Determination of Paeoniflorin in Rat Plasma by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry and its Application to a Pharmacokinetic Study.
In this study, a rapid and highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was established for the quantification of paeoniflorin in rat plasma. The analyte and the internal standard (IS), hyperoside, were extracted from rat plasma via liquid-liquid extraction with ethyl acetate. LC separation was carried on a Phenomenex Luna C18 column within 2.5 min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization and operated in the multiple reaction monitoring and negative ion modes. The precursor to product ion transitions monitored for paeoniflorin and the IS were m/z 524.8→449.0 and 463.1→300.0, respectively. The assay was validated with a linear range of 0.02~25.6 μg/mL for paeoniflorin. The intra- and inter-day precisions (relative standard deviation%) were within 9.7%, and the recoveries were greater than 65.2%. Paeoniflorin was proven to be stable during all sample storage, preparation and analytical procedures. The method was successfully applied to a pharmacokinetic study of the Baixiangdan Capsule in eight female rats.
Pharmacokinetic study of multiple active constituents after oral gavage of Guizhi decoction in rats using a LC–MS/MS method
Guizhi decoction (GZD) is a classic traditional Chinese medicine formula, clinically used for the treatment of influenza, common cold, and other pyretic conditions. A sensitive, specific, and validated liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed to investigate the pharmacokinetic properties of cinnamic acid, hippuric acid, paeoniflorin, and glycyrrhetic acid in rat. After single dose oral administration of 7.9 g extract/kg body weight GZD in rats, plasma concentrations of cinnamic acid, hippuric acid, paeoniflorin, and glycyrrhetic acid were measured by LC–MS/MS. Pharmacokinetic parameters were calculated from the plasma concentration–time data. The values of AUC0−t, half-life (t1/2), and Cmax were 7.2 ± 2.3 μg h/mL, 1.2 ± 0.3 h, and 9.2 ± 5.2 μg/mL for cinnamic acid, 53 ± 31 μg h/mL, 2.8 ± 2.0 h, and 17 ± 3 μg/mL for hippuric acid, 1.1 ± 0.5 μg h/mL, 1.9 ± 1.1 h, and 0.6 ± 0.3 μg/mL for paeoniflorin, and 11 ± 6 μg h/mL, 6.6 ± 2.5 h, and 0.9 ± 0.6 μg/mL for glycyrrhetic acid, respectively. The results would offer useful information for effective components of GZD in vivo.
New method for analysis of enzymatic reactions: Thermolysin-catalyzed synthesis of aspartame precursor
Aspartame is an artificial sweetener which is 200 times sweeter than sucrose. A precursor of aspartame, N-(benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester (Z-APM) was synthesized and hydrolyzed with thermolysin in an aqueous monophasic system. Graphical plotting methods and/or nonlinear regression have been used to analyze enzymatic reactions because of the nonlinearity of enzymatic rate equations. There are, however, problems: the former require accurate measurement of the initial reaction rate and the latter gives no analytical information. The authors proposed a method to solve the nonlinear enzymatic rate equations with respect to the concentration of a product with the Taylor expansion, especially for the reversible Theorell-Chance mechanism (Murakami, Y., Hirata, M., and Hirata, A., J. Ferment. Bioeng., 82, 246–252, 1996). This solution, however, is complex for practical use. In this paper, a simpler solution to explain the thermolysin-catalyzed synthesis of Z-APM is obtained. New methods to determine the kinetic parameters, especially for the reversible Theorell-Chance mechanism, based on the approximated solution are proposed. The results calculated using the obtained kinetic parameters can satisfactorily explain the experimental results, which demonstrates the effectiveness of the proposed analysis. We must know the relation between the concentrations of substrates and a product, and accurately evaluate of the result obtained using Taylor expansion when we determine the kinetic parameters using our newly developed methods. The methods to determine the kinetic parameters of the reversible Theorell-Chance mechanism described in this paper is, however, expected to be applied to general enzymatic reactions.