Identification of metabolites of gardenin A in rat liver microsomes using ultra-high performance liquid chromatography coupled with linear ion-trap Orbitrap mass spectrometry

Purpose: To identify the metabolites of gardenin A (GA) in rat liver microsomes (RLMs) using ultra-high performance liquid chromatography coupled with linear ion-trap Orbitrap mass spectrometry (UHPLCLTQ-Orbitrap). Methods: The sample was prepared by incubating GA (100 μg/mL) with RLMs (0.5 mg/mL) for 8 h. Then 5 μL of the sample was injected into UHPLC-LTQorbitrap mass spectrometer. The metabolites of GA were tentatively identified based on accurate mass measurements, fragmentation patterns, chromatographic retention times, and bibliography data. Results: A total of 12 metabolites were detected and identified. Based on their structures, the main reactions in the metabolism of GA are de-methoxylation and de-methylation. Conclusion: This is the first report on in vitro metabolites of GA. These results are considered very helpful for better comprehension of the metabolism of GA and its pharmacological effects.

Research on drug metabolism research is very important in the early phases of drug discovery and development.It is also important for minimizing the adverse effect of pharmaceuticals, and for maximizing their therapeutic value [10,11].A previous report [12] demonstrated that 26 metabolites of GA were observed in vivo; these might be responsible for the pharmacological actions of GA.However, it is hard to establish the reliability of this observation, due to difficulties involved in obtaining enough metabolites in vivo.Thus, it is important to obtain GA metabolites in vitro.To the best of our knowledge, metabolites of GA have not been investigated in vitro.
High performance liquid chromatography coupled with mass spectrometry has become a popular, and indeed the main method for the structural characterization of drug metabolites in vivo and in vitro due to its high efficiency, sensitivity, and selectivity.Among several different LC/MS platforms, ultra-high performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) [13][14][15] such as UHPLC-LTQ-Orbitrap is especially useful for the characterization of drug metabolites due to its higher and faster separation and resolution capacities.
The present study was carried out identify the metabolites of GA in rat liver microsomes (RLMs) with a view to providing better understanding of the metabolism of the drug and its pharmacological effects.

Microsome incubation
Incubation conditions of microsome incubation experiment were established and controlled to provide a reproducible and linear rate of the metabolism in vitro.The incubation mixture in final volume of 1 mL contained GA (100 µg/mL), phosphate buffer (0.1 mol/L, pH 7.4), magnesium chloride (5 mM) and RLMs (0.5 g/mL).The incubation mixture was pre-incubated for 5 min in a water bath at 37 °C and reactions were initiated by addition of NADPH (1 mM).The reaction was terminated by adding 1 mL of ice-cold acetonitrile, vortexing and centrifuging at 15,000 rpm for 10 min at 4 °C.Aliquots of the supernatant were subjected to UHPLC-LTQ-Orbitrap MS to identify the metabolites.Blank samples were of the same composition as the test samples, but without GA.All samples were prepared in triplicate.
The optimized operating parameters in the positive ion mode were as follows: capillary voltage of 25 V, source voltage of 4.0 kV, capillary temperature of 350 °C, sheath gas flow rate of 40 (arbitrary units), auxiliary gas flow rate of 20 (arbitrary units), and tube lens of 110 V. Metabolites were detected by full-scan mass analysis from 100 to 800 m/z at a resolving power of 30,000 with data-dependent MS 2 analysis triggered by the three most-abundant ions from the precursor list of predicted metabolites followed by MS 2 analysis of the most−abundant product ions.Collision-induced dissociation (CID) was performed with an isolation width of 2 Da.The collision energy was set to 35 %.

Data processing
Thermo Xcaliber 2.1 workstation (Thermo Fisher Scientific) was used for data acquiring and processing.In order to obtain as many fragment ions as possible, the peaks detected with intensity over 50,000 were selected for identification.The chemical formulas of all parent and fragment ions of the selected peaks were calculated from the accurate mass using a formula predictor by setting the parameters as follows: and Ring Double Bond (RDB) equivalent value [0 -15].Other elements such as P and Br were not considered as they were rarely present in the complex matrix.The maximum mass errors between the measured and the calculated values were < 5 ppm.Blank RLMs samples were used as control to compare with the analyzed samples, and they were all processed under the same conditions.

Fragmentation pathway of GA
The parent ion showed a protonated ion [ spectrum of GA is shown in Figure 1.

Identified metabolites
The high-resolution extracted Ion Chromatography (HREIC) of blank, GA incubated in RLMS samples in 30 min are shown the Figure 2.For the first time, a total of 12 metabolites of GA were detected and identified based on accurate mass measurements, fragmentation patterns, and chromatographic retention times in positive ion mode.The detailed information is illustrated in Table 1.

DISCUSSION
In the study, we identified metabolites of GA in vitro for the first time.These metabolites are polymethoxylated flavonoids with many methoxy groups (OH-PMFs), which facilitate their detection by ESI in positive mode.MS conditions were optimized on an UHPLC-ESI-LTQ-Orbitrap instrument using standard solution of GA (10 μg/mL).In order to obtain an appropriate elution system, two different mobile systems, acetonitrile -water and methanol -water were tested.It was found that for GA, [M+H] + ions of basically the same intensity were commonly detected with high sensitivity with these two solvent conditions.However, the mobile systems of acetonitrilewater with a gradient elution mode afforded much lower column pressure and better resolution of chromatographic peaks among the metabolites and endogenous components than that of water-methanol.
To our best knowledge, not much has been done on the elucidation of the metabolites of GA, although some studies on the metabolism of GA  have been done in rats [12].For example, 26 metabolites of GA were unambiguously and tentatively identified in Sprague-Dawley rats by comparison of retention times and mass spectrometry.However, it is worth mentioning that in vitro metabolites of GA; and the differences between in vivo and in vitro metabolites of GA were still unclear until now.In our study, 12 metabolites were detected and identified in RLMs.There was a good agreement between these metabolites and those obtained in in vivo studies.This clearly shows that it is feasible to obtain metabolites of GA in vitro.

CONCLUSION
This study has successfully elucidated the in vitro metabolism of GA in RLMs using UHPLC-LTQ-Orbitrap mass spectrometry.Among the 12 metabolites detected, 3 have been unambiguously confirmed, while the others are tentatively identified.The structures clearly indicate that de-methoxylation and demethylation are the major metabolic fates of GA.These metabolites of GA in RLMs would facilitate better understanding of the metabolism and its pharmacological effects.