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A reversed-phase HPLC method for the quantitative determination of total topotecan in human whole blood and unwashed erythrocytes has been developed and validated in terms of sensitivity, specificity, precision and accuracy. Linear calibration curves were constructed in the range of 0.20 to 50.0 ng/ml. The sample pre-treatment for whole blood involved a two-step extraction with methanol and perchloric acid. Prior to extraction, erythrocytes were separated from other blood components by centrifugation in MESED instruments. Separations were achieved on an Inertsil ODS-80A analytical column (150×4.6 mm, 5 μm particle size), eluted at 50°C and a flow-rate of 1.00 ml/min, with a mixture of 100 m M ammonium acetate (pH 6.0)–tetrahydrofuran (94.6:5.4, v/v). Fluorescence detection was performed using excitation and emission wavelengths of 381 and 525 nm, respectively. With the applied method, 80% of topotecan was extracted out of whole blood. The lower limit of quantitation in whole blood was established at 0.20 ng/ml with within-run and between-run precisions, respectively, ranging from 1.7 to 9.3% and 1.5–6.1%, while the accuracy ranged from 100 to 113%. The described method will be used in clinical studies to explore the role of erythrocytes in the overall kinetic behavior of topotecan.

A new HPLC method has been developed for the quantitative determination of methotrexate (MTX) and its 7-hydroxyl metabolite in human plasma. Samples were purified by protein precipitation with acetone and methanol, and a sample clean-up with a mixture of n-butanol and diethyl ether. The analytes were separated on an RP Inertsil ODS-80A column and eluted in a solvent system containing 5% (v/v) tetrahydrofuran in water (pH 2.0). UV absorption measurement was performed at 313 nm, and the detector response was linear in a concentration range of 10-10,000 ng/ml. The lower limit of quantitation of MTX was 10 ng/ml using 1 ml sample aliquots. Values for accuracy and (within-run and between-run) precision were between 95.5-111% and 3.69-11.0%, respectively, at four concentrations analyzed in quintuplicate on four separate occasions. The assay was applied to study the effects of docetaxel co-administration on the pharmacokinetics and metabolism of MTX in cancer patients.

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-microl 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 microM 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 microM 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 microM (approximately 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.

Publisher Summary This chapter elaborates probabilities of predictors of residual mass histology in abdominal masses. An international dataset was collected comprising patient data from five research groups for uni- and multivariate meta-analysis. Patients with extragonadal tumors were excluded and patients who had elevated tumor markers, AFP or HCG, at the time of surgery. Uni- and multivariate odds ratio (OR) were estimated with logistic regression analysis. For cancer, the ORs of these predictors were generally smaller than one. A number of factors in the international dataset predict the histology of resected residual masses. These predictors were also found in a meta-analysis of 415 patients from 12 publications. It appears that all factors remain predictive for the histology of resected tissue. The primary tumor histology, initial tumor marker values, residual mass size, and shrinkage of the mass can, thus, be used to estimate the probability of necrosis, mature teratoma, and cancer.