Lecture 16 - Mycotoxins Flashcards
what are mycotoxins?
chemical toxins produced by molds
what species are the main producers of mycotoxins?
aspergillus, fusarium, penicillin
what percentage of the world’s total crops are affected with unacceptable levels of mycotoxins?
25%
what are the 5 major classes of mycotoxins?
- aflatoxins
- ochratoxins
- trichothecenes
- fumonisins
- zearalenone
name some aflatoxins
B1, B2, M1 M2, G1 and G2
name some ochratoxins
ochratoxin A, OTA
name some trichothecenes
- deoxynivalenol (DON)
- T2
- HT-2
name some fumonisins
- FBs (FB1, FB2, FB3)
- patulin
what’s the abbreviation for zearalenone?
ZEN
in what kind of feedstuffs can aflatoxins be present?
sorghum, soybeans, corn, wheat, barley
in what kind of feedstuffs can trichothecens be present?
barley, oats, sorghum, soybeans, corn, wheat
in what kind of feedstuffs can ZEN be present?
wheat, sorghum, corn, barely, silage
describe the solubility of aflatoxins
- soluble in polar organic solvents (chloroform, methanol, ethanol, propyl alcohol)
- poorly soluble in water
- insoluble in petroleum ether, ethyl acetate, and hexane
describe aflatoxin stability depending on pH
- stable in neutral solutions
- resistant to strong acids
- rapid decomposition in alkaline solutions
what happens to aflatoxins under UV light?
- groups B & G fluoresce
- destructive for low concentration of AFB
describe aflatoxins’ heat stability
- stable in 200C
- no decomposition until 268C
- hard to destroy under normal temps
what methods can be used to control and detox?
physical - grinding & rinsing - heat treatment - irradiation - inorganic absorption chemical methods - alkalization - ozone degradation biological - microbial adsorption - microbial degradation - biological degradation
solubility of ZEN?
- water insoluble
- slightly soluble in hexane
increasingly soluble in benzene, acetonitrile, methylene chloride, methanol, ethanol, and acetone - soluble in aqueous alkali
pH stability of ZEN?
- stable when neutral
- ester bond open in alkaline environment
ZEN response to UV light?
exhibits blue-green fluorescence (360 nm), but also intense green fluorescence (260 nm)
ZEN heat stability?
- melting point is 161C
- difficult to destroy with normal cooking
expand DON
deoxynivalenol
solubility of DON?
- easily soluble in water and polar solvents
- insoluble in hexane and diethyl ether
pH stability of DON?
- stable in neutral AND acid environments
- sensitive to alkaline
DON response to UV light?
- absorption peak under short-wave UV light
- decomposed under high tense UV light
DON heat resistance?
resistant
- stable in 120C for 1 hour
- decomposed 170C for 14 mins under pH 10
- hard to destroy by normal cooking
true or false: mycotoxins can be detected from the body
false, because they can be transformed
health effect of aflatoxin
carcinogenic
health effect of ctrinin
nephrotoxic
health effect of fumonisin
carcinogenic, hepatotoxic
health effect of trichothecenes
cytotoxic, immunosuppressive
health effect of ochratoxin
carcinogenic, nephrotoxic, hepatotoxic, teratogenic
health effect of patulin
carcinogenic, immunotoxic, genetoxic
health effect of zearalenone
estrogenic activity, potential carcinogenic and teratogenic
what are the general steps for mycotoxin analysis
- sampling
- pre-treatment/extraction
- clean-up
- detection
what are some ways mycotoxins can be extracted?
- liquid-liquid extraction
- supercritical fluid extraction
- solid phase extraction
- solid phase micro-extraction
what are some ways mycotoxins can be detected?
- TLC
- GC
- HPLC
a mycotoxin-sampling plan is defined by a mycotoxin test procedure and a(n) _____/_____ limit
accept/reject
true or false: traditional methods of sampling an be applied to mycotoxins
false, because population can be heterogeneous
how do you determine sample number for mycotoxins in large and small lots?
- square root of total number of containers for large lots
- one fourth of the containers for small
liquid-liquid extraction makes use of partitioned liquid phases to separate analytes with different affinities. what solvents can be used to extract mycotoxins?
polar solvents
- methanol
- acetone
- acetonitrile
- ethyl acetate
- diethyl ether
- toluene
- chloroform
what does water do to mycotoxins?
breaks up complexes along with acid
solid phase extraction is based on _____
molecularly imprinted polymers (MIP)
what are MIPs?
- molecularly imprinted polymers
- synthetic receptors for mycotoxins
- used for extraction, cleanup, and concentration
what kinds of solid phases are out there?
- C-18
- silica gel
- anionic and cationic exchange
- immunosorbents
- molecularly imprinted polymers
what’s an immunoaffinity column?
- analyte (mycotoxins) are bound selectively to antibodies
- used for solid phase concentration
how do immunoaffinity columns concentrate mycotoxins?
- mycotoxins bind to antibodies
- rinsing step removes most possible interferences
- toxin eluted by antibody denaturation
true or false: immunoaffinity columns are highly selective
true
why do we bother doing sample clean-up?
- remove interference
- concentrate analyte for better detection
describe TLC
- clean, smooth glass plate has a thin layer of silica gel applied
- sample is applied as a spot just above the solvent level
- solvent rises by capillary action
- compounds separate out based on affinity for solvent and silica
what is TLC used for?
- qualitative analysis (characterization)
- semi-quantitative measurement using fluorescence and comparing it with a standard
how is detection achieved using gas chromatography?
- linking system to MS, flame ionization, or fourier transform infrared spec
true or false: mycotoxins are often volatile so they are suitable for analysis using GC
false - they’re not volatile and need to be derivatized. GC isn’t really used for commercial purposes
what kind of reactions can derivatize mycotoxins?
- silylation
- polyfluoroacylation
how is detection accomplished using HPLC?
by linking the system to often one of the following detectors
- UV
- fluorescence
- amperometric
- spectrofluorimetric
since mycotoxins have natural fluorescence, they can be directly detected with what?
HPLC-fluorescence (HPLC-FD)
which detectors have greater sensitivity than HPLC-FD?
MS with electrospray or pressure chemical ionization
the LOD for HPLC-MS can be between _____ and _____ ug/kg
0.1 and 1
advantages of HPLC?
- high resolution
- improved limit of detection
- possibility of being coupled to multiple detection automated
which method of detection is widely accepted as an official method for the determination of mycotoxins?
HPLC
limitations of chromatographic assays
- $$$ equipment
- time-consuming
- requires clean-up procedures
expand ELISA
enzyme linked immunosorbent assay
describe the principle of ELISA
based on the ability of a specific antibody to distinguish the 3-D structure of a specific mycotoxin
what kind of ELISA is commonly used in mycotoxin analysis?
direct competitive
what’s a competitive assay for ELISA like?
we got mycotoxin. and we got enzyme-toxin conjugate. we let those fight to bind for antibodies
the enzyme conjugate makes a colored, fluorescent or chemiluminescent product (so more mycotoxin = less color)
