Quantitative determination of risedronate in urine by spe-lc-ms-ms

Abstract

The present invention is directed to a SPE-LC-MS-MS method for quantitatively determining risedronate in a urine sample.

1 . A method for quantitatively determining risedronate in a urine sample comprising:
 a) adding an internal standard solution to the urine sample;   b) applying the urine sample to an Oasis HLB cartridge, wherein the cartridge has been pre-conditioned with methanol;   c) washing the cartridge with about 1% (v/v) TEA in water and about 1% (v/v) formic acid in methanol;   d) eluting risedronate, at least once, with a mixture of about 60% (v/v) methanol and about 40% (v/v) water containing about 3 mM EDTA under vacuum;   e) evaporating the eluted solution and reconstituting with a mixture of 10% (v/v) methanol and 90% (v/v) 0.05 M NH 4 Ac—NH 4 OH buffer to provide a sample mixture of risedronate and the internal standard; and   f) analyzing the sample mixture with a LC-MS/MS system.   
     
     
         2 . The method according to  claim 1 , wherein the urine sample is a mouse or rat urine sample. 
     
     
         3 . The method according to  claim 1 , wherein the internal standard is deoxy-risedronate. 
     
     
         4 . The method according to  claim 1 , wherein the internal standard is deoxy-risedronate that is added as a solution in water. 
     
     
         5 . The method according to  claim 1 , wherein the internal standard is deoxy-risedronate that is added as an about 10 μg/mL solution in water. 
     
     
         6 . The method according to  claim 1 , wherein the Oasis HLB cartridge is a 30 mg Oasis HLB cartridge. 
     
     
         7 . The method according to  claim 1 , wherein the amount of the urine sample is about 0.4 mL. 
     
     
         8 . The method according to  claim 1 , wherein the cartridge has been pre-conditioned with about 1 mL of methanol. 
     
     
         9 . The method according to  claim 1 , 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. 
     
     
         10 . The method according to  claim 1 , wherein the cartridge is washed with about 0.5 mL of about 1% (v/v) TEA in water and about 0.5 mL of about 1% (v/v) formic acid in methanol. 
     
     
         11 . The method according to  claim 1 , wherein the amount of the mixture of about 60% (v/v) methanol and about 40% (v/v) water containing about 3 mM EDTA is about 1.5 mL. 
     
     
         12 . The method according to  claim 1 , wherein risedronate is eluted twice with about 0.75 mL of the mixture of about 60% (v/v) methanol and about 40% (v/v) water containing about 3 mM EDTA. 
     
     
         13 . The method according to  claim 1 , wherein the amount of the mixture of 10% (v/v) methanol and 90% (v/v) 0.05 M NH 4 Ac—NH 4 OH buffer is about 100 μL. 
     
     
         14 . The method according to  claim 1 , wherein the internal standard is deoxy-risedronate, and analyzing the sample mixture with a LC-MS/MS system comprises separating risedroante and deoxy-risedronate 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.