Quantification of annonacin in Rat plasma: Application to pharmacokinetic study
Annonaceous acetogenins  are neurotoxic molecules distributed in the Annonaceae family. They have been found in several parts of these plants, including worldwide consumed fruits from Annona muricata. A link between its consumption and a high prevalence of atypical parkinsonism has been established by epidemiological studies . The most abundant acetogenin in A. muricata is annonacin; hence it has become a main concern to evaluate its bioavailability. We developed a quantification method for annonacin in rat plasma, using UPLC-ESI-TQ, with its analogue annonacinone as internal standard. We used SRM mode with a focus on 112 uma loss, which is characteristic of this type of molecules. We used an Acquity UPLC BEH C18 column, with a 5 minutes water/acetonitrile gradient. We prepared calibration standards from 0.1 ng/mL to 100 ng/mL of annonacin in rat plasma, containing 10 ng/mL of IS. Sample preparation was performed by two consecutive liquid/liquid extractions with 0.5 mL of ethylacetate, evaporation of the supernatant under stream-flow, then reconstitution in 100µL of acetonitrile. 5µL were injected in 3 replicates.
The method was applied to plasma samples collected after intravenous and oral administration on 6 rats per route, at the concentration of 0.5 mg/kg by intravenous route, and 10 mg/kg by oral route. Blood samples were collected periodically during 48 hours. At these doses, we were able to quantify annonacin in plasma until 48 and 32 hours after i.v. and oral administration, respectively. Pharmacokinetic parameters (mean ± SD) were obtained by non-compartmental analysis, using PK Solver add-in. They were determined as Cmax15 ± 8 ng.mL-1, T1/2 15 ± 3h, Vd 43 ± 17 L, Cl 2 ± 1 L.h-1, AUC0-∞ 86 ± 31h.ng.mL-1 for i.v. route, and Cmax 8 ± 4 ng.mL-1, T1/2 5 ± 2h, Vd 389 ± 158 L, Cl 55 ± 9 L.h-1, AUC0-∞ 55 ± 8h.ng.mL-1 for oral route. The bioavailability of annonacin was estimated to be 3%.
Keywords: Acetogenin, LC-MS quantitation, Rat plasma, bioavailability, Annonaceae, neurotoxin.
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