Larry A. Sternson

Factors affecting the stability of fluorescent isoindoles derived from reaction of o-phthalaldehyde and hydroxyalkylthiols with primary amines

The stability of a series of fluorescent isoindole derivatives formed in situ under analytical conditions following the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol (2-ME) with a series of primary amines are reported. Increasing the bulk and degree of substitution at C-10 of the resulting isoindole resulted in substantial increases in product stability. The effects of excess OPA and 2-ME on isoindole stability were examined and OPA was observed to catalyze isoindole degradation while 2-ME had no effect. Previously proposed degradation mechanisms were reexamined in light of the present data and an alternate degradation pathway is proposed. 3-Mercapto-1-propanol (3-MP) was found to be a superior thiol for use in the fluorogenic OPA reaction. The OPA/3-MP reagent combination was utilized to derive several amino acids and offered detection limits (S/N = 2) of less than 200 fmol.

Urine analysis of platinum species derived from cis-dichlorodiammineplatinum(II) by high-performance liquid chromatography following derivatization with sodium diethyldithiocarbamate.

A clinically useful method is described for the quantitative analysis of platinum species derived from cis-dichlorodiammineplatinum(II) in urine. The drug and its biodegradation products are derivatized directly in urine by reaction with sodium diethyldithiocarbamate (DDTC) to form a common product, a 2:1 DDTC-platinum adduct. This complex is stable and can be quantitatively extracted into 0.1 volumes of chloroform. An aliquot of the chloroform layer is then subjected to high-performance liquid chromatography on a muBondapak CN column and the eluent monitored spectrophotometrically at 254 nm. At this wavelength the DDTC-platinum adduct has a molar absorptivity of 43,000, and platinum levels of 25 ng/ml or urine can be detected with a precision of +/- 2.5% and an accuracy of +/- 4%.

Clinical pharmacology of tamoxifen in patients with breast cancer: Correlation with clinical data

Blood tamoxifen levels were determined for patients with metastatic breast cancer following initial and chronic dosing at twice daily 10 mg/m2 or a 20 mg/m2 single dose. Median time to response was six weeks. Blood tamoxifen levels at that time were ten‐fold greater than those obtained after an initial single dose; however, steady‐state values were not achieved until 16 weeks of chronic dosing. On a loading dose schedule of 40 mg/m2 twice daily for seven days and 20 mg/m2 daily thereafter, blood levels ≥ 10 mg/m2 twice daily steady‐state values were reached in one week. Levels drawn at peak and trough times suggest that tamoxifen may be given on a once‐daily basis. Tamoxifen half‐life was 9‐12 hours after the initial dose and seven days after chronic dosing.

High-pressure liquid chromatographic analysis of aniline and its metabolites.

A high-pressure liquid chromatographic method has been developed for the determination of nanomole quantities of aniline; its metabolites o- and p-aminophenol, phenylhydroxylamine, nitrosobenzene and nitrobenzene; and azobenzene and azoxybenzene which form non-enzymatically by condensation of reactive metabolites. These compounds were separated by reverse-phase chromatography (mu-Bondapak C18 column) and detected spectrophotometrically. The first four components were eluted using methanol-water (15:85) containing 0.26 M ammonium acetate and 0.015 M nickel acetate as mobile phase. The remaining compounds were eluted with methanol-water (50:50). The stabilities of the metabolites were studied electrochemically and results were used in the development of the chromatographic system.

High-performance liquid chromatography of cis-dichlorodiammineplatinum(II) using chemically-bonded and solvent-generated ion exchangers

cis-Dichlorodiammineplatinum(II) (cisplatin), a neutral square planar platinum(II) complex useful in the clinical management of a variety of neoplasms, was found to be retained on chemically-bonded and solvent-generated anion exchangers. The solvent-generated anion exchanger was prepared by the adsorption of hexadecyltrimethylammonium bromide onto the surface of a hydrophobic stationary phase. Investigations into the effects of ionic strength, organic modifiers and temperature revealed certain fundamental differences between the two systems. However, the retention mechanism of cisplatin on both types of cationic stationary phases was most readily explained in terms of ion-dipole interaction.

Paired-ion chromatographic analysis of tamoxifen and two major metabolites in plasma

A method is described for the clinical analysis of the non-steroidal anti-estrogenic, antineoplastic agent, tamoxifen and its 4-hydroxy and N-desmethyl metabolites in human plasma. The analytes are extracted from biological fluid with diethyl ether and subsequently converted to fluorescent phenanthrene derivatives by irradiation with UV light. The fluorophores are separated by paired-ion chromatography on a reversed-phase (C18) column. Spectrofluorometric monitoring of the column eluent allows quantitation of analytes as their phenanthrene derivatives to levels of 100 pg/ml of plasma.

Nonenzymatic reduction of nitrosobenzene to phenylhydroxylamine by NAD(P)H

Abstract The nonenzymatic reduction of nitrosobenzene by NADPH and NADH in aqueous buffer solution at 25°C is described. Both reactants quantitatively convert nitrosobenzene to phenylhydroxylamine. Rate constants for reduction ( k r ) were determined spectrophotometrically and found to be identical at pH 5.7 and 7.4 and independent of buffer concentration. The values of k NADH (124–149 M −1 sec −1 ) and k NADPH (131–170 M −1 sec −1 ) are essentially identical. The reaction is not subject to general catalysis or specific salt effects. The oxidation of phenylhydroxylamine by NAD(P) to nitrosobenzene is only stimulated by a factor of 1.2 over oxidation in its absence (when the ratio of NADP: phenylhydroxylamine was 8:1).

Rational design and evaluation of improved o-phthalaldehyde-like fluorogenic reagents

Evidence was presented suggesting that the fluorescent isoindole produced by reaction of o-phthalaldehyde (OPA), ethanethiol, and primary amine was formed by initial imine formation followed by conversion to an alpha-alkylaminobenzylsulfide and subsequent ring closure to form the isoindole nucleus. This mechanism suggested that the minimum structural requirement for condensation to an isoindole was an o-diacyl benzene in which one of the carbonyl groups was aldehydic. A major drawback of OPA as an analytical reagent is the limited stability of the fluorescent 1,2-disubstituted isoindole. Since isoindole instability is related to autoxidation at C-3, the use of o-(formyl) arylketones as alternatives to OPA is attractive in increasing the lifetime of the fluorescent species in that such reagents would form 1,2,3-trisubstituted isoindoles. Two compounds, o-acetylbenzaldehyde (OAB) and o-benzoylbenzaldehyde (OBB), were synthesized and evaluated as potential fluorogenic reagents. Both formed fluorescent products. The rate of formation of isoindole from the latter was too slow to make it of practical analytical value; however, OAB formed isoindoles with t1/2 less than 10 s and offered markedly improved stability over that observed with OPA.

Influence of tamoxifen and its N-desmethyl and 4-hydroxy metabolites on rat liver microsomal enzymes

Tamoxifen (Nolvadex; TAM) and its major metabolites, N-desmethyl- (DMT) and 4-hydroxy-tamoxifen (HT), were shown to be potent inhibitors of hepatic cytochrome P-450-dependent mixed function oxidations. From in vitro experiments, all three were found to be potent inhibitors of oxidation of Type-I substrates (ethylmorphine and aminopyrine) and less potent, non-competitive inhibitors of Type-II substrates (aniline and dimethylnitrosamine). TAM, DMT and HT were of essentially equal potency and had a much more pronounced effect on Type-I substrates than on Type-II compounds studied. Their action appears to parallel SKF-525A in type and potency of inhibition produced. Spectral binding studies suggest that TAM and its metabolites exert their effects by occupying the Type-I binding site of cytochrome P-450 and thus limiting the accessibility of other substrates to the active site of the enzyme. TAM (and its metabolites) also inhibits its own metabolism, altering the distribution and elimination half-lives of tamoxifen-derived species. In addition, tamoxifen metabolism was found to be sensitive to the presence of other drugs. These results raise concern regarding the role that continued administration of tamoxifen plays in changing its own disposition as well as in the detoxification of drugs administered with it.

Assessment of cisplatin reactivity with peptides and proteins using reverse-phase high-performance liquid chromatography and flameless atomic absorption spectroscopy.

Abstract Methodology based on gradient elution reverse-phase high-performance liquid chromatography has been developed to permit monitoring of reactions of cisplatin, a noble metal-containing antineoplastic agent, with peptides, polypeptides, and proteins. Such reactions have been implicated in biotransformation of eisplatin. Specificity is provided by both the chromatographic column and the use of on-line uv and off-line atomic absorption spectroscopic detectors placed in series postcolumn. chromatographic conditions were optimized to maximize resolution of nitrogenous components. In some cases, however, resolution of platinum-containing components and those devoid of metal was not possible. This chromatographic overlap could be deconvoluted by sequentially monitoring the eluant with a uv detector (responsive to all proteinaceous material) and on atomic absorption spectrophotometer (specific for platinum detection). This technique has been applied to a kinetic investigation of cisplatin reactivity toward Met-enkephalin.