title: Determination and control of TEMPO, a potentially genotoxic free radical reagent used in the synthesis of filibuvir.
abstract: The synthesis of filibuvir, a hepatitis C virus polymerase inhibitor candidate, involves use of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), a potentially genotoxic free radical reagent. A headspace gas chromatographic method utilizing selected-ion monitoring (SIM) mode mass spectrometric detection was developed, validated and applied to the determination of low levels of TEMPO in filibuvir. The GC-MS method was validated in terms of specificity, linearity, precision, accuracy/recovery, limit of quantitation (LOQ) and limit of detection (LOD). The method was shown to be specific for detection of TEMPO in the presence of filibuvir and exhibited acceptable linearity (r ≥ 0.997) over the range of 4-60 ppm vs. filibuvir (0.4-6.0 μg/mL). The system precision was 14% and 8% relative standard deviation (RSD) at the 4 ppm and 8 ppm levels, respectively. Method repeatability was 15% and 13% RSD at the 4 ppm and 8 ppm levels, respectively. Recovery was approximately 50-80% across the method range. Accuracy was 135% and 91% vs. nominal at the 4 and 8 ppm levels, respectively. The LOQ and LOD are 4 ppm and 2 ppm, respectively. Thirteen batches of filibuvir drug substance had no detectable TEMPO (≤ 2 ppm). Purge studies demonstrated that the synthetic process has an extremely high capability to remove TEMPO and consistently delivers filibuvir drug substance with TEMPO levels well below the staged threshold of toxicological concern.
authors.affiliation: Analytical Research & Development, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA.
authors.forename: Holly E
authors.initials: HE
authors.lastname: Strohmeyer
authors.forename: Gregory W
authors.initials: GW
authors.lastname: Sluggett
mesh_terms: D000998:Antiviral Agents / Q000138:chemical synthesis*; D003497:Cyclic N-Oxides / Q000032:analysis* / Q000737:chemistry; D005609:Free Radicals; D008401:Gas Chromatography-Mass Spectrometry; D007202:Indicators and Reagents / Q000737:chemistry*; D057230:Limit of Detection; D009153:Mutagens / Q000032:analysis / Q000737:chemistry; D011753:Pyrones / Q000138:chemical synthesis*; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors; D014230:Triazoles / Q000138:chemical synthesis*; D011743:Pyrimidines
chemical_list: D000998:Antiviral Agents; D003497:Cyclic N-Oxides; D005609:Free Radicals; D007202:Indicators and Reagents; D009153:Mutagens; D011753:Pyrones; D012324:RNA-Dependent RNA Polymerase; D014230:Triazoles; C550357:filibuvir; D011743:Pyrimidines; C003959:TEMPO