Development and validation of a UFLC–MS/MS method for the determination of anhydrosafflor yellow B in rat plasma and its application to pharmacokinetic study

• A sensitive UFLC–MS/MS method for the quantification of anhydrosafflor yellow B (AHSYB) in rat plasma has been developed and validated.
• The method was successfully applied to the pharmacokinetic study of AHSYB in normal and acute blood stasis syndrome rats.
• The oral bioavailability of AHSYB was very low and the AUC0–t, AUC0–∞ and F were all significantly lower (P < 0.05) in acute blood stasis syndrome rats.

A sensitive ultrafast liquid chromatography coupled with triple quadrupole mass spectrometric (UFLC–MS/MS) method for the quantification of anhydrosafflor yellow B (AHSYB), a major active water-soluble pigment from Carthamus tinctorius, in rat plasma has been developed and validated. Sample preparation was achieved by protein precipitation of plasma with four volumes of methanol. Rutin was used as the internal standard (IS). The analytes were separated using a C18 column with an 8 min gradient elution, followed by mass spectrometric detection using negative electrospray ionization (ESI−) in multiple reaction monitoring (MRM) mode. The method was linear in the concentration range of 25–10,000 ng/mL for AHSYB. Intra-day and inter-day precision variation was less than 6.5%. The relative error of accuracy was within ±9.4%. The mean recovery of AHSYB was higher than 70.9%. The established method was successfully applied to the pharmacokinetic study after intravenous (2.5 mg/kg) and oral (30 mg/kg) dosing of AHSYB in normal rats. And the pharmacokinetic properties of AHSYB in rats with acute blood stasis and the differences between normal and acute blood stasis syndrome rats were also investigated. The results showed that the compound was poorly absorbed (∼0.3%) and the AUC0–t, AUC0–∞ and F were all significantly lower (P < 0.05) in acute blood stasis syndrome rats, suggesting that disease condition may alter the body metabolism by enhancing metabolite enzyme activity.