Optimization and Validation of a Method Without Alkaline Clean-Up for Patulin Analysis on Apple Puree Agar Medium (APAM) and Apple Products

A sensitive high-performance liquid chromatography-UV (HPLC-UV) method, based on the Association of Analytical Communities (AOAC) Official method 2000.02, was developed and validated for the high-throughput analysis of patulin in in vitro experiments on apple puree agar medium (APAM). The importance of repeating the ethyl acetate extraction step at liquid-liquid extraction (LLE) was examined, as well as the extent of patulin degradation during the sodium carbonate clean-up. In addition to this alkaline clean-up, the efficiency of using an Oasis HLB or C-18 cartridge as solid-phase extraction (SPE) clean-up was compared. This resulted in a two-step ethyl acetate LLE, followed by an Oasis HLB SPE clean-up, without alkaline clean-up conditions. The method was fully validated for APAM, cloudy apple juice, and apple puree. Average patulin recoveries at levels of 100, 500, and 1000 mu g kg(-1) of APAM varied between 95 and 113 % over 3 independent days, with an interday precision (RSDR) of 5 to 10 %. Recovery experiments carried out with the spiked apple juice (at 50 mu g kg(-1)) and apple puree (10 mu g kg(-1)) showed average recovery rates laying between 80-101 % (RSDR = 12 %) and 77-100 % (RSDR = 9 %), respectively. This method offered a detection limit of 3-4 mu g kg(-1) and a quantification limit of 5-8 mu g kg(-1) for APAM, apple juice, and puree.