Structural Elucidations of derivatized Sterols using Electrospray Ionization/Atmospheric Pressure Chemical Ionization-Quadrupole Ion Trap-Mass spectrometry

Iyad. A. Hailat

Faculty of Pharmacy, Middle East University, Amman-Jordan.

*Corresponding Author E-mail: ihailat@meu.edu.jo

ABSTRACT:

Free sterols are neutral molecules that are difficult to ionize by Electrospray Ionization (ESI)  or Atmospheric Pressure Chemical Ionization (APCI). Therefore, in order to increase their ionization efficiency, sterols were converted into their corresponding picolinyl esters. In this study we examined the possibility of analyzing picolinyl ester of sterols derivatives using flow injection ESI-quadrupole ion trap (QIT) MS and APCI-QIT MS and we investigated their fragmentation pathways using low energy collision induced dissociation-tandem mass spectrometry (CID-MS²). This study also aimed to examine the possibility of using ESI-QIT MS³ to identify sterol isomers. The picolinyl esters readily formed protonated molecular ions ([M+H]⁺) in ESI and APCI sources except for the picolinyl ester of 7-dehydrocholesterol which was  detected as the radical cation ion [M]^.+ using  APCI-QIT MS. The ester bonds of picolinyl esters cleaved during CID MS², resulting in diagnostic fragments corresponding to steryl cation moieties [M+H-C₆H₅NO₂]⁺. The CID MS³ of [M+H]⁺ → [M+H-C₆H₅NO₂]⁺ of picolinyl esters was found to be useful for structural elucidation and for distinguishing among steryl isomers.

KEYWORDS: Sterols, Picolinyl ester of sterols, QIT-MS, Fragmentation pathways, Sterol isomers.

1. INTRODUCTION: 

Sterols are natural chemical compounds present in many Bio-Orgnisims such as plants, animals, fungai and        algae ¹. The most abundant sterols in plants are β-sitosterol, stigmasterol and 24-methylenecholesterol, while, cholesterol is the dominant in animals, and in fungai, the most abundant sterol is ergosterol². Sterols are synthesized in bio-organisms via cyclization of squalene epoxide³. This cyclyzation process produce a tetracyclic chemical structures which are biologically transform giving many bio-chemical compounds which have a common steroid nucleus (perhydrocyclopentano-phenanthrene) and differing in the structure of the alkyl side chain⁴. Phytosterols are non-cholesterol sterols which can be found mainly in plants⁵. The chemical structures of phytosterols are similar to cholesterol but they are differing in the chemical structure of the alkyl side chain.

Phytosterols are one of the plant membrane constituents which are responsible of stabilizing the phospholibid bilayers. This function is similar to that of cholesterol in animal cell membrane⁵.

It has been found that the levels of cholesterol decrease in blood as a result of having food enriched with phytosterols^6,7. Moreover, the consumption diet with high levels of phytosterols can protect from cardiovascular and heart diseases⁸. Due to the health benefits of phytosterols, many nutraceutical companies extract these beneficial compounds from plants and add them to prepared food as health ingredients⁸.

Chromatographic techniques coupled with mass spectrometry have been used for identification and structural elucidations of bio-chemical compounds such as Liquid Chromatography-Electrospray Ionization-Tandem mass spectrometry (LC-ESI-MS/MS)^9,10 and Gas Chromatography-mass spectrometry (GC-MS)^11,12

Many instruments have been used for identification and quantification free sterols including gas chromatography mass spectrometry (GC/MS)^13,14,15. Gas chromatography-flame ionization detection            (GC/FID) ^15,16, liquid chromatography/ultraviolet detection (LC-UV)¹⁷, Liquid chromatography/ atmospheric pressure chemical ionization detection (LC/APCI-MS)^17,18, LC/APCI-MS/MS¹⁹, Liquid Chromatography/ atmospheric pressure photoionization detection (LC/APPI)-MS/MS²⁰, high-performance liquid chromatography/electrospray ionization detection (HPLC-ESI-MS)²¹, LC-MS²², GC-MS/MS²³ and UV-VIS spectrophotometer²⁴.

Many researchers have used IR and NMR in addition to mass spectroscopy for Identification and quantification of phytosterols in plants and fruits^25,26,27. However, sterols content was determined in leaf and fruit of MalaysionMengkudu using aqueous and organic solvent extracts²⁸.

Atmospheric pressure chemical ionization (APCI) is appropriate for profiling of non-polar and low molecular weight compounds such as sterols. However, ESI can be used for identification of sterols with lower sensitivity than APCI²⁹. Therefore, it is necessary to derivatize sterols with a suitable reagent to improve their ionization efficiency and thus selectivity³⁰. In addition to derivatization of sterols as their TMS ethers before GC analysis,other derivatization methods have been used to produce derivatives with more in polarity. These include the analysis of sterols in human serum by LC/ESI-MS/MS as their picolinyl esters³⁰.

Direct flow injection-Electrospray ionization with quadrupole ion trap mass spectrometry ((ESI-QIT MSⁿ) can be a very efficient method for analysis mixtures containing isomers, as the technique give more data about structural elucidation using collision-induced dissociation (CID) tandem MS.Contrary to triple quadrupole MS, QIT MS can be used to carry out MS³ thus giving more information about structural chemical elucidation³¹.

The objectives of this study were to examine the possibility of using flow injection ESI-QIT MS and APCI-QIT MS for analyzing picolinyl ester of sterols derivatives and to investigatetheir fragmentation pathways using low energy CID-MS/MS. This study also aimed to examine the possibility of using ESI-QIT MS³to identify sterol isomers. To the best of my knowledge, this is the first study which     investigates the analysis of picolinyl esters of sterols using ESI/APCI-QIT MSⁿ and to demonstrate its ability to differentiate among sterol isomers.

2. EXPERIMENTS AND METHODS:

2.1 Chemicals and reagents:

Picolinic acid, 2-methyl-6-nitrobenzoic anhydride, 4-dimethylaminopyridine, triethylamine and pyridine were obtained from Sigma Chemical Co. (St. Louis, Mo, USA). Cholesterol, β-sitosterol, ergosterol, cholestanol, brassicasterol, fucosterol, desmosterol, stigmasterol and 7-dehydrocholesterol were obtained from Steraloids, Inc. (New Port, RI, USA). All other chemicals and solvents were of analytical grade.

2.2 Derivatization of sterols:

Picolinyl ester derivatization was carried out using a procedure reported by Yamshitaet al.³² with minor modifications.  A 200μL aliquot of a reagent mixture consisting of picolinic acid (80mg), 4-dimethylaminopyridine (30mg), 2-methyl-6-nitrobenzoic anhydride (100mg), pyridine (1.5mL), and triethylamine (250μL) was added immediately to 2.0mg of sterol standards. The reaction mixture was heated at 60°C for 20 min followed by addition of 750μL of hexane and vortexed for 1 min. The reaction mixture was then centrifuged for 5 min. The supernatant was transferred to a vial and evaporated to dryness under nitrogen flow. The residue was re-dissolved in 2mL of dichloromethane and analyzed immediately.

2.3 Instrumentation:

ESI-QIT MS and APCI-QIT MS of picolinyl ester of sterols:

Picolinyl esters of sterols were detected as protonated ions [M+H]⁺ using a flow injection-ESI/APCI-MS system (Agilent 1100, SL LC/MSD (Trap) CA.USA) equipped with an ion trap mass selective detector operating in positive mode. 10µL of picolinyl sterol esters in dichloromethane were injected into an acetonitrile eluent at a flow rate of 0.4mL/min. The ESI operating parameters were as follow: nebulizer gas was nitrogen at a flow rate of 9L/min and pressure of 60 psi, drying temperature 350 ºC, capillary voltage 3500 volts, capillary exit 109.4 volts and skimmer potential 40 volts. Full scan ESI mass spectra were acquired in positive mode and the mass range was set at m/z 50-600. In addition, picolinyl sterol esters were analyzed using an APCI source operating in positive mode. 10µL of picolinyl sterol esters in dichloromethane were injected into a methanol eluent at a flow rate of 0.4mL/min. The operating parameters were as follows: nebulizer gas was nitrogen with a flow rate of 7 L/min and with pressure of 60 psi, drying gas temperature was 350ºC, APCI source temperature was 250ºC, capillary voltage 3500 volts and corona discharge current was 5000 nA.

ESI/APCI-QIT MS² and ESI/APCI-QIT MS³ spectra of picolinyl ester of sterols were acquired in positive mode using helium as the collision gas with a fragmentation amplitude voltage of 1 volt and a mass window of 1.5 Da.

3. RESULTS AND DISCUSSIONS:

3.1 Analysis of sterols as their picolinyl esters using both flow-injection ESI-QIT MS andAPCI-QIT MS:

Sterol standards were converted into their picolinyl esters to investigate their ionization behavior and fragmentation pathways using ESI/APCI-QIT MS. The chemical structures of sterol standards as their picolinyl esters are shown in Figure (1). Picolinyl esters of sterols were found to be easily detected as protonated molecular ion [M+H]⁺. Figure 2 (a) shows flow injection ESI-QITMS analysis of cholesterol picolinyl ester which was detected at m/z 492.4. In addition to the protonated molecular ion, an abundant fragment ion [M+H-C₆H₅NO₂]⁺at m/z 369.4 was observed indicating that picolinyl esters can lose picolinic acid under ESI conditions. The analysis of cholesterol picolinyl esters by APCI-QITMS showed similar ionization and fragmentation behaviors. However, sterols containing an additional double bond in the B ring of the sterol skeleton such as 7-dehydrocholesterol (Figure 1) behaved differently under both ESI and APCI conditions. Its molecular ion cannot be observed by ESI-QIT MS and facile cleavage occurred, giving an abundant fragment ion ([M+H-C₆H₅NO₂]⁺) at m/z 367.4. (fig. 3 (b)). However, in APCI-QITMS, 7-dehydrocholesterol derivative could be detected as a radical cation [M]^.+ (Fig. 3 (a)). An abundant fragment ion corresponding to [M+H-C₆H₅NO₂]⁺ at m/z 367.4 was observed (Fig. 3(a)). This observation is in agreement with the results reported by Herrera et al. ³³ as they observed benzothiophene formed radical cations when analyzed by APCI. The analysis of other picolinyl sterol esters, using ESI-MS and APCI-MS, showed similar ionization and fragmentation behaviors to the picolinyl ester of cholesterol.

Figure 1: Chemical structures of picolinyl ester of sterols used in this study

Figure 2: (a) Flow injection ESI-QIT MS spectrum of cholesteryl picolinyl ester detected as [M+H]⁺, (b) The CID MS² spectrum of the precursor ion m/z 492.3, and (c) the CID MS³ spectrum of the precursor ion m/z 369.3 which is the cholesteryl cation.

Figure 3: (a) Flow injection APCI-QIT MS spectrum of picolinyl ester of 7-dehydrocholesterol detected as radical cation [M]^.+, (b) flow injection ESI-QIT MS spectrum of picolinyl ester of 7-dehydrocholesterol with no molecular ion observed.

3.2 ESI/APCI-CID MS² and ESI/APCI-CID MS³ of picolinyl sterol esters:

ESI-CID MS² and APCI-CID MS² of picolinyl esters were carried out to investigate the fragmentation pathways and to confirm the identity of the sterols. CID- MS² spectra were obtained for cholesterol picolinyl ester using both ESI-QIT and APCI-QIT. As shown in Fig. 2 (b), the cholesterol picolinyl ester cleaves at the ester bond, during CID MS², exhibiting product ion [M+H-C₆H₅NO₂]⁺ (steryl cation moiety) at m/z 369.3. ESI-QIT MS³ and APCI-QIT MS³ were performed on cholesterol picolinyl ester. The results showed that both ESI-CIDMS³ and APCI-CID MS³ of [M+H]⁺ → [M+H-C₆H₅NO₂]⁺ gave informative, unique fragments that can be utilized for structural elucidation of the sterol carbon skeleton and for distinguishing among sterol isomers Fig. 2 (C) as will be discussed in the following section. The analysis of other picolinyl sterol esters, using ESI/APCI-MS², showed similar ionization and fragmentation behaviors to the picolinyl ester of cholesterol.

According to Hailatet al³⁴, MALDI-TOF MS of derivatized sterols showed different ionization and fragmentation behaviours since picolinyl sterol esters showed very weak protonated molecular ion signals. However, sodiated adducts, with excellent intensity, were formed after addition of sodium acetate (20mM). Product ion scanning high energy CID MALDI-TOF/TOF resulted in the sodiated picolinyl moiety ([C₆H₅NO₂+Na]⁺), m/z 146.1, while product ion scanning low energy ESI/APCI QIT MS² resulted in the steryl cation as mentioned previously.

A method for analysis of sterols in serum using liquid chromatography tandem mass spectrometry (LC-ESI-triple quadruple-MS/MS) after derivatization to picolinyl esters was developed by Honda et al. ³⁰. It was found that picolinyl sterol esters formed adduct ions with sodium and acetonitrile [M+Na+CH₃CN]⁺ and the CID-MS/MS spectra of these adducts gave a base peak ([M+Na]⁺) and a sodiated picolinyl moiety with a low intensity at m/z 146.

3.3 ESI-MS³ of sterol picolinyl esters:

ESI-CID MS³ mass spectra of four sterol picolinyl ester derivatives were obtained in positive ion mode as shown in Fig. (3.10) those being cholesterol (Fig. 4(a)), cholestanol (Fig. 4(b)), β-sitosterol (Fig, 4(c)) and brassicasterol (Fig. 4(d)). The CID of [M+H]⁺ → [M+H-C₆H₅NO₂]⁺ showed unique and multi-pathway fragmentation. Figure (5) shows a proposed fragmentation pathway of the β-sitosteryl moiety using ESI-QIT MS³. The most abundant ions are at m/z of 161.1, 147.1, 135.1, 243.2, 257.2, 315.3, and 297.2. Fragment ions at m/z of 315.3, 327.3, 341.2, 355.3 and 369.3 are diagnostic of the side chain cleavage, and other fragment ions arise from multi carbon ring cleavage. Fragment ions at m/z 147.1 and 161.1 are diagnostic of free sterols that have a double bond in the B ring (i.e. β-sitosterol and cholesterol). However, most of fragment ions observed in the saturated sterol (cholestanol) are shifted by 2 mass units; i.e. in cholestanol, fragment ions at m/z 163.1 and 149.1 were observed instead of ions at m/z 161.1 and 147.1 in  cholesterol (Fig. 4 (a)). The peak at m/z 257.2 probably arises from losing the side chain of the steryl cation. However, the CID spectrum of brassicasteryl showed different fragmentation pathway where the fragment ion at m/z 255.1 was observed as a base peak which is diagnostic of sterols that have a double bond between carbons 22 and 23. However, the fragment ion at m/z 255.1 could result either from loss of the side chain or the cleavage in the A ring and the side chain as shown in Figure (5. (b)

Figure 4. ESI MS³ spectra of precursor ions of the steryl cationic fragment moieties of the picolinyl ester of sterols. (a) picolinyl ester of cholesterol, (b) picolinyl ester of cholestanol, (c) picolinyl ester of β-sitosterol, and (d) picolinyl ester of brassicasterol.

Figure 5. (a) Proposed CID fragmentation pathway of t β-sitosterol cationic moiety resulting from QIT MSⁿ as revealed in spectra of Figure 3.10 (c), (b) fragmentation pathway for the generation of the base ion m/z 255.2 from stigmasteryl cationic moiety as revealed in the spectra of Figure 3.12(aˋ).

3.4 Distinguishing among isomers using CID-ESI MS³:

CID-ESI/APCI MS³ experiments resulted in unique fragmentation patterns that allow structural elucidation and differentiation among sterol picolinyl ester isomers. Figure (6) shows CID-ESI MS³ spectra of two sets of sterol picolinyl ester isomers. The first set consisted of the picolinyl esters of fucosterol (Fig. 6(a)), and stigmasterol (Fig. 6(aˋ)), m/z 517.4, and the second set included picolinyl esters of desmosterol (Fig. 6(b)), and 7-dehydrocholesterol (Fig. 6(bˋ)), m/z 489.4. Significant differences in ion intensities were observed in the CID-MS³ spectra of the picolinyl esters of fucosterol and stigmasterol. In the spectrum of the picolinyl ester of fucosterol, fragment ions at m/z 161.1, 255.2, 297.2 and 311.2 were found to be significant fragments (Fig. 6(a)) while, the fragment ion at m/z 255.2 was found to be the base peak in the CID spectrum of the picolinyl ester of stigmasterol  (Fig. 6(aˋ)).

The picolinyl esters of desmosterol and 7-dehydrocholesterol can also be distinguished. The CID spectrum of the picolinyl ester of desmosterol showed major fragment ions at m/z 257.2, 161.1, 147.1, 203.1 and 285.2 (Fig. 6(b)), while, the picolinyl ester of 7-dehydrocholesterol showed only two major fragment ions at m/z 159.1 and 145.1 (Fig. 6(bˋ)). The fragment ions at m/z 161.1 and 147.1 in the CID-MS³ spectrum of the picolinyl ester of desmosterol and those at m/z 159.1 and 145.1 in the CID-MS³ spectrum of the picolinyl ester of 7-dehydrocholesterol arise from the cleavage of the C ring. However, the shifting by 2 mass units in the CID-MS³ spectrum of picolinyl ester of 7-dehydrocholesterol confirm the presence of a second double bond in the B ring of 7-dehydrocholesterol (Fig. 6(bˋ).

Figure 6. ESI MS³ spectra of precursor ions of the steryl cationic fragment moieties of the picolinyl ester of sterol isomers. (a) picolinyl ester of fucosterol, (aˋ) picolinyl ester of stigmasterol, (b) picolinyl ester of desmosterol, and (bˋ) APCI MS³ of picolinyl ester of 7-dehydrocholesterol.

4. CONCLUSION:

A new and rapid method has been developed for analysis of free sterols using ESI/APCI-QIT MSⁿ. Free sterols are poorly suited for direct MS analysis, thereforesterols were converted to their corresponding picolinyl esters.

Picolinyl esters of sterols were analyzed using direct injection ESI-QIT MSⁿand  APCI-QIT MSⁿ as well. The picolinyl esters formed protonated molecular ions ([M+H]⁺) in ESI and APCI sources except for the picolinyl ester of 7-dehydrocholesterol which was  detected as the radical cation ion [M^.]⁺using  APCI-QIT MS. The ester bonds of picolinyl esters cleaved during CID MS² resulting in diagnostic fragments corresponding to steryl cation moieties [M+H-C₆H₅NO₂]⁺. The CID MS³ of [M+H]⁺→ [M+H-C₆H₅NO₂]⁺ of picolinyl esters spectra were found to be useful for structural elucidation and to distinguish among steryl isomers.

5. ACKNOWLEDGMENT:

The author is grateful to Middle East University-Amman-Jordan for the financial support granted to cover the publication fees of this research article.

6. CONFLICT OF INTEREST:

The author declare that there is no conflict of interest.

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Received on 25.09.2022            Modified on 31.12.2022

Accepted on 01.02.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(7):3385-3392.

DOI: 10.52711/0974-360X.2023.00560