| Ochratoxin A (OTA) is the toxic metabolites mainly produced by Penicillium verrucosum, Aspergillus ochraceus and A. carbonarius. Chemically, OTA is a colorless crystal with molecular weight of 403.8 and fusion point of 169℃. It is solvable in both organic solvents (chloroform and methanol) and NaHCO3 solution, while minimally solvable in water. OTA shows green fluorescence with peak absorption wavelength at 333 nm under ultraviolet exposure. OTA is usually found in food and foodstuffs, such as cereals, grains, vegetables, spices, coffee, beer, wine and dried fruits. It may also be found in products of animal origin as a carry-over from animals fed with contaminated feed.OTA contamination of food and animal feed poses a high risk for human and animal health. This toxin is a potent nephrotogen, teratogenen and carcinogen with strong kidney and liver toxicity, and has immunosuppressive effects. OTA is suspected of being a possible cause of a chronic kidney disease known as "Balkan endemic nephropathy". Accordingly, the International Agency for Research on Cancer (IARC) has classified OTA into group 2B as a possible human carcinogen in 1993. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) and EU member states have studied OTA thoroughly, and set the maximal limitation in foodstuffs. The maximal limitation of 5μg/kg is demanded by the codex committee on food additives and contaminants (CCFAC) early in 2000. The national standard in China (GB/T 23502-2009) established determination of OTA in food by high performance liquid chromatographic method with immunoaffinity column clean-up in 2009. This standard is used to determine the concentration of OTA in food and food products, wine, soy, vinegar and sauce and sauce products. The detection limit of OTA is 1.0μg/kg in food and food products,0.1μg/kg in wine and 0.5μg/kg in soy, vinegar and sauce and sauce products. The levels of OTA in foodstuffs are regulated in several countries. Therefore, a reliable and sensitive method is necessary for the determination of OTA.Various analytical methods have been developed for the determination of OTA, including thin-layer chromatography (TLC), enzyme-linked immunosorbent assays (ELISA), capillary electrophoresis (CE), liquid chromatography with mass spectrometry (LC/MS) and high performance liquid chromatography (HPLC) coupled with fluorescence detection (FLD). Because OTA has natural fluorescence, HPLC with fluorescence detection (HPLC-FLD) is the most frequently used technique.The researches on contamination and limits of OTA usually concentrated on foodstuff, wine and feed and so on. There is little or no information in the literature on analysis of OTA in traditional Chinese medicine (TCM). In fact, the subject in this field is still blank. Due to their provenance, TCM may be contaminated by various fungal species, including ochratoxigenic fungi, during their growth, collection, transportation and storage, and will affect the quality and safety of TCM. Therefore, more and more attention will be paid in contamination of OTA in TCM. As a result, it is urgent to establish the fast and efficient method for determination of OTA in TCM in order to ensure the quality of TCM.High-performance liquid chromatography (HPLC) with fluorescence detection (FLD) method was established for the analysis of ochratoxin A in TCM in this paper. This method involved the extraction of OTA by methanol-water (80:20, v/v), immunoaffinity (IAC) clean-up and HPLC quantification with fluorescence detection (FLD). The mobile phase consisted of 0.5% acetic acid with water-acetonitrile (52:48, v/v) at a flow rate of 0.8 ml/min. The column was kept at 30℃. For the fluorescence detection of OTA, the excitation wavelength was set at 333 nm and emission wavelength at 460 nm. The method for the determination of ochratoxin A (OTA) in TCM of different origins was developed. The limit of detection (LOD) for OTA was 0.3μg/kg based on a signal-to-noise ratio of 3:1. The limit of quantification (LOQ) was determined by the signal-to-noise 10:1 and it was 0.8μg/kg. The linearity for OTA was good. The equations of the calibration curves is Y=4460.3X - 1221.4 (Y:peak area, X: concentration, r=0.9999), Three different traditional Chinese medicines, liquorice root, medicated leaven and foxtail millet sprout were selected for the recovery test, because they represent TCM of different origins and matrixes. The recoveries for three TCMs ranged from 89.33 to 99.12% and the RSD was from 1.16 to 6.65% within the spiking range of 1.0～50.0μg/kg. The results showed that the moldy samples were more easily contaminated with OTA than the normal samples. But OTA levels and degree of moldiness were not correlated in most cases. Among total 57 TCM samples collected from six different places (Beijing, Jiangxi, Xinjiang, Henan, Zhejiang, Ningxia),23 among 31 visibly moldy samples and 2 among 26 not visibly moldy samples were contaminated with OTA, at levels ranging from 1.2 to 158.7 and 2.5 to 5.7μg/kg, respectively. The results of this study suggest that incidence and occurrence of OTA in moldy samples were higher than those of normal samples.
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