Multi-mycotoxin detection and human exposure risk assessment in medicinal foods.

Mycotoxin contamination in medicinal foods has attracted increasing global attention. In this study, a simple and sensitive ultrasonication assisted one-step extraction based ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed for simultaneous detection of multi-mycotoxins in five kinds of medicinal foods rich in starch. Under optimal conditions, the developed technique displayed excellent analytical performances. Limits of detection and quantitation for the six mycotoxins were 0.04-0.25 ng/mL and 0.10-0.67 ng/mL, respectively. Average recoveries at three fortified levels ranged from 75.33 % to 118.0 %. Real-world application in 103 batches of medicinal foods displayed that 58 samples were positive with one or more mycotoxins at an occurrence rate of 56.31 % (58/103). Coix seed gave the highest positive rate of 96.15 %, followed by Lily (90 %), Chinese yam (50 %), Lotus seed (34.04 %) and Malt (30 %). Zearalenone had the highest positive rate of 28.16 % with contents in 5 Coix seeds exceeding the maximum residue limit (MRL), followed by aflatoxin B1 of 27.18 % (28/103) with contents in 7 Coix seed and 10 Lotus seeds over its MRL, and ochratoxin A (OTA) of 11.65 % with contents in 1 Lotus seed and 5 Lily samples greater than its MRL. Exposure risk assessment indicated that Coix seed and Lotus seeds that were susceptible to aflatoxins posed great threats to human health. Long-term consumption of Lily that was easily contaminated with OTA were also harmful. This work provides a robust platform for multi-mycotoxin monitoring in medicinal foods to protect the consumers from potential health risks.

Development of an economic ultrafast liquid chromatography with tandem mass spectrometry method for trace analysis of multiclass mycotoxins in Polygonum multiflorum.

Rapid, economic, and highly effective determination of multiple mycotoxins in complex matrices has given huge challenges for the analytical method. In this study, an economic analytical strategy based on sensitive and rapid ultrafast liquid chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometry technique was developed for the determination of seven mycotoxins of different chemical classes (aflatoxin B1 , B2 , G1 , and G2 , ochratoxin A, T-2 toxin, and HT-2 toxin) in Polygonum multiflorum. Target mycotoxins were completely extracted using a modified quick, easy, cheap effective, rugged, and safe method without additional clean-up steps. The types of extraction solvents and adsorbents for the extraction procedure were optimized to achieve high recoveries and reduce coextractives in the final extracts. Due to significant matrix effects for all analytes (≤68.9% and ≥110.0%), matrix-matched calibration curves were introduced for reliable quantification, exploring excellent linearity for the seven mycotoxins with coefficients of determination >0.9992. The method allowed high sensitivity with limit of detection in the range of 0.031-2.5 µg/kg and limit of quantitation in the range of 0.078-6.25 µg/kg, as well as satisfactory precision with relative standard deviations lower than 8%. Recovery rates were between 74.3 and 119.8% with relative standard deviations below 7.43%. The proposed method was successfully applied for 24 batches of P. multiflorum samples, and six samples were found to be positive with aflatoxin B1 , B2 , G1 , or ochratoxin A. The method with significant advantages, including minimum analytical time, low time and solvent consumption, and high sensitivity, would be a preferred candidate for economic analysis of multiclass mycotoxins in complex matrices.

Regeneration and Reuse of Immunoaffinity Column for Highly Efficient Clean-Up and Economic Detection of Ochratoxin A in Malt and Ginger.

Immunoaffinity columns (IACs) are most popularly used for mycotoxin clean-up in complex matrices prior to chromatographic analysis. But, their high cost has limited their wide application and the regeneration of IACs for multiple instances of reuse is important. This study aimed to investigate the feasibility of regeneration and reuse of IACs for purification of ochratoxin A (OTA) in spiked raw malt and dried ginger samples followed by high performance liquid chromatography-fluorescence detection. After each use, the IACs were filled with phosphate buffer saline (PBS) as the preservation solution and stored at 8 °C overnight for regeneration and reuse until the recovery rate was <70%. The results showed that matrix type, preparation procedure, and pH value of sample extraction exhibited major effects on the reuse of IACs for OTA clean-up. While, after modifying the sample preparation procedure using water as the diluent and the solution at a pH of 7 to 8, the IACs could be used eight and three times for the spiked raw malt and dried ginger samples with OTA after regeneration. Regarding the traditional procedure recommended in Chinese Pharmacopoeia (2015 edition), the IACs could be used for three and two times for the spiked raw malt and dried ginger samples with OTA, respectively. Therefore, the corresponding experimental cost could be reduced to one-eighth and one-third of the original cost. This is the first study on the regeneration and reuse of IACs for OTA clean-up in complex Chinese herbal medicines, providing a green and economical tool for a large number of samples analysis with low cost.

Assessment of critical points and development of a practical strategy to extend the applicable scope of immunoaffinity column cleanup for aflatoxin detection in medicinal herbs.

Although extraction methods based on immunoaffinity column (IAC) cleanup have been used to detect aflatoxins in medicinal herbs, they do not yield satisfactory results for all sample matrices. The difficulty arises from the chemical complexity of the herbs, and there is a pressing need to determine which steps in IAC cleanup limit the scope of aflatoxin detection in many different kinds of medicinal herbs. In this work, we found that there were two main factors that severely decreased antibody-antigen recognition and led to serious nonspecific adsorption: (1) high extract acidity and (2) high co-extraction of interfering compounds. We therefore carried out a systematic study to optimize extraction efficiency. We found that dilution of samples in 0.1M phosphate buffer solution (pH 7.8, 2% Tween-20) at a 1:8 dilution ratio mitigated the effect of high acidity, decreased co-precipitation of compounds and nonspecific adsorption, and ameliorated the matrix effect. To validate this finding, and test if our method is widely applicable to in different kinds of herbal materials, we analyzed several representative complex sample matrices including fructus, cortex, and radix with varying extract pH values. The recovery efficiency was generally higher than 70%. We further validated our method by testing a certified reference material, and found that our approach accurately quantified aflatoxin concentration. After validation, we successfully used this method to determine the aflatoxin concentration of real samples. The approach described here could potentially be used to extract aflatoxin from other complex matrices with varying acidity.

Occurrence of toxigenic fungi and determination of mycotoxins by HPLC-FLD in functional foods and spices in China markets.

Twenty-four samples including 14 functional foods and 10 spices obtained from Chinese markets were examined for their mould profile. The mycotoxin contamination levels were also determined by an optimized HPLC-FLD method. 124 fungal isolates belonging to four different genera were recovered with Aspergillus and Penicillium as predominant fungi, with an incidence of 66.1% and 15.3%, respectively. In functional foods Aspergillus niger section (57.1%) was isolated more frequently, followed by Aspergillus flavi section (50.0%) and Aspergillus ochraceus section (21.4%), with the most contaminated samples being Coix seeds. Similar fungal presence and frequency were encountered in spice with A. niger section group (60.0%) and A. flavi section (40.0%) as main fungi. Cumin and Pricklyash peel samples showed the highest fungal contamination. Four functional foods and three spices were found to be positive at low levels for mycotoxins including aflatoxin B1 (up to 0.26µg/kg) and ochratoxin A (OTA) (5.0µg/kg). The more frequently detected mycotoxin was AFB1 (16.7%).

Natural occurrence of ochratoxin A in wolfberry fruit wine marketed in China.

Wolfberry fruit wine (WFW) is widely used as a global functional food to improve the immune system and prevent human disease. A total of 36 bottled WFWs were randomly collected in China between 2005 and 2010. Samples were analysed for the presence of ochratoxin A (OTA) using immunoaffinity column (IAC) clean-up and high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Positive results were confirmed by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). The limit of detection (LOD), based on a signal-to-noise ratio of 3, was 0.05 ng mL⁻¹. Recoveries ranged from 78.3% to 94.7% and relative standard deviations from 1.1% to 4.3% within the spiking range of 0.2-20 ng mL⁻¹. OTA was detected in one sample, below the maximum allowable limit as established by the European community.