Recent Advances in Analytical Methodology for Cyclopiazonic Acid

1
Recent Advances in Analytical Methodology for
Cyclopiazonic Acid

• Joe W. Dorner
• USDA, ARS
• National Peanut Research Laboratory

2
Cyclopiazonic Acid
3
CPA-Producing Fungi
Penicillium commune
P. cyclopium
P. griseofulvum
P. crustosum
P. camembertii
Aspergillus flavus
A. tamarii
A. oryzae
A. versicolor
4
Natural Occurrence of CPA
Corn
Sunflower
Mixed Feeds
Peanuts
Cheese
Eggs
Millet
Milk
5
Mycotoxicoses Involving CPA
Turkey X Disease (peanut meal)
Kodua Poisoning (millet)
Quail Death (mixed feed)
6
Analytical Methods for CPA
TLC
Colorimetry/spectrophotometry
HPLC
ELISA
Capillary electrophoresis
7
Quantitation Limits for CPA
TLC
100 ng/g
Spectro.
80 ng/g
HPLC
50-100 ng/g
ELISA
100-600 ng/g
8
Typical Cleanup Protocolfor CPA Extracts
Extraction
Filtration/centrifugation
Liquid-liquid extraction (3-5 times)
Filtration
Column chromatography or SPE
Concentration
9
Normal Phase HPLC of CPA
Std.
Corn, 300 ng/g QL 100 ng/g
Fungal culture
Goto et al., 1987
10
Reverse Phase HPLC of CPA (linear
gradient)
Corn, 230 ng/g QL 50 ng/g
Peanuts, 430 ng/g QL 100 ng/g
Urano et al., 1992
11
Cleanup with Immunoaffinity Column(IAC)
Extraction
Filtration
Cooling
Filtration
Concentration
IAC cartridge
12
HPLC Conditions
Column Zorbax Rx-SIL 250 x 4.6 mm
Mobile Phase 50027516 hexane/
reagent alcohol/TRIS
Flow Rate 1.2 mL/min
Detector Diode Array
13
Normal Phase Partition HPLC of CPA
CPA standard 20 ng
14
Naturally contaminated peanuts, 130 ng/g
15
Recovery of CPA from PeanutsWith IAC Cleanup and
HPLC
Added (ng/g)
CV ()
Recd
10
90.8
7.6
100
83.7
6.6
Values are the mean of 5 determinations. Limit of
quantitation 2.5 ng/g.
16
Natural Contamination of Peanuts with CPA
and Aflatoxin
Aflatoxin (ng/g)
Seed Size
CPA (ng/g)
26.5
Jumbo
3.0
0.0
Medium
44.9
122.4
Number 1
145.6
0.6
Oil Stock
170.5
7,094.3
Damage
8,105.0
17
CPA Analysis by ELISA
Detection Limits (ng/g)
Commodity
No Cleanup
IAC Cleanup
Corn
100
2.0
600
Peanuts
4.7
300
Mixed Feed
4.4
Yu et al., 1998
18
Immunoaffinity Column Cleanup
Advantages

• Cleaner extract for quantitative step
• Improved recoveries
• Good reproducibility
• Lower quantitation limits

19
Immunoaffinity Column Cleanup
Advantages

• Cleaner extract for quantitative step
• Improved recoveries
• Good reproducibility
• Lower quantitation limits

Disadvantage

• Not readily available

20
Capillary Electrophoresis Method
Commodities Tested
Milk and dairy products
Cleanup Liq-liq part. SPE cartridge
Recovery 78-81
Quantitation Limit 20 ppb
Prasongsidh et al., 1998
21
Summary

• Many methods used for CPA analysis

22
Summary

• Many methods used for CPA analysis
• Problems associated with sample cleanup,
  reproducibility, and high quantitation limits

23
Summary

• Many methods used for CPA analysis
• Problems associated with sample cleanup,
  reproducibility, and high quantitation limits
• Immunoaffinity columns address most of the
  problems, but are not readily available

24
Summary

• Many methods used for CPA analysis
• Problems associated with sample cleanup,
  reproducibility, and high quantitation limits
• Immunoaffinity columns address most of the
  problems, but are not readily available
• Capillary electrophoresis recently applied for
  successful CPA analysis