A simple and sensitive method for determination of vitamins D₃ and K₁ in rat plasma: application for an in vivo pharmacokinetic study

Abstract

Purpose: To develop and to validate a simple but sensitive method for determination of vitamins D₃ and K₁ in rat plasma.

Methods: The sample treatment included protein precipitation by cold acetonitrile, evaporation, reconstitution with methanol and filtration. The chromatography conditions included Xterra RP18 3.5 µm 4.6 × 100 mm column at ambient temperature and mobile phase consisting of methanol/water (93/7, v/v) at 0.5 mL/min flow rate. Vitamin D₃ and probucol were detected at 265 nm and vitamin K₁ at 239 nm. Rats were administered intravenously by 0.1 mg/kg of vitamin D₃ or K₁ and the blood samples were withdrawn pre-administration and at pre-determined time points post-administration. The pharmacokinetic analysis was performed using a non-compartmental approach.

Results: The calibration curves in rat plasma were linear up to 5000 ng/mL for both vitamins. The limit of quantification (LOQ) was 20 ng/mL for vitamin D₃ and 40 ng/mL for K₁. Inter- and intra-day precision and accuracy were below 15%. The pharmacokinetic parameters of vitamin D₃ following intravenous administration were: AUC_0−∞ = 11323 ± 1081 h × ng/mL, V_d = 218 ± 80 mL/kg, CL = 8.9 ± 0.8 mL/h/kg, t_1/2 = 16.8 ± 5 h; and of vitamin K₁: AUC_0−∞ = 2495 ± 297 h × ng/mL, V_d = 60 ±24 mL/kg, CL = 40.5 ± 5.1 mL/h/kg, t_1/2 = 1.1 ±0.5 h.

Conclusion: The developed HPLC–UV assay is a simple and sensitive method for the determination of vitamins D₃ and K₁ in rat plasma. A higher dose of vitamin K₁ should be used in future studies for accurate estimation of pharmacokinetic parameters. The data show the suitability of the assay for pharmacokinetic studies in rats.

• Bioavailability
• drug interactions
• HPLC
• intestinal absorption
• lipid-soluble vitamins