A simple and sensitive method for determination of vitamins D3 and K1 in rat plasma: application for an in vivo pharmacokinetic study.
OBJECTIVE
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 Rats were administered intravenously by 0.1 mg/kg of vitamin D3 or K1 and the blood samples were withdrawn pre-administration and at pre-determined time points post-administration.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: AUC0-∞ = 11323 ± 1081 h × ng/mL, Vd = 218 ± 80 mL/kg, CL = 8.9 ± 0.8 mL/h/kg, t1/2 = 16.8 ± 5 h; and of vitamin K1: AUC0-∞ = 2495 ± 297 h × ng/mL, Vd = 60 ±24 mL/kg, CL = 40.5 ± 5.1 mL/h/kg, t1/2 = 1.1 ±0.5 h.


CONCLUSIONS
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.
Faculty of Pharmaceutical Sciences, University of British Columbia , Vancouver, BC , Canada .
Pavel
P
Gershkovich
Fady
F
Ibrahim
Olena
O
Sivak
Jerald W
JW
Darlington
Kishor M
KM
Wasan
D000818:Animals; D019540:Area Under Curve; D002138:Calibration; D002762:Cholecalciferol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006207:Half-Life; D057230:Limit of Detection; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D010837:Vitamin K 1 / Q000097:blood*
D002762:Cholecalciferol; D010837:Vitamin K 1