Liquid Chromatography LC

HPLC methods are the most commonly employed methods for the quanti-tation of patulin in fruit juices. HPLC requires a large initial cash investment, but provides good sensitivity, precision, and ease of use. In addition, a skilled and experienced staff is required to operate and maintain the HPLC equipment.

Almost all published methods involve liquid-liquid extraction of patulin into ethyl acetate, a cleanup step using a sodium carbonate solution to remove interfering phenolic compounds, and HPLC with UV detection to separate and detect patulin [25,26]. HPLC columns typically are reversed phase (C18), and the mobile phases tend to be predominately mixtures of water and acetonitrile (up to 10% v/v) or tetrahydrofuran (up to 5% v/v). Although patulin can be detected with single-wavelength UV detectors (276 nm), many laboratories use photodiode array (PDA) detectors to detect patulin and spectrally distinguish the compound from coextracted compounds such as polyphenols and hydroxymethyl furfural (HMF).

The HPLC procedure described by Brause et al. [26] was subjected to an interlaboratory study on method reproducibility and accuracy. In this collaborative study, 22 laboratories analyzed apple juice spiked with 20 to

200 mg patulin/l as well as naturally contaminated juice containing 31 mg patulin/l [25,26]. Mean recovery of patulin spiked into juice was 96%. Based on the results of this collaborative study, the method was adopted as a first action method by AOAC International (AOAC Official Method 995.10) [27]. A second AOAC official method for determination of patulin by HPLC describes the analysis of clear and cloudy apple juices and apple purees. The method (AOAC Official Method 2000.02) [28] is based on a publication by MacDonald et al. [29]. This method differs from that of Brause et al. [26] in the use of pectinase prior to extraction to remove the cloudiness present in some juice samples.

Solid phase extraction (SPE) methods recently have been developed for extracting and purifying patulin from apple juice. In the method developed by Trucksess and Tang [30], patulin was extracted from undiluted apple juice with a reversed-phase SPE (Oasis, Waters, Milford, MA) column. The column was washed to remove interfering compounds, and patulin was eluted and then detected by HPLC with a recovery of 93 to 104% [30]. Recently, Eisle and Gibson [31] modified the method of Trucksess and Tang [30] and reduced analysis time to approximately 1 hour including extraction and HPLC analysis steps.

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