Novel HPLC and Solid-Phase Extraction Methods for Quantitating Methadone and its Metabolite in Human Urine

Library Number:
Yung-Fong Cheng;Robert Bonin;Ziling Lu;Uwe Neue;Laura Woods;Pamela Iraneta;Edouard Bouvier;Dorothy Phillips
HPLC''99, Granada, Spain, June 2, 1999
Content Type:
Content Subtype:
SPE Format:
Oasis HLB 30 mg 1 cc Cartridge Oasis HLB 10 mg 96-well Plate Oasis MCX 60 mg 3 cc Vac Cartridge
Methadone; EDDP [metabolite]; Diphenhydramine [IS]
Plasma; Urine
Analytical Techniques:
SymmetryShield(™) RP18 5 µm Steel 3.9 mm 150 mm SymmetryShield(™) RP18 3.5 µm Steel 3.9 mm 150 mm SymmetryShield(™) RP18 5 µm Sentry Guard N/A N/A
ABSTRACT: A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200µL) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrices were greater than 91% with RSDs less than 4.0%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP18 column with a mobile phase of 0.1% TFA/methanol at 60:40. Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signall-to-noise enhancement was achieved by diluting the final SPE eluates four-fold with water.

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