*2.7. LC-HRMS Analysis of Catalposide and Metabolites*

To separate and identify catalposide and its metabolites, we used a Q-Exactive Orbitrap mass spectrometer coupled to an Accela UPLC system (Thermo Scientific, Waltham, MA, USA). Catalposide and its metabolites were optimally separated on a Halo C18 column via gradient elution using 5% (*v*/*v*) methanol in 1 mM ammonium formate (pH 3.1) (mobile phase A) and methanol (mobile phase B) at a flow rate of 0.5 mL/min: 5% mobile phase B for 2 min, 5–20% mobile phase B over 11.5 min, 20–90% mobile phase B over 0.5 min, 90% mobile phase B for 3 min, 90–5% mobile phase B over 0.5 min, and 5% mobile phase B for 2.5 min. The column and the autosampler were maintained at 40 and 6 ◦C, respectively. Accurate mass measurements of catalposide and its metabolites were derived via electrospray ionization in the negative mode using the following electrospray source settings: ion transfer capillary temperature, 330 ◦C; needle spray voltage, −3000 V; capillary voltage, −47.5 V; nitrogen sheath gas, 50 arbitrary units; auxiliary gas, 15 arbitrary units. The resolution and automatic gain control were scaled to 70,000 and 1,000,000, respectively. MS data were obtained using external calibration over the scan range *m*/*z* 100–700 and processed using Xcalibur software version 2.2 (Thermo Scientific). The Q-Exactive Orbitrap MS was calibrated using MSCAL5 and MSCAL6 for the positive and negative ion modes, respectively. Nitrogen gas was employed for higher-energy collision dissociation (HCD) at an energy of 25 eV to obtain product ion spectra of catalposide and its metabolites. Structures were determined using Mass Frontier software (version 6.0; HighChem Ltd., Bratislava, Slovakia). We used the extracted ion monitoring mode for quantification: *m*/*z* 481.1349 for catalposide, *m*/*z* 657.1674 for catalposide glucuronide, *m*/*z* 561.0921 for catalposide sulfate, *m*/*z* 137.0239 for 4-hydroxybenzoic acid, *m*/*z* 313.0569 for 4-hydroxybenzoic acid glucuronide, and *m*/*z* 175.0410 for 4-methylumbelliferone (the internal standard). The peak areas of all components were integrated using Xcalibur software. The calibration curve was linear over the catalposide concentration range 0.5–200 pmol. The concentrations of catalposide glucuronide and catalposide sulfate were calculated using the calibration curve for catalposide because we had no authentic standards.

#### *2.8. Data Analysis*

All results are the average of two determinations obtained using pooled human intestinal microsomes, pooled human liver S9 fractions, UGTs, and SULTs. The apparent kinetic parameters (*K*m, *V*max, n, and *K*<sup>i</sup> ) for formation of catalposide glucuronide or catalposide sulfate by human intestinal microsomes, liver S9 fractions, UGTs, or SULTs were determined by fitting the Hill equation model [*V* = *V*maxS n /(*K*<sup>m</sup> <sup>n</sup> + S n )], the substrate inhibition model [*V* = *V*max/(1 + *K*m/S + S/*K*<sup>i</sup> )], or the single enzyme model [*V* = *V*maxS/(*K*<sup>m</sup> + S)] to the unweighted formation rates of catalposide glucuronide and catalposide sulfate, respectively, over a range of catalposide concentrations using Enzyme Kinetics software (version 1.1 SPSS Science Inc., Chicago, IL, USA). In the above equations, *V* is the velocity of the reaction at substrate concentration [S], *V*max is the maximum velocity, n is the Hill constant, *K*<sup>m</sup> is the substrate concentration at which the reaction velocity is 50% of *V*max, and *K*<sup>i</sup> is the dissociation constant of the substrate binding to the inhibitory region within the enzyme active site.

#### **3. Results**

#### *3.1. In Vitro Metabolic Profiles of Catalposide Incubated with Human Hepatocytes and Intestinal Microsomes*

LC-HRMS analysis of extracts after incubation of catalposide with human hepatocytes revealed three metabolites (M1–M3) and residual catalposide (Figure 1A). LC-HRMS analysis of reaction mixtures after incubation of catalposide with human intestinal microsomes in the presence of UDPGA yielded M2, M3, and a new metabolite M4 (Figure 1B).

μ **Figure 1.** Extracted ion chromatograms of catalposide and its possible metabolites after incubation of <sup>200</sup> <sup>µ</sup>M catalposide with (**A**) human hepatocytes for 2 h at 37 ◦C in a CO<sup>2</sup> incubator and (**B**) human intestinal microsomes in the presence of UDPGA at 37 ◦C for 1 h (mass accuracy: 5 ppm). The extracted ion chromatograms were reconstructed based on the [M–H]<sup>−</sup> ions: *m*/*z* 481.1349 for catalposide, *m*/*z* 561.0921 for M1 (catalposide sulfate), *m*/*z* 137.0239 for M2 (4-hydroxybenzoic acid), *m*/*z* 313.0569 for M3 (4-hydroxybenzoic acid glucuronide), and *m*/*z* 657.1674 for M4 (catalposide glucuronide).

− − The formulae, deprotonated molecular ions ([M−H]−), mass errors, and retention times of catalposide and its four metabolites, M1–M4, are shown in Table 1. The four metabolite peaks were identified using the accurate mass values and the characteristic product ions of the product scan spectra (Table 1, Figure 2). The mass errors between the theoretical and observed *m*/*z* values for each metabolite were less than 5 ppm, indicating good correlations between the calculated theoretical masses and the experimentally observed masses obtained after full-scan MS analysis.

**Table 1.** Molecular formulae, deprotonated molecular ions ([M−H]−), mass errors, retention times (tR), and product ions of catalposide and its metabolites were identified after incubation of catalposide with human hepatocytes and intestinal microsomes.


**Figure 2.** Product scan spectra of catalposide and its four metabolites, M1–M4 obtained via liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of reaction mixtures obtained after incubation of catalposide with human intestinal microsomes in the presence of uridine 5 ′ -diphosphoglucuronic acid (UDPGA) or human hepatocytes. Glc: glucose; Glu: glucuronosyl.

The product scan spectra of catalposide exhibiting the [M−H]<sup>−</sup> ion at *m*/*z* 481.1349 generated characteristic product ions at *m*/*z* 319.0822 (reflecting loss of glucose from the [M−H]<sup>−</sup> ion), *m*/*z* 205.0497 (loss of C5H6O<sup>3</sup> caused by the breakdown of the iridoid moiety of *m*/*z* 319.0822), and *m*/*z* 137.0239 (the 4-hydroxybenzoyl moiety) (Figure 2). − − − −

M1 exhibited an [M−H]<sup>−</sup> ion at *m*/*z* 561.0921, that is, 80 amu higher than the [M−H]<sup>−</sup> ion of catalposide, indicating that M1 was catalposide sulfate. The product scan spectra of M1 generated the characteristic product ions at *m*/*z* 481.1349 (loss of SO<sup>3</sup> from the [M−H]<sup>−</sup> ion), *m*/*z* 319.0822 (loss of glucose from *m*/*z* 481.1349), *m*/*z* 205.0499, and *m*/*z* 137.0239 (Figure 2). M1 was also formed from catalposide after incubation with human liver S9 fractions in the presence of PAPS. Thus, M1 was identified as catalposide sulfate. − <sup>−</sup> − − − −

M2 exhibited an [M−H]<sup>−</sup> ion at *m*/*z* 137.0239 and generated a characteristic product ion at *m*/*z* 93.0339 (reflecting loss of a carboxyl group from the [M−H]<sup>−</sup> ion)(Figure 2). M2 was identified as 4-hydroxybenzoic acid by comparison with the mass, retention time, and product ion of the authentic standard. − − − −

M3 exhibited an [M−H]<sup>−</sup> ion at *m*/*z* 313.0569, that is, 176 amu higher than the [M−H]<sup>−</sup> ion of M2 (4-hydroxybenzoic acid), reflecting glucuronidation of M2. In the product scan spectrum of M3, characteristic product ions were observed at *m*/*z* 137.0239 (reflecting loss of the glucuronosyl moiety from the [M−H]<sup>−</sup> ion), *m*/*z* 175.0240 (the glucuronosyl moiety), and *m*/*z* 113.0239 (loss of CO<sup>2</sup> and H2O from *m*/*z* 175.0240) (Figure 2). Incubation of 4-hydroxybenzoic acid (M2) with human liver S9 fractions or intestinal microsomes in the presence of UDPGA yielded M3, which was identified as 4-hydroxybenzoic acid glucuronide by comparison with the mass, retention time, and product ions of the authentic standard. − <sup>−</sup> − − − −

M4 exhibited an [M−H]<sup>−</sup> ion at *m*/*z* 657.1674, that is, 176 amu higher than the [M−H]<sup>−</sup> ion of catalposide, reflecting catalposide glucuronidation. M4 yielded characteristic product ions at *m*/*z* 481.1353 (reflecting loss of the glucuronosyl moiety from the [M−H]<sup>−</sup> ion), *m*/*z* 319.0823 (loss of glucose from *m*/*z* 481.1353), *m*/*z* 205.0499, *m*/*z* 175.0240, *m*/*z* 113.0238, and *m*/*z* 85.0283 (Figure 2). Thus, M4 was identified as catalposide glucuronide. − <sup>−</sup> − − − −

The possible in vitro metabolic pathways of catalposide in humans are shown in Figure 3. Catalposide is metabolized to catalposide sulfate (M1), catalposide glucuronide (M4), and 4-hydroxybenzoic acid (M2); the latter is then further metabolized to 4-hydroxybenzoic acid glucuronide (M3).

**Figure 3.** Possible in vitro metabolic pathways of catalposide incubated with human hepatocytes and intestinal microsomes. H: human hepatocytes; I: human intestinal microsomes; Sul: sulfate; Glu: glucuronic acid; CES: carboxylesterase; UGT: UDP-glucuronosyltransferase; SULT: sulfotransferase.
