Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC-MS/MS method

Abstract

The purpose of this research was to develop a sensitive and reproducible UPLC-MS/MS method to analyze matrine, an anticancer compound, and to use it to investigate its biopharmaceutical and pharmacokinetic behaviors in rats. A sensitive and fast UPLC-MS/MS method was successfully applied to determine matrine in rat plasma, intestinal perfusate, bile, microsomes, and cell incubation media. The absolute oral bioavailability of matrine is 17.1+/-5.4% at a dose of 2mg/kg matrine. Matrine at 10microM was shown to have good permeability (42.5x10(-6)cm/s) across the Caco-2 cell monolayer, and the ratio of P(A-B) to P(B-A) was approximately equal to 1 at two different concentrations (1 and 10microM). Perfusion study showed that matrine displayed significant differences (P<0.05) in permeability at different intestinal regions. The rank order of permeability was ileum (highest, P(w)=6.18), followed by colon (P(w)=2.07), duodenum (P(w)=0.61) and jejunum (P(w)=0.52). Rat liver microsome studies showed that CYP and UGTs were not involved in matrine metabolism. In conclusion, a sensitive and reliable method capable of measuring matrine in a variety of matrixes was developed and successfully used to determine absolute oral bioavailability of matrine in rats, transport across Caco-2 cell monolayers, absorption in rat intestine, and metabolism in rat liver microsomes.

Copyright 2009 Elsevier B.V. All rights reserved.

Figures

Fig. 1

The structure of matrine.

Fig. 2

Sample chromatograms of different analytes in current UPLC system with MS/MS detection. (a) A blank plasma sample spiked with matrine (2500 ng/ml) and testosterone (500 ng/ml). (b) A blank microsome sample spiked with matrine (10 μM) and rutin (0.5 μM). (c) A blank microsome sample spiked with rutin (0.5 μM), 6β-hydroxytestosterone (1 μM) and testosterone (0.1 μM). (d) A blank microsome sample spiked with rutin (0.5 μM), genistein 7-O-glucuronide (1 μM) and genistein (0.1 μM).

Fig. 3

Plasma concentration–time curves of matrine after i.v. and p.o. administration of matrine (2 mg/kg). Each point represents an average of four determinations and the error bars are standard deviations of the mean. The point in the circle represents the average concentration, which is an approximate value since not all samples contained quantifiable matrine.

Fig. 4

Effect of different pH on transport of matrine across Caco-2 cell monolayer. Each data point is the average of three determinations and the error bar is the standard deviation of the mean.

Fig. 5

Amounts of matrine absorbed in a four-site rat intestinal perfusion model (n = 3). Each bar is the average of three determinations and the error bar is the standard deviation of the mean.

Fig. 6

Concentrations of matrine in bile sample in a four-site rat intestinal perfusion model (n = 3). The average volume of bile is 0.5 ml every 30 min, which means that at steady state conditione, a bile concentration of 1 μM will be equivalent to 0.5 nmol/30 min.

Fig. 7

Chromatograms of various CYP and UGT reaction substrates before and after incubation with rat liver microsomes for 1 h. The detector used was MS/MS and the methods were described in details in the Method section. (a) MRM chromatograms of testosterone after 1 h incubation in a CYP reaction system lacking NADPH (negative control); (b) MRM chromatograms of testosterone after 1 h incubation in a CYP reaction system; (c) MRM chromatograms of matrine after 1 h incubation in a CYP reaction system lacking NADPH (negative control); (d) MRM chromatograms of matrine after 1 h incubation in a CYP reaction system; (e) MRM chromatograms of genistein after 1 h incubation in a UGT reaction system lacking UDP-GA (negative control); (f) MRM chromatograms of genistein after 1 h incubation in a UGT reaction system; (g) MRM chromatograms of matrine after 1 h incubation in a UGT reaction system lacking UDP-GA (negative control); (h) MRM chromatograms of matrine after 1 h incubation in a UGT reaction system.