Xiaowen Dou

An aptamer based lateral flow strip for on-site rapid detection of ochratoxin A in Astragalus membranaceus

An aptamer based lateral flow strip based on competitive format was developed for on-site rapid detection of ochratoxin A (OTA) in Astragalus membranaceus. Some crucial parameters that might influence the sensitive detection, such as the characterization of the colloidal gold, size and shape of gold nanoparticles (AuNPs), amount of AuNPs-aptamer conjugate, migration rate and the addition amount of methanol, were investigated to provide the optimum assay performance. To perform the test, 1g sample was extracted with 2.5mL of methanol-water (80:20, v/v) and diluted by 4-fold running buffer to eliminate the matrix and methanol interferences. Under optimized conditions, the aptamer-based assay showed a visual limit of detection (LOD) of 1ngmL(-1), and with no significant cross-reactivity with several homologous toxins. The whole detection could be completed within 15min without special equipment because of available visual results. One out of nine A. membranaceus samples was found to be positive of OTA, which was in a good agreement with those obtained from LC-MS/MS analysis. The results demonstrated that the aptamer-based lateral flow assay could be used as a rapid, reliable, cost-effective and robust on-site screening technique for mycotoxins at trace level in complex matrices without special instrumentation.

High-throughput determination of multi-mycotoxins in Chinese yam and related products by ultra fast liquid chromatography coupled with tandem mass spectrometry after one-step extraction

A simple, accurate and sensitive ultra fast liquid chromatography coupled with tandem mass spectrometry (UFLC-MS/MS) method was developed for high-throughput determination of aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), fumonisins (FB1 and FB2) and zearalenone (ZEA) in Chinese yam, yam flours and yam-derived products. Mycotoxins were extracted from the samples with methanol-water-formic acid (79:20:1, v/v/v) and no further cleanup step before analysis. After optimization of some crucial parameters including sample preparation, chromatographic separation and MS/MS conditions, the method was successfully validated to exhibit excellent performance in terms of satisfactory linearity (r≥0.9977), limits of detection (≤0.15ngmL(-1)) and quantification (≤0.5ngmL(-1)) with good precision (RSD for intra- and inter-day variations of ≤4.65% and 6.31%, respectively), good accuracy (recoveries of 71.0-106.0%) and robustness, together with short run time (8min/sample). The developed method was applied for simultaneous detection and quantification of the above 8 mycotaxins in 27 batches of Chinese yam and related products collected from different markets and pharmacies in China. The results revealed that 1 normal sample and 4 moldy samples were found to be contaminated with different mycotoxins. The detected concentrations of AFB1 in 2 moldy samples exceeded the regulatory maximum residue levels. The proposed method was capable for simultaneous determination of mycotoxins in this and other types of complex matrices.

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.

Quantitative and fingerprinting analysis of Pogostemon cablin based on GC-FID combined with chemometrics.

In this study, a simple, sensitive and reliable gas chromatography-flame ionization detection (GC-FID) method is established for quantitative chemical fingerprinting of essential oils from Pogostemon cablin. Oil samples are prepared by hydrodistillation, with yields ranging from 0.73% to 2.02%. The two main components of the oil, patchouli alcohol and pogostone, were detected simultaneously in 36 samples and were found to have average contents of 43.07% and 7.84%, respectively. The method was validated in terms of linearity, sensitivity, precision, stability, and accuracy. All calibration curves showed excellent linearity (r(2)>0.9992) within the test ranges, and the relative standard deviation (RSD) values for intra- and inter-day precision were less than 1.5%, indicating a high degree of precision. The GC-FID chemical fingerprints of the 36 samples were established using 12 common peaks which account for over 90% of the total peak area. Chemometric techniques, including similarity analysis and hierarchical cluster analysis, were also employed to explore the similarities and outstanding consistencies among different P. cablin oil samples. The results demonstrate that chromatographic fingerprinting and quantitative analysis can be achieved simultaneously when evaluating quality and authenticating samples of P. cablin.

An indirect competitive fluorescence assay for ochratoxin A based on molecular beacon

A novel, simple and efficient method based on a molecular beacon (MB) probe was developed to detect ochratoxin A (OTA) in malts, which is a common starting material in the brewery industry. With the critical site for OTA binding in capture aptamer in mind, a MB probe containing 20 bases with a fluorophore–quencher pair at the stem ends was designed and synthesized. In the “off” configuration, the fluorescein (FAM) is internally quenched due to close contact with dabcyl and the fluorescence signal is recovered after hybridization with the capture aptamer at the “on” state. In the presence of OTA, the MB probe competes for binding at the loop region of the aptamer, resulting in a decrease in fluorescence signal. Using this indirect competitive assay, the detection of OTA in malt samples was accomplished for the first time. In addition, the effect of binding affinity of the capture aptamer and OTA on the assay performance was investigated. Under optimal conditions, this method allowed for OTA detection at a linear range of 0.0001–1 μg mL−1 (correlation coefficient, R = 0.9920) with superior sensitivity and a detection limit as low as 0.05 ng mL−1. The sensing system also displayed an excellent selectivity and perfect anti-interference capacity in the matrix. Moreover, the entire process of detection was accomplished in less than 20 min. The recovery from spiked malt samples ranged from 81.0% to 95.2% with RSDs below 5.4%. The performance of our method was further validated by ultra-fast liquid chromatography coupled with tandem mass spectrometry. Compared with similar fluorescence assays, the proposed method is simple, efficient and does not require complicated conjugation steps. Taken together, this novel detection strategy could be a promising tool for hand-held devices used during on-site monitoring of contaminants.

Carbon nanotube-based QuEChERS extraction and enhanced product ion scan-assisted confirmation of multi-pesticide residue in dried tangerine peel

A sorbent package consisting of a combination of multiwalled carbon nanotubes (MWNTs) and a primary secondary amine (PSA) has been used for a modified quick, easy, effective, rugged, and safe (QuEChERS) extraction of 104 pesticides from dried tangerine peel samples. MWNTs and graphitized carbon black (GCB) have been compared in terms of purification efficiency and recovery; the best results were achieved with MWNTs. The pesticides were quantified on a liquid chromatography tandem mass spectrometry (LC-MS/MS) system in scheduled multiple reaction monitoring mode, and identified on the basis of product ion abundance ratios as well as characteristic fragments in enhanced product ion spectra. Calibration curves for most of the analytes showed correlation coefficients better than 0.9916. Detection limits ranged from 0.2 to 40 μg kg−1. Good precision was achieved with relative standard deviations of less than 20%. Results of accuracy in spiked samples were in the range 71.1–117.6%, except for pesticides such as thiabendazole, teflubenzuron, hexaflumuron, and methomyl. The proposed method has been applied to 57 dried tangerine peel samples from the Chinese market; 16 pesticides were detected, including carbendazim, chlorpyrifos, isoprothiolane and methidathion, at levels that exceeded the recommended maximum residue limits in some samples. The newly established method has advantages of good recovery and a rapid clean-up procedure, showing enhanced product ion scan-assisted confirmation to be a useful tool for obtaining reliable results.

Rapid Detection of Ochratoxin A in Malt by Cytometric Bead Array Based on Indirect Competition Principle

Abstract A cytometric bead array (CBA) method based on indirect competition principle was developed for the sensitive and rapid detection of ochratoxin A (OTA) in malt. The malt samples were extracted by 60% methanol/PBS and the extracts were diluted five times with 20% methanol/PBS. After centrifugation, the supernatant was collected to prepare sample solution for analysis. The fluorescence-encoded microsphere surface was labeled with bovine serum albumin OTA (BSA-OTA) to compete with OTA in samples for anti-OTA monoclonal antibody (mAb). Then, FITC-IgG was added to bind with the captured mAb on the microsphere. After centrifugation and washing, the mean fluorescence intensity from FITC on the surface of microsphere was detected by a BD FACSCalibur analyzer for accurately qualitative and quantitative analysis of OTA. The results showed that the half inhibitory concentration (IC 50 ) was 1.20 ng mL −1 with the correlation coefficient ( R 2 ) of 0.989, and the limit of detection (LOD) for OTA was 0.12 ng mL −1 . The average recovery rates in malt samples were 93.9%–97.4% with relative standard deviations (RSDs) less than 3.6% at three spiking levels. Sixteen malt samples were analyzed and OTA was found in two samples with the contents less than 3.83 μg kg −1 which was lower than the maximum permitted residue level (5 µg kg −1 ) proposed by the European Union. All the positive samples were confirmed by LC-MS/MS. In this study, the CBA technique based on indirect competition principle was developed for the first time for successful detection of OTA in malt samples. The method was easy, rapid, sensitive and reliable with high potential for the qualitative and quantitative of multiple mycotoxins in other complex matrices.

Enhancement effect of essential oils from the fruits and leaves of Alpinia oxyphylla on skin permeation and deposition of indomethacin

Essential oils from plants are gaining increasing attention as potential chemical penetration enhancers. This study aimed to investigate the enhancement effect of essential oils from the fruits and leaves of Alpinia oxyphylla on skin permeation and deposition of indomethacin. In vitro permeation experiments were performed in Franz-type cells through rat skin, and the amount of drug passing through into the receptor phase was analyzed by ultra performance liquid chromatography-photodiode array (UPLC-PDA). Ultra fast liquid chromatography coupled with tandem mass spectrometry (UFLC-MS/MS) was used to analyze the plasma drug concentration of indomethacin to examine the enhancement effect of essential oils in an in vivo rat model of drug delivery. Both oils demonstrated a significant enhancement effect on drug delivery and skin deposition (p < 0.05). Particularly, at 3% concentration, enhancement ratios of fruit oil and leaf oil were 10.16 and 4.61, respectively, which were both significantly higher than that of the commonly used enhancer, Azone (2.04). Major constituents of both oils were identified by gas chromatography-mass spectrometry (GC-MS). It may be deduced that higher content hydrocarbon terpenes in the fruit oil contribute to the increased enhancement effect relative to leaf oil. The skin irritation test indicated that both oils at certain concentrations (1%, 3%, and 5%) did not cause obvious erythema or edema in rabbits. Considering their enhanced drug permeation and low skin irritation, essential oils from Alpinia oxyphylla could be novel penetration enhancers and have promising applications in transdermal drug delivery and cosmetics.

Accumulation of Arsenic Speciation and In Vivo Toxicity Following Oral Administration of a Chinese Patent Medicine Xiao-Er-Zhi-Bao-Wan in Rats

Realgar-containing traditional Chinese medicines such as Xiao-Er-Zhi-Bao-Wan (XEZBW), have been widely used for thousands of years. However, events associated with arsenic-induced ailments have increasingly become a public concern. To address the toxicity of XEZBW, we studied the histopathology and blood biochemistry of rats exposed to XEZBW using technology like high-performance liquid chromatography-inductively coupled mass spectrometry to determine arsenic speciation. Our results demonstrated that dimethylarsinic acid (DMA) increased from 18.57 ± 7.45 to 22.74 ± 7.45 ng/g in rat kidney after oral administration for 7 and 14 days, which was 10-fold higher than the levels observed in controls. Trivalent arsenite As(III) showed a large increase on day 7 (26.99 ± 1.98 ng/g), followed by a slight decrease on day 14 (13.67 ± 6.48 ng/g). Total arsenic levels on day 7 (185.52 ± 24.56 ng/g) and day 14 (198.57 ± 26.26 ng/g) were nearly twofold higher than that in the control group (92.77 ± 14.98 ng/g). Histopathological analysis showed mild injury in the liver and kidney of rats subjected to oral administration of realgar for 14 days. As in the XEZBW groups, a mild injury in these organs was observed after administration for 14 days. This study inferred that the toxicity of arsenic was concentration- and time-dependent. The accumulation of DMA, a byproduct of choline metabolism, was responsible for inducing higher toxicity. Therefore, we concluded that measuring the levels of DMA, instead of total arsenic, might be more suitable for evaluating the toxicity of realgar-containing traditional Chinese medicines.

Development of a broad-specificity antibody-based immunoassay for triazines in ginger and the quantitative structure-activity relationship study of cross-reactive molecules by molecular modeling

In the present study, molecular modeling and principle component analysis (PCA) were used to select appropriate haptens for group detection of triazine herbicides. Four new structures together with three reported triazine derivatives were chosen for the screening of immunizing and coating haptens. A total of 31 triazines coupled with a 3D-QSAR methodology were employed to investigate the relationship between antigen-antibody recognition and molecular structures, the results of which revealed that the antibodies may recognize triazines from the side of molecules with the distinguishing atom and a steric volume matching with the spatial structure of antibodies. Finally, a broad-specificity heterologous immunoassay was developed for determining 10 triazine herbicides in ginger, where the detection limits were 2.5-15.1 μg kg-1 and recoveries were 67.9-102.6%. This study may broaden insight into triazine-antibody interactions and benefit designing novel performance-enhanced antibodies. The developed immunoassay can be further used for triazine detection in other complicated matrices.