HPLC-MS/MS Determination of Estriol in Canine Plasma

Estriol (E₃) in canine plasma sample (500 μL) with 50 μL of 20.0 μg·L^-1 chloramphenicol -C₃HOH solution added as internal standard, was extracted twice with 1.00 mL of methyl tert-butyl ether (MTBE) in each extraction. After mixing well for 2 min, the mixture was centrifuged for 10 min. The supernatants from each extraction were collected, combined and evaporated to dryness by N₂-blowing. 100 μL methanol was added to dissolve the residue, and the solution obtained was centrifuged for 10 min. An aliquot of 10.0 μL of the supernatant was taken and introduced into the instrument for HPLC separation on Agilent XDB-C₁₈ column, with mixtures of acetonitrile (A) and water (B) in various ratios as mobile phase for gradient elution. In MS/MS analysis, ESI source with negative ion scanning mode and MRM were adopted. Linearity range for E₃ was found between 0.2 to 40.0 μg·L^-1, with value of lower limit of determination (10S/N) of 0.2 μg·L^-1. Tests for recovery and precision were made by adding E₃ standard solutions to blank canine plasma sample and analyzed by the proposed method. Values of recovery (inter-day) found were in the range from 93.3% to 110%, and values of RSDs (n=5) found were in the ranges of 1.6% to 3.1% (intra-day) and 4.2% to 5.9% (inter-day).