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Ultra sensitive method for the determination of 9-(2-phosphonylmethoxyethyl)adenine in human serum by liquid chromatography-tandem mass spectrometry.

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Drug Development Department, Ribapharm Inc., 3300 Hyland Avenue, Costa Mesa, CA 92626, USA.


An ultra sensitive method for the direct measurement of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), an antiviral agent for hepatitis B, in human serum using high performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed. This method involves the addition of [13C]PMEA (contains 5 13C) as internal standard, the purification and enrichment by a MCX solid phase extraction (SPE) cartridge, and quantitative analysis using LC-MS/MS. The MS/MS is selected to monitor the m/z 272 --> 134 and m/z 277 --> m/z 139 transitions for PMEA and [13C]PMEA, respectively, using negative electrospray ionization. The MS/MS response is linear over a concentration of 0.1-10 ng/ml with a lower limit of quantitation (LLOQ) of 0.1 ng/ml. The mean inter-assay accuracy (%Bias) for quality control (QC) at 0.1, 0.25, 1.0, and 10 ng/ml are 10, 1.6, -0.8, and 0.0%, respectively. The mean inter-assay precision (%CV) for the corresponding QCs is 3.9, 3.8, 5.3, and 3.4%, respectively. The method has been used to determine PMEA concentration in human serum following a single oral administration of a PMEA pro-drug at dose of 10 and 30 mg.

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