Structural Investigation of Betulinic Acid Plasma Metabolites by Tandem Mass Spectrometry

Betulinic acid (BA) has been extensively studied in recent years mainly for its antiproliferative and antitumor effect in various types of cancers. Limited data are available regarding the pharmacokinetic profile of BA, particularly its metabolic transformation in vivo. In this study, we present the screening and structural investigations by ESI Orbitrap MS in the negative ion mode and CID MS/MS of phase I and phase II metabolites detected in mouse plasma after the intraperitoneal administration of a nanoemulsion containing BA in SKH 1 female mice. Obtained results indicate that the main phase I metabolic reactions that BA undergoes are monohydroxylation, dihydroxylation, oxidation and hydrogenation, while phase II reactions involved sulfation, glucuronidation and methylation. The fragmentation pathway for BA and its plasma metabolites were elucidated by sequencing of the precursor ions by CID MS MS experiments.


Introduction
Betulinic acid (BA) (3β, hydroxy-lup-20(29)-en-28-oic acid) ( Figure 1) is a lupaneskeleton pentacyclic triterpene secondary plant metabolite. BA is widely distributed in natural occurring sources, considerable amounts being found throughout Betulaceae species, in the outer layers of birch bark [1]. Some vegetal sources with high amounts of BA are an important part of natural remedies that include traditional Chinese medicine such as Ziziphi Spinosae semen (Rhamnaceae) [2] or Indian medicinal remedies such as Nyctanthes arbor-tristis [3]. In recent decades, BA has been extensively investigated during in vitro and in vivo studies that demonstrated a wide range of biological and pharmacological activities, the most high profile being its anticancer potential, already recognized by the National Cancer Institute of the USA [4]. BA displays well documented cytotoxicity activities mainly by triggering the mitochondrial pathway of apoptosis in cancer cells [5] of BA and its metabolites. All combined, the current study contributes to elucidate the metabolomic profile of BA and can represent a support for future studies.

Screening of BA and BA Metabolites by Negative Ion Mode ESI Orbitrap MS in Plasma Samples
Aliquots of 10 µL of deproteinized plasma samples were loaded into the LTQ Orbitrap Velos Pro TM mass spectrometer for negative ion mode screening experiments. Screening mass spectra were recorded in 100-1000 mass range. Betulinic acid was identified as the deprotonated ion [M-H]-at m/z 455. 35. MS screening experiments enabled the identification of 13 phase I and phase II metabolic products of BA. The main phase I metabolic reactions BA underwent were oxidation, monohydroxylation, dihydroxylation and hydrogenation, while the major metabolic pathways during phase II reaction were sulfation, glucuronidation and methylation. The proposed metabolic products and their molecular modifications are depicted in Table 1.

Screening of BA and BA Metabolites by Negative Ion Mode ESI Orbitrap MS in Plasma Samples
Aliquots of 10 µL of deproteinized plasma samples were loaded into the LTQ Orbitrap Velos Pro TM mass spectrometer for negative ion mode screening experiments. Screening mass spectra were recorded in 100-1000 mass range. Betulinic acid was identified as the deprotonated ion [M-H] − at m/z 455. 35. MS screening experiments enabled the identification of 13 phase I and phase II metabolic products of BA. The main phase I metabolic reactions BA underwent were oxidation, monohydroxylation, dihydroxylation and hydrogenation, while the major metabolic pathways during phase II reaction were sulfation, glucuronidation and methylation. The proposed metabolic products and their molecular modifications are depicted in Table 1. Phase I metabolism of natural phytocompounds, as for medicinal drugs, aims to convert liposoluble molecules into more polar ones by introducing or liberating a polar substituent. The resulting metabolites may have their biological activity both altered or unchanged and are the outcome of complex metabolic reactions such as oxidation, reduction and hydrolysis catalyzed by oxidases, reductases and esterases [27]. Oxidation reactions are mostly dependent and mediated by the isoforms of cytochrome P450, out of which CYP2C, CYP2D6 and CYP3A are responsible for the metabolic fate of nearly 75% of drugs. They are present in the endoplasmic reticulum of hepatocytes and intestines, involve a large variety of reactions such as hydroxylations, epoxidations or oxidative dealkylations, and exert by the addition of one or more oxygen atoms to the structure of the parent drug [28]. Since BA is a highly hydrophobic molecule, it is expected to undergo phase I metabolic changes as well.
The screening of plasma samples revealed the presence of the [M-H] − ion at m/z 457.37 (M1), which has a molecular weight (Mw) 2 Da more than BA, corresponding to the addition of 2 H atoms and was assigned according to the mass calculations to the product of a hydrogenation reaction by double bond reduction occurring at the isopropenyl group (C 20 -C 29 ) of ring E. Hydrogenation is a common metabolic reaction reported both for human steroid metabolism and for the metabolism of phytochemicals that bear double bonds in their structure, such as flavonoids [29], terpenes [30] or phenylpropanoid glycosides [31]. Next, the precursor ion identified as [M-H] − at m/z 471.35 (M2 ) has a Mw 16 Da more than BA, corresponding to the addition of an O atom as a result of a metabolic reaction that modified most likely one of the methyl substituents to hydroxymethyl, as reported both for medicinal drugs [32] and phytocompounds [23]. Hence, m/z 471.35 was assigned as the monohydroxylated metabolite of BA. The purpose of hydroxylation, an oxidative metabolic reaction, is to increase the hydrosolubility of less polar compounds, in most cases generating active compounds. Moreover, monohydroxylated metabolites were already reported as in vivo metabolic products for asiatic and madecassic acid [33], maslinic acid [34] and betulinic acid [23]. The precursor ion identified as [M-H] − at m/z 485.33 (M3) has a Mw 30 Da more than BA, corresponding to the addition of 2 O atoms while eliminating 2 H atoms. This second step of the oxidation reaction most likely altered the already available hydroxymethyl substituent; the end result of this two-step oxidation is the conversion of the methyl substituent to carboxyl, generating an inactive metabolite, as previously reported [23,32]. Furthermore, the MS screening spectrum indicated also the formation of the precursor ion at m/z 487.34 (M4), which has a Mw 32 Da more than BA, corresponding to the addition of two O atoms as a result of a two-site hydroxylation metabolic reaction, most likely converting two methyl substituents to hydroxymethyl ones. A dihydroxylation metabolic reaction was previously reported by other studies that investigated the in vivo or in vitro metabolism of: (i) triterpenes, such as maslinic acid [34], asiatic and madecassic acid [33], betulinic acid [35], (ii) diterpenes [36] and (iii) alkaloids [37].

Phase II Metabolic Products
Phase II metabolism consists in conjugation reactions such as glucuronidation, sulfation, acetylation, methylation and glycine conjugation or glutathione conjugation of the parent drug or the already formed phase I metabolites with endogenous substrates such as UDP glucuronic acid (GluA), phosphoadenosyl phosphosulfate, Acetyl-CoA, gluthatione, glycine or S-adenosyl-methionine, catalyzed by specific transferases [38]. Glucuronidation and/or sulfation are the most common types of metabolism for phytocompounds such as flavonoids, terpenoids or alkaloids [27]. The resulting metabolic products are generally inactive, more polar and readier to undergo renal or biliary excretion.
The screening mass spectra of analyzed samples indicated the presence of the precursor ion identified as [M-H] − at m/z 469.37 (M5),which has a Mw 14 Da more than BA, and was assigned according to the mass calculations to the methylated metabolite of BA, which is consistent with previous findings that reported methylated metabolites for echinocystic acid, a triterpene [39], as well as for BA [22,23]. Although methylation is a minor conjugation reaction for xenobiotic transformation, being more common for endogenous neurotransmitters, it still remains a metabolic pathway for a large number of medicinal drugs and phytocompounds [38]. It is catalyzed by methytransferases and the resulting metabolic products are either active or inactive, while their solubility is almost the same as that of the parent drug. Since BA possesses a hydroxyl substituent, most probably the parent ion underwent O-methylation.
In addition, the [M-H] − ion identified at m/z 535.31 (M6) has a Mw 80 Da more than BA and was assigned according to the mass calculations to the sulfoconjugate metabolite of BA. Sulfoconjugation is one of the most important phase II metabolic reactions affecting a great variety of compounds such as hormones, bile acids, medicinal drugs and various classes of phytocompounds, including triterpenes. Addition of the sulfonate group occurs both directly to the parent compound or to its phase I metabolite, leading to the formation of a non-toxic water-soluble compound ready for excretion [38]. The reaction is catalyzed by sulfotransferases (SULTs) that are capable to transfer (SO 3 ) from 3 -phosphoadenosine 5 -phosphosulfate (PAPS) to hydroxyl or amino groups [40]. Hence, in the case of BA, the reaction most likely occurred at the -OH substituent present on ring A. These findings are consistent with the data of Li et al. [23] on the in vivo metabolism of BA, the sulfoconjugate being one of the reported metabolites. In addition, the result of sulfoconjugation after in vivo administration in rats was also reported for betulin [41].
Next, the [M-H + ] − ion detected at m/z 551.30 (M7), has a Mw 96 Da more than BA and 16 Da more than M5. Hence, M7 resulted most likely from both phase I and phase II metabolic reactions and consequently was assigned according to mass calculations to the monohydroxylated and sulfoconjugated BA metabolite, as depicted in Table 1. Moreover, the MS screening revealed the presence of a precursor ion detected at m/z 565.28 (M8), which has a Mw 110 Da more than BA and 30 Da more than M5. Thus, M8 is probably the product of both metabolic phases aswell, and was assigned according to mass calculations as the oxidated sulfoconjugated metabolite of BA; in this case the oxidation reaction converted the methyl group into carboxyl, as mentioned above. Moreover, the [M-H + ] − ion detected at m/z 567.30 (M9) which has a Mw 112 Da more than BA and 32 Da more than M5, presumably underwent both metabolic phases as well and thereupon was assigned according to mass calculations as the dihydroxylated sulfoconjugated metabolite of BA. M7, M8 and M9 were also described and associated with the in vivo metabolism of BA by Li et al. [23] following the intake of Ziziphi Spinosae semen extract in rats. Following these findings it must be emphasized that both BA and its phase I metabolites undergo sulfoconjugation during SKH1 female mice metabolism, data that are in line with previously published reports regarding in vivo triterpene metabolism [42] and, in particular, BA [23].
Next, the [M-H] − identifiedat m/z 631.39 (M10) has a Mw 176 Da more than BA and was associated according to mass calculations to the glucuronide conjugate of BA. Glucuronidation is the most frequent phase II metabolic reaction and the most important detoxification pathway for both endogenous compounds and xenobiotics. Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs) that transfer UDP-glucuronic acid to nucleophilic atoms in the acceptor molecule [43]. Glucuronidation occurs at Olinked moieties such as hydroxy, phenolic or acyl and the resulting conjugates exhibit high hydrosolubility and consequently are easily eliminated via renal excretion or biliary excretion [38]. This type of conjugation also occurs both at the newly formed functional group during phase I reactions or directly with the parent ion that already possesses such a functional group. Hence, in the case of BA, most likely conjugation with GluA altered the already available hydroxyl group, as was reported before [23,44]. A large variety of phytocompounds undergo glucuronidation after in vivo administration in rodents, such as flavonoids [45], alkaloids [37] or triterpenes such as ursolic acid [44], betulinic acid [23] and betulin [46], thus enabling their excretion.
Inspection of the MS screening spectrum also indicated the formation of the [M-H] − precursor ion at m/z = 647.38 (M11), which has a Mw 192 Da more than BA and 16 Da more than M10. Hence, according to the mass calculations, M11 is most likely the monohydroxylated glucuronoconjugate of BA. In a similar manner, the [M-H] − ion detected at m/z 661.37 (M12) has a Mw 206 Da more than BA and 30 Da more than M10. Upon mass calculation, M12 has the features of being the oxidated glucuronoconjugated metabolite of BA, where the oxidation reaction altered the methyl group into carboxyl. Last, the [M-H] − ion detected at m/z 663.37 (M13) has a Mw 208 Da more than BA and 32 Da more than M10 and thus was considered upon calculation as the dihydroxylated, glucuronoconjugated metabolite of BA. M10, M11 and M12 compounds associated with BA metabolism were also described by Li et al. [23], following the metabolomic profiling in rats subsequent to administration of traditional Chinese medicine remedies. As in the case of sulfoconjugates, it seems that both BA and its phase I metabolites undergo glucuronidation, as reported before for these types of molecules [42,47].  [22,32,40,43,47,48], but also for flavonoids [49] and other plant-derived glycosides [50].

Structural Analysis of Phase II Metabolites
The precursor ions related to phase II metabolism of BA resulting from sulfation, glucuronidation and methylation were isolated and successfully sequenced by CID MS/MS. ESI CID MS/MS spectra are depicted in Figure 6

Proposed Metabolic Pathway for BA in SKH1 Female Mice
In the current research, we were able to identify a total of 13 metabolites of BA after intraperitoneal administration to SKH1 female mice, as depicted in Figure 11. Out of the total metabolites, four (M1-M4) were associated with phase I metabolism resulting from metabolic reactions such as monohydroxylation, dihydroxylation, oxidation and hydrogenation. During these reactions, the targets for metabolic alterations were the isopropenyl and methyl substituents of BA. In addition, nine (M5-M13) more metabolites, correlated with phase II metabolism, were identified. The main phase II metabolic reactions for BA were sulfation, glucuronidation and methylation, out of which: (i) three resulted directly from phase II metabolism (M5, M6, M10) and (ii) six resulted from sulfoconjugation and glucuronidation of phase I metabolites (M7-M9, M11-M13) yielded by monohydroxylation, dihydroxylation and oxidation reactions. The metabolic site for M5, M6, M10 was the only available hydroxyl substituent of BA, as described before [38]. Since BA metabolism exhibited both compounds resulting from direct conjugation with endogenous substrates and compounds conjugated with phase I metabolites, as disclosed in a previous study [23], we may assume that the metabolic target for the conjugation of M7-M9 and M11-M13 metabolites could also be the original hydroxyl substituent of BA.
Although during the CID MS/MS structural investigations, a lot of diagnostic fragments for the proposed structures were exhibited, more confirmation is needed for the exact metabolic sites and alterations. For a future perspective, the current research will be completed with multistage sequencing (MS n ) of the metabolic products and NMR confirmation of their structure.

Chemicals
BA was purchased from Sigma-Aldrich (Taufkirchen, Germany). Acetone, methanol, flax-seed oil and egg phosphatidylcholine were purchased from Merck (Darmstadt, Germany) and used without further purification.

BA Nanoemulsion
BA nanoemulsion was obtained by reproducing a previously reported technique [53]. Briefly, BA was prepared as an oil-in-water nanoemulsion by homogenizing flaxseed oil (the internal oil phase) with BA dissolved in chloroform. The aqueous phase containing dissolved egg phosphatidylcholine in deionized ultrapure water was added under constant stirring, followed by homogenizing cycles.

In Vivo Experiment
For the in vivo studies, SKH1 adult female mice (n = 4 mice, age: 20-24 weeks, weight: 25 ± 2 g) were acquired from Charles River Laboratory (Budapest, Hungary) and used. The animals were kept in the University animal facility in standard conditions, as follows: food and water ad libitum, a 12 h/12 h light-dark cycle, an ambient temperature of 22-24 Figure 11. Proposed metabolic pathway for betulinic acid (BA) (3β, hydroxy-lup-20(29)-en-28-oic acid) in SKH1 female mice.

Chemicals
BA was purchased from Sigma-Aldrich (Taufkirchen, Germany). Acetone, methanol, flax-seed oil and egg phosphatidylcholine were purchased from Merck (Darmstadt, Germany) and used without further purification.

BA Nanoemulsion
BA nanoemulsion was obtained by reproducing a previously reported technique [53]. Briefly, BA was prepared as an oil-in-water nanoemulsion by homogenizing flax-seed oil (the internal oil phase) with BA dissolved in chloroform. The aqueous phase containing dissolved egg phosphatidylcholine in deionized ultrapure water was added under constant stirring, followed by homogenizing cycles.

In Vivo Experiment
For the in vivo studies, SKH1 adult female mice (n = 4 mice, age: 20-24 weeks, weight: 25 ± 2 g) were acquired from Charles River Laboratory (Budapest, Hungary) and used. The animals were kept in the University animal facility in standard conditions, as follows: food and water ad libitum, a 12 h/12 h light-dark cycle, an ambient temperature of 22-24 • C, and humidity around 55%. The experimental procedures were performed in compliance with the European Directive 2010/63/EU, the AVMA Guidelines for the Euthanasia of Animals (2013 Edition) and the National Law 43/2014 regarding the protection of animals used for scientific purposes and were analyzed and approved by the University Research Ethics Committee. The following protocol was applied: each mouse was intraperitoneally injected with a dose of 40 mg/kg body weight BA nanoemulsion. Blood samples were collected after 2 h post administration by using the periorbital technique also known as orbital venous plexus bleeding, after a standardized protocol [54]. All the procedures were performed under anesthesia provided by isoflurane inhalation. At the end of the experiment, the mice were euthanized by anesthesia and cervical dislocation.

Plasma Samples
Blood was collected in sterile vials on ethylenediaminetetraacetic acid (EDTA), centrifuged for 10 min at 10,000 rpm and then plasma was collected and stored at −20 • C. All plasma samples were deproteinized with methanol by dissolving 1 part plasma with 3 parts methanol, respectively (v/v). The supernatant was collected and subjected to another round of purification with acetone by the same protocol. All sample solutions were homogenized with a WisdVM-10vortex mixer (Witeg Labortechnik, Wertheim, Germany) and centrifuged for 2 min at 10,000 rpm in a ThermoMicro CL17 microcentrifuge (Thermo Fisher Scientific, Massachusetts, MA, USA). The supernatant was collected and submitted to MS measurements.

Orbitrap Mass Spectrometry
The nanoESI MS experiments were conducted on a LTQ Orbitrap Velos Pro TM mass spectrometer, from Thermo Fisher Scientific (Bremen, Germany), equipped with the offline nanoES source ES 259 (Thermo Fisher, Massachusetts, MA, USA). Ten microliters from each plasma sample was loaded into the borosilicate emitters ES380 (Proxeon, Roskilde, Denmark) and directly infused into the instrument through the offline nanoES source connected to the instrument using the Nanospray Flex Ion Source (Thermo Scientific, Massachusetts, MA, USA) at a spray current of 0.08 µA, obtained by applying a spray voltage of 0.70 kV, keeping the capillary temperature at 275 • C and the S-lens RF level at 60%. All mass spectra were subsequently screened in negative ion mode, which was previously demonstrated to be the best option for molecules containing carboxylic moiety [55], and detected under identical conditions, with no sheath, sweep or auxiliary gas, in an m/z range of 100 to 1000. Prior to experiments, the m/z scale was calibrated with external standard Pierce ESI Negative Ion Solution (Thermo Scientific, Massachusetts, MA, USA). The mass spectrometer was operated and controlled by the LTQ Tune Plus v2.7 build 1112 SP2 software (Thermo Scientific, Massachusetts, MA, USA) running under Windows 7, while the MS data acquisition and processes were achieved using Xcalibur 3.0.63 software (Thermo Scientific, Massachusetts, MA, USA). MS/MS experiments were carried out in the LTQ sector by CID with Orbitrap detection. Ion selection and fragmentation were performed manually, using variable collision energies within 0-35 eV. The precursor ions were selected within an isolation width of 2 m/z units.

Conclusions
The current study was focused on the screening and sequencing of BA metabolites by MS-based techniques, following in vivo administration. To this end, a nanoemulsion containing BA was prepared and administered intraperitoneally to SKH1 female mice. Plasma samples were collected post-treatment and were analyzed by HRMS. Screening experiments of BA and BA metabolites in plasma samples were conducted by ESI Orbitrap MS in the negative ion mode, while structural characterization was conducted by CID MS/MS. The study design combined with the applied analytical strategy enabled the identification of 13 phase I and phase II metabolites of BA in plasma samples. The main phase I metabolic reactions BA underwent were monohydroxylation, dihydroxylation, oxidation and hydrogenation, while phase II metabolism was represented by a conjugation reaction with sulfate or GluA and methylation. In addition, detailed structural information were collected by sequencing of the precursor ions by CID MS MS, data that enabled elucidation of the fragmentation pathway for BA and its plasma metabolites. The main fragmentation pattern for BA and its phase I metabolites revealed during MS/MS experiments consisted mainly in neutral losses of H 2 O, CO 2, HCOOH, C 3 H 4 , O and CH 2 accompanied in some cases by ring cleavage fragmentation. In addition, phase II metabolites exhibited diagnostic fragments by the cleavage of sulfate and GluA. The whole set of information obtained during the detailed structural investigation of BA metabolites contributes to framing a solid database of BA's metabolic pathway and provides in-depth knowledge of their CID MS/MS sequencing behavior. The obtained results enrich and complete BA's ADME parameters, in particular its metabolic profile, mandatory for all molecules that are considered to be developed as medicinal drugs. Moreover, some of the identified metabolites during the present study may be active metabolites, which are most likely more polar than BA and possess superior bioavailability. Hence, this work represents a foundation for developing BA derivatives that share its biological and pharmacological activities but have the credentials to display a superior pharmacokinetic profile, suitable for cancer treatment in humans.