TY - JOUR
T1 - Determination of 5-fluorouracil in microvolumes of human plasma by solvent extraction and high-performance liquid chromatography
AU - Loos, Walter J.
AU - De Bruijn, Peter
AU - Van Zuylen, Lia
AU - Verweij, Jaap
AU - Nooter, Kees
AU - Stoter, Gerrit
AU - Sparreboom, Alex
PY - 1999/12/10
Y1 - 1999/12/10
N2 - In the present study, a new reversed-phase HPLC method has been developed and validated for the quantitative determination of 5-fluorouracil (5-FU) in human plasma using only 100-μl samples. The sample extraction and clean-up procedure involved a simple liquid-liquid extraction after addition of 5-chlorouracil (5-CU), used as internal standard, with 5 ml ethyl acetate. Chromatographic separations were performed on an Inertsil ODS-3 column (250x4.6 mm ID; 5 μM particle size), eluted with a mobile phase composed of acidified water (pH 2.0). The column effluent was monitored by UV absorption measurement at a wavelength of 266 nm. The calibration curves were constructed over a range of 0.20-50.0 μM and were fitted by weighted (1/x) linear regression analysis using the ratio of peak heights of 5-FU and 5-CU versus concentrations of the nominal standards. Extraction recoveries over the total range averaged 92 and 93% for 5-FU and 5-CU, respectively. The lower limit of quantitation was established at 0.20 μM (~26 ng/ml), with within-run and between-run precisions of 4.2 and 7.0%, respectively, and an average accuracy of 109.3%. The within-run and between-run precisions at four tested concentrations analyzed in quintuplicate over a time period of four days were <1.4 and <4.4%, respectively. The accuracy at the tested concentrations ranged from 98.4 to 102.3%. Compared to previously described validated analytical methods for 5-FU, our present assay provides equivalent to superior sensitivity using only microvolumes of sample. Copyright (C) 1999 Elsevier Science B.V.
AB - In the present study, a new reversed-phase HPLC method has been developed and validated for the quantitative determination of 5-fluorouracil (5-FU) in human plasma using only 100-μl samples. The sample extraction and clean-up procedure involved a simple liquid-liquid extraction after addition of 5-chlorouracil (5-CU), used as internal standard, with 5 ml ethyl acetate. Chromatographic separations were performed on an Inertsil ODS-3 column (250x4.6 mm ID; 5 μM particle size), eluted with a mobile phase composed of acidified water (pH 2.0). The column effluent was monitored by UV absorption measurement at a wavelength of 266 nm. The calibration curves were constructed over a range of 0.20-50.0 μM and were fitted by weighted (1/x) linear regression analysis using the ratio of peak heights of 5-FU and 5-CU versus concentrations of the nominal standards. Extraction recoveries over the total range averaged 92 and 93% for 5-FU and 5-CU, respectively. The lower limit of quantitation was established at 0.20 μM (~26 ng/ml), with within-run and between-run precisions of 4.2 and 7.0%, respectively, and an average accuracy of 109.3%. The within-run and between-run precisions at four tested concentrations analyzed in quintuplicate over a time period of four days were <1.4 and <4.4%, respectively. The accuracy at the tested concentrations ranged from 98.4 to 102.3%. Compared to previously described validated analytical methods for 5-FU, our present assay provides equivalent to superior sensitivity using only microvolumes of sample. Copyright (C) 1999 Elsevier Science B.V.
KW - 5-Fluorouracil
UR - http://www.scopus.com/inward/record.url?scp=0032754717&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/S0378-4347(99)00414-4
DO - https://doi.org/10.1016/S0378-4347(99)00414-4
M3 - Article
C2 - 10670744
SN - 1387-2273
VL - 735
SP - 293
EP - 297
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
IS - 2
ER -