High Performance Liquid Chromatographic Determination of Fumaric Acid in Rat Plasma, Urine, and Fecal Samples

Fumaric acid, a Krebs cycle intermediate, is a potential cancer chemoprevention agent. A high performance liquid chromatographic procedure with UV detection for determination of fumaric acid in large numbers of rat plasma, urine and fecal samples was developed. Fumaric acid was extracted from plasma, urine, and fecal samples utilizing solid phase extraction using Clean Up® Quaternary Amine 1 mL (plasma, fecal samples) and 3 mL (urine) extraction columns followed by reverse phase high performance liquid chromatography with UV detection at 215 nm. Standard curves for plasma (1 μg/mL - 200 μg/mL), urine (5 μg/mL - 200 μg/mL), and fecal material (25 μg/g - 500 μg/g) were analyzed and replicate analysis of controls were used to determine intra-day and inter-day variability. Chlorofumaric acid was used as an internal standard for plasma and fecal samples; trans-glutaconic acid for urine samples. Precision and accuracy were studied using control solutions (low and high) prepared in naive rat plasma, urine, and fecal material. Intra-day variability was determined using 3−6 replicates of each control solution on a single day. Coefficient of variation (CV) for 20 μg/mL control (low) in plasma was 6.11% and for 80 μg/mL (high) was 7.07%; relative accuracy (RA) was 0% and 6.01%, respectively. CV for 15 μg/mL low control in urine was 38.95% and for 150 μg/mL high control was 8.84%; RA values were -12.2% and -15.16%, respectively. For the fecal material, CV for low control (100 μg/g) was 0.92% and for the high control (400 μg/g) was 4.67%. RA values were -4.02% and -4.14%, respectively. Inter-day variability was determined over a four day period. For the 20 and 80 μg/mL plasma control solutions, CVs were 12.97% and 9.09%, respectively, and RA values were 1.1% and 4.04%, respectively. For the 15 and 150 μg/mL urine control solutions, CVs were 26.85% and 14.17%, respectively, and RA values were 33.07% and -1.24%, respectively. For the 100 and 400 μg/g fecal material controls, CVs were 2.5% and 4.39%, respectively, and RA values were -4.07% and -3.87%, respectively. The standard curves for plasma, urine, and fecal samples were linear over the range of fumaric acid assayed and the means for the regression coefficient were 0.9939, 0.9972, and 0.9956, respectively. The limits of quantitation for plasma, urine, and fecal material were 1 μg/mL, 5 μg/mL and 25 μg/g, respectively.