Validated LC-MS/MS Method for the Determination of Scopoletin in Rat Plasma and Its Application to Pharmacokinetic Studies

Abstract

A rapid, sensitive and selective liquid chromatography-electrospray ionization-tandem mass spectrometric method was developed and validated for the quantification of scopoletin in rat plasma. After the addition of the internal standard xanthotoxin, plasma samples were pretreated by a simple one-step protein precipitation with acetonitrile-methanol (2:1, v/v). Chromatographic separation was achieved on a Diamonsil ODS chromatography column using gradient elution with the mobile phase consisting of acetonitrile and 0.1% formic acid. The determination was performed by positive ion electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear over the concentration range of 5-1000 ng/mL (r = 0.9996). The intra- and inter-day precision (RSD%) was less than 6.1%, and the accuracy (RE%) was from -3.0%-2.5%. This method was successfully applied to the pharmacokinetic research of scopoletin in rats after intravenous (5 mg/kg) or oral (5, 10 and 20 mg/kg) administration. The result showed that oral bioavailability with a dose of 5 mg/kg was 6.62% ± 1.72%, 10 mg/kg, 5.59% ± 1.16%, and 20 mg/kg, 5.65% ± 0.75%.

Keywords: LC-MS/MS; pharmacokinetics; rat plasma; scopoletin.

Figure 1

Chemical structures of scopoletin and xanthotoxin internal standard.

Figure 2

Fragment ion spectra of (A) scopoletin and (B) xanthotoxin.

Figure 3

Representative multiple reaction monitoring (MRM) chromatograms of scopoletin and xanthotoxin in rat plasma: (A) a blank plasma sample; (B) a blank plasma spiked with 100 ng/mL of scopoletin and internal standard; (C) a plasma sample collected 5 min after oral administration of scopoletin; (a) scopoletin-glucuronide conjugate; (b) scopoletin-sulfate conjugate.

Figure 4

Mean scopoletin plasma concentrations over time after oral (p.o., 5, 10 and 20 mg/kg) or intravenous (i.v., 5 mg/kg) administration in rats (n = 5).