A New Look at Citrinin: Quantitative Analysis by HPLC and LCMSMS Utilizing Immunoaffinity Purification
Jordon Bierbaum, Julie Brunkhorst, Heather Henderson, Ronald Niemeijer
Trilogy Analytical Laboratory, Washington, MO 63090
Citrinin, a naturally occurring toxin produced by Penicillium, Monascus, and Aspergillus species of fungus, is most commonly considered a nephrotoxic compound. It is a mycotoxin that typically occurs in the storage processes of grains such as corn, soybeans, wheat, barley, and rice (red yeast rice). Evaluations were conducted through an R-Biopharm Rhone immunoaffinity column. This method allowed a single-extraction validation of both HPLC and LCMSMS with acceptable recoveries. This analysis uses the same purified extract to be injected onto the HPLC and/or LCMSMS. Use of this method has shown limits of quantification at 10ppb for several different matrices giving a better look at detection of this toxin. EFSA (European Food and Safety Authority) recommends levels of daily Citrinin consumption in children to be below 53 µg/kg, and adults 100 µg/kg.
|Procedure for Analyzing Citrinin by HPLC and LCMSMS|
Four Commodities (Feed, Pet Food, Red Yeast Rice, and Corn) were analyzed by both HPLC and LCMSMS under the below conditions. The study was to prove that both Accuracy and Precision analysis would meet validation guidelines:
Weigh out 25 g of sample into a 250 ml flask.
Add 100 ml of 3/1 (MeOH/H2O). Shake for 1 hour. Filter sample after extraction.
Dilute 2 ml extract with 18 ml PBS solution.
Filter extract through glass fiber.
Take 10 ml of filtered sample through the column at a flow rate of 1-2 drops/second
Wash Column with 10 ml of 10 mM Phosphoric Acid (pH 7.4).
Elute column with 1 ml of 100% Methanol, followed by 1 ml of DI Water for a 2 ml total.
If using HPLC, transfer 500 µl into silanized 2ml clear vial. Inject 50 µl
If using LCMSMS, pipette 400 µl of Standards/Matrix Spikes/Samples and add 400 µl H2O to all Standards/Matrix Spikes/Samples into a clear silanized 2ml LCMSMS vial, for a total of 800 µl. Vortex. Inject 50 µl onto the LCMSMS system.
HPLC Conditions – Equipment or Equivalent
Shimadzu Sil-20AXR Injector
Shimadzu RF-20AXS Fluorescence Detector
LC-20AD XR Pump
LC Solutions Chromatography Software
Column: Phenomenex Prodigy 5µ 0DS3-100A, 250 x 4.6 mm 5 micron
Mobile Phase: 59.7/39.5/0.009, Water/Acetonitrile/Acetic Acid
Flow Rate: 2.5ml/min
Fluorescence Detector: Ex - 331nm Em - 500
Approximate Retention Time: 8.0 minutes
LCMSMS Conditions – API3000
Equipment or Equivalent LC Conditions
Shimadzu Sil-20A HT Injector
Analyst LCMSMS Software
Column: Phenomenex Kinetex 2.6u C18, 100A 100 x 3.0mm
Column temp: 40oC
Flow rate: 0.5ml/min
Mobile Phase A: LCMSMS grade water with 0.1% Formic Acid
Mobile Phase B: LCMSMS grade Methanol with 0.1% Formic Acid
Time (min) %A %B
1.0 95 5
2.0 50 50
5.0 50 50
10.49 5 95
10.50 95 5
Total Run Time: 12.0 minutes
Injection Volume: 50µl
MS system: API3000
Ionization: ESI positive
Source Temp: 500oC
Curtain Gas: 15
Nebulizer Gas: 7
CAD Gas: 5
Acquisition: Analyst 1.6.2 (MRM with full scan)
Citrinin was analyzed, using the same extract, by both HPLC and LCMSMS. This allows for confirmation of positive results to be determined by two different systems of analysis simultaneously.
For Feed Analysis:
For Red Yeast Rice Analysis:
Thus, both HPLC and LCMSMS analysis of Citrinin can be quantitatively reported at levels of 10 ppb.
* Special thanks to R-Biopharm Rhone for supplying columns for analysis purposes
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