Validation of a reversed-phase high performance liquid chromatography (RP-HPLC) method for analysis of ¹¹C-Nicotine injectable

Objectives: The objective of the present study was to validate a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method for determination of radiochemical purity, chemical purity, and specific activity of ¹¹C-Nicotine Injectable.

Methods: The method involved a model 1100 Agilent HPLC system interfaced with a UV/Vis and a radioisotope detectors, a 150 x 4.6 mm, 5 µm XTerra C18 column at 40°C, and an isocratic mobile phase containing 80:20 mixture of 6.5 mM ammonium acetate pH 10 and acetonitrile at a flow rate of 1.0 mL/min. The method was validated for specificity, linearity, precision, robustness, the Limit of Detection (LOD), and the Limit of Quantitation (LOQ).

Results: The HPLC analytical method was specific with excellent resolution of ¹¹CH₃I, (+) Nornicotine, and ¹¹C-Nicotine peaks, i.e., 1.5, 3.1, and 5.5 minutes retention times, respectively; linear (R² > 0.99) for detection of (+) Nornicotine, Nicotine and ¹¹CH₃I and ¹¹C-Nicotine using a UV/Vis and a radioisotope detectors, respectively; and had acceptable precision (within USP limits of < 2.0% RSD for five injections). The estimated LOD and the LOQ were 1 and 3.3 ng for (+) Nornicotine and 2 and 6.6 ng for Nicotine. The robustness of the method was tested by the impact of small but deliberate variation of various method parameters including, column temperature, mobile phase composition and pH, flow rate, and wavelength.

Conclusion: An RP-HPLC method for analysis of ¹¹C-Nicotine injectable was developed and validated successfully. Research Support: