Riboflavin as an oral tracer for monitoring compliance in clinical research

Abstract

We studied urinary riboflavin as an objective biomarker of compliance in clinical research using a simplified method amenable to high throughput analysis. Six healthy women not taking vitamin supplements ingested a study pill containing riboflavin (32 mg) as an inactive tracer and the soy isoflavones daidzin (0.243 mmole) and genistin (0.222 mmole) as active ingredients once daily for four days. Riboflavin and metabolites of the isoflavones were measured in urine samples obtained before and after each pill. Urinary excretion of riboflavin and metabolites of both isoflavones peaked within 8 hrs and remained higher than baseline for 24 hrs. Urinary excretion of riboflavin was also measured in 152 additional women with unrestricted dietary supplement intakes. Mean and median urinary riboflavin concentrations in these women were 0.42 and 0.31 μg/mL, respectively, compared to 0.2 μg/mL during a riboflavin-restricted diet. Receiver operating characteristics (ROC) curves indicated that urinary riboflavin within 24 hrs after a 32 mg dose would perform well as a measure of compliance (all areas under the ROC curves ≥0.84. Samples collected during the initial 8 hrs after pill ingestion performed better as a compliance measure than later collections. In summary, compliance in a clinical study can be monitored in real time by incorporating 32 mg of riboflavin into study pills, with compliance indicated by urinary riboflavin levels increasing over individual baselines or to ≥1.0 μg/mL, with a false positive rate of being classified as compliant at <5%.

Conflict of interest statement

Disclosures: V.M. Sadagopa Ramanujam, K.E. Anderson, J.J. Grady, F. Nayeem, and L-J.W. Lu have no conflicts of interest.

Figures

Figure 1

Mean urinary excretion rates, mg/hr, (z-axis) of riboflavin (A), daidzein (B), and genistein (C) after ingesting each of the four study pills (y-axis). Urine collected during baseline and at timed intervals after pill ingestion is shown on the x-axis. Each subject (n=6) ingested a total of 32 mg riboflavin, 0.243 mmole of daidzin, and 0.222 mmole of genistin once every day for four days. Comparisons among each time point of an analyte after four different daily doses by repeated measures ANOVA showed no difference (all P>0.05).

Figure 2

Receiver operating characteristic (ROC) curves of urinary riboflavin as a tracer for medication compliance: comparison between different measures of urinary levels (μg/ml vs. μg/hr) and between different intervals of pill ingestion to time of urine sampling (0–8 hrs vs. 8–24 hrs). AUC, area under the ROC curve.