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A new method of analysis has been developed and validated for the determination of hydromorphone in bearded dragon plasma. Following SPE, samples were separated by reversed-phase HPLC on an Atlantis T3 column (4.6 x 150 mm, 3 μm) and detected by MS. The mobile phase was a mixture of water with 0.1% formic acid, and acetonitrile with 0.1% formic acid (91:9), with a flow rate of 0.7 mL/min. The procedure produced a linear curve over the concentration range of 1 to 500 ng/mL with an LOQ of 1 ng/mL and LOD of 0.5 ng/mL. Intra- and inter-assay variability ranged from 3.9% - 9.3% and the average recovery for hydromorphone was 91%. The average recovery for naltrexone, which was the internal standard, was 90%. This method has been developed in bearded dragon plasma and should be applicable to other species, it should also be useful to those investigators dealing with small volumes, particularly when conducting PK studies that require multiple sampling from the same animal.

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