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Simultaneous determination of four aflatoxins and ochratoxin A in ginger and related products by HPLC with fluorescence detection after immunoaffinity column clean-up and post-column photochemical derivatizationJing Wen a,b, Weijun Kong b, Jian Wang a*, Meihua Yang b*a School of Pharmacy, Chengdu University of TCM, Chengdu 611137, Chinab Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 100193, ChinaAbstractGinger, a widely used spice or traditional Chinese medicine, is prone to be contaminated by mycotoxins. A simple, sensitive and reproducible method based on immunoaffinity column clean-up coupled with high performance liquid chromatography and on-line post-column photochemical derivatization-fluorescence detection was developed for simultaneous determination of aflatoxins B1, B2, G1, G2 and ochratoxin A in 25 batches of gingers and related products marketed in China for the first time. The samples were firstly extracted by ultrasonication with methanol-water (80:20, v/v) and then cleaned up with immunoaffinity columns for analysis. Under the optimized conditions, the limits of detection and quantification for the five mycotoxins were 0.03-0.3 g/kg and 0.1-0.9 g/kg, respectively. The average recoveries ranged from 81.3% to 100.8% for aflatoxins and from 88.6% to 99.5% for ochratoxin A at three spiking levels. Good linearity was observed for the analytes with correlation coefficients all higher than 0.9995. All moldy gingers were contaminated with at least one kind of the five investigated mycotoxins, while none of them were found in normal gingers. Ginger powder samples were contaminated slightly with the contamination levels below the limits of quantification, while Ginger tea bags were mainly contaminated by ochratoxin A at 1.05-1.19 g/kg and ginger black tea bags were mainly contaminated by aflatoxins at 3.37-5.76 g/kg. All the contamination levels were below the legally allowable limits.Keywords: Aflatoxins; Ochratoxin A; Immunoaffinity column; High performance liquid chromatography; Fluorescence detection; Photochemical derivatization; Ginger and related productsAbbreviations: IAC, immunoaffinity column; HPLC, high performance liquid chromatography; PCD, photochemical derivatization; FLD, fluorescence detection; AFs, aflatoxins; OTA, ochratoxin A; LOD, limit of detection; LOQ, limit of quantification.1 IntroductionGinger (Zingiber officinale Rosc., Shengjiang in Chinese) has been regarded as one of the “medicinal and edible” herbs/spices since ancient times. First of all, Ginger is a frequently-used traditional Chinese medicine and has been officially recorded in the Chinese Pharmacopoeia for its therapeutic benefits to treat common cold especially nausea and vomiting 1. Modern pharmacological and clinical experiments have proved that ginger has the functions of antioxidant 2, anti-microbial 3, anti-diabetic 4, anti-hyperlipidemia 5, anti-cholesterol 6, anti-inflammation 7, antineoplastic 8, etc. Ginger was included in many ancient prescriptions as well as modern proprietary Chinese medicine. In addition, ginger is a spice widely used as a food condiment or a nutritional supplement all over the world. As a spice, ginger is marketed in many forms such as fresh or dried products, liquid or solid extracts, tablets or capsules, powders and tea bags. People can easily and freely buy these ginger-related products from the pharmacies or markets. However, its worth noting that ginger is prone to be contaminated by mycotoxins, which are toxic secondary metabolites produced by toxigenic fungi. These mycotoxins pose a serious threat to human health through ingestion because of their carcinogenicity, mutagenicity, teratogenicity and immunosuppression. A few publications have reported that aflatoxins (AFs) and ochratoxin A (OTA) have been found in ginger and its products with various contamination levels 9-10.AFs are secondary metabolites produced by Aspergillus flavus and A. parasiticus, and among which AFB1, AFB2, AFG1, AFG2 are more frequently found in foodstuffs, spices and medicinal plants and AFB1 is the most toxic. While, OTA, mainly produced by A. ochraceus and Penicillium verrucosum, is one of the most toxic ochratoxins. AFB1 and OTA have been designated as a class I carcinogen by the International Agency for Research on Cancer (IARC) because of their hepatotoxicity 11-13. Since AFs and OTA can threaten human health and ginger can be easily contaminated by AFs and OTA, the European Union (EU) has set the legal limit of 5 g/kg for AFB1, 10 g/kg for total AFs and 15 g/kg for OTA in spices 14-15.As many herbs or spices usually can be contaminated simultaneous with some different fungal species which might produce various mycotoxins 16-18, simultaneous determination of multiple mycotoxins in a single test is required. Meanwhile, because of the complexity of herbs or spices matrices, as well as the wide range of physical and chemical properties of mycotoxins, a selective and sensitive detection technique is demanded. Hitherto, a few methods for simultaneous analysis of AFs and OTA in different matrices can be found in the publications, such as thin-layer chromatography (TLC) 19, enzyme-linked immunosorbent assay (ELISA) 20, gas chromatography (GC) 21-22 and liquid chromatography (LC) with various detectors 23-24. However, so far, high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) 25-26 is still the most frequently used technique for its high sensitivity and selectivity and relatively lower cost.Moreover, the increasing multi-mycotoxin analysis has promoted the needs of adequate and effective extraction and cleanup procedures. First of all, the extraction should allow good recoveries for all mycotoxins of interest in the specific matrix. Several publications 16-18, 27 have reported that high contents (75%) of organic solvent (methanol or acetonitrile) are suitable for the extraction of most mycotoxins including AFs and OTA. After that, the extract should be cleaned up and enriched to remove matrix components and enhance sensitivity prior to analysis. In recent years, in order to get high sensitivity, various techniques have been used in purification of mycotoxins such as solid phase extraction (SPE) 28, solid-phase microextraction (SPME) 29 and liquid-liquid extraction (LLE) 30. Among them, immunoaffinity column (IAC)-based SPE is the most widely used technique in the pretreatment of mycotoxins for its high specificity and selectivity towards selected mycotoxins in many complex matrices 31-32, 10. Some publications have reported the determination of AFs or OTA in ginger or its products with two different separation conditions 9-10. However, until now, there was no report on the simultaneous quantitative determination of five mycotoxins in ginger and its products in a single test using IAC-based SPE technique coupled with HPLC-FLD. The purpose of this study is to develop a simple, sensitive, and reproducible method based on IAC clean-up for simultaneous multiple determination of AFs (AFB1, AFB2, AFG1, AFG2) and OTA in ginger and its related products by HPLC and on-line post-column photochemical derivatization-fluorescence detection (HPLC-PCD-FLD).2 Experimental2.1 Chemicals and reagentsAflatoxins standard (2 g of AFB1, 2 g of AFG1, 0.5 g of AFB2, 0.5 g of AFG2 in 1 mL of acetonitrile) was purchased from Pribolab (Singapore), and OTA standard (1mg, powder) was purchased from ALEXIS (Lausen, Switzerland). The IAC-SEP IAC-AFLA/OTA immunoaffinity columns and GF/A glass microfiber filter (1.5m) were all purchased from VICAM (Waterworn, MA, USA). A KQ-500 ultrasonic clean bath (503035 cm) was bought from Kunshan Ultrasonic Instrument Co. Ltd (Jiangshu, China). HPLC-grade methanol was purchased from Honeywell (Burdick & Jackson, USA). Other reagents and chemicals were all analytical grades and purchased from Beijing Chemical Works (Beijing, China). Water used in this experiment was Wahaha purified water (Wahaha, Hangzhou, China).Phosphate-buffered saline was prepared by dissolving 0.2 g of KCl, 0.2 g of KH2PO4, 8 g of NaCl and 1.2 g of Na2HPO4 in 1000 mL of water (PH was adjusted to 7.0 with 0.1M HCl). 2% tween-20-PBS solution was prepared by dissolving 0.2 g of KCl, 0.2 g of KH2PO4, 8 g of NaCl and 1.2 g of Na2HPO4 and 20 mL of tween-20 in 1000 mL of water.2.2 Standard solutionsA series of working standard solutions (0.25, 0.5, 1, 5, 10, 20, 40, 50 ng/mL for AFG1, AFB1, OTA and 0.0625, 0.125, 0.25, 1.25, 2.5, 5, 10, 12.5 ng/mL for AFG2, AFB2) were prepared freshly with the solution of methanol-water (50:50, v/v). The standard solutions were used to calculate the LC detector response and recovery studies.2.3 samplesTwenty-five batches of fresh gingers and related products including 10 batches of fresh ginger (of which, 5 batches were moldy), 5 batches of ginger powder, 5 batches of ginger tea bags and 5 batches of ginger black tea bags were all purchased from several different local markets (Beijing, China). They were packaged in plastic bags and stored at 4 C in a fridge prior to analysis. All the samples were extracted and analyzed in triplicate.2.4 sample preparation2.4.1 Extraction procedureTen grams of tested sample (fresh ginger should be chopped) and 2 g NaCl were weighed and placed in a 100 mL Erlenmeyer flask, and then 50 mL of methanol-water (80:20, v/v) solution were added and the mixtures were extracted by ultrasonication in a ultrasonic clean bath at 500 W for 20 min. After the ultrasonication, the mixture was filtered through a filter paper and 10 mL of the filtrate was diluted by 40 mL of 2% tween-20-PBS solution in a 50 mL Erlenmeyer flask. At last, the diluent was filtered through the GF/A glass microfiber filter and the final filtrate was obtained for analysis.2.4.2 Immunoaffinity column cleanupTwenty-five milliliters of the final filtrate was passed through an IAC-SEP IAC-AFLA/OTA immunoaffinity column. The column was washed with 20 mL of PBS solution (pH 7.0) until 2-3 mL of air passed through it and the investigated toxins were finally eluted with 3 mL of pure methanol. The elutes were evaporated to dryness under a stream of N2 at 50C and the residue were redissolved in 1 mL of methanol-water (50:50, v/v). 50 L of the solution was filtered through a 0.22 m syringe filter prior to analysis.2.5 Apparatus and HPLC conditionsHPLC analysis was performed on a Shimadzu LC-20 AT HPLC system connects with a RF-10 AXL fluorescence detector (Shimadzu, Kyoto, Japan) and a post-column photochemical derivatization reactor. HPLC separations were performed on an ACCHROM Unitary C18 column (4.6 150 mm, 5 m). The optimized conditions of HPLC-PCD-FLD were as follows: flow rate, 1.0 mL/min; temperature, 30 C; and injection volume, 50 L. The gradient elution solution and detection wavelength were shown in Table 1. The investigated mycotoxins could be well separated in 20 min under the above conditions.Table 12.6 Analytical method performanceThe HPLC-FLD analytical method was assessed in reference to stability, linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, reproducibility and precision. 2.6.1 Stability, precision and reproducibilityThe stability of spiked ginger samples (5 g/kg for AFB1, AFG1 and OTA, 1.25 g/kg for AFB2 and AFG2) were determined by monitoring the peak area of each analyte over a period of 24 h on the same day stored at 4 C for analysis.The precision of the established method was determined by measuring intra- and inter-day precision from analysis of mixed standard solutions of 5 ng/mL for AFB1, AFG1 and OTA, 1.25 ng/mL for AFB2 and AFG2. The intra-day precision was determined by six consecutive injections of the mixed standard solution within one day. The inter-day precision was determined by five consecutive injections of the mixed standard solution during five consequent days. Intra- and inter-day precisions were expressed by the relative standard deviation (RSD) of replicate results.The reproducibility of the method was determined by analyzing the blank ginger samples spiked with 5 g/kg of AFB1, AFG1 and OTA, 1.25 g/kg of AFB2 and AFG2 and separately treated by the above-described procedure. Each analysis was repeated six times and the peak area of each analyte was recorded. Then the RSD of six analyses was calculated.2.6.2 Linearity, LOD and LOQCalibration curves were constructed with seven levels of investigated standard analytes in the range of 0.3-50 ng/mL for AFB1, 0.1-12.5 ng/mL for AFB2, 0.6-50 ng/mL for AFG1, 0.2-12.5 ng/mL for AFG2 and 0.9-50 ng/mL for OTA, each standard solution was injected triplicate. The calibration curves were constructed by plotting the peak area (y) versus concentration (x) of each analyte obtained from HPLC analysis. The linearity was determined by linear regression analysis and expressed as correlation coefficient (R).The LOD and LOQ were used to assess the sensitivity of the HPLC-FLD method, which were determined by injecting serial diluted the blank ginger samples spiked with a certain concentration of mixed standard solutions and were calculated as signal-to-noise (S/N) ratio of 3 and 10.2.6.3 RecoveryThe recovery was used to evaluate the accuracy of the established method. The recovery experiments were performed at three spiking levels (1.0, 5.0, 25.0 g/kg for AFB1, AFG1, OTA and 0.25, 1.25, 6.25 g/kg for AFB2, AFG2) by adding an appropriate amount of AFs and OTA standard solutions to the blank ginger samples. The spiked samples were extracted, cleaned-up, derivatized and analyzed by HPLC-FLD as previously described. The recovery was calculated by the following equation:Recovery = (measured concentration for spiked sample/spiked concentration) 100%3 Results and discussion3.1 Optimization of the extraction conditionsThe sample extraction should allow good recoveries for all analytes of interest in the specific matrix. As reported previously, a high percentage of organic solvent is widely used for the extraction of AFs and OTA from real samples. Acetonitrile based extractant could lead to absorption of water by the dry matrix, and methanol-water mixtures could prevent this 33. Moreover, methanol-water (80:20, v/v), which was used the most commonly providing the highest recoveries for AFs and OTA as reported 34, was chosen as the extraction solvent in this study.The amount of extraction solvent was then investigated, in order to get high recovery. For 10 g of ginger samples, 20 mL and 50 mL of extraction solvent were compared, and it turned out that 10 g of samples extracted with 50 mL of extraction solvent could provide higher recovery. Therefore, 50 mL of extraction solvent was chosen for the extraction of 10 g of ginger samples.Sodium chloride was added in the step of extraction for the purpose of salting out. In order to reduce the matrix effects caused by the complex matrices, purification was performed with IAC-SEP AFLA/OTA immunoaffinity clean-up column due to its efficiency and specificity for aflatoxins and OTA.Owing to the complexity of ginger-related products, including ginger powder and ginger tea bags, yellowish to reddish precipitate would appear in the process of diluting sample extract, which would influence the recovery of analytes. It is reported that 17 adding an appropriate amount of tween-20 to the PBS solution can restrain the appearance of precipitate and raise the recovery of investigated mycotoxins in ginger samples. So, different ratios (1%, 2% and 5%) of tween-20 adding into the PBS solution were compared, and we found that 2% of tween-20 was the most appropriate amount for the dilution of ginger-related products. But, it would not generate precipitate in the process of diluting fresh ginger samples, so PBS solution without tween-20 were used to dilute the extracting solution of fresh ginger samples.3.2 Optimization of HPLC-PCD-FLD conditionsIn order to get a high separation efficiency of the five investigated analytes, some important parameters of chromatographic conditions including the proportion and flow rate of mobile phase, kind of column, column temperature and elution procedure were investigated. After the investigation, the aforementioned chromatographic separation condition was obtained. Under the optimized chromatographic condition, the five investigated analytes, were well separated in less than 20 min, which was shorter than most of the published methods 26, 35. The typical HPLC-FLD chromatogram was shown in Fig. 1a.Fig. 1In this study, a fluorescence detector was used to detect the five analytes as all of them have fluorescence absorption, and the excitation as well as emission wavelengths are the most important parameters to be optimized in fluorescence detection. According to the results of repeated preliminary experiments, the optimal excitation and emission wavelengths were 360 and 440 nm for aflatoxins during the first 9 min, and 333 and 460 nm for OTA after 9 min.A post-column photochemical derivatization (PCD) device was adopted to enhance the native fluorescence of AFB1 and AFG1 and further to improve the detection limits. Although pre-column and post-column derivatization can also reach that goal, both of them present several defects such as time-consuming, use of harm reagents and additional pumps or electrochemical cells. However, the on-line PCD technique does not need any chemical reagents or additional devices, and makes derivational fast and easy. Therefore, a photochemical reactor was added between the chromatographic column and the fluorescence detector to enhance fluorescence detection and quantifica