Fluorogenic derivatization of peptides with naphthalene-2,3-dicarboxaldehyde/cyanide: optimization of yield and application in the determination of leucine-enkephalin spiked human plasma samples.

Initial attempts to derivatize the alpha-amino site of several tripeptides with naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN) resulted in poor yields of the expected N-substituted 1-cyanobenz[f]isoindole (CBI) products. Examination of the CBI-formation mechanism, in conjunction with knowledge of the general structure-reactivity properties of the tripeptides, led to the recognition of a competing non-productive reaction pathway. Through the use of model reactions and the isolation and structural elucidation of a predicted side-product the viability of the competing pathway was confirmed. From an understanding of the key features of both the productive and non-productive reaction pathways, a rational approach for the optimization of CBI-derivative yield was proposed and confirmed experimentally. This information led, in turn, to the development of HPLC methodology suitable for the determination of leu-enkephalin spiked into human plasma; fluorescence detection was used in conjunction with leu-enkephalin amide as the internal standard. The method enabled leu-enkephalin to be determined at a concentration of 0.31 nmol ml-1 with an error of less than 4% for 25 pmol injected.

Liquid chromatographic determination of cyclophosphamide enantiomers in plasma by precolumn chiral derivatization.

On the basis of reactions described in the synthetic literature, a two-step chiral derivatization sequence was developed for the anticancer agent cyclophosphamide (CP). The sequence involves amidoalkylation of CP with anhydrous chloral containing 1% dimethylformamide followed by acylation of the resulting secondary alcohol with a chiral carboxylic acid chloride, (+)-6-methoxy-alpha-methyl-2-naphthaleneacetyl chloride, to form a diastereomeric pair. Derivatized (-)-CP and (+)-CP exhibited retention times of 17.2 and 20.7 min, respectively, when chromatographed on Hypersil ODS with acetonitrile/phosphate buffer (pH 6.8) as the mobile phase. Preparation of the individual diastereomers from enantiomerically pure CP enabled correlation of the chromatographically observed peaks with a particular enantiomer. Various aspects of the overall assay methodology have been systematically investigated (derivatization solvents, temperatures, reaction time, and work-up procedure) and optimized on the scale required for trace analysis in biological fluids. Calibration curves were established for each enantiomer in spiked human plasma over the therapeutically relevant concentration range of 0.99-49.94 micrograms/mL.

Suitability of DTAF as a fluorescent labelling reagent for direct analysis of primary and secondary amines--spectral and chemical reactivity considerations.

DTAF has been used successfully to prepare fluorescent labelled reagents for fluorescence polarization immunoassays. Its applicability as a derivation reagent for direct fluorescence analysis of primary and secondary amines was evaluated. DTAF was shown to have spectral properties that closely resemble those of fluorescein and that are apparently insensitive to the presence of the triazine nucleus. Spectrally determined pKa values also closely resemble those of fluorescein and other amino aryl-s-triazines. DTAF is prone to hydrolytic degradation with the rate of reaction increasing with increasing pH, until a pH value is reached at which ionization of the amine bridging the two aromatic nuclei occurs; at this pH the rate reaches a plateau value. Both primary and secondary amines react efficiently with DTAF, and the reactivity increases with the increasing basicity of the amine reactants. The reaction is pH-dependent, proceeding most efficiently at pH values at which both the "bridging amine" of DTAF and the amine substrate are unionized. Methyl substituted secondary amines were consistently more reactive than the corresponding primary amine, but further imposition of steric bulk about the amine nitrogen significantly reduced the reactivity of amines toward DTAF. In cases where such steric bulk is minimal. DTAF appears to be a suitable fluorescent labelling reagent for direct analytical applications.

Analytical challenges to the pharmaceutical industry in developing products of biotechnology.

Problems associated with the analysis of peptides and proteins in pharmaceutical products produced by biotechnology are discussed. Analytical techniques for the determination of peptides and proteins in such products are classified into four categories: bioassays, binding assays, enzyme assays and physical/chemical methods.

Chemical derivatization as a strategy to enhance detectability of agents used in cancer management.

The bioanalysis of drugs used in the management of cancer is often complicated by the lack of selectivity and sensitivity. Chemical derivatization of these drugs prior to their chromatographic analysis represents a viable strategy to improve chromatographic resolution and to enhance detectability. This review provides examples of how this approach can meet these objectives. Derivatization of racemic cyclophosphamide with a chiral acylating agent, following hydroxyalkylation to introduce a reactive centre into the molecule, provides the basis for its stereospecific analysis. The analysis of dianhydrogalactitol is described, in which diethyldithiocarbamate is used as a nucleophilic derivatizing agent that improves chromatographic behaviour and analytical sensitivity. The final example that is described is the design and preparation of improved fluorogenic reagents (o-phthalaldehyde analogues) for the derivatization of peptides and application of these reagents to the trace analysis of leu-enkephalin in plasma.

Analysis by liquid chromatography of desipramine in aqueous solutions and plasma application of dichlorotriazinylaminofluorescein (DTAF) as a pre-column fluorescent derivatising agent for selected secondary amines.

Dichlorotriazinylaminofluorescein (DTAF) has been proposed as a fluorescent derivatising agent for secondary aliphatic amines. In this report, its applicability as a pre-column reagent is demonstrated by its use in the analysis of aqueous solutions and plasma samples of the tricyclic antidepressant, desipramine. Samples were processed by liquid-solid extraction onto XAD-2 resin, followed by DTAF derivatisation of the fraction of the column effluent containing analyte. The reaction mixture was then subjected to HPLC using a dual column switching configuration that was designed to (a) separate excess DTAF from the reaction product, and to (b) achieve the necessary analytical resolution. Fluorometric detection of the analyte in column effluent afforded a sensitivity of 150 fmol of desipramine injected on-column, a 2-3 order of magnitude increase in sensitivity relative to previously described methods. DTAF appears to be a convenient reagent for fluorescence derivatisation of N-methyl secondary amines prior to their HPLC analysis.

The application of chemical derivatization to clinical drug analysis.

The sensitivity and selectivity achievable in the analysis of drug substances from biological matrices is often limited by the physical and chemical properties of the analyte. These limitations are further exacerbated by the inherent reactivity of most drugs in biological systems (i.e., their propensity for undergoing biotransformation). One very powerful approach that has been taken to improve the quality of the analytical methodology is to alter the physico-chemical properties of the drug through chemical modification (derivatization) during some stage of the analytical sequence. This approach has been successfully applied to situations and has resulted in improved chemical stability, analytical selectivity and sensitivity. In most cases, drug analysis from biological fluids involves a chromatographic step; the derivatization reaction can be carried out either prior or subsequent to chromatography. In this paper, examples of the advantages (and limitations) offered by the introduction of a chemical derivatization step in clinical drug analysis will be presented. Specifically, focus will be placed on analysis of chemically-reactive antineoplastic agents and peptides/proteins. The latter represent an emerging class of drugs which present significant analytical challenges. The use of o-phthalaldehyde analogues offering improved derivative stability and increased sensitivity will be described.

Design and synthesis of a theophylline bonded-phase column for HPLC--application to separation of aromatic carboxylic acids.

A stationary phase has been designed and synthesized in which theophylline residues are covalently bonded to a silica support through an eight carbon hydrocarbon linkage. The phase offers improved resolution in the separation of aromatic carboxylic acids over that available with conventional reversed phase supports. The column is relatively stable. Retention can be modified by adjusting mobile phase composition with respect to pH, electrolyte type and concentration, and organic modifier as well as by manipulating the temperature at which chromatography is carried out. The capacity factors, k', for a series of ring substituted benzoic acids were correlated with the complexation constants previously reported for these compounds with theophylline in bulk phase solution.

Aspects of the stability of isoindoles derived from the reaction of o-phthalaldehyde-ethanethiol with primary amino compounds.

The stability of a series of fluorescent isoindoles formed under analytical conditions following the reaction of o-phthalaldehyde (OPA) and ethanethiol (ET) with a series of primary amines is reported. Increasing the bulk and degree of substitution of the isoindole N-substituent resulted in substantial increases in isoindole stability. The effects of excess reagents on isoindole stability is examined and OPA is observed to accelerate isoindole degradation whilst ET provides a stabilizing effect. Comparison with previously reported data involving the use of 2-mercaptoethanol revealed that ET clearly forms the more stable isoindole derivatives, i.e. a minimum of five-fold improved stability based on the time for 10% degradation to occur. Identification of the major degradation product together with kinetic data suggests that degradation proceeds via autoxidation.

Phase I evaluation and pharmacokinetics of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC 286193).

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, TCAR, Riboxamide, NSC 286193) is a novel C-nucleoside with antitumor activity against several murine tumor models, including Lewis lung carcinoma. The mechanism whereby this compound exerts its antineoplastic effects is most likely related to a state of guanine nucleotide depletion whereby the anabolite, thiazole-4-carboxamide adenine dinucleotide, potently inhibits inosine-5'-monophosphate dehydrogenase. This Phase I study was designed to determine the maximally tolerated dose of Tiazofurin administered on a 5-day, every-28-day schedule. Tiazofurin levels were measured using a high-pressure liquid chromatography assay, and pharmacokinetic studies were performed in patients treated at each dose level. Nineteen patients received a total of 24 courses of the drug in doses ranging from 550 to 2200 mg/sq m. The dose-limiting toxicities were pleuropericarditis and a general illness best described as a "viral-like" syndrome (manifested by severe malaise, headaches, myalgias, fever, nausea, vomiting, and diarrhea). Other toxicity included myelosuppression, hyperuricemia, elevated serum creatine phosphokinase and serum glutamic oxaloacetic transaminase, conjunctivitis, mucositis, and desquamation of the palms of the hands. Plasma clearance of Tiazofurin followed a biexponential pattern with a harmonic mean terminal half-life of 7.6 h. The mean volume of distribution at steady state was 30 liters/sq m, and the mean plasma clearance was 3 liters/h/sq m. The total cumulative urinary excretion ranged from 15 to 49%. The maximally tolerated dose of Tiazofurin on a 5-day schedule was 1650 mg/sq m. The recommended dose for Phase II evaluations is 1100 mg/sq m for 5 days. However, exploration of other schedules which might allow administration of more Tiazofurin combined with biochemical studies including thiazole-4-carboxamide adenine dinucleotide measurements would be desirable.

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.

Effect of impaired renal function on tamoxifen.

There is no information available in the literature on the blood levels of tamoxifen in patients with decreased renal function. As serious side effects of tamoxifen administered at high doses have been reported, a patient with decreased renal function and metastatic breast cancer was studied to determine the blood levels of tamoxifen while under therapy. Since no abnormally elevated levels of tamoxifen were found in this patient during the month of therapy, results of this study indicate that tamoxifen can be administered to patients with some degree of renal impairment without the risk of giving rise to abnormally elevated blood levels.

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.

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.