ([M]⁺→[M-Py]⁺→), and (h) ([M]⁺→[M-Py-18]⁺→) from the analysis of C-3α,7α,12α,26,27-pentol. Data was generated on the Orbitrap-Elite mass spectrometer as in Figure 1. Fragment ions are described in Table 1 and postulated structures are shown in Supplemental Schemes S5 and S6.

Figure 6

LC–(MS)MSⁿ analysis of oxidized/GP-derivatized C-3α,7α,12α,23,25-pentol. (a) RIC, (b) MS² ([M]⁺→), (c) MS³ ([M]⁺→[M-Py]⁺→), and (d) ([M]⁺→[M-Py-18]⁺→). Data was generated on the Orbitrap-Elite mass spectrometer as in Figure 1. Fragment ions are described in Table 1 and postulated structures are shown in Supplemental Schemes S7.