Pickering Laboratories


Method Abstract 220



The Mycotoxin Ochratoxin A is found in many matrices, such as grapes. As red and white wines are produced from grapes, the mycotoxin can be found in the final product. Therefore, a maximum limit for Ochratoxin A in red, white and rosé wine, other wine and/or grape-must-based beverages was set at 2.0 μg/kg by Commission Regulation (EC) No. 123/2005. Thus, these beverages must be analyzed for their Mycotoxin content before they are sold on the market.

The standard procedure for analysis of Ochratoxin A in matrices is to use immunoaffinity column cleanup. The cleanup may either be performed manually or much more convenient by an automated system. The following application note shows how wine samples can be processed with the OtaCLEAN™ immunoaffinity columns in combination with the fully automated sample preparation system AcceCLEAN™ (LCTech, Germany) producing high recovery rates and repeatability.


For the analysis, standard laboratory equipment such as beakers, funnels, filters, and stirrers are required. Furthermore, a centrifuge or a syringe with PVDF filters may be needed. The HPLC system should at least have an injection system (manually or automated), an isocratic pump, a column oven with guard and HPLC column, and a sensitive fluorescence detector. Using a post-column derivatization system further increases the sensitivity by a factor of 6 to 8, and the increases selectivity as interfering peaks are removed significantly.


  • Extraction solution, 5 % NaHCO3 with 1 % PEG 8000 (e. g. Sigma 4423)
  • PBS buffer pH 7.2
  • Water (HPLC grade)
  • Methanol (HPLC grade)
  • Acetonitrile (HPLC grade)
  • Glacial acetic acid p. a.Sodium hydroxide 1.0 N

Acceclean System




Sample Preparation

Mix 10 mL of wine thoroughly with 10 mL of extraction solution for 3 min.

Pass the extract through a plaited filter.

10 mL of the filtered extract are diluted with 40 mL PBSbuffer (pH 7.2). If there is any precipitation during mixing with the buffer, the sample volume has to be filtered by means of a syringe filter (PVDF) or alternatively, centrifuged.

25 mL are applied on the immunoaffinity column by LCTech program 2 in the AcceCLEAN™ (25 mL, flow rate 2mL/ min); please follow the manual for using the instrument.

Dilute or concentrate the eluate to your requirements and analyze by HPLC.

Chromatographic Conditions

Eluant: Water/Methanol/Acetonitrile/glacial acetic acid40/55/5/1 (v/v)
HPLC Eluent: 125 x 3 mm; RP C18; 3 μm; 120 Å
Flow Rate: 0.6 mL/min
Injection Volume: 10 – 100 μL
Column Temperature: 40 °C
Post-column Reagent: 1 N NaOH at 0.3 mL/min
FLD wavelength: 390 Ex; 440 Em (with derivatization)
                          335 Ex; 460 Em (no derivatization)

M 218

Black        Wine blank containing OTA below 1 ppb

Red          Wine sample spiked with 5 ppb after extraction

Orange    Wine sample spiked prior to extraction with 5 ppb

Brown      OTA standard (12.5 ng / 2 mL diluted to HPLC eluant to 5 ppb)


The recovery rates obtained at levels of 1 and 5 ppb were 99 ± 6 %.

Retention Times

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