J.A.F. de Silva

Determination of 1,4-benzodiazepines in biological fluids by differential pulse polarography

The determination of various 1,4-benzodiazepines and their metabolites by differential pulse polarography is reviewed and compared with that by other methods, and the general applicability of the polarographic methods, in terms of simplicity and flexibility, is demonstrated.

Determination of an antisecretory trimethyl prostaglandin E2 analog in human plasma by combined capillary column gas chromatography—negative chemical ionization mass spectrometry

A method is described for measuring a trimethyl prostaglandin E2 analog, TM-PGE2, in human plasma. Trideuterated and monofluorinated analogs of TM-PGE2 and added to plasma as internal standard and carrier, respectively. The plasma is adjusted to pH 3.0 and is extracted with a mixture of benzene-dichloromethane (9:1). The residue, following removal of the extracting solvent, is reacted consecutively with pentafluorobenzyl bromide and bistrimethylsilyltrifluoroacetamide. The excess derivatizing reagents are removed by evaporation, and an aliquot of the reconstituted residue is analyzed by capillary column gas chromatography using methane as the carrier gas. A quadrupole mass spectrometer is set to monitor in the gas chromatographic effluent the (M - C7H2F5)-fragment ion of TM-PGE2 (m/e 449) and trideuterated TM-PGE2 (m/e 452) generated by methane negative chemical ionization. Quantitation of unknowns is based on a comparison of the m/e 449 to m/e 452 ion ratio in each unknown to that obtained from the analysis of control plasma spiked with known amounts of TM-PGE2 and fixed amounts of internal standard and carrier. The sensitivity limit of the assay is approximately 100 pg ml-1, which is equivalent to 1 pg injected. The assay was used to measure the concentration of TM-PGE2 in the plasma of two subjects following a single 10 micrograms kg-1 oral dose of the drug.

A differential pulse polarographic examination of the 1,4-benzodiazepines.

Abstract A differential pulse polarographic examination of 1,4-benzodiazepines structurally related to medazepam. chlordiazepoxide and N-desmethyldiazepam is presented. The data are used to correlate polarographic peak potentials (E p ) and Hammett substituent constants for the three series of compounds. Good correlations were found for the reduction of the 4,5-azomethine functional group for the three series of compounds in pH 3 and pH 7 supporting electrolyte. No correlation could be established for the reduction of the 1,2-azomethine and N 4 -oxide in the series of compounds structurally related to chlordiazepoxide. Polarographic data are also presented for two groups of benzodiazepines structurally related to medazepam and N-desmethyldiazepam with heterocyclic substituents in the 5-position.

Analytical strategies for therapeutic monitoring of drugs in biological fluids

Therapeutic drug monitoring can involve quantitation in either microgram, nanogram or picogram concentrations present in a complex biological matrix (whole blood, urine or tissue). The chemical structure of a compound influences not only the analytical method best suited to its quantitation, but also its acid/base character (pKa) and its extractability. The dose administered, the bioavailability of the dosage form, and the pharmacokinetic profile of the drug govern the circulating concentrations of either the parent drug and/or its metabolites present in vivo, and dictate the ultimate sensitivity and specificity required of the analytical method. The degree of sample preparation required is dependent on the analytical method used (gas--liquid chromatography, thin-layer chromatography, high-performance liquid chromatography) and on the tolerance of the specific type of detection system to contamination. Factors leading to compound losses during sample preparation (adsorption, stability) are critical at low concentrations and can adversely affect the reliability of an assay, therefore maximizing the overall recovery of the assay is essential not only for high sensitivity but also for good precision and accuracy. Therefore, the criteria to be used in sample preparation should aim to optimize all of the above factors in the overall development of a reliable and validated method for the compound suitable for use in clinical therapeutic monitoring.

Analytical strategies for therapeutic monitoring of drugs and metabolites in biological fluids. Plenary lecture.

Therapeutic drug monitoring can involve quantitation in either microgram, nanogram or picogram concentrations present in a complex biological matrix (whole blood, urine or tissue). The chemical structure of a compound influences not only the analytical method best suited to its quantitation, but also its acid/base character (PKa) and its extractability. The dose administered, the bioavailability of the dosage form, and the pharmacokinetic profile of the drug govern the circulating concentrations of either the parent drug and/or its metabolites present in vivo, and dictate the ultimate sensitivity and specificity required of the analytical method. The degree of sample preparation required is dependent on the analytical method used (gas—liquid chromatography, thin-layer chromatography, high-performance liquid chromatography) and on the tolerance of the specific type of detection system to contamination. Factors leading to compound losses during sample preparation (adsorption, stability) are critical at low concentrations and can adversely affect the reliability of an assay, therefore maximizing the overall recovery of the assay is essential not only for high sensitivity but also for good precision and accuracy. Therefore, the criteria to be used in sample preparation should aim to optimize all of the above factors in the overall development of a reliable and validated method for the compound suitable for use in clinical therapeutic monitoring.

Determination of the antiallergenic agent, N-[4-(1H-imidazol-1-YL)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[2,1,-b]quinazoline-8-carboxamide, in plasma by reversed-phase high-performance liquid chromatographic analysis using fluorometric detection

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[ 2,1-b] quinazoline-8-carboxamide (I), and its major metabolite, 2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxylic acid (I-A), in plasma. The assay involves precipitation of the plasma proteins with acetonitrile--methanol (9:1), followed by the analysis of an aliquot of the protein-free filtrate by reversed-phase ion-pair HPLC with fluorescence detection for quantitation. The analogous compound, N-[6-(1H-imidazol-1-yl)hexyl]-2-(1-methylethyl)-11-oxo-11H-pyrido [2,1-b]-quinazoline-8-carboxamide (II), is used as the internal standard. The overall recovery of compounds I and I-A from plasma is 107.0 +/- 8.6% and 107.0 +/- 10.0%, respectively. The sensitivity limits of quantitation are 20 ng of I, and 10 ng of I-A per ml of plasma using a 0.5-ml aliquot. The assay was used to monitor the plasma concentrations of I and of I-A in a dog following a 5 mg/kg intravenous infusion of I . 2HCl, a 10 mg/kg oral dose of I . 2HCl and of metabolite I-A.

Determination of diclofensine, an antidepressant agent, and its major metabolites in human plasma by high-performance liquid chromatography with fluorometric detection

A sensitive and selective high-performance liquid chromatographic assay was developed for the determination of diclofensine (I) and its key metabolites in human plasma. The assay involves deproteinization of plasma, overnight Glusulase incubation to hydrolyze the major metabolite (I-B-glucuronide), extraction of the parent compound and its deconjugated metabolites (I-A, I-B and I-C) from the alkalinized aqueous phase into diethyl ether-ethanol (95:5), the residue of which (containing compounds I, I-A, I-B and I-C) is alkylated with 2-iodopropane dissolved in acetone, using solid potassium hydroxide as a catalyst. The compounds are extracted from the reaction mixture into diethyl ether, after adding ethanol-water-acetic acid (55:40:5), the residue of which is dissolved in 0.05 M sulfuric acid, and reacted with mercuric acetate at 100 degrees C, which oxidizes tertiary tetrahydroisoquinolines to their 3,4-dihydroisoquinoline derivatives, followed by a photochemical reaction in the same solution to form intensely fluorescent isoquinolinium derivatives. An aliquot of this reaction mixture is injected onto a reversed-phase high-performance liquid chromatography column (5-microns Nova-Pac C13 phase in a radial compression cartridge, 10 cm X 8 mm), using the mobile phase 0.25 M triethylammonium phosphate (pH 2.5)-0.25 M acetic acid-methanol-acetonitrile-tetrahydrofuran (150:350:125:375:25). The void volume (Vo) is approximately 1.4 min and the retention times (tR) of the respective isoquinolium derivatives of diclofensine (I) are ca. 3.5 min, internal standard (II) ca. 4.2 min, nordiclofensine (I-A) ca. 5 min, while the phenolic metabolites I-B and I-C give peaks at 6.4 min and 10.4 min, respectively. The derivatives are detected by fluorescence. The method was used to determine plasma concentrations of the parent drug (I) and its major phenolic metabolite I-B (aglycone) in plasma in two normal volunteers following a single oral 45-mg dose and following seven consecutive days of oral dosing of 45 mg three times a day as part of a multiple ascending dose tolerance study.

Determination of coumermycin A1 in plasma by reversed-phase high-performance liquid chromatographic analysis

Coumermycin A1 is an antibiotic isolated from Streptomyces hazeliensis var. hazeliensis nov. sp. as a sodium salt which exhibits antistaphylococcal activity. A sensitive and selective high-performance liquid chromatographic method was developed for the determination of the compound and three known homologues which are extracted from plasma buffered to pH 6.5 into methyl-tert.-butyl ether-2-propanol (97.5:2.5), the residue of which is dissolved in the mobile phase and analyzed by automated reversed-phase high-performance liquid chromatography using UV detection at 330 nm for quantitation. Novobiocin is used as the internal standard. The method was used to determine the plasma concentration--time profile of coumermycin A1 in the dog following a single intravenous administration of a 12 mg/kg dose of a solubilized dosage form of the bulk drug substance.

Validation of Bioanalytical Procedures: An Example

The present example concerns GC and RIA determination of midazolam and a metabolite in plasma. The validation description complements that given for a midazolam analogue (assayed by HPLC in this laboratory) in Vol. 10 of this series [1], in an article ‘GLP in Bioanalytical Method Validation’. That article, which should be consulted as the foundation for the present one, dealt with analytical policies for implementing FDA stipulations for ‘Non-clinical Laboratory Studies’. These bear on impending regulations for ‘Good Clinical Practices’ (GCP) focused on pharmacokinetic studies that need analytical back-up.

Determination of 3-Phenoxy-N-Methylmorphinan and Metabolites in Plasma

The drug (I, Fig. 1) is one of the phenoxymorphinan analogues synthesized by Mohacsi [1] and is under development as a non-narcotic analgesic. It is extensively metabolized in the rat [2] and in the dog [3] byN-demethylation to form the nor-analogue (I-A),and byp-hydroxylation of the 3-phenoxy ring to yieldI-Band cleavage of the ether linkage to yield the morphinanI-Cand itsN-demethylation produces nor-levorphanol (I-D) (Fig. 1). The presence of four metabolites besides the drug necessitated a strategy for the development of sensitive and specific assays for their quantitation. Of the approaches tried, with dog plasma during pre-clinical drug development, GC-MS (+ve CI) proved the most satisfactory.