Abstract
Glucuronidation is the most common phase II metabolic pathway to eliminate small molecule drugs from the body. However, determination of glucuronide structure is quite challenging by mass spectrometry due to its inability to generate structure-informative fragments about the site of glucuronidation. In this article, we describe a simple method to differentiate acyl-, O-, and N-glucuronides using chemical derivatization. The idea is that derivatization of acyl-, O-, or N-glucuronides of a molecule results in predictable and different numbers of derivatized functional groups, which can be determined by the mass shift using mass spectrometry. The following two reactions were applied to specifically derivatize carboxyl and hydroxyl groups that are present on the aglycone and its glucuronide metabolite: Carboxyl groups were activated by thionyl chloride followed by esterification with ethanol. Hydroxyl groups were derivatized via silylation by 1-(trimethylsilyl)imidazole. The mass shift per derivatized carboxyl and hydroxyl group was +28.031 Da and +72.040 Da, respectively. This approach was successfully validated using commercial glucuronide standards, including benazepril acyl-glucuronides, raloxifene O-glucuronide, and silodosin O-glucuronide. In addition, this approach was applied to determine the type of glucuronide metabolites that were isolated from liver microsomal incubation, where alvimopan and diclofenac acyl-glucuronides; darunavir, haloperidol, and propranolol O-glucuronides; and darunavir N-glucuronide were identified. Lastly, this approach was successfully used to elucidate the definitive structure of a clinically observed metabolite, soticlestat O-glucuronide. In conclusion, a novel, efficient, and cost-effective approach was developed to determine acyl-, O-, and N-glucuronides using chemical derivatization coupled with liquid chromatography–high resolution mass spectrometry.
SIGNIFICANCE STATEMENT The method described in this study can differentiate acyl-, O-, and N-glucuronides and allow for elucidation of glucuronide structures when multiple glucuronidation possibilities exist. The type of glucuronidation information is particularly useful for a drug candidate containing carboxyl groups, which can form reactive acyl-glucuronides. Additionally, the method can potentially be used for definitive structure elucidation for a glucuronide with its aglycone containing a single carboxyl, hydroxyl, or amino group even when multiple types of functional groups are present for glucuronidation.
Introduction
Metabolite profiling and identification play essential roles in discovery and development of new chemical entities. Identification of metabolites in nonclinical and clinical species is required in both in vitro preparations and in vivo biological matrices to understand the metabolic fate of a drug. Such applications include: 1) metabolic stability assessment in discovery lead optimization, 2) identification of bioactivation potential, 3) safety study species selection, 4) detection of the presence and determination of coverage of disproportionate metabolites in nonclinical safety evaluation, and 5) evaluation of clearance mechanism (circulating and excretory metabolites) of a drug in radiolabeled absorption, distribution, metabolism, and excretion studies. Structure elucidation of a drug metabolite is usually assisted with the mass-to-charge ratio obtained in full-scan mass spectrometry (MS) analysis and product ion information from subsequent fragmentation analysis of the metabolite using high-resolution mass spectrometry. High-resolution mass spectrometry can provide the elemental composition data, but generally does not provide sufficient information for definitive structure elucidation, e.g., the exact location of a given modification of the structure by metabolism. Other tools are therefore used to assist structure characterization such as NMR spectroscopy (Subramanian et al., 2010), chemical derivatization (Liu and Hop, 2005), online hydrogen/deuterium exchange technique (Ohashi et al., 1998; Liu and Hop, 2005), and stable isotope labeling (Yan and Caldwell, 2004; Ly et al., 2009). Among them, chemical derivatization can be used to determine the specific functional group of a molecule. For example, one of the classic chemical derivatizations is to use TiCl3 to identify the presence of N-oxide, which can be selectively reduced to amines by TiCl3 in biological matrices such as plasma and urine (Kulanthaivel et al., 2004). Similarly, dansyl chloride can react with primary and secondary amine as well as the aromatic hydroxyl group to form sulfonamide and sulfonate, respectively (Dalvie and O’Donnell, 1998).
Glucuronidation is the most common phase II metabolic pathway. The reaction is mediated by uridine 5′-diphosphate-glucuronosyltransferase, which is mainly located in the endoplasmic reticulum. Glucuronidation can occur on nucleophilic functional groups such as carboxylic acid, alcohol, and amine, leading to the formation of O-acyl- (will be called acyl- hereafter), O-, and N-glucuronide, respectively. These glucuronide metabolites are more polar and therefore more water soluble than their aglycones, thus facilitating the elimination of the drugs from the body. O- and N-glucuronides are generally stable and considered bioinactivated metabolites. In contrast, acyl-glucuronide is usually unstable at neutral or slightly basic conditions and is considered a reactive metabolite, although the reactivity is dependent on the structure of the molecule. Reactive acyl-glucuronide can undergo intramolecular rearrangement and intermolecular transacylation (Hyneck et al., 1988), leading to covalent binding to proteins (van Breemen and Fenselau, 1985; Smith et al., 1990). As a result, formation of acyl-glucuronide can be a safety concern for a drug candidate and may need additional risk assessment (Smith et al., 2018; Walles et al., 2020). It is therefore critically important to determine if acyl-glucuronide is formed from a carboxyl-bearing drug candidate.
Structure elucidation of glucuronide metabolites is generally not achievable by liquid chromatography-tandem mass spectrometry (MS/MS). The glucuronyl moiety (176.032 Da) can be readily lost upon the collision-induced disassociation (CID) of a glucuronide metabolite. Therefore, the information on the glucuronidation site is typically missed in the MS/MS spectrum of a glucuronide metabolite, except in some rare cases. However, there are some liquid chromatography-mass spectrometry (LC-MS)–based methods to differentiate different types of glucuronides following derivatization. An O-glucuronide of an oxidative metabolite, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid from N-(3,5-dichlorophenyl)succinimide, was identified after chemical derivatization with 3-pyridinylcarbinol in conjunction with LC-MS (Cui and Harvison, 2000). Another study demonstrated that acyl-glucuronide could be identified after derivatization to a hydroxamic acid with hydroxylamine coupled with LC-MS (Vaz et al., 2010). Recently, it has been reported that O- and N-glucuronides could be differentiated by a tandem mass spectrometric method coupled with gas phase ion-molecule reactions of deprotonated glucuronide with trichlorosilane (Kong et al., 2018). However, acyl-glucuronide could not be differentiated from O- and N-glucuronides using this approach. A later report from the same group demonstrated that migrated acyl-, N-, and O-glucuronides can be differentiated using tandem mass spectrometry based on gas phase ion-molecule reactions between the glucuronide and BF3 (Niyonsaba et al., 2019). Nevertheless, unmigrated acyl- and O-glucuronide cannot be differentiated using this methodology. More often, the structure of the glucuronide metabolite is determined by NMR, which is a time- and resource-intensive process. NMR analysis typically needs at least micrograms of purified material. Preparation of glucuronide metabolites can be achieved by biosynthesis or organic synthesis and is sometimes quite challenging to find an appropriate biosynthetic system or a synthetic route. Thus, identification of the nature of glucuronidation, particularly when multiple possibilities (different functional groups) exist, remains a major challenge in drug discovery and development.
At present there is no published methodology to simultaneously characterize acyl-, O-, and N-glucuronides using chemical derivatization. The objective of this study is to demonstrate a new consolidated approach which uses chemical derivatization toward carboxyl and hydroxyl groups coupled with liquid chromatography–high-resolution mass spectrometry (LC-HRMS) to efficiently determine the type of acyl-, O-, and N-glucuronides simultaneously. Four commercial acyl- and O-glucuronide metabolites (Table 1) were first used to validate the method. The approach was then used to determine acyl-, O-, or N-glucuronides of six compounds (Table 1) generated from liver microsome incubation. The chemical derivatization reaction and the strategy to differentiate different types of glucuronides are described herein.
Materials and Methods
Chemical and Biologic Reagents
Alvimopan, benazepril hydrochloride, darunavir, diclofenac sodium salt, haloperidol, propranolol, raloxifene hydrochloride, thionyl chloride (SOCl2), anhydrous ethanol (EtOH), 1-(trimethylsilyl) imidazole - pyridine mixture (TMSI), uridine 5′-diphosphoglucuronic acid trisodium salt (UDPGA), alamethicin, and anhydrous dimethylformamide were obtained from Sigma (St. Louis, MO). Silodosin was purchased from Toronto Research Chemical (Toronto, Canada). Benazepril acyl-β-D-glucuronide (Ben-A-Gluc), diclofenac acyl-β-D-glucuronide, raloxifene 4’-glucuronide (Ral-4’-O-Gluc), raloxifene 6-glucuronide (Ral-6-O-Gluc), and silodosin β-D-glucuronide (Sil-O-Gluc) were purchased from Cayman Chemical (Ann Arbor, MI). Pooled human liver microsomes (HLMs) and rat liver microsomes (RLMs) were purchased from Xenotech (Kansas City, KS). Soticlestat was synthesized at Takeda Pharmaceutical Company Limited (Fujisawa, Kanagawa, Japan). Soticlestat O-glucuronide (Sot-O-Gluc) was prepared by Wuxi AppTec Co., Ltd (Tianjin, China).
Isolation of Glucuronide Metabolites from Microsomal Incubation
1-mL of reaction mixture was prepared by adding 250 µL of 40-µM substrate solution, except darunavir, 100 µL of 20-mg/mL pooled HLMs, 397.5 µL of 0.1-M phosphate buffer containing 3 mM of MgCl2 (pH 7.4), 2.5 µL of 20-mg/mL alamethicin stock solution prepared in methanol, and 250 µL of 12-mg/mL UDPGA solution. Substrate, alamethicin, and UDPGA were prepared in 0.1-M phosphate buffer containing 3 mM of MgCl2 (pH 7.4). Incubation was conducted at 37°C for 1 hour in ThermoMixer (F1.5; Eppendorf, Enfield, CT), which was subsequently quenched by adding 1 mL of ice-cold acetonitrile (ACN). After brief vortexing and centrifuging at 14,000g for 30 minutes, the supernatant was collected. 40 µL of sample was injected into high-performance liquid chromatography, and the corresponding glucuronide metabolite peak was isolated via fractionation and dried down using SpeedVac vacuum centrifugation (SPD1010; Thermo Fisher Scientific Inc., San Jose, CA).
For darunavir, an approximate 1 mL of reaction mixture was prepared by adding 5 µL of 5-mM darunavir prepared in DMSO, 100 µL of 20-mg/mL pooled HLMs and RLMs, 695 µL of 0.1-M phosphate buffer or 0.1-M tris buffer containing 10 mM of MgCl2 (pH 7.4), 2.5 µL of 20-mg/mL alamethicin stock solution prepared in methanol, and 200 µL of 25-mM UDPGA solution prepared in 0.1-M phosphate buffer or 0.1-M tris buffer containing 10 mM of MgCl2 (pH 7.4). The remaining procedure was the same as above.
Derivatization of Carboxyl Groups
To derivatize carboxyl groups, the isolated glucuronide metabolite from liver microsomal incubation, or 10 nmol of substrate if the authentic standard is available, was first dried down using SpeedVac (SPD1010; Thermo Fisher Scientific Inc.) and reconstituted in 40 µL of EtOH. A reaction was initiated by adding 10 µL of ACN that contains 2% SOCl2 (v/v). After incubating at 4°C in Thermomixer (900 rpm) for 30 minutes, reactions were stopped by adding 100 µL of water. 10 µL of the reaction mixture was then injected into liquid chromatography–high-resolution mass spectrometer.
Derivatization of Hydroxyl Groups for Alvimopan, Raloxifene, Silodosin, and Their Glucuronide Metabolites
The derivatization of hydroxyl groups followed the published procedure with modified conditions (Watanabe et al., 2003). To derivatize hydroxyl groups, the isolated glucuronide metabolite from liver microsomal incubation, or 20 nmol of substrate if the authentic standard is available, was dried using SpeedVac (SPD1010, Thermo Fisher Scientific Inc.) and reconstituted in 100 µL of ACN. 100 µL of 1-(Trimethylsilyl) imidazole (TMSI)-Pyridine mixture was then added and incubated at 60°C for 2 hours. 10 µL of sample was then directly injected into liquid chromatography–high-resolution mass spectrometer.
Derivatization of Hydroxyl Groups for Haloperidol, Propranolol, Soticlestat, and Their Glucuronide Metabolites
After isolating glucuronide metabolites of haloperidol, propranolol, or soticlestat as described above, each glucuronide sample was completely dried. Subsequently, 50 µL of dimethylformamide and 50 µL of TMSI were added to the dried mixture. The reaction was incubated overnight for haloperidol and soticlestat glucuronides or 1 hour for propranolol glucuronide at 60°C. Haloperidol and soticlestat glucuronide samples were dried and reconstituted in 100 µL of 80:20 (v/v) water:ACN before being subjected to LC-HRMS analysis. Propranolol glucuronide was analyzed by LC-HRMS directly. As a positive control, 1 µL of 10 mM haloperidol, propranolol, or soticlestat was added to a test tube and diluted with 9 µL of ACN and dried under N2 flow. After the samples were completely dried, they were treated the same way as their isolated glucuronide metabolites.
LC-HRMS Analysis
All samples except soticlestat-related samples were analyzed using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography (Agilent Technologies Inc., Wilmington, DE) that was composed of a binary pump, an autosampler, a temperature-controlled column compartment, and a diode-array detector. The liquid chromatography separation was achieved with an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm; Waters Corp., Milford, MA) maintained at 40°C at a flow rate of 0.4 mL/min using mobile phase A (water, 0.1% formic acid) and mobile phase B (ACN, 0.1% formic acid). The following gradient program was used: 0–1 minute (5% B), 1–7 minutes (5%–95% B), 7–12 minutes (95% B), 12–12.5 minutes (95%–5% B), 12.5–16 minutes (5% B). Samples were analyzed using both a diode-array detector and Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Inc.). For MS analysis, a full MS scan (m/z 100–1000, resolution 30,000) was first performed in the positive ion mode, followed by an MS/MS scan (resolution 15,000) of a list of parent masses or the most intense ions if the former were absent. The list contains theoretical m/z of a parent compound, corresponding glucuronide metabolites, and derivatized products. Other experiment parameters were set as follows: capillary temperature, 350°C; N2 sheath gas flow, 60 (arbitrary units); N2 auxiliary gas flow, 20 (arbitrary units); source voltages, 3.0 kV; S-Lens RF level, 66.2%; normalized collision energy for collision-induced dissociation (CID), 40%; and normalized collision energy for higher-energy collisional dissociation, 30%. The data were acquired and processed using Xcalibur 2.2 (Thermo Fisher Scientific Inc). For darunavir, 40 µL of darunavir HLM and RLM incubations were injected using the same method described above, except with a change in mobile phases (mobile phase A: 10 mM ammonium formate containing 0.1% formic acid; solvent B: methanol containing 0.1% formic acid) to allow for better separation of N- versus O-glucuronides.
Analysis of soticlestat, its glucuronides, and derivatized products were performed by injecting 5 µL of sample into a microLC system (M5 MicroLC system; SCIEX, Framingham, MA) coupled to a TripleTOF 6600 mass spectrometer (SCIEX). Samples were separated using a Kinetex C18 column (0.3 × 50 mm, 2.6 µm; Phenomenex Inc., Torrance, CA) heated to 45°C at a flow rate of 10 µL/min using mobile phase A (water, 0.1% formic acid) and mobile phase B (ACN, 0.1% formic acid). The following gradient program was used: 0–1 minute (5% B), 1–5 minutes (5%–95% B), 5–8.5 minutes (95% B), 8.5–9 minute (95%–5% B), 9–10 minute (5% B). The HPLC elute was introduced to electrospray ionization source using the positive ion mode on a TripleTOF 6600. The electrospray ionization parameters were set as follows: declustering potential, 60 V; Gas 1, Gas 2, and curtain gas, 30, 45, and 30 (arbitrary units), respectively; ion spray voltage, 4500 V; and source temperature, 300°C. The samples were analyzed in full scan mode with a mass range of 300 to 1000 Da. MS/MS data were analyzed with a mass range of 100 to 1000, with data-dependent acquisition triggered by the mass defect filter list. Collision energy for full scan and MS/MS analysis were 10 and 35 V, respectively. The data were acquired using AnalystTF 1.8 (SCIEX) and processed using PeakView 2.2 (SCIEX).
Results
Method Validation Using Acyl- and O-Glucuronide Standards
Four commercial glucuronide metabolite standards, Ben-A-Gluc, Ral-4’-O-Gluc, Ral-6-O-Gluc, and Sil-O-Gluc, were used for the validation of the method. After Ben-A-Gluc was treated with SOCl2 in the presence of EtOH, the only carboxyl group on the glucuronide moiety was converted to the ethyl (Et) ester (Fig. 1A). After LC-HRMS analysis, the 1 × Et–derivatized Ben-A-Gluc was detected (Fig. 1B; Table 1), while the 2 × Et–derivatized peak was absent in the carboxyl-derivatized sample (Fig. 1C).
After Ral-4’-O-Gluc and Ral-6-O-Gluc were treated with TMSI, the 4 × trimethylsilyl (TMS)–derivatized glucuronide metabolite was detected for each of them, while the 5 × TMS–derivatized glucuronide metabolite was not detected, suggesting that four hydroxyl groups were present on both glucuronides, which was consistent with their structures (Table 1). After Sil-O-Gluc was treated with TMSI, the 3 × TMS–derivatized glucuronide metabolite was detected, while the 4 × TMS–derivatized glucuronide metabolite was not detected by LC-HRMS, suggesting that three hydroxyl groups were present on this metabolite, which was also consistent with its structure (Table 1).
Determination of Acyl-, O-, or N-Glucuronides Isolated from Liver Microsomal Incubation
After human and/or rat liver microsomal incubation with alvimopan, diclofenac, darunavir, haloperidol, and propranolol, an alvimopan glucuronide, a diclofenac glucuronide, two darunavir glucuronides, a haloperidol glucuronide, and a propranolol glucuronide were isolated, and the type of glucuronide was determined by carboxyl and hydroxyl derivatization using LC-HRMS. Each of those isolated glucuronide metabolites was treated with SOCl2 in the presence of EtOH and/or TMSI separately. The maximum number of Et- and/or TMS-derivatized groups was determined by LC-HRMS and their proposed glucuronide type was listed in Table 1.
Alvimopan glucuronide was proposed to be acyl-glucuronide based on both carboxyl and hydroxyl derivatization result. Diclofenac glucuronide was proposed to be acyl-glucuronide based on carboxyl derivatization result. Its structure was confirmed by the comparison of the retention time and MS/MS data to those from the authentic standard (data not shown). Darunavir glucuronides 1 and 2 were proposed to be N- and O-glucuronides, respectively, based on hydroxyl derivatization results. Both structures were confirmed by the MS/MS spectra (Fig. 2). The product ion m/z 464.232 from 4 × TMS–derivatized glucuronide 1 indicated that the TMS was added to the darunavir moiety, suggesting that the only hydroxyl group on the darunavir moiety was silylated and was not conjugated to glucuronide (Fig. 2A). Additionally, the product ion m/z 332.042 from darunavir glucuronide 1 suggested that glucuronidation occurred on the phenylamine (Fig. 2B). Therefore, darunavir glucuronide 1 structure was determined to be the N-glucuronide as shown in Table 1. In contrast, the product ion m/z 392.199 from 3 × TMS–derivatized glucuronide 2 suggested that the hydroxyl group on the darunavir moiety was not silylated and was thus conjugated to glucuronide (Fig. 2C), which was consistent with the O-glucuronide assignment. Haloperidol glucuronide isolated from HLM incubation was proposed to be O-glucuronide based on hydroxyl derivatization results. This was corroborated by the fact that haloperidol O-glucuronide has been reported as the major glucuronide metabolite observed in HLM incubation (Kato et al., 2012). Propranolol glucuronide was proposed to be O-glucuronide based on hydroxyl derivatization results. Although the structure of the propranolol glucuronide was not confirmed further by other experiments, propranolol O-glucuronide has been reported as the major human plasma metabolite (Walle et al., 1979).
Determination of Sot-O-Gluc
A soticlestat glucuronide was isolated from HLM incubation and subsequently derivatized with TMSI. Since soticlestat does not contain a carboxyl group, the isolated compound is either O- or N-glucuronide. After TMSI derivatization, the three hydroxyl groups on the glucuronide moiety can be derivatized for O-glucuronide (Fig. 3A). Three potential N-glucuronides can be formed for this compound. A total of four hydroxyl groups would be derivatized for any of the N-glucuronides (Fig. 3A). After LC-HRMS analysis, the 3 × TMS–derivatized soticlestat glucuronide was detected (Fig. 3B), while the 4 × TMS–derivatized peak was absent in the hydroxyl-derivatized sample (Fig. 3C). Therefore, the isolated glucuronide metabolite was proposed to be Sot-O-Gluc (Table 1). The assigned structure was later confirmed by comparison of the retention time and MS/MS data to those from the authentic standard (data not shown).
Positive Control Samples
Each of the aglycone compounds was treated the same way as their respective glucuronide metabolites. The maximum number of Et- and TMS-derivatized groups was determined by LC-HRMS and listed in Table 2. All carboxyl and/or hydroxyl groups on those compounds were successfully derivatized by either SOCl2 in the presence of EtOH or TMSI.
Discussion
Carboxyl, hydroxyl, and amine are common functional groups for marketed drugs, drug candidates under development, and drug metabolites. A carboxyl-, hydroxyl-, and amine-bearing molecule can potentially form acyl-, O-, and N-glucuronides in vitro and in vivo. If glucuronidation is a major metabolic pathway for clearance of a drug, identification of the type of glucuronidation can be important for the project team for optimization of clearance. Additionally, the identification of the presence of acyl-glucuronides is particularly important to understand if the reactive metabolite is formed for a drug. If a drug only contains a single type of the functional group, such as carboxyl, hydroxyl, or amine, that can be glucuronidated, the type of glucuronidation is self-explained and does not need further study to elucidate it. However, if a drug contains multiple types of the functional groups (e.g., carboxyl and hydroxyl; carboxyl and amine; hydroxyl and amine; or carboxyl, hydroxyl, and amine), the method described in this study can be easily and effectively used to differentiate the type of its glucuronide metabolite. If a specific type of glucuronide has only one possible glucuronidation site, the definitive structure is determined for this glucuronide metabolite even though the aglycone contains a combination of carboxyl, hydroxyl, and amino groups. For instance, soticlestat contains a hydroxyl, an amide, and two amine groups. The isolated soticlestat glucuronide was determined to be O-glucuronide in this study. Consequently, its definitive structure was unambiguously assigned (Table 1).
Different types of glucuronides from an aglycone compound contain different, predictable numbers of carboxyl and/or hydroxyl groups. For example, alvimopan acyl-glucuronide has only one carboxyl group (Fig. 4A), while alvimopan O- or N-glucuronide has two carboxyl groups (Fig. 4, B and C). On the other hand, alvimopan O-glucuronide has three hydroxyl groups while alvimopan acyl- or N-glucuronide has four hydroxyl groups (Fig. 4). Therefore, the number of carboxyl and hydroxyl groups on the glucuronide metabolite can inform the type of glucuronidation. The number of carboxyl or hydroxyl groups on a glucuronide metabolite can be determined by the number of corresponding derivatized groups using specific derivatization reactions. In this study, carboxyl groups are selectively derivatized with SOCl2 in the presence of EtOH to form the Et ester with a mass shift of +28.031 Da per derivatized group under the described condition. Hydroxyl groups are selectively derivatized with TMSI to form trimethylsiloxy group with a mass shift of +72.040 Da per derivatized group. After derivatization, the type of glucuronide can then be determined by the maximum number of derivatized carboxyl or hydroxyl groups calculated from the mass shift detected by LC-HRMS (Table 3). For example, in case of alvimopan glucuronides, detection of 1 × Et–derivatized group (i.e., a positive mass shift of 1 × 28.031 Da) after carboxyl derivatization and 4 × TMS–derivatized groups (i.e., a positive mass shift of 4 × 72.040 Da) after hydroxyl derivatization means it is an acyl-glucuronide (Fig. 4A). Similarly, detection of 2 × Et– and 3 × TMS–derivatized groups indicates an O-glucuronide (Fig. 4B) and detection of 2 × Et– and 4 × TMS–derivatized groups indicates a N-glucuronide (Fig. 4C). It is worth noting that the method is expected to work for the migrated acyl-glucuronide as well, since the number of the carboxyl and hydroxyl groups is identical between unmigrated and migrated acyl-glucuronides.
Based on this strategy, a decision tree is proposed to determine the type of glucuronide metabolites (Fig. 5). The decision tree provides guidance on how to determine acyl-, O-, and N-glucuronides using chemical derivatization.
In this study, we first used four commercial metabolite standards—Ben-A-Gluc, Ral-4’-O-Gluc, Ral-6-O-Gluc, and Sil-O-Gluc—to validate the method. The assigned glucuronide types determined using the above strategy was consistent with the structure of all those commercial standards (Table 1). Subsequently, five commercial compounds, alvimopan, diclofenac, darunavir, haloperidol, and propranolol, known to generate different types of glucuronide metabolites, were incubated in HLMs or RLMs. A total of six glucuronide metabolites were isolated from those liver microsomal samples. The types of glucuronide metabolites were then proposed based on the result from chemical derivatization (Table 1). Among those proposed glucuronide metabolites, the structures of diclofenac acyl-glucuronide were confirmed by the authentic standard, diclofenac acyl-β-D-glucuronide. The structures of darunavir N-glucuronide and O-glucuronide were confirmed by their respective MS/MS spectra. The assignment of haloperidol O-glucuronide and propranolol O-glucuronide was supported by the literature data. The assignment of alvimopan acyl-glucuronide was corroborated with the result from both carboxyl and hydroxyl derivatization. Therefore, the method was successfully applied to determine the acyl-, O-, and N-glucuronides generated in vitro. Additionally, the method was also used for the characterization of the structure of a glucuronide metabolite from soticlestat observed in clinical samples. Soticlestat is a potent and highly selective inhibitor of cholesterol 24-hydroxylase and is being tested in clinical trials for the treatment of rare developmental and epileptic encephalopathies. A soticlestat glucuronide was formed in HLM incubation, which was also observed in human plasma and urine. The structure of this metabolite was characterized with hydroxyl derivatization using the above strategy (Fig. 5). The derivatization results suggested that it was an O-glucuronide. This proposed structure was later targeted for synthesis (as opposed to synthesizing all potential positional isomers) and confirmed to be Sot-O-Gluc. All those data indicate that the type of glucuronide metabolite can be determined by the chemical derivatization method described in this study.
It is worth noting that when the chemical derivatization experiment is conducted, it is critically important to run the derivatization reaction under the same conditions on the aglycone compound as the positive control simultaneously. This method only works when the derivatization is successful on all carboxyl or hydroxyl groups on the aglycone. If any carboxyl or hydroxyl groups on the aglycone cannot be derivatized, the maximum number of the derivatized groups may be identical for acyl-, O-, and N-glucuronides, which may lead to an incorrect assignment of glucuronide type. For example, compound A contains an inert hydroxyl group, which cannot be derivatized by TMSI. Although N-glucuronide of compound A has four hydroxyl groups, only the three on the glucuronide moiety can be derivatized. As a result, a maximum of 3 × TMS–derivatized groups will be detected for the N-glucuronide of compound A. That observation will result in a misleading conclusion that this is an O-glucuronide of compound A, according to the decision tree (Fig. 5). In this study, the chemical derivatization on the aglycone compounds listed in Table 2 were all successful. However, the chemical derivatization on some other compounds was unsuccessful (data not shown). As a result, determination of the types of glucuronide metabolites for the latter compounds was not pursued. To use this method efficiently, a good strategy is to first run the method development of the desired chemical derivatization on the aglycone compound. The derivatization conditions described in this study have been successfully applied for multiple compounds, which can be a good starting point for the method development. If carboxyl or hydroxyl derivatization is effective on the aglycone compound, the method can then be used for the determination of the type of its glucuronide metabolite. The carboxyl and hydroxyl groups on the glucuronide moiety are relatively reactive and their reactivity should be similar for any glucuronide metabolite regardless of the aglycone structure. Therefore, method development of the chemical derivatization is not needed for the glucuronide metabolite. Additionally, the derivatization reaction was selective on either carboxyl or hydroxyl group under the conditions used in this study. The method will not work if the derivatization reaction is not selective on a specific functional group.
In summary, the type of 11 glucuronide metabolites from nine compounds were successfully determined by chemical derivatization coupled with LC-HRMS. Among them, the definitive structures of eight glucuronide metabolites were unambiguously assigned, as there was only one possible glucuronidation site for the type of glucuronide. The method utilizes the maximum number of derivatized carboxyl and/or hydroxyl groups to differentiate acyl-, O-, and N-glucuronides. Carboxyl and hydroxyl groups were derivatized by SOCl2 in the presence of EtOH and TMSI, respectively, in this study. However, the method can be extended to any other chemical derivatization as long as the reaction is specific to carboxyl or hydroxyl group.
Acknowledgments
The authors thank Drs. Suresh K. Balani and T. Eric Ballard for their critical review of this manuscript.
Authorship Contributions
Participated in research design: Guo, Shah, Zhu.
Conducted experiments: Guo, Shah, Oh.
Contributed new reagents or analytic tools: Guo, Shah, Zhu.
Performed data analysis: Guo, Shah, Zhu.
Wrote or contributed to the writing of the manuscript: Guo, Shah, Oh, Chowdhury, Zhu.
Footnotes
- Received January 15, 2022.
- Accepted February 24, 2022.
↵1 Current affiliation: ClearView Healthcare Partners, Newton, Massachusetts
↵2 Current affiliation: Boston Pharmaceuticals, Inc., Cambridge, Massachusetts
↵3 Y.G. and A.S. contributed equally to this work.
This work received no external funding.
The work was supported by Takeda Pharmaceutical Company Limited. All authors were Takeda employees when the work was performed. Some of the authors are shareholders in Takeda. All authors declare no competing interest.
Abbreviations
- ACN
- acetonitrile
- Ben-A-Gluc
- benazepril acyl-β-D-glucuronide
- CID
- collision-induced dissociation
- Et
- ethyl
- EtOH
- ethanol
- HLM
- human liver microsome
- LC-HRMS
- liquid chromatography–high-resolution mass spectrometry
- LC-MS
- liquid chromatography-mass spectrometry
- MS
- mass spectrometry
- MS/MS
- tandem mass spectrometry
- Ral-4’-O-Gluc
- raloxifene 4’-glucuronide
- Ral-6-O-Gluc
- raloxifene 6-glucuronide
- RLM
- rat liver microsome
- Sil-O-Gluc
- silodosin β-D-glucuronide
- SOCl2
- thionyl chloride
- TMS
- trimethylsilyl
- TMSI
- 1-(trimethylsilyl) imidazole - pyridine mixture
- UDPGA
- uridine 5′-diphosphoglucuronic acid trisodium salt
- Copyright © 2022 The Author(s)
This is an open access article distributed under the CC BY Attribution 4.0 International license.