Gas chromatographic—mass spectrometric determination of plasma 5-fluorouracil after administration of 1-hexylcarbamoyl-5-fluorouracil to dogs and humans

A simple, sensitive and specific method for determining 5-fluorouracil (5-FU) in plasma after the administration of 1-hexylcarbamoyl-5-fluorouracil (HCFU) was developed using gas chromatography—mass spectrometry. Thymine was used as the internal standard. After removal of interfering substances with chloroform, diethyl ether and Amberlite XAD-2 resin, 5-FU and thymine were extracted with 16% n-propanol in diethyl ether and methylated with trimethylanilinium hydroxide. Fragment ions at m/e 158 and 154, the molecular ion of the dimethyl derivatives of 5-FU and thymine, respectively, were used to monitor 5-FU and thymine. The sensitivity of the method is 10 ng/ml, which is sufficient to determine the 5-FU levels in plasma after the administration of therapeutic doses of HCFU to patients.     

Quantitative gas-liquid chromatographic determination of ftorafur and 5-fluorouracil in biological specimens

A method developed for measuring the antitumor agent ftorafur and its biotransformation product 5-fluorouracil was applied to biological specimens. After extraction with ethylacetate, ftorafur, 5-fluorouracil, and the internal standard 2-methyl-4-hydroxy-6-chloromethylpyrimidine are converted to their chloromethyldimethylsilyl derivatives and assayed by glc, using either an electron-capture or a flame ionization detector. The minimum detectable amount is 100 pg/injection for ftorafur and 50 pg/injection for 5-fluorouracil employing electron-capture detection. Linearity was found up to microgram amounts of both substances, without any interference from endogenous substrates. Preliminary data are reported on the comparative serum kinetics of ftorafur and 5-fluorouracil in mice.     

Quantitative high-performance liquid chromatographic determinations of aminopyrine and its metabolites in man

A quantitative high-performance liquid chromatographic method, using a polystyrene—divinyl benzene (Hitachi No. 3010 gel) column and aqueous methanol as the mobile phase, was employed for the determination of aminopyrine and its related compounds, 4-acetylaminoantipyrine, 4-aminoantipyrine and 4-monomethylaminoantipyrine. Baseline separation could be achieved within 25 min. The method was applied to the recovery of these materials from control urine and human urine. Before separation human urine was adjusted to pH 9 and extracted with ethyl acetate, chloroform and diethyl ether.     

Quantitative determination of tulobuterol and its metabolites in human urine by mass fragmentography

A method is described for the simple and simultaneous determination of tulobuterol and its metabolites in human urine by gas chromatography-mass spectrometry. Quantification was achieved by single-ion monitoring at m/e 86 derived from trimethylsilyl-tulobuterol and its metabolites using a column packed with a mixed phase, 2% OV-1–2% QF-1 (1 : 1, w/w). The detection limits were estimated to be 2 ng/ml in urine for tulobuterol and 5 ng/ml for metabolites, respectively.     

Determination of the concentrations of 5-fluorouracil and its metabolites in rabbit plasma and tissues by high-performance liquid chromatography

The concentrations of 5-fluorouracil, 5-fluoro-5,6-dihydrouracil, 5-fluorouridine and 5-fluoro-2′-deoxyuridine in plasma, liver, kidney, lung and heart of rabbits were determined by high-performance liquid chromatography (HPLC) after drug administration by two different routes. HPLC was carried out by using a Spherisorb 5 ODS 2 column and 0.05 M phosphate buffer as the mobile phase with UV detection at 200 nm. The pH of the mobile phase, organic modifier content and column temperature were found to have a profound influence on the results, hence it was necessary to optimize a procedure for each matrix. A comparison of the efficiency of intravenous and peritoneal administration revealed that the latter provides higher drug concentrations in the liver and minimal contents in plasma and all other tissues studied.     

Methods for the complete analysis of 5-fluorouracil metabolites in cell extracts

Methodology that permits complete analysis of the intracellular metabolites of 5-fluorouracil (FUra) has been developed. A high-pressure liquid chromatography system that is capable of separating all metabolites of FUra found in acid-soluble cell extracts is described. In addition to the expected FUra metabolites, FUDP-hexoses were found to be present in large amounts in L121O cells treated with FUra. Improved procedures that permit quantitation of the FdUMP which is covalently bound to dTMP synthetase, as well as the total intracellular FdUMP levels are described; the latter is accomplished by dissociation of the FdUMP-dTMP synthetase complex in sonicated cell extracts followed by phosphatase treatment and subsequent high-pressure liquid chromatography analysis of FdUrd. An example of the integrated methodology in which all metabolites of FUra metabolism are analyzed over a 6-h exposure period of L1210 cells to [6-³H]FUra is provided.     

Quantitative determination of trimebutine maleate and its three metabolites in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective HPLC-MS-MS method was developed for the determination of trimebutine maleate (TM) and its major metabolites N-monodemethyltrimebutine (TM-MPB), N-didemethyltrimebutine (APB) and 3,4,5-trimethoxybenzoic acid (TMBA) in human plasma. The analytes were extracted from plasma samples by liquid-liquid extraction and chromatographed on a YMC J'sphere C(18) column. The mobile phase consisted of 2 mM ammonium acetate buffer (pH 6.5)-methanol (20:80, v/v), and at a flow-rate of 0.2 ml/min. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive-negative switching electrospray ionization (ESI). The method was validated over the concentration range of 1-100 ng/ml for trimebutine maleate and APB, 1-500 ng/ml for MPB, and 50-10,000 ng/ml for TMBA. Inter- and intra-day precision (RSD%) for trimebutine maleate and its three metabolites were all within +/-15% and the accuracy was within 85-115%. The limit of quantitation was 1 ng/ml for trimebutine maleate, TM-MPB and APB, and 50 ng/ml for TMBA. The extraction recovery was on average 58.2% for trimebutine maleate, 69.6% for MPB, 51.2% for APB and 62.5% for TMBA. The method was applied to the pharmacokinetic study of trimebutine maleate and its metabolites in healthy Chinese volunteers.     

Automated determination of 5-fluorouracil and its metabolite in urine by high-performance liquid chromatography with column switching

We report a quantitative assay of 5-fluorouracil (FU) and its metabolite, 5-fluorodihydrouracil (FDHU) in human urine by used a column-switching high-performance liquid chromatographic method. The analyses were carried out using a molecular exclusion column for sample purification, and a cation-exchange column for separation. Each sample required only 40 min to analyze, and required no preparation other than filtration. Linearity was verified up to 1000 nmol/ml (r>0.993). The recovery of FU was 96–101%; recovery of FDHU was 96–105%. The imprecision (RSD) for FU (10–100 nmol/ml) was <1.5%, same-day (n=5), and <1.8%, day-to-day (n=5). The imprecision (RSD) for FDHU (10–100 nmol/ml) was <3.2%, same-day (n=5), and <4.0%, day-to-day (n=5). The detection limits were, respectively, 0.1 nmol/ml. We measured FU and FDHU in urine of seven cancer patients after oral administration of FU. The cumulative quantity ratio of the FDHU and FU (FDHU/FU) excreted in their urine within 120 min after FU administration was a constant value in all seven patients. Based on these results, we believe that our method provides a useful tool for evaluating FU metabolism.     

Validated liquid chromatographic determination of 5-fluorouracil in human plasma

A sensitive, reproducible, selective and accurate high performance liquid chromatographic (HPLC) method for the quantitative determination of 5-flurorouracil in plasma has been developed and validated using isocratic elution and UV detection. The method provides a selective quantifications of 5-flurorouracil without any interference of the endogenous uracil. The assay is performed after a double extraction of 5-flurorouracil and thymine (internal standard) from human plasma using ethyl acetate. The drug and the internal standard were eluted from a Genesis C(18) analytical column at ambient temperature with mobile phase consisting of methanol:water (10:90, v/v) adjusted to pH 3.2 with perchloric acid at a flow rate of 1.0 ml/min. The effluent was monitored with an ultraviolet detector at 260 nm. Quantification was achieved by the measurement of the peak-height ratios and the limit of quantification for 5-flurorouracil in plasma was 30 ng/ml. The retention times for 5-flurorouracil, uracil, and thymine were 4.5, 6.0, and 9.0, respectively. The intra-day coefficient of variation (CV) ranged from 1.35 to 4.53% at three different concentrations and the inter-day CVs varied from 1.29 to 4.98%. The relative and absolute recoveries varied from 96 to 101%. Stability tests showed that 5-flurorouracil is stable for at least 72 h in plasma after freezing. The simple method may permit the assessment of 5-flurorouracil plasma concentrations for pharmacokinetic studies in combination with clinical trials.     

Rapid determination of 5-fluorouracil in plasma using capillary electrophoresis

A rapid, simple and sensitive capillary electrophoresis (CE) method used for the determination of 5-fluorouracil in rabbit plasma is described in the present paper. In this method, samples were simply pretreated by a solvent extraction procedure prior to injection. With a running buffer composed of 30 mM Tris-H(3)PO(4) (pH 7.0) and 5% isopropanol, 5-fluorouracil was easily separated from the external standard alpha-phenethylol as well as other substances existed in the plasma. A linearity of 5-fluorouracil was determined in the range from 0.17 to 42.50 microg/ml with a correlation coefficient of 0.999. A limit of quantitation (LOQ) corresponding to signal-to-noise ratio of 10 was obtained (LOQ=0.08 microg/ml). The method was successfully used for determining the 5-fluorouracil in real plasma samples from rabbits.     

Quantitative determination of 5-aminolaevulinic acid and its esters in cell lysates by HPLC-fluorescence

The development of a reliable sensitive method for the HPLC determination of 5-aminolaevulinic acid (ALA) and ALA esters in cell lysates is described. The method relies on the quantification of a fluorescent derivative of ALA following its derivatisation with acetylacetone and formaldehyde. Following this procedure it is possible to quantify ALA in cell lysates with no need for pre-purification of the sample. The method has been validated in two ranges of concentration (0.6-65 microM, 0.1-10 microg/mL, and 30-600 microM, 5-100 microg/mL), and has also been extended and validated for the determination of ALA released from ALA prodrugs after acidic hydrolysis.     

Quantitative determination of histamine metabolites by capillary gas chromatography

A method using capillary gas chromatography is described for the determination of histamine and eight of its basic and acid metabolites in a single biological sample of serum, urine, or gastric juice. Ion-exchange chromatography and extraction with organic solvents are used for isolation and purification, and gas chromatography for identification and quantitation. The heptafluorobutyryl derivatives of histamine and some basic metabolites are compatible with nitrogen-phosphorus and electron capture detection modes and offer an excellent sensitivity (detection limit 0.1 pmol with electron capture). The acid metabolites are quantitated after esterification. The linearity range, the sensitivity, a partial study of reproducibility and typical chromatograms show that the method is adaptable to a variety of applications.     

Rapid determination of sparteine and its metabolites in urine

A method is presented for the isolation, separation and determination of sparteine and its metabolites in urine. The isolation is based on rapid extraction with dichloromethane and pentane in a glass separator. For the separation and determination, capillary gas chromatography with nitrogen—phosphorous detection was used. The recovery of the method ranged from 81.6% to 94.8%, and the limit of determination varied between 0.2 and 0.5 μg ml⁻¹. For quantification, 17-ethylsparteine was used as the internal standard.     

Determination of 5-fluorouracil and its main metabolites in plasma by high-performance liquid chromatography: Application to a pharmacokinetic study

This paper describes a relatively simple and sensitive high-performance liquid chromatographic assay (HPLC) with ultraviolet absorbance detection for 5-fluorouracil (5-FUra) and its two main metabolites, 5-fluorouridine (5-FUrd) and 5-fluoro-2′-deoxyuridine (5-FdUrd), in plasma. In this study, two plasma clean-up procedures involving addition of internal standard, solid-phase and liquid-liquid extractions have been developed. A reversed-phase Kromasil C₁₈ column was used. The detection was performed at 268 nm for 5-FUra and at 275 nm for the two metabolites. Linear detection responses were obtained for concentrations ranging from 25 to 1000 ng/ml. The average recovery from plasma was 35, 42 and 48% for 5-FUra, 5-FUrd and 5-FdUrd, respectively. Precision, expressed as C.V., ranged from 2.7 to 13% and the mean recovery from 94 to 105%. The limits of quantitation and detection of the three analytes were 20 and 10 ng/ml, respectively. The method was used to monitor the pharmacokinetic profile of 5-FUra and its two metabolites in patients with metastatic colorectal cancer.     

Quantitative determination of butorphanol and its metabolites in human plasma by gas chromatography-electron capture negative-ion chemical ionization mass spectrometry

Two separate analytical methods have been developed for the determination of butorphanol and its metabolites in human plasma. One method is specific for butorphanol (I) while the other determines the metabolites, hydroxybutorphanol (II) and norbutorphanol (III). Both procedures incorporate solid-phase extraction, chemical derivatization and separation, and detection using gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry (GC-ECNCI-MS). Both methods use the cyclopropyl analog of I (BC-2605, IV) as the internal standard and the procedures for extraction of the analytes from plasma are identical. However, following extraction, either the pentafluorobenzoyl ester of I or the tris- and bis-trifluoroacetyl esters of II and III, respectively, were prepared. The derivatives were analyzed by GC-ECNCI-MS with selected-ion monitoring of the molecular ions. The standard curves were linear over the concentration ranges of 20–2000, 20–1000 and 50–1000 pg/ml for I, II and III, respectively. All standard curves from the assay validation had r² values of ≥0.994, 0.991 and 0.985 for I, II and III, respectively. For all three compounds, the intra- and inter-assay precisions (CV) and inter-assay accuracy (deviation from nominal) were within 12% for plasma quality control samples. All derivatives were stable in the reconstitution solvent for at least 24 h. The assays are being used for the determination of plasma concentrations of I, II and III in humans following repeated administration of nasal spray.