Derivatization of 5-fluorouracil with 4-bromomethyl-7-methoxycoumarin for determination by liquid chromatography-mass spectrometry.

A robust new analytical method has been developed for the determination of 5-fluorouracil (5-FU) in human plasma samples using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The method is based on a liquid-liquid extraction procedure, precolumn derivatization, reversed-phase HPLC separation, and detection using atmospheric pressure chemical ionization and selected reaction monitoring. The derivatization agent used was 4-bromomethyl-7-methoxycoumarin. The internal standard for the assay procedure was a stable isotope labeled analog of 5-FU. The lower limit of quantitation was 1.0 ng/mL using 500 microL aliquots of plasma. Sample throughput on the mass spectrometer was approximately 17 samples/h (3.5 min/sample). The method was fully validated. The recovery of 5-FU averaged 76.1%. The accuracy of the assay, assessed from quality control samples, ranged from 99.1% to 104.3% (% theoretical). The overall interassay precision (% RSD) was 2.7%, and the intraassay precision (% RSD) ranged from 1.5% to 3.9%. The derivatized samples were found to be stable under sample analysis conditions and during refrigerator storage. The method was specific for the determination of 5-FU.

An electron-capture dienophile derivatization agent for increasing sensitivity: determination of a vitamin D analog (Ro 24-2090) in plasma samples with liquid chromatography/mass spectrometry.

A practical analytical method has been developed for the determination of an analog of vitamin D3 (Ro 24-2090) in plasma samples. The method employs liquid-liquid extraction, precolumn derivatization, HPLC separation using an automated column-switching system, and particle-beam negative ionization MS determination. An electron-capture derivatization reagent, 4-pentafluorobenzyl-1,2,4-triazoline-3,5-dione, is utilized to attain high sensitivity through a unique ionization mechanism. The method achieved a lower limit of quantitation of 25 pg (63 fmol) of Ro 24-2090 in plasma aliquots ranging from 0.125 to 1.0 ml. Method validation data were obtained for the quantitation of Ro 24-2090 in plasma from humans and seven animal species. Intraassay precision [mean percent relative standard deviation (%RSD)] ranged from 1.49 to 7.12% among the various species. Interassay precision (mean %RSD) ranged from 4.28 to 17.5%. Mean overall recovery ranged from 71 to 89%. The method has been successfully used to analyze plasma samples from several pharmacokinetic studies. The potential use of this method for determination of vitamin D2, vitamin D3, 1 alpha, 25-dihydroxyvitamin D3, and other drugs containing a diene structural moiety is discussed.

Determination of Ro 19-6327 (Lazabemide) in human plasma and urine by gas chromatography-negative chemical ionization mass spectrometry.

A sensitive and specific analytical method was developed for determination of Ro 19-6327 (Lazabemide) in human plasma and urine samples to provide pharmacokinetic data from clinical trials. The new method employs a simple liquid-liquid extraction to isolate the drug from biological samples. The extract is reacted to form the trifluoroacetyl derivative of Ro 19-6327 and then analyzed by gas chromatography-negative chemical ionization mass spectrometry (GC-NCIMS). The lower limit of quantitation of the assay is 0.05 ng/ml for plasma and 5.0 ng/ml for urine, based on 1-ml aliquots. No interferences from anticoagulants, collection devices, or endogenous constituents of plasma and urine were observed. Recovery (64.3%), inter-assay precision (< 8% R.S.D.), and accuracy (> 85%) of the method were considered acceptable. The assay proved reliable enough to be automated for unattended sample analysis of approximately 50 samples daily. In an additional series of tests, Ro 19-6327 was shown to be stable under conditions that might be encountered during the analysis of samples from clinical trials.

Quantitation of the enantiomers of rimantadine and its hydroxylated metabolites in human plasma by gas chromatography/mass spectrometry.

A gas chromatographic/mass spectrometric procedure has been developed for the quantitation in human plasma of the enantiomers of rimantadine and its three hydroxylated metabolites. The assay utilized derivatization of all analytes with the optically active reagent S-alpha-methyl-alpha-methoxy(pentafluorophenyl)acetic acid, selective ion monitoring, methane negative ion chemical ionization mass spectrometry and stable isotope dilution techniques. This method has been used to measure plasma concentrations of the enantiomers of rimantadine, m-hydroxyrimantadine and p-hydroxyrimantadine (equatorial and axial epimers) in the ranges 2.5-250, 2.5-50, 1.25-62.5 and 1.25-62.5 ng/mL, respectively, in six subjects given a single 200 mg dose of racemic rimantadine. Although there are no significant differences in the concentration-time profiles of R- and S-rimantadine, large stereospecific differences in the disposition of their metabolites are observed.

Identification of a hydroxy metabolite of diflunisal in rat and human urine.

1. A new metabolite of diflunisal, a hydroxy derivative, has been identified in rat and human urine following administration of diflunisal. 2. This hydroxy metabolite of diflunisal is excreted in urine of both species as a polar conjugate, most likely a sulphate. 3. Attempts to isolate the polar conjugate in pure form were unsuccessful due to its rapid hydrolysis in the presence of acid, and organic solvents such as diethyl ether. Its breakdown product, however, was more stable and was isolated and purified by semi-preparative h.p.l.c. Unequivocal identification as 3-hydroxy-diflunisal (i.e. hydroxylation in position 3 of the salicylic acid ring) was accomplished by means of FAB-mass spectrometry and n.m.r. spectroscopy. 4. The contribution of this oxidative metabolic pathway to the overall elimination scheme of diflunisal is more important in rat than in man. Gunn rats excrete more of the hydroxy diflunisal conjugate in urine (20-30% of a 50 mg/kg i.v. dose of diflunisal) than Wistar rats. In healthy humans, hydroxylation of diflunisal contributes only to a small extent to the overall biotransformation of diflunisal.

Determination of norgestimate and its metabolites in human serum using high-performance liquid chromatography with tandem mass spectrometric detection.

A rapid and reliable analytical method is described for the simultaneous determination of a synthetic progestin norgestimate (NGM), and its metabolites, 17-deacetylnorgestimate (17-DA-NGM), 3-ketonorgestimate (3-keto-NGM) and norgestrel (NGL) in human serum using reversed phase high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The assay was linear over the concentration ranges of 0.1-5.0 ng/ml for 17-DA-NGM and NGL and 0.5-5.0 ng/ml for NGM and 3-keto-NGM. The inter-assay reproducibility was consistently less than 10%. The overall recovery of the analytes ranged from 72 to 92%. Serum profiles following oral administration of norgestimate to female volunteers are presented.