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Objective To investigate the contamination of aflatoxin B1 in traditional Chinese medicine decoction pieces.and to provide evidence for the development of sdandards and scientific management.Methods Immunoaffinity column and post-column photochemical derivatization were used to detect and quantify aflatoxin B1 in 35 traditional Chinese medicines.Results A total of 48.57％(17 out of 35 batches) traditional Chinese medicine were contained aflatoxin B1.The contents of aflatoxin B1 in all contaminated varieties were less than 1μg/kg,except for Sterculia lychnophorae Semen,Foeniculi Fructus,Corydalis Rhizoma,which exceeded the standard.Conclusions The tested traditional Chinese medicine are highly contaminated of aflatoxin,it is necessary to further study the increase of aflatoxin content under the examination of Chinese Pharmacopoeia Foeniculi Fructus and Corydalis Rhizoma to better control its quality.The degree of aflatoxin B1 pollution is reated to the site of drug use and the place of origin.
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Objective: To determine the aflatoxins residue of animal medicines by immunoaffinity column HPLC method with post column photochemical derivatization and fluorescence detection, and to evaluate the feasibility of this method. The contamination status of aflatoxins in animal medicines was evaluated according to the determination data of aflatoxin contamination in animal medicines. Methods: After extraction by organic solvent and purification by immunoaffinity column, aflatoxins samples were analyzed by HPLC with fluorescence detection after photochemistry derivation. The recovery rates of aflatoxins in animal medicines, especially the species easily contaminated by aflatoxin, were then determined by adding aflatoxin standard mixtures. Finally, aflatoxins in animal medicines were determined and the results were analyzed. Results: Recovery rates of aflatoxin B1, B2, G1, and G2 were from 70% to 120%. Twenty-four batches of six kinds in 64 batches of 16 kinds of animal medicines were contaminated by aflatoxins, and the contamination rate was 37.5%. Thirteen batches of four kinds of animal medicines exceeded the limit of Chinese Pharmacopoeia of (2015 Edition), and the rate was 20.3%. Conclusion: This method can be used to determine aflatoxins in animal medicines. Some species of animal medicines are likely to be contaminated by aflatoxins, so the aflatoxin control in those animal medicines should be put forward to ensure the safety of drug use.
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The three-in-one immunoaffinity column ( IAC ) for the determination of aflatoxin B1 ( AFB1 )-zearalenone (ZEN)-deoxynivalenol (DON) was prepared with rProtein A-sepharose 4B as the column matrix. The comprehensive performance ( such as nonspecific adsorption, column blank, column capacity, column efficiency and sample standard addition recovery rate) was evaluated and investigated. The results showed that, the column capacities of AFB1 , ZEN, DON were 295 ng per 0. 25 mL gel, 905 ng per 0. 25 mL gel, 2342 ng per 0. 25 mL gel, respectively, and the column blank was 0. The average recoveries of AFB1 , ZEN and DON were 97. 4%, 98. 0% and 98. 4%, respectively. By optimizing conditions, the samples were extracted using the mixture of methanol and water (80:20, V/V), and diluted with phosphate buffered saline (contain 0. 1% Tween-20, PBST). The detection results of FAPAS (Food Analysis Performance Assessment Scheme) by different batch three-in-one columns were close to the target value. The prepared three-in-one immunoaffinity column which could take the place of conventional single immunoaffinity column was able to meet the requirement for treatment of food and feed samples, and lay a foundation for one step enrichment, purification and detection of multi-mycotoxins.
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A high performance liquid chromatography-tandem mass spectrometric ( HPLC-MS / MS) method coupled with an immunoaffinity clean-up column was successfully developed for determination of aflatoxins (AFB1 , AFB2 , AFG1 , AFG2 , AFM1 and AFM2 ) and zeranols ( α-zeranol, β-zeranol, α-zearalenol,β-zearalenol, zearalanone and zearalenone ). The sample was extracted with methanol-acetonitrile (20∶ 80, V/ V) after enzymatic digestion by β-glucuronidase / sulfatase, and the extraction solution was passed through glassy fiber filter paper and then diluted with phosphate buffer solution (PBS). The reconstituted solution was cleaned up with IAC-AZ immunoaffinity column, and then analyzed by HPLC-MS / MS in multiple reaction monitoring (MRM) mode. The results indicated that the linear detection range was 0. 03-6. 0 μg / L for AFB2 and AFG2 , and 0. 05-20 μg / L for the rest compounds. The correlation coefficients were above 0. 999. The limits of detection (LOD) and limits of quantitation (LOQ) were 0. 01-0. 03 μg / kg and 0. 04-0. 09 μg / kg, respectively. The recoveries of the aflatoxins and zeranols were in the range of 73. 6% -98. 4% at the spiked levels of 0. 5, 1 and 5 μg / kg, and the relative standard deviations (RSDs) were in the range of 1. 9% -11. 2% . The method was proved to be simple and accurate, and suitable for the rapid determination of aflatoxins and zeranols in animal-originated foods.
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Two hundred and fifty-seven samples of milk proceeding from different geographical regionsof Brazil were analyzed for determining the presence of aflatoxin M1 (AFM1). The AFM1extraction was carried out using immunoaffinity column, separated by reversed-phase (C-18) high performance liquid chromatography (HPLC), and quantified by fluorescence detector.The Limits of Quantification (LOQ) were 0.008 µg/kg and 0.080 µg/kg to the fluid and the powder milk, respectively. AFM1 were detected in 209 (81.3 %) samples, being 26 (63.4 %), 105(84.0 %) and 78 (85.7 %) of pasteurized, UHT (Ultra-high Temperature) and powder milk,respectively. The highest concentration of AFM1 in powder milk was found in one sample from Minas Gerais (1.210 µg/kg). In UHT and pasteurized milk, the highest levels were detected inone sample from Sergipe (0.120 µg/kg) and one sample from Goiás (0.050 µg/kg), respectively.None of the samples analyzed in this study exceeded the Brazilian legal limits for AFM1.
Duzentas e cinquenta e sete amostras de leite provenientes das diferentes regiões geográficasdo Brasil foram analisadas para realizar a determinação de aflatoxina M1 (AFM1). As AFM1foram extraídas por meio de colunas de imunoafinidade, separadas por cromatografia líquidade alta eficiência em fase reversa (C-18) e quantificadas por detector de fluorescência (CLAE-FL).Os limites de quantificação (LQ) foram de 0,008 µg/kg e 0,080 µg/kg para o leite fluidoe em pó, respectivamente. AFM1 foi detectada em 209 (81,3 %) amostras, sendo 26 (63,4 %), 105(84,0 %) e 78 (85,7 %) para o leite pasteurizado, UHT (Ultra-high Temperature) e em pó,respectivamente. A maior concentração de AFM1 no leite em pó foi encontrada em umaamostra proveniente de Minas Gerais (1,210 µg/kg). No leite UHT e pasteurizado, os maioresníveis foram encontrados em uma amostra de Sergipe (0,120 µg/kg) e Goiás (0,050 µg/kg),respectivamente. Nenhuma amostra analisada ultrapassou os limites da legislação brasileira emvigor para AFM1.
Subject(s)
Humans , Aflatoxin M1 , Brazil , Breast-Milk Substitutes , Chromatography, High Pressure LiquidABSTRACT
Two hundred and fifty-seven samples of milk proceeding from different geographical regions of Brazil were analyzed for determining the presence of aflatoxin M1 (AFM1). The AFM1 extraction was carried out using immunoaffinity column, separated by reversed-phase (C-18) high performance liquid chromatography (HPLC), and quantified by fluorescence detector. The Limits of Quantification (LOQ) were 0.008 µg/kg and 0.080 µg/kg to the fluid and the powder milk, respectively. AFM1 were detected in 209 (81.3 %) samples, being 26 (63.4 %), 105 (84.0 %) and 78 (85.7 %) of pasteurized, UHT (Ultra-high Temperature) and powder milk, respectively. The highest concentration of AFM1 in powder milk was found in one sample from Minas Gerais (1.210 µg/kg). In UHT and pasteurized milk, the highest levels were detected in one sample from Sergipe (0.120 µg/kg) and one sample from Goiás (0.050 µg/kg), respectively. None of the samples analyzed in this study exceeded the Brazilian legal limits for AFM1.
Duzentas e cinquenta e sete amostras de leite provenientes das diferentes regiões geográficas do Brasil foram analisadas para realizar a determinação de aflatoxina M1 (AFM1). As AFM1 foram extraídas por meio de colunas de imunoafinidade, separadas por cromatografia líquida de alta eficiência em fase reversa (C-18) e quantificadas por detector de fluorescência (CLAE-FL). Os limites de quantificação (LQ) foram de 0,008 µg/kg e 0,080 µg/kg para o leite fluido e em pó, respectivamente. AFM1 foi detectada em 209 (81,3 %) amostras, sendo 26 (63,4 %), 105 (84,0 %) e 78 (85,7 %) para o leite pasteurizado, UHT (Ultra-high Temperature) e em pó, respectivamente. A maior concentração de AFM1 no leite em pó foi encontrada em uma amostra proveniente de Minas Gerais (1,210 µg/kg). No leite UHT e pasteurizado, os maiores níveis foram encontrados em uma amostra de Sergipe (0,120 µg/kg) e Goiás (0,050 µg/kg), respectivamente. Nenhuma amostra analisada ultrapassou os limites da legislação brasileira em vigor para AFM1.
Subject(s)
Aflatoxin M1/analysis , Milk/microbiology , Mycotoxins , Cattle , Chromatography, High Pressure LiquidABSTRACT
A method was developed for the determination of tetrodotoxin in marine organisms by high perfor-mance liquid chromatography-mass spectrometry with immunoaffinity column. The samples were extracted with 1% acetic acid methanol solution and diluted with phosphate buffer at pH 7-8. After cleaned up by immuno-affinity column, the samples were analyzed by LC-MS/MS and quantitatively determined by external standard method. The chromatographic separation was performed on an ACQUITY UPLC BEH Amide column with gradient elution by using acetonitrile and 5 mol/L ammonium acetate solution containing 0. 1% formic acid as mobile phase. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode. Linear ranges of TTX was in the range of 0. 3 -20. 0 μg/L with correlation coeffi-cient more than 0. 997. The quantification limit of the method was 0. 3 μg/kg. The recoveries of standard addition for tetrodotoxin were 88. 7%-102. 3%, and the relative standard deviation was 2. 0%-6. 4%. The method could be used to identify and quantify tetrodotoxin in marine organisms with satisfactory reproducibility and sensitivity.
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Anultra-performancehydrophilicinteractionliquidchromatography-triplequadrupolemass spectrometric ( UPLC-MS/MS) method was developed for the determination of tetrodotoxin ( TTX) in human urine and plasma. After the sample was cleaned-up and concentrated by immunoaffinity column, the separation of the TTX was carried out on an Acquity UPLC BEH amide column (100 mm×2. 1 mm, 1. 7 μm) with gradient elution using mobile phases of 0. 1% ( V/V) formic acid in water and acetonitrile. The analyte was detected by positive electrospray ionization mass spectrometry in the multiple reaction monitoring ( MRM) mode, and quantified by external solvent standard calibration. The measuring ranges of TTX in urine and plasma were 0. 05-400 μg/L. The average recoveries were 92%-95% and 91%-96% for TTX respectively spiked in urine and plasma with relative standard deviations of 3 . 3%-7 . 2% and 3 . 9%-7 . 8% ( n=5 ) . The limits of detection (LOD, S/N=3) and limits of quantitation (LOQ, S/N=10) of TTX were 0. 02 μg/L and 0. 05μg/L for urine and plasma, respectively. This method is suitable for the detection of TTX in urine and plasma for both forensic and clinical purposes.
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In this study, we developed a novel tool for purifying two mycotoxins, aflatoxin B1 (AFB1) and zearalenone (ZEN), in feed. This system utilized monoclonal antibodies (mAbs) against AFB1 and ZEN, and magnetic nanoparticles (MNPs). Among ten MNPs with different diameters and functional groups, a 100-nm diameter MNP (fMA) conjugated to an amine group (-NH2) was found to be optimum for coupling with mAbs. The optimal mAb concentrations for coupling to the fMA along with mycotoxin purification capacities of the fMA-mAb conjugates (fMA-AFB1 and fMA-ZEN) were determined. A comparison of mean recovery rates (from corn and product X feed) between the fMA-mAb conjugates and immunoaffinity columns (IAC-AFB1 and IAC-ZEN) showed that the rate for fMA-AFB1 (90~92% and 81~88%) was higher (p > 0.05) than that of IAC-AFB1 (81~84% and 72~78%) for AFB1 (5, 10, 15 ng/mL), and the rate for fMA-ZEN (99~100% and 92~94%) was significantly higher (p 30 min). This study suggests that the novel purification system we developed would be a useful tool for monitoring and regulating mycotoxin contamination in feed, and replace IAC methods.
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Aflatoxin B1 , Antibodies, Monoclonal , Magnetics , Magnets , Mycotoxins , Nanoparticles , Zea mays , ZearalenoneABSTRACT
Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-beta-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.
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Animals , Mice , Antibodies, Monoclonal/biosynthesis , Antigens, Viral/analysis , Capsid Proteins/genetics , Chicken anemia virus/genetics , Chickens , Circoviridae Infections/blood , Escherichia coli/genetics , Immunohistochemistry/veterinary , Liver/virology , Mice, Inbred BALB C , Microscopy, Fluorescence/veterinary , Poultry Diseases/blood , Specific Pathogen-Free Organisms , Thymus Gland/virologyABSTRACT
AIM:To bring forward a method of determining aflatoxin G_2、G_1、B_2、B_1 in six kinds of traditional Chinese drugs by HPLC.METHODS:After being extracted by 70% methanol,purified by immunoaffinity column,aflatoxins were analysed by HPLC with fluorescence detection.RESULTS:Aflatoxin G_2、B_2 showed a good linear relationship at a range of 1.5-60pg,and Aflatoxin G_1、B_1 at a range of 5-200 pg,r＞0.999 9.The recovery was between 60%-120%.CONCLUSION:The method is simple,accurate and can be used to determine aflatoxin G_2、G_1、B_2、B_1 in Naoliqing Pill,Renshen Yangrong Pill,Rensen Jiapi Pill,Sanqi Tablet,Jinshuibao Capsule and Bailine Capsule.
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Produtos derivados de leite, como o queijo, podem estar com aflatoxina M1 (AFM1) quando o gado leiteiro consome raçäo contendo aflatoxina B1 (AFB1). Amostras de queijo tipo prato e tipo parmesäo ralado foram coletadas na cidade de Belo Horizonte pela Vigilância Sanitária de Minas Gerais - Brasil. Foi obtido um extrato purificado através de extraçäo com diclorometano, seguido de lavagem com n-hexano e purificaçäo em coluna de imunoafinidade. A quantificaçäo da AFM1 foi feita por cromatografia líquida de alta eficiência (CLAE) usando detetor de fluorescência. AFM1 foi detectada em todas as marcas de queijo tipo prato analisadas, em uma faixa de concentraçäo de 0,02 a 0,54 ng/g e média de 0,15 ng/g. Em queijo ralado, tipo parmesäo, AFM1 foi detectada em 13 das 14 marcas de amostras analisadas (93 por cento), em faixa de concentraçäo de 0,04 a 0,30 ng/g e média de 0,14 ng/g. (AU)
Aflatoxin M1 was determined in Soft and Parmesan cheese by immunoaffinity columnand liquid chromatography. Milk products such as cheese may be contaminated by aflatoxin M1 (AFM1)when dairy cattle have consumed feeds contaminated with aflatoxin B1 (AFB1). Samples of Soft andParmesan cheese were collected in Belo Horizonte city, by the Inspection Service of Minas Gerais Brazil. A purified extract was obtained by extraction with dichloromethane followed by a washing withn-hexane and immunoaffinity column clean-up. The quantification of AFM1 was done by highperformance liquid chromatography (HPLC) using a fluorescence detector. AFM1 was detected in allsoft cheese brands analysed, in concentrations ranging between 0.02 and 0.54 ng/g and mean level of0.15 ng/g. In grated cheese, Parmesan variety, AFM1 was detected in 13 of 14 brands analysed (93%),in the range 0.04-0.30 ng/g with mean level of 0.14 ng/g. (AU)
Subject(s)
Cheese , Chromatography, Liquid , Aflatoxin M1 , Aflatoxin B1ABSTRACT
Objective:To observe the effect of CD34 immunoaffinity column on the isolation of hematopoietic stem and progenitor cells of cord blood and the proliferatory and expansion characteristics of CD34 + cells in vitro.Methods:CD34 +cells were isolated from cord blood using CD34 immunoaffinity column.Cell surface antigens were analysed by FACS.The separated and un separated cells were cultured with human hematopoietic growth factors(HGFS)in liquid culture system and CFU GEMM culture system.Results:CD34 + cells were enriched with a purity of 49.62%?17.69% and a recovery of 54.38%?11.91% using the immunoaffinity column.The separated CD34 +cells and cord blood MNC expanded to 561.00 folds and 44.44 folds respectively after cultured with HGFS for 20 days.The percentages of CD34 + cells in separated group and control were 53.38% and 7.91% respectively after cultured for 12 days.The number of CFU GEMM in separated group was significantly higher than that in control(P
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Objective To develop a method for the determination of aflatoxins B1,B2,G1 and G2 in Chinese herbal medicine.Methods The herbal medicine samples were cleaned up on immunoaffinity columns and analyzed by high performance liquid chromatography(HPLC) with fluorescence detection.For chromatographic separation,a Zorbax SB C18(4.6 mm?150 mm,5 ?m) column was employed.The separation was carried out as the mobile phase of methanol-0.01 mol/L KH2PO(44+6)with a flow rate of 0.5 ml/min at column temperature of 35 ℃.The reaction tube temperature of postcolumn derivatization system was 70 ℃.The detection was observed by fluorescence with excitation at 360 nm and emission wavelength at 425 nm.Results The calibration curve showed good linearity in the range of 12.00-300.00 ?g/L for aflatoxin B1,and 24.00-600.00 ?g/L for aflatoxin B2,G1 and G2.The lowest limits of detection for aflatoxins in Chinese herbal medicine were 0.025 ?g/L for AFB1,0.085 ?g/L for AFB2,0.060 ?g/L for AFG1,and 0.055 ?g/L for AFG2,and the correlation coefficients were ≥0.996.Recoveries were 81.7%-101.2% for aflatoxin B1,B2,G1 and G2 spiked to Fructus amomi,Radix angelicae sinensis,Rhizoma polygonati,Rhizoma atractylodis macrocephalae and Rhizoma dioscoreae at the level of 5 ?g/L,and the relative standard deviations were 0.7%-4.9% in all instances.Conclusion The method has good stability,high sensitivity and high selectivity,and it is applicable to the determination of aflatoxins in Chinese herbal medicine.
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0.999 9.The recovery was between 60%-120%.CONCLUSION:The method is simple,accurate and can be used to determine aflatoxin G2、G1、B2、B1 in Naoliqing Pill,Renshen Yangrong Pill,Rensen Jiapi Pill,Sanqi Tablet,Jinshuibao Capsule and Bailine Capsule.