Resazurin as System Suitability Indicator for Radio-TLC Analysis of [18F] Fludeoxyglucose

1061 Objectives: Our goal was to develop an alternative method for the determination of radiochemical purity of [18F] Fludeoxyglucose by radio-TLC. Our current method specifies the use of a [19F] Fludeoxyglucose standard with p-anisidine as the radiochemical identity indicator solution. This method was previously cross-validated against the method described in the United States Pharmacopeia (USP41-NF36) monograph for Fludeoxyglucose F18 Injection. Until recently, p-anisidine was available as a prepared reagent. The discontinuation of the commercially available p-anisidine reagent necessitated the preparation of the reagent in-house. We successfully validated the in-house preparation using p-anisidine in the crystalline form solubilized in 1-butanol. P-anisidine in the solid, crystalline form is a hazardous substance with extensive PPE and special handling requirements for the preparation of the reagent. We developed an alternative to the currently used method using FDG reference standard and p-anisidine. Previously, we gained approval from the FDA for the use of resazurin as a system suitability indicator for N-13 Ammonia Injection. Based on this approval, we chose to apply a similar method to [18F]-FDG. We demonstrate that using resazurin as a system suitability indicator is a suitable replacement for the FDG reference standard with p-anisidine. Methods: A novel method was developed using resazurin as a system suitability indicator. Resazurin is a blue dye that when developed, is easily viewable, being within the visible spectrum. Resazurin is prepared from 1 mg/mL mixture of resazurin sodium salt (Sigma-Aldrich) solubilized in sterile water for injection, USP. The Radio-TLC method is performed on a stationary phase consisting of 15 x 100 mm aluminum-backed silica gel plate (EMD) with the origin marked at 15mm and the solvent front marked at 90mm. The mobile phase is 95:5 acetonitrile:water. With a pipette, a 0.2µL sample of FDG sample is placed at origin. Likewise, a 0.2µL sample of resazurin 1 mg/mL solution is also placed at the origin (Diagram A). The TLC plate is air dried for 5 minutes and then placed in the development chamber. When the solvent migrates to the solvent front mark of the TLC plate, the plate is removed from the developing chamber and dried. The resazurin standard migration is easily visualized and can be quantitated prior to Radio-TLC analysis, providing an early indication of system suitability. The TLC plate is then analyzed on a Radio-TLC scanner (LabLogic Scan-Ram or Bioscan AR-2000, both controlled with LabLogic Laura Software.) The results of the Radio-TLC chromatogram and the resazurin migration are reported. The Rf of resazurin should be approximately 0.35 to indicate a suitable test. Results: Radiochemical purity analysis was performed on [18F] Fludeoxyglucose with the newly developed method using resazurin as a system suitability indicator solution. Sample result data (See Table 1) show a mean Rf of 0.37 (range 0.35 - 0.39) compared to an average Rf of 0.43 for [18F]-FDG. On 9 recent [18F]-FDG production runs, the data show a mean Rf 0.43 for FDG Standard using p-anisidine reagent as indicator compared to an average Rf of 0.42 for [18F]-FDG. The migration of the resazurin is approximately 85% when compared to the Rf of [18F]-FDG. Though the Rf of the resazurin is not equal to that of [18F]-FDG that data show that the Rf value is a consistent and reliable marker for system suitability. Conclusions: A novel method to rapidly determine system suitability has been developed for [18F] fludeoxyglucose radio-TLC testing utilizing regents that are readily available and safe to use. As an additional benefit, this method enables the performance of test suitability prior to performance of radio-TLC analysis, which can allow for test acceptance or rejection based on resazurin system suitability result.