Liquid-chromatographic measurement of riboflavin in serum and urine with isoriboflavin as internal standard.

We describe how riboflavin can be precisely and accurately measured in serum and urine. A structural analog, isoriboflavin, is used as an internal standard. Urine samples are prepared by adding the internal standard and trichloroacetic acid. For serum, proteins are denatured with trichloroacetic acid. A simple Sep-pak treatment of the supernate removes contaminating interferents. Within- and between-run precision is reflected by respective CVs of 2.2 and 4.9% for urine at 180 micrograms/L and 4.4 and 7.3% for serum at 10 micrograms/L. The standard curve is linear far beyond the concentrations encountered in serum and urine. The detection limit is estimated to be 10 micrograms/L and 1 microgram/L for urine and serum, respectively. The normal reference interval, as determined from 50 results for each matrix, is 36 to 349 micrograms/g of creatinine (mean 112 micrograms/g) for urine and 5.5 to 14.4 micrograms/L (mean 8.8 micrograms/L) for serum. Both distributions are skewed. The method is suited for routine use.