Analysis of Ochratoxin A in Coffee

During the last decade the analytical methods for ochratoxin A have been considerably improved with the use of immunoaffinity columns and high-performance liquid chromatography [359]. The first report (SCOOP-1) of the Scientific cooperation on questions relating to food (SCOOP), which was initiated by the European Commission (EC) in 1995, stated that 19% of the commodities on the European market were contaminated with OTA [360]. After initiation of a second SCOOP task in 1999, the SCOOP-2 report showed that 49 % of the commodities were contaminated with OTA, which is the consequence of the improvement of the analytical methods used [361].

3.2.1 Sampling and Sample Preparation

Since fungal contamination of raw materials, such as cereals, coffee and fruits, occurs randomly, it is very difficult to obtain representative samples, particularly in the case of cereals, since these are handled in bulk amounts [362]. Based on statistical sampling plans, multiple sub-samples are taken and pooled. Thereafter, this single sample has to be thoroughly ground to a fine powder (coffee, cereals) or minced (dried fruits) and slurred prior to analysis [363, 364]. In contrast, sampling of retail products is less problematic, since potentially contaminated commodities are already mixed and homogenised during food processing. But again, large numbers of individual samples need to be bulked together.

Therefore, sampling that encompasses not only the choice of sample size and source but also sample extraction techniques and subsequent clean-up are of fundamental importance and determine length of the analytical procedure, accuracy, recovery and achievable detection

limits. Moreover, a wrong sampling plan can greatly affect the reliability of the measured levels of mycotoxin and can, in turn, even result in legal disputes and trade restrictions.

Miraglia et al. [368] have given a holistic view of sampling for mycotoxins in general, assuming that given considerations could be adapted to OTA in most cases. Therein, primary sampling schemes (‘why, where and when’ to collect samples), purposes of sample collection (monitoring, surveillance and targeted sampling) as well as secondary sampling schemes (‘how’ to collect samples) are discussed. In addition, Vargas et al. [369] reported on the design of a sampling plan for OTA detection in green coffee.

Replacing conventional liquid-liquid extraction (LLE) techniques, preparation of samples for subsequent analysis is achieved by several efficient clean-up and preconcentration procedures based on immunoaffinity columns (IAC) and solid-phase extraction (SPE), typically using underivatised silica, C8, C18 and CN stationary phases [325, 358]. In fact, underlying interactions in SPE are relatively unselective and the resulting clean-up levels might be insufficient for some challenging matrices. Therefore, sample clean-up and enrichment with immobilised antibodies that exclusively retain OTA became increasingly popular for samples that can be directly loaded on IAC, including coffee, beer and wine [365, 366]. Application of IACs results in the production of cleaner extracts with a minimum level of interfering matrix components and excellent signal-to-noise ratios compared to less selective SPE sorbent materials [365, 367]. However, Castegnaro et al. investigated the advantages and drawbacks of IAC in mycotoxin analyses and found OTA to be underestimated in more complex matrices such as breakfast cereals and coffee [368]. Indeed, roasted coffee tends to be the most problematic foodstuff regarding OTA analysis, and in some protocols an additional clean-up step is included prior to the affinity column step [369].

3.2.2 Analytical Methods

Several methods for the determination of OTA in foods have been established and make use of the strong native fluorescence of this mycotoxin. The methods include spectrofluorometry [370], thin-layer chromatography (TLC) [371-373] and high-performance liquid chromatography (HPLC) [325, 358, 374-378]. HPLC is nowadays the preferred routine analysis technique for ochratoxins and their metabolites, since it offers better selectivity and sensitivity compared to other detection methods [30]. Positive results are sometimes confirmed by methylation of OTA and a second HPLC experiment [65]. Due to its robustness

and its easy and cost-effective handling TLC in combination with fluorescence detection is still routinely used in countries outside North America and Europe. Moreover, the official OTA analysis method of the Association of Official Analytical Chemists (AOAC) in green coffee beans is based on TLC for separation [379]. Mono-dimensional TLC has two main sources of error; the supposed mycotoxin spot might be a co-extracted impurity or the amount of mycotoxin present might be incorrectly assessed because of background interference. Both errors could be overcome by use of two-dimensional TLC [372]. Nevertheless this method is inapplicable to roasted coffee beans and coffee products since the detection in coffee products requires a much higher selectivity and sensitivity, which could be achieved by the establishment of HPLC for the determination of OTA in coffee beans and coffee products [380]. Most HPLC methods use a reversed-phase column and an acidic mobile phase, so that the carboxyl group of the toxin is in the undissociated form. Post-column addition of a 10 % ammonia solution may increase the fluorescence emission of OTA ten-fold at alkaline pH [381]. Recently, Fujii et al. reported on an indirect competitive and monoclonal antibody-based ELISA (ic-ELISA) for OTA-screening in green coffee that does not need a clean-up or concentration step but results in a fourfold increased detection limit as compared to HPLC [382]. New and innovative approaches based on immunochemistry and surface plasmon resonance, that are capable of simultaneous detection up to four mycotoxins, have been developed [383]. Additional sensitive methods for OTA detection based on mass spectrometry in combination with liquid chromatography (LC-MS) as well as capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) have been developed [384].

Results obtained therewith are comparable with those obtained by using a conventional LC-fluorescence (LC-FD) method. Becker et al. and Lau et al. reported on the development of a LC-ESI-MS/MS method for OTA detection in several foods and feeds, which turned out to be especially helpful to confirm doubtful ‘OTA positive’ results obtained by LC-FD [385, 386].

The problem of coelution of interfering compounds could be overcome by the structural information provided by tandem mass spectrometry. Recently, Zöllner et al. reviewed the application of LC-API-MS in the analysis of frequently occurring and highly toxic mycotoxins [30]. There are additional official and validated methods available for OTA analysis in several food matrices besides coffee, that have been recently reviewed by Visonti et al. [387].