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Sensitive and rapid radlolmmunoassay (RIA) procedures for the determination of plasma concentrations of psychotroplc drugs--chlorpromazlne, Imipramlne, desipramlne, amltrlptyline, and nortrlptyllne--are These procedures employing 20 to 200FI of plasma can determine subnanogram concentrations of these drugs and are shown to be suitable for therapeutic monitoring of these drugs In patients. For the antidepressant drugs, the RIA procedures were found to compare favorably with gas chromatographic procedures employing alkali flame ionization detectors. The RIA procedure for chlorpromazine measures r as well as Its active metabolite, 7-hydroxychlorpromazlne. Cross-reactivity profiles for the antiserum of each drug were obtained, and In each case the antiserum of the drug dlstlnguished the drug from its inactive metabolites and most of the other psychotropir drugs tested.

A gas-liquid chromatographic procedure employing an alkali flame ionization detector (GLC-AFID) for the quantitation of imipramine and its major active metabolite, desipramine, in plasma was developed. This method validated a previously reported radioimmunoassay for imipramine and desipramine. The GLC-AFID method was also compared with a GLC-mass spectrometric procedure which employed selected ion monitoring (GLC/MS-SIM) in which specific ions for imipramine and desipramine were monitored. The two methods using gas chromatographic separation were shown to be suitable for therapeutic monitoring of these drugs in patients. The direct immunoassay, which measures the total imipramine plus desipramine, is very useful when large numbers of samples are to be analysed rapidly and it compares favorably with the other two analytical procedures.

For the development of radioimmunoassay procedures for tricyclic antidepressants, two drug haptens were synthesized for each of the two amitriptyline-nortriptyline and imipramine-desipramine groups. In one case, nortriptyline or desipramine was treated with succinic anhydride to yield N -(3-carboxypropionyl) derivatives; in the other case, the haptens were novel, N -(2-carboxyethyl) derivatives. The hapten and its corresponding ester were characterized by GLC-mass spectrometry, PMR spectrometry, and IR spectrophotometry. Each hapten was coupled to bovine serum albumin, and the number of hapten residues per mole of bovine serum albumin was determined by UV spectrophotometric methods. Antibodies to each hapten protein conjugate were developed in rabbits, and titers of the antiserums were checked by evaluating their binding characteristics to tritiated drug.

A recently described GLC method for the determination of phenylbutazone and its major metabolite, oxyphenbuta- zone [1 -phenyl-2- (p -hydroxyphenyl) -3,5-dioxo-4-n -butylpyrazoli- dine], in plasma was extended to the estimation of the second major metabolite, γ-hydroxyphenylbutazone [l,2-diphenyl-3,5-dioxo-4-(3-hydroxybutyl)pyrazolidine]. The method is sensitive to 1.0 μg/ml.

Sensitive specific methods are described for the determination of phenylbutazone and its metabolite oxyphenbutazone from the same plasma sample. The sample, to which an internal standard 5-(4-hydroxyphenyl)-5-phenylhydantoin (Standard II) is added, is first extracted with ether to remove interfering substances and then with n-heptane under acidic conditions to separate phenylbutazone, which is determined on a gas chromatograph by flash methylation (310°) with trimethylanilinium hydroxide using 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane as the external standard (Standard I). The aqueous acidic residue from which the phenylbutazone has been selectively removed is shaken again with ether to extract the oxyphenbutazone, which is analyzed with a different GLC system with flash methylation (310°) against Standard II. The methods are of sufficient sensitivity to determine plasma levels in humans after a 200-mg dose of phenylbutazone (phenylbutazone, 1 μg/ml; oxyphenbutazone, 0.5 μ/gml).

A method for the quantitative estimation of quinidine in biological fluids is described. Plasma containing quinidine, to which a known amount of cinchonidine is added as the internal standard, is extracted at pH 12.0 with benzene. The residue from the extract is mixed with 25 μl of trimethylanilinium hydroxide in methanol, and aliquots (1–2 μl) are injected into a gas chromatograph in which the injection port is held at 350°. The methyl derivatives of quinidine and the internal standard give well-separated symmetrical peaks. Detection by flame ionization gives a linear response over the range of 0.2–12.0 μg quinidine/ml plasma. The limit of detectability is 0.05 μg/ml and the method is adequate for following blood profiles of 200-mg quinidine sulfate doses in humans.

A high-performance liquid chromatographic procedure is described for the determination of disopyramide and its mono-N-dealkyl metabolite which offers simplicity of extraction with excellent selectivity, sensitivity and reproducibility. The drug and metabolite, following basic diethyl ether extraction and back-extraction with acetic acid, are injected into a reversed-phase high-performance liquid chromatographic column and the absorbance of the eluate measured at 254 nm. Detectability limits of 0.05 μg/ml were obtained with both compounds, and studies of the reproducibility, precision, recovery, stability during storage and effect of time in separating plasma from erythrocytes are described. Applications of this high-performance liquid chromatographic procedure to plasma samples from patients on disopyramide therapy and to plasma and urine from a healthy dog administered single doses are reported.

Samples of ethacrynic acid were treated with methanol-hydrochloric acid or with diazomethane. GLC and mass spectrometric analysis indicated that the methanol-hydrochloric acid reaction gave the expected methyl ester, whereas diazomethane treatment gave a compound containing an additional 14 mass units. Accurate mass measurement and PMR and IR spectra showed that this product was a cyclic derivative of the methyl ester of ethacrynic acid, methyl 4-(2,3-dihydro-4-ethyl-5-furyl)-2,3-dichlorophenoxyacetate. Either derivatization method can be used for development of an assay for ethacrynic acid.

A GLC procedure was developed for the quantitative estimation of intact chlorpropamide and tolbutamide concentrations in plasma; the drugs are used as mutual internal standards. After extraction of plasma containing the drug and internal standard with toluene, the dried residue is treated with ethereal diazomethane to form the methyl derivatives of tolbutamide and chlorpropamide. Aliquots of the ethereal solution are injected into a gas chromatograph equipped with a glass‐lined injection port and glass column packed with a phenyl methyl silicone fluid (OV‐25) on Chromosorb W, which facilitates the intact determination of the methyl derivatives of the drugs. The response to the flame‐ionization detector was linear over a range of 0.20–25 μg/ml, with a 0.05–μg/ml limit of detectability for both drugs. The method compares favorably with a recently developed high‐pressure liquid chromatographic procedure and is adequate for following blood level profiles of single doses of chlorpropamide (125 mg) and tolbutamide (250 mg). Mass spectral evidence showing that intact sulfonylureas are measured is presented.

For the development of radioimmunoassay procedures for tricyclic antidepressants, two drug haptens were synthesized for each of the two amitriptyline-nortriptyline and imipramine-desipramine groups. In one case, nortriptyline or desipramine was treated with succinic anhydride to yield N -(3-carboxypropionyl) derivatives; in the other case, the haptens were novel, N -(2-carboxyethyl) derivatives. The hapten and its corresponding ester were characterized by GLC-mass spectrometry, PMR spectrometry, and IR spectrophotometry. Each hapten was coupled to bovine serum albumin, and the number of hapten residues per mole of bovine serum albumin was determined by UV spectrophotometric methods. Antibodies to each hapten protein conjugate were developed in rabbits, and titers of the antiserums were checked by evaluating their binding characteristics to tritiated drug.