Characterization of metabolites in rat plasma after intravenous administration of salvianolic acid A by liquid chromatography/time‐of‐flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

Abstract

Salvianolic acid A (SalA) is one of the main active constituents in Salvia miltiorrhiza (Danshen). Although the pharmacokinetics of SalA in rats after intravenous (i.v.) administration of Danshen injection has been reported, the information relevant to the metabolites of SalA in vivo is absent so far. In this study, by means of liquid chromatography with time-of-flight mass spectrometry (LC/TOFMS) and liquid chromatography with ion trap mass spectrometry (LC/MS(n)) techniques, the unknown metabolites of SalA in rat plasma after i.v. administration of the purified SalA at the dose of 20 mg/kg body weight were identified. A liquid-liquid extraction method was established to separate the metabolites from the plasma and the chromatographic separations were performed on a Xterra MS C(18) column (100 mm x 4.6 mm i.d., 3.5 microm) with acetonitrile/methanol/water/formic acid (20.5:19.5:64: 0.05, v/v/v/v) as the mobile phase at a constant flow rate of 0.2 mL/min. Based on the data obtained from the LC/TOFMS determination (the total ion chromatograms, MS spectra and extracted ion chromatograms), in combination with the characteristic fragment ions acquired from the LC/MS(n) determination, five metabolites were identified as SalA-monoglucuronide, monomethyl-SalA-monoglucuronide, mono-methyl-SalA, dimethyl-SalA and dimethyl-SalA-monoglucuronide, and the possible chemical structures were deduced. The results indicated that SalA might mainly undergo two metabolic pathways in vivo in rats, which were methylation and glucuronidation. The present studies have laid a solid foundation for the metabolic mechanism of SalA in vivo.

Metabolism of Salvianolic Acid A and Antioxidant Activities of Its Methylated Metabolites

“…For more exact qualitative characterization, the extracted ion current (XIC) chromatograms created via a special data-mining process were recovered from the entire data set for a chromatographic run, the total ion current (TIC) chromatogram. In view of the two main in vivo metabolic pathways of SAA in rats, methylation and glucuronidation (Shen et al, 2009), the extracted m/z values of [M-H] 2 ions representing SAA and all of its conjugative metabolites of interest included the following: 493 (SAA); 507, 521, and 535 (monomethyl SAA, dimethyl SAA, and trimethyl SAA); and 669, 683, 697, and 711 (glucuronides of SAA, and monomethyl, dimethyl, and trimethylmethyl SAA, respectively). The HPLC-UV, TIC and XIC chromatograms of the drug-containing bile and blank bile samples are shown in Fig.…”

“…It has been shown that SAA is metabolized in rats by methylation and glucuronidation into a series of methyl conjugates and their glucuronides, which were previously found in plasma (Shen et al, 2009). Such unique metabolic fate produces a rather complicated metabolism profile of SAA, and isolating these metabolites from excreta is quite challenging.To thoroughly understand the metabolism mechanism of SAA and the activities of its metabolites thus becomes a new challenge.…”

“…All of these findings suggest that metabolism is an important elimination pathway of SAA, and pharmacological activities in vivo may be related to its metabolic fate. Five conjugated metabolites of SAA (including methylated, glucuronidated, and both) were identified from rat plasma, indicating methylation and glucuronidation as the two main in vivo metabolic pathways of SAA in rats (Shen et al, 2009). The kinetics of SAA glucuronidation by pooled human microsomes and recombinant UDP-glucuronosyl transferase (UGT) isozymes were recently studied, and UGT1A1 and UGT1A9 were identified as the major human UGT isoforms that catalyze the glucuronidation of SAA (Han et al, 2012).…”

In vivo metabolism study of polygalic acid in rat using HPLC‐ESI‐MSn

“…After losing two glucuronic acid groups, the main fragment ions of M1 in the MS 3 ( m/z 487) and MS 4 spectra ( m/z 469) were identical to those of polygalic acid in the MS 1 and MS 2 , respectively. Because of the action of uridine‐5′‐diphosphate glucuronosyltransferases in the biosystem, glucuronidation was a very common metabolic pathway of drug in vivo ; and the glucuronic acid was prone to connect with the acidic groups such as carboxyl group and phenolic hydroxyl group (Shen et al , ). Based on the above analysis, M1 was presumably elucidated as the diglucuronidation product of polygalic acid at its two carboxyl group positions.…”

Active Hydrophilic Components of the Medicinal HerbSalvia miltiorrhiza(Danshen) Potently Inhibit Organic Anion Transporters 1 (Slc22a6) and 3 (Slc22a8)

“…It is known that after oral administration in healthy volunteers, the majority of RMA exists as such conjugated forms, as well [30, 31]. Currently, there are no reports on characterization of LSA and SAA metabolites in human plasma, while SAA might undergo methylation and glucuronidation in rats [32]. Therefore, studies elucidating the metabolic pathways of these compounds are needed such that potential interactions of their metabolites with Oat1 and/or Oat3 can be examined.…”