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HPLC-column : C-18 column for aflatoxin 150 x 4.6 mm;
Flow phase : methanol-water 45:55
Flow rate : 1.0 mL/min      Â
Injection volume: 20μL
Fluorescence detector : λ-excitation wavelength: 365 nm λ-emission wavelength: 455 nm
An overview of the detection technology of aflatoxin in Chinese herbal medicine
First, the status quo of Chinese herbal medicine quality and safety
Chinese herbal medicine is an important basis for Chinese medicine to prevent and cure diseases. Chinese medicinal materials in the field of growth, harvesting processing, storage and transportation, etc. will be mildewed due to contamination of mold. The toxic metabolites secreted by these molds make the Chinese medicinal materials moldy and degenerate, directly threatening the safety of the people. The mycotoxins in traditional Chinese medicine mainly include aflatoxin, ochratomycin, zearalenone, hyalin toxin, fumonisin and the like. The type of Chinese medicinal materials is the main factor affecting the contamination of mycotoxins. The easily contaminated Chinese medicinal materials can produce mycotoxins under different conditions of temperature and humidity. Temperature and humidity conditions can also affect the growth of mycotoxins. For example, aflatoxins can be produced under different conditions of temperature and humidity; ochratoxin needs to be produced under high humidity conditions.
In recent years, as Chinese herbal medicines continue to heat up in the international market, their safety has attracted more and more attention. Mycotoxin residues are traces of exogenous toxic and harmful substances in Chinese herbal medicines. They generally do not exhibit acute toxicity, but usually have strong accumulation, causing cancer, teratogenicity and mutagenic effects, which seriously affect the safety of Chinese herbal medicines.
2. National regulations related to the control of mycotoxin in Chinese herbal medicines
In recent years, food and drug supervision departments at all levels have continuously increased the supervision of Chinese medicines and strived to maintain the overall stability of Chinese medicine quality.
The State Food and Drug Administration's Notice on Regulating the Order of Traditional Chinese Medicines to Strictly Investigate Violations of Laws and Regulations (Guo Shi Jian Jian [2012] No. 187 Document) requires strengthening aflatoxin, heavy metals and harmful elements, pesticide residues, etc. The detection and control of safety indicators ensure the quality and safety of Chinese herbal medicines. In addition, enterprises are required to have inspection equipment and capabilities that are compatible with the production varieties, and strictly control quality inspection.
"National Drug Safety "Twelfth Five-Year Plan", the plan clearly states "to carry out rapid drug testing technology research, build a platform for testing technology sharing", "accelerate the application of rapid drug testing technology at the grassroots level, and configure rapid testing equipment", Strengthen the requirements of county-level institutions for rapid inspection capacity building.
Third, Chinese herbal medicine mycotoxin testing items and limit standards
The State Food and Drug Administration has 14 kinds of medicinal materials such as Baiziren, lotus seeds, gentian, betel nut, malt, nutmeg, cassia seed, Yuanzhi, Huanren, jujube, earthworm, medlar, leeches, and glutinous rice. Add a "aflatoxin" test item, the limit is "aflatoxin B1 must not exceed 5 μg / kg; aflatoxin G2, aflatoxin G1, aflatoxin B2 total amount must not exceed 10 μg / kg".
Appendix IX V of the 2010 edition of the Pharmacopoeia The determination of aflatoxin was performed by high performance liquid chromatography, and the supplemental pharmacopoeia described the method to establish an increased post-column photochemical derivatization method.
Fourth, Chinese herbal medicine aflatoxin detection program introduction
(1) Instrumental analysis
Characteristics
advantage
Disadvantage
Accurate, sensitive, and anti-interference, the method of confirmation by the national inspection department.
The instrument has high cost, high technical requirements for operators, complicated operation and long time.
Immunoaffinity column - high performance liquid chromatography : in accordance with the 2010 edition of the Pharmacopoeia Appendix IX V aflatoxin determination high performance liquid chromatography, additional Pharmacopoeia description of the method to establish an increased post-column photochemical derivatization method.
1. Equipment and consumables configuration
Serial number
Product name
Specifications and parameters
Quantity
use
1
High performance liquid chromatography
One
Riddle
2
Fluorescence detector
One
Reading fluorescent signal
3
Pribolab mycotoxin-specific column
150/250mm
Item No.: PRC-18
One
Compatible with various chromatographs for detection of mycotoxins
4
PriboFast® aflatoxin total immunoaffinity column
3ml, 25pcs/box, fast column type,
Item No.: IAC-011-3
Several
High specificity, purified sample
5
PriboFast® KRC photochemical post-column derivatization reactor
Item No.: EQ-KRC
One
Suitable for aflatoxin detection
6
High speed homogenizer
Up to 22000rpm, item number: EQ-WR-1L
One
Corrosion resistant, high speed, homogeneous
7
PriboFast® fiberglass filter paper
100P, 110mm, 1.5μm
Item No.: GMF-110
a box
Mycotoxin-specific filtration
8
PriboFast® eight-position pump flow operator
Item No.: EQ-PUMP-8
One
Used to control the flow rate of the immunoaffinity column
9
Aflatoxin mixed standard
2 μg/ml aflatoxin B1, G1; 0.5 μg/ml aflatoxin B2; G2 (4/1/4/1) dissolved in acetonitrile,
Item No.: STD#1081
1ml
For the preparation of quantitative analysis standards
2. Sample preparation: Preweang provides different treatment solutions for Chinese herbal medicines (for detailed scheme, please contact Puribang, Puri State has developed an optimized treatment plan for various Chinese herbal medicine substrates)
3. Immunoaffinity column purification :
Enrichment - Wash - Elution - Collect all eluents for chemical derivatization.
4. Chemical Derivatization
Why use a photochemical derivative? Due to its strong UV absorption and fluorescence-producing properties, Aspergillus flavus can be subjected to HPLC-ultraviolet or fluorescence detection, but at the same time, B1 and G1 in aflatoxin are prone to fluorescence quenching in the aqueous mobile phase, so Derivatives are used to determine aflatoxins in many experiments. There are also many methods of derivatization: pre-column, post-column, and online. Due to the obvious advantages of post-column photochemical derivatization, it is now supplemented as a pharmacopoeia detection method. The main advantage lies in the fact that no chemical reagents or personnel are needed. No damage; no direct manipulation by personnel, avoiding errors; no need to worry about the derivative corrosion detector.
5. High performance liquid chromatography analysis
PriboFast KRC Photochemical Derivative Wavelength : 254 nm
Column temperature : 35 °C
HPLC configuration; high performance liquid chromatography with fluorescence detector; aflatoxin dedicated column C18 150 x 4.6 mm
(two) enzyme-linked immunosorbent assay
Characteristics
advantage
Disadvantage
High sensitivity, short time-consuming, simple sample processing, low requirements for technicians, low detection cost, and simultaneous detection of large quantities of samples. Suitable for rapid quantitative screening of large numbers of samples.
There may be a certain false positive rate (cross-reaction rate).
The PriboFast R Aflatoxin Total Kit from Pribolab has high sensitivity and accuracy for quantitative analysis of large numbers of samples.
1. The principle of the method
The aflatoxin antigen was pre-coated on the microplate by direct competition enzyme-linked immunosorbent assay, and the sample (or aflatoxin standard solution) and the horseradish peroxidase-labeled aflatoxin-specific antibody were added. The antigen in the sample or standard solution competes with the antigen pre-coated on the plate well to bind to the enzyme-labeled antibody. Unbound enzyme-labeled antibodies are removed upon washing. Add TMB coloring solution and read the absorbance. The absorbance of the sample is inversely related to the total amount of aflatoxin contained in it, and the total amount of aflatoxin in the sample can be obtained by comparison with the standard curve.
2. Equipment and reagent configuration:
Serial number
Product name
Specifications and parameters
Quantity
use
1
PriboFast R Aflatoxin Total Kit
96T, sensitivity: 0.1ppb
Item No.: EKT-011
a box
Quantitative analysis
2
PriboSpin centrifugal cartridge
25T
a box
Purification sample
3
Microplate reader (enzyme-linked immunosorbent assay)
Filter: 450nm
One
Compatible with various chromatographs for detection of mycotoxins
3. Sample pretreatment: Chinese herbal medicine: crushing, extracting, centrifuging, taking the supernatant and adding it to the PriboSpin centrifugal column for purification.
Why use the PriboSpin centrifugal cartridge? The matrix of Chinese medicinal materials is complex, and the sample liquid is darker in extraction and is not easily removed. The organic reagent extraction and purification method is cumbersome, and the reagent volatilization causes harm to the environment and the human body, and the purification effect is unstable. By using the PriboSpin centrifugal column, the extract can be centrifuged to remove impurities in the extract, which is convenient and quick, and has a good purification effect, and the recovery rate can reach more than 85%.
4. Detection steps
4.1 The immune reaction was carried out for 30 min; the plate was washed 5 times; the color reaction was carried out for 30 min; the reaction was terminated, and the OD value was read by the microplate reader.
5. Expression of analysis results
The results were interpreted using a microplate reader.
(3) Rapid detection method of immune gold standard
Characteristics
advantage
Disadvantage
The operation is simple, the cost is low, the time is short, and the detection is fast. Suitable for rapid qualitative screening of samples
There are certain false positives, data is inconvenient to save and analyze.
Pribolab's total amount of PriboStrip TM aflatoxin immunoassay is well-suited for rapid detection of test strips. It is suitable for detecting large sample sizes; short-term test results and control of funds can be quickly characterized and screened.
1. The principle of the method
The aflatoxin total rapid detection strip uses the principle of competitive inhibition immunochromatography. The total amount of aflatoxin in the sample binds to the colloidal gold-labeled specific anti-aflatoxin monoclonal antibody in the enzyme-labeled well, inhibiting the antibody. Binding to the AF-BSA conjugate on the NC membrane detection line. If the total content of aflatoxin in the sample is greater than 0.5 ug/L, the test line does not show color and the result is positive; otherwise, the test line is red and the result is negative.
2. Equipment and reagent configuration
Serial number
Product name
Specifications and parameters
Quantity
use
1
PriboStrip TM aflatoxin total immunogold standard rapid test strip
25T
Detection limit: 10ppb
Item No.: PR-011
a box
Qualitative analysis
3. Analysis step: After the Chinese medicinal materials are pulverized and dissolved, the supernatant is taken for detection. Pipette the sample into the well and read the results for 8-15 minutes. In the case of C-line color development: T-line color development is negative or deeper than C-line. The T line is lighter than the C line, or no coloration is a positive result.
Five, warm TIPS:
1. The favorable conditions for the growth and reproduction of fungi in traditional Chinese medicine are mainly suitable temperature and moisture. If the traditional Chinese medicine can be stored below 10 ° C and the water content is kept below 10%, it can effectively prevent mildew.
2. Personnel engaged in the research and testing of mycotoxins must pay attention to protection. For example, wear a protective coat cap. When performing fungal separation and culture work, wear a mask and try to prevent spores from flying.
3. If the operating table is leaking, it should be disinfected immediately with the new 5% sodium hypochlorite. When treated with 5% sodium hypochlorite (NaOCl), aflatoxin is destroyed within a few seconds and is a commonly used disinfectant.
4. There are also reports on the application of biological methods to detoxification. The low cost and high efficiency of biological methods may be a promising detoxification measure.
In view of the harm of mycotoxins to the human body, teachers who are struggling to fight on the front line of anti-drug must pay attention to protect themselves!
About Pribolab®
Pribolab® is one of the leading providers of mycotoxin detection solutions worldwide, with a strong R&D team and specialized mycotoxin detection technology for global agricultural production, food processing and food, feed industry The industry provides professional mycotoxin detection technology and product services, and provides testing and analysis of mycotoxins and food ingredients for food, feed and beverages.