Tarhana is a traditional fermented, sun-dried Turkish food containing yogurtand cereals. There are several potential sources of mycotoxins in tarhana, such as contamination of ingredients or formation during preparation, when water activity is suitable for fungal growth and may lead to mycotoxin production during fermentation or subsequent sun-drying. To optimize an immunoaffinity column method and carry out single-laboratory validation for the determination of aflatoxins B, B₂, G, and G₂ together with ochratoxin A (OTA) in tarhana. A homogenized sample was extracted with methanol-acetonitrile-water (25 + 25 + 50) using a high-speed blender. The sample extract was filtered, diluted with phosphate buffered saline (PBS) solution, and applied to a multi-immunoaffinity column (AFLAOCHRA PREP®). Aflatoxins and OTA were removed with neat methanol and then directly analyzed by reverse-phase LC with fluorescence detection using post-column bromination (Kobra Cell). Test portions of blank tarhana were spiked with a mixture of total aflatoxins and OTA to give levels ranging from 2.5 to 10.0 and 1.5 to 6.0 μg/kg, respectively. Recoveries for total aflatoxins and OTA ranged from 82 to 93 and 78 to 94%, respectively, for spiked samples. Based on results for spiked tarhana (30 replicates, each at three levels), the relative standard deviation for repeatability ranged from 1.4 to 7.2 and 3.6 to 7.7% for total aflatoxins and OTA, respectively. The performance characteristics for recovery, repeatability, and sensitivity have demonstrated that the method meets method performance criteria for use for official purposes. The method was demonstrated as being applicable to naturally contaminated samples of tarhana of varied composition obtained from local markets in Turkey. This is the first immunoaffinity column method for simultaneous analysis of aflatoxins and OTA in traditional Turkish food (tarhana). Suitability was demonstrated by single-laboratory validation for official purposes in Turkey. The method was demonstrated as suitable for naturally contaminated samples of tarhana of varied composition.

: Pekmez and pestil are traditional Turkish foods made from concentrated grape juice, which can be contaminated with mycotoxins such as aflatoxins and ochratoxin A (OTA). : To carry out a single-laboratory validation of a method to simultaneously determine aflatoxins B, B₂, G, and G₂ and ochratoxin A in pekmez and pestil. : The homogenized sample is extracted with methanol-water (80 + 20) using a high-speed blender. The (sample) extract is filtered, diluted with phosphate-buffered saline solution, and applied to a multi-immunoaffinity column (AFLAOCHRA PREP®). Aflatoxins and ochratoxin A are removed with (neat) methanol and then directly analyzed by reversed-phase LC with fluorescence detection using post-column bromination (Kobra cell®).: Test portions of blank pekmez and pestil were spiked with a mixture of aflatoxins and ochratoxin A to give levels ranging from 2.6 to 10.4 μg/kg and 1.0-4.0 μg/kg, respectively. Recoveries for total aflatoxins and ochratoxin A ranged from 84 to 106% and 80-97%, respectively, for spiked samples. Based on results for spiked pekmez and pestil (30 replicates each at three levels), the repeatability RSD ranged from 1.6 to 12% and 2.7-11% for total aflatoxins and ochratoxin A, respectively. : The method performance in terms of recovery, repeatability, and detection limits has been demonstrated to be suitable for use as an Official Method.: First immunoaffinity column method validated for simultaneous analysis of aflatoxins and ochratoxin A in pekmez and pestil. Suitability for use for official purposes in Turkey, demonstrated by single-laboratory validation. Co-occurrence of aflatoxins and OTA in mulberry and carob pekmez reported for the first time.