Analytical HPLC method validation for C-11 palmitic acid

Objectives C-11 Palmitic acid (PA) is a fatty acid metabolism imaging agent. We describe herein a method for validation of an analytical HPLC procedure following FDA guidance. Both UV and radio-detectors were validated for linearity, accuracy, precision, range, quantitation limit (QL) and detection limit(DL).

Methods : 1) UV detector: HPLC, Phenomenex Luna 3µ C-8(2) 100A column 4.6mm X 150mm, MeCN/0.1%TFA water (90/10), 1.0 mL/min, 10µL loop, UV 215nm. A series of reference standards in methanol (1.0 µg/mL-1000.0 µg/mL) were prepared for testing. 2) Radio-detector: Flow Count, Range 200K cps. A series of radioactive concentrations of F18 fluoride in water (76 µCi/mL-66.7 mCi/mL@injection) were prepared for testing.

Results 1) UV detector: R2 = 0.9999. Recovery for 25.0, 250.0 and 1000.0 µg/mL standard concentration are 102.12%, 98.30% and 100.31% respectively. Precision is shown as coefficient of variation (COV), and is < 3.85%. Accuracy, precision and linearity data for PA are acceptable in the range of 25.0 and 1000.0 µg/mL. The QL is 25.0 µg/mL and DL is 5.0 µg/mL. 2) Radio-detector: R2 = 0.9992 for 1.92 to 53.4 mCi/mL @injection. The recovery for 1.92, 24.73 and 53.4 mCi/mL standards were 97.7%, 100.48% and 100.40% respectively. The COV is 1.38% from different time injections of 9.57 mCi/mL standard; The radioactive concentration range between 1.92-53.4 mCi/mL is acceptable. QL and DL are 1.107 and 0.332 mCi/mL respectively.

Conclusions We have validated the HPLC procedure for chemical and radiochemical purity for C-11 PA. PA is not water soluble and it has very low response to UV or refractive index detectors. The QL is too high to accurately determine the mass of PA and therefore specific activity, but it can be used for radiochemical identity confirmation.