Determination of urinary thiamine by high-pressure liquid chromatography utilizing the thiochrome fluorscent method

A sensitive, reproducible, and specific method for the determination of urinary thiamine has been established. Unique to the use of high-pressure liquid chromatography (HPLC) to separate the fluorescent thiamine derivative from interfering fluorescent compounds. Urine samples were passed through a Decalso catoin-exchange column, washed with 0.5 M KCl to remove some interfering compounds, and eluted with 3.4 M KCl. The eluted thiamine was converted to the fluorescent derivative, thiochrome, by reaction with alkaline potassium ferricyanide. The reaction mixture was extracted with isobutanol and subjected to HPLC monitored by a fluorescent detector. Within-day and day-to-day coefficients of variation proved to be 2.5% and 1.2% respectively. Recovery of added thiamine (range 0.04 to 2.0 microgram/ml) averaged 99.9 +/- 5.3%. The sensitivity of this method was 0.03 microgram/ml.