Thiamin Phosphates in Egg Yolk Granules and Plasma of Regular and Embryonated Eggs of Hens and in Five- and Seven-Day-Old Embryos

The thiamin (TH), thiamin monophosphate (TMP), thiamin diphosphate (TDP), and thiamin triphosphate (TTP) content was analyzed in yolk plasma and yolk granules of unincubated and incubated chicken eggs and in 5- and 7-day-old chick embryos. Analytes were extracted from samples weighing approximately 1 g, with trichloroacetic acid (0.4 g/L), isolated with solid-phase extraction, and determined with HPLC. Before solid-phase extraction with a C-18 cartridge and a strong anion exchange cartridge, analytes were converted to thiochromes with cyanogen bromide. For HPLC, a C-8 pre-column and a C-8 column connected to a NH₂ column were used with a fluorescence detector. No thiamin phosphates were found in egg yolk plasma or granules in native and embryonated eggs. The TH content in DM was 3,400 ± 700 ng/g for yolk plasma, 1,500 ± 140 ng/g for yolk granules of unincubated eggs, and 4,400 ± 1,100 ng/g for yolk plasma and 2,100 ± 450 ng/g for yolk granules of 5-d embryonated eggs. In chick embryos, TH as well as thiamin phosphates were found. The mean content in 5-d-old embryos was 1,200 ± 400 ng of TH and 8,000 ± 1,000 ng of TDP/g of DM. In 7-d-old embryos, the content of TH was 900 ± 200 ng, TMP was 400 ± 200 ng, and TDP was 5,500 ± 1,300 ng/g of DM. Thiamin triphosphate was not detected, and the minimum detectable limit for T was 0.3 ng/mL, for TMP 0.5 ng/mL, and for TDP 0.9 ng/mL, respectively. Recovery was 71% for TMP and 64% TDP in plasma and 58% TMP and 47% TDP in granules. It was demonstrated that only TH is present in egg yolk fractions in native and embryonated eggs of hens and that thiamin phosphates can be found in 5- and 7-d-old embryos, which indicates that they are synthesized by the chick embryo. According to its assumed unique role in highly specialized tissues, TTP was not found in early embryos.