Identification of metabolites of Buyang Huanwu decoction in rat urine using liquid chromatography-quadrupole time-of-flight mass spectrometry

Abstract

In the present study, rapid resolution liquid chromatography was coupled with quadrupole time-of-flight tandem mass spectrometry (RRLC-Q-TOF-MS) to identify the absorbed components and metabolites in rat urine after oral administration of Buyang Huanwu decoction (BYHWD). After oral administration of BYHWD, urine samples were collected and pretreated by solid phase extraction. The mass measurements were accurate within 5 ppm of error for all the protonated molecules, and subsequent fragment ions offered higher quality structural information for interpretation of the fragmentation pathways of various compounds. A total of 50 compounds were detected in rat urine samples within 20 min, including 12 parent compounds and 38 metabolites. Except for three prototype components (Hydroxysafflor yellow A, Paeoniflorin, and Amygdalin), the metabolites identified mainly came from Radix Astragali, Radix Angelicae Sinensis, and Rhizoma Chuanxiong. The results indicated that glucuronidation and sulfation were the major metabolic pathways of isoflavonoids, while glutathione conjugation, glucuronidation and sulfation were the main metabolic pathways of phthalides. No saponin-related metabolites were detected. The present study provided important structural information on the metabolism of BYHWD. Furthermore, the results of this work have demonstrated the feasibility of the RRLC/ESI-Q-TOF-MS approach for rapid and reliable characterization of metabolites from herbal medicines.

Figure 1

Chemical structures of 12 parent compounds of DBT identified in the drug-containing urine.

Figure 2

TICs of urine samples of rat by RRLC-QTOF/MS (a): The blank urine in positive mode; (b): The drug-containing urine in positive mode (*The parent constituents in Buyang Huanwu Decoction).

Figure 3

Proposed major metabolic pathways of G1(calycosin) -related metabolites.

Figure 4

Proposed major metabolic pathways of G2 (formononetin) -related metabolites.

Figure 5

Proposed major metabolic pathways of G4 ((3R)-7,2′-dihydroxy-3′,4′-dimethoxyiso-flavan) -related metabolites.

Figure 6

Proposed major metabolic pathways of senkyunolide-related metabolites.

Figure 7

Proposed major metabolic pathways of butylidenephthalide-related metabolites.