Validated LC-MS/MS method for the determination of maackiain and its sulfate and glucuronide in blood: application to pharmacokinetic and disposition studies

Abstract

The purpose of this study was to develop a simultaneous, sensitive and reproducible UPLC-MS/MS method to quantify maackiain and its phase II metabolites, maackiain-sulfate (M-7-S) and maackiain-glucuronide (M-7-G). A Waters BEH C18 column was used with acetonitrile/water as mobile phases. Analysis was performed under negative ionization electrospray mass spectrometer via the multiple reaction monitoring (MRM). The one-step protein precipitation by methanol was used to extract the analytes from plasma. The results showed that the linear response range was 5000-9.75 nM for maackiain, M-7-S, and M-7-G. The lower limit of detection (LLOD) was 4.88 nM for these three analytes. The intra-day variance is less than 12.4% and accuracy is in 85.7-102.0%. The inter-day variance is less than 11.2% and accuracy is in 89.6-122.2%. The analysis was done within 4.0 min. Only 20 μl of blood is needed for the analysis due to the high sensitivity of this method. The validated method was used for pharmacokinetic study in A/J mouse, maackiain Caco-2 cell culture model experiment, and maackiain glucuronidation/sulfation metabolism studies. The applications revealed that this method can be used for maackiain, M-7-S, and M-7-G analysis in both bioequivalent buffer and in blood.

Copyright © 2011 Elsevier B.V. All rights reserved.

Figures

Figure 1

Chemical structures of maackiain, M-7-S, and M-7-G

Figure 2

The chromatograms of maackiain, M-7- G, M-7-S, and formononetin (IS) in a mouse blood sample. The retention time of M-7-S, M-7-G, IS, and maackiain were 1.37, 1.67, 2.32, and 2.68 min respectively. The molecular weight these four compounds were m/z 364, 460, 268, and 284.

Figure 3

The MS/MS chromatograms for M-7-G (a) and M-7-S (b). The results showed the molecular ion 459 [M-H]− and its MS2 fragment at 283 [M-H-Glu]− for M-7-G and molecular ion 363 [M-H]− and its MS2 fragment at m/z 283[M- H-Sul]− for M-7-S.

Figure 4

Plasma concentrations of maackiain (diamond), M-7-G (square), and M-7-S (triangle) after i.v. administration of 2.5mg/kg maackiain in A/J mice (n=5). Blood sample (20 µl) was spiked into KPI (20 µl), and IS (80 µl, formononetin in methanol, 1 µM). The mixture was vortexed and centrifuged for 15 min at 15,000rpm. The supernatant was taken out and reconstituted in 80 µl of 50 % methanol and centrifuged for another 15 min at the same speed. Then 10 µl of supernatant were injected into the UPLC-MS/MS system. Each point is average of five determinations and the error bars are standard deviations of the mean. The circled points represented data points where concentrations for some mouse blood samples fell below the LLOD.

Figure 5

Transport of maackiain in the Caco-2 cell culture model. The buffer used in both donor and receiver sides was HBSS (pH = 7.4). The donor side maackiain concentration was 10 µM for both apical to basolateral and basolateral to apical directions. The experiment was performed at 37°C. Each data point is the average of three determinations. Bars are standard deviations of the mean

Figure 6

Excretion rates of M-7-G and M-7-S in the Caco-2 cell culture model. The buffer used in both donor and receiver sides was HBSS (pH = 7.4). The donor side maackiain concentration was 10 µM for both apical to basolateral and basolateral to apical transport and metabolism studies. The experiment was performed at 37°C. Each data point is the average of three determinations, and the error bar is the standard deviation of the mean. The asterisk (*) indicates a statistically significant difference between products and control (p < 0.05, One-way ANOVA).

Figure 7

Maackiain (1µM) glucuronidation and sulfation rates in A/J mouse liver S9 fraction reaction. Each data point is the average of three determinations, and the error bar is the standard deviation of the mean.