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.
Highlights
The idea is that derivatization of acyl, O- or Nglucuronide of a molecule results in predictable and different numbers of derivatized functional groups, which can be determined by the mass shift using mass spectrometry
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 (Figure 1A)
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 4 hydroxyl groups were present on both glucuronides, which was consistent with their structures (Table 1)
Summary
Metabolite profiling and identification play essential roles in discovery and development of new chemical entities. Identification of metabolites in non-clinical and clinical species is required both in in vitro preparations and in in vivo biological matrices to understand the metabolic fate of a drug. Its application includes: a) metabolic stability assessment in discovery lead optimization, b) identification of bioactivation potential, c) safety study species selection, d) detection of the presence and determination of coverage of disproportionate metabolites in nonclinical safety evaluation, and e) evaluation of clearance mechanism (circulating and excretory metabolites) of a drug in radiolabeled ADME studies. One of the most 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). 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)
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