Patent ID: 8168437

Claim:
A method for quantitatively determining risedronate in a mouse or rat urine sample, comprising: a) adding about 100 μL of about 10 μg/mL dexoy-risedronate solution in water to about 0.4 mL of the mouse or rat urine sample; b) applying the mouse or rat urine sample to a 30 mg Oasis® HLB cartridge, wherein the cartridge has been pre-conditioned with about 1 mL of methanol and followed by about 0.5 mL of 1% (v/v) TEA in water; c) washing the cartridge with about 0.5 mL of 1% (v/v) TEA in water and about 0.5 mL of 1% (v/v) formic acid in methanol; d) eluting risedronate with about 1.5 mL of a mixture of about 60% (v/v) methanol and about 40% (v/v) water containing about 3 mM EDTA; e) evaporating the eluted solution and reconstituting with about 100 μL of a mixture of about 10% (v/v) of methanol and about 90% (v/v) of about 0.05 M NH 4 Ac—NH 4 OH buffer to provide a sample mixture of risedronate and dexoy-risedronate; and f) analyzing the sample mixture with a LC-MS/MS system, wherein risedronate and dexoy-risedronate are separated by HPLC on an Inertsil Phenyl-3 HPLC column by gradient elution with a mixture of solution A and solution B, wherein the amount of solution A is increased from about 10% (v/v) to about 95% (v/v), and wherein the solution A is about 90% (v/v) methanol in water, and solution B is about 10 mM ammonium acetate-acetic acid buffer with 2% (v/v) triethylamine.