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.
Mix thoroughly.
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

LC-20AD Pumps

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


Procedure continued

MS Conditions

MS system: API3000

Ionization: ESI positive

Source Temp: 500oC

Curtain Gas: 15

Nebulizer Gas: 7

CAD Gas: 5

IS: +3000V

Acquisition: Analyst 1.6.2 (MRM with full scan)

Citrinin - Feed and Pet Food.pngCitrinin - Red Yeast Rice and Corn.png

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:
Accuracy percent recovery ranged from 70.3% - 74.2% with an average of 72.1%
Precision spiked at 50 ppb ranged from 35.78 - 38.99, with a %RSD= 3.02
Accuracy percent recovery ranged from 70.6% - 84.5% with an average of 78.2%
Precision spiked at 50 ppb ranged from 38.45 - 41.79, with a %RSD= 2.99

For Pet Food Analysis:
Accuracy percent recovery ranged from 70.32% - 81.20% with an average of 73.17%
Precision spiked at 50 ppb ranged from 34.3 - 37.94, with a %RSD= 4.31
Accuracy percent recovery ranged from 75.9% - 82.4% with an average of 78.3%
Precision spiked at 50 ppb ranged from 36.9 - 40.7, with a %RSD= 3.70

For Red Yeast Rice Analysis:
Accuracy percent recovery ranged from 71.4% - 87.3% with an average of 80.3%
Precision spiked at 50 ppb ranged from 37.5 - 44.0, with a %RSD= 6.01
Accuracy percent recovery ranged from 76.2% - 84.7% with an average of 77.1%
Precision spiked at 50 ppb ranged from 38.8 - 42.6, with a %RSD= 3.36

For Corn Analysis:
Accuracy percent recovery ranged from 80.3% - 84.1% with an average of 82.5%
Precision spiked at 100 ppb ranged from 76.3 - 83.6, with a %RSD= 3.15
Accuracy percent recovery ranged from 75.1% - 91.6% with an average of 81.5%
Precision spiked at 25 ppb ranged from 20.7 - 25.0, with a %RSD= 10.9

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

** Poster display created by Kelley Siem



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