Rapid and sensitive liquid chromatography-tandem mass spectrometry for the quantitation of epirubicin and identification of metabolites in biological samples

A highly sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of epirubicin in serum and cell specimens using daunorubicin as an internal standard. Using atmospheric pressure chemical ionisation (APCI), the epirubicin metabolites were readily distinguishable by their fragmentation pattern in the mass spectrometer. Selected reaction monitoring (SRM) mode was employed for quantitation of epirubicin and the metabolites. Following extraction, chromatography was performed on a C18 column with a mobile phase consisting of water-acetonitrile-formic acid, pH 3.2, with a flow rate of 200 μl/min. The limit of detection (LOD) and the limit of quantitation (LOQ) of this method in serum were determined to be 1.0 and 2.5 ng/ml, respectively. Linearity of the method was verified over the concentration range of 2.5-2000 ng/ml, with a high correlation coefficient (R² ≥ 0.998). For the extraction procedure, an aliquot of 500 μl serum, spiked with internal standard, was extracted using a chloroform-2-isopropanol (2:1, v/v) mixture. The method has been applied to the analysis of epirubicin in cancer cell samples and the identification of known and unknown metabolites in clinical trial patient serum samples.     

Highly sensitive method for the determination of a new anthracycline: Pirarubicin

Summary A new and highly sensitive HPLC method for the simultaneous determination of pirarubicine (THP-doxorubicin) and its metabolites, adriamycin and adriamycinol, in human plasma, is described. Samples were treated by liquid-liquid extraction, the organic phase removed and the residue dissolved in methanol. Separation was on a Lichrocart Supersher RP 8 column, (250×4 mm) 4 m, with a mobile phase of acetonitrile/methanol/formate-buffer.     

Simultaneous determination of anthracyclines and taxanes in human serum using online sample extraction coupled to high performance liquid chromatography with UV detection

An online SPE‐LC method that can determine both anthracyclines and taxanes simultaneously in human serum samples is reported. The entire method of extraction, separation and UV detection was achieved online by column switching between an SPE column (Biotrap 500 (20×4 mm)) and an analytical column (Zorbax XDB C18, 150×4.6 mm, 5 μm) with a 23 min total cycle time. The method is linear (r²>0.998) over the range of 0.5–25 μg/mL. The analytes of interest are retained on the SPE column with good recovery (84–117%), while proteins and other serum components elute to waste. This online clean‐up is much faster (150 s) and less manual than traditional off‐line extraction methods. Using 0.1 mL spiked serum samples, the LOQ was 0.5 μg/mL. Intra‐ and inter‐day precision were acceptable (≤15% RSD) at and above the LOQ. The method was applied to the analysis of serum samples from patients undergoing chemotherapy with these agents.     

Inclusion complex formation of anthracycline antibiotics with cyclodextrins; a proton nuclear magnetic resonance and molecular modelling study

The inclusion complexation of the anthracyline antibiotics doxorubicin and daunorubicin with cyclodextrins has been studied by proton NMR and molecular modelling. The anthracyclines were found to complex with -cyclodextriny-cyclodextrin, whereby the aglyconic part of the molecule is included in the cyclodextrin cavity. Job ratio plots based on NMR data indicate that the complex has a stoichiometry of 1 : 1. Complex constant values of 345 M^–1 and 323 M^–1 (at pH 3) were found for doxorubicin and daunorubicin, respectively.     

Studies directed towards the total synthesis of anthracycline antibiotics

At present anthracycline antibiotics have proven to be the most exciting agents in cancer chemotherapy. Both adriamycin and daunomycin are proven to be effective against a variety of human tumour cells, despite their cardiotoxicity. However, some synthetic analogues, such as 4-demethoxydaunomycin, are shown to be better therapeutic ratios compared to adriamycin or daunomycin. Various approaches have been successfully made for the total synthesis of (±) 4-demethoxydaunomycinone, the aglycone of (±) 4=demethoxydaunomycin, starting from benzoquinone, napthalene or anthraquinone precursors. Finally an elegant approach for the stereoconvergent synthesis of (+) 4-demethoxydaunomycinone has been worked out. New methods involving both Diels-Alder and Friedal-Crafts acylation, have been developed for the total synthesis of daunomycinone and 11-deoxydaunomycinone. The synthesis of L-daunosamine, the amino sugar unit present in the antitumor anthracyclines has been successfully elaborated starting either from D-glucose or D-glucosamine.     

Electron affinities of p-benzoquinone, p-benzoquinone imine and p-benzoquinone diimine, and spin densities of their p-benzosemiq

Restricted and unrestricted (U) Hartree–Fock (HF), second-order Møller–Plesset perturbation (MP2), density functional (DF), hybrid HF/DF and semiempirical (half-electron (HE) method) models have been used to calculate adiabatic electron affinities (EAad values) of p- benzoquinone (I), p-benzoquinone imine (VI) and p-benzoquinone diimine (XI), as well as expectation values (S2) and spin density distributions in the radical anions of I, VI and XI. The AM1/AM1-HE and ab initio calculated structures are found to be in accord with each other. The ROHF/6-31G(d) method gave the poorest EAad result. The UHF and UMP2 wave functions were found to be substantially spin contaminated (for the radicals) and the accuracies of the EAad values calculated were also poor. The use of molecular energies obtained after spin annihilation did not lead to significant improvement of the UHF and UMP2 results. In contrast to the ROHF, UHF and UMP2 results, the DF(USVWN, UBVWN, UBLYP) and hybrid HF/DF(UB3LYP) methods, as well as the AM1-HE, gave much better results. The calculated EAad values decreased, as predicted by most of the models, in the order EAad(I) > EAad(VI) > EAad(XI). The differences in the EAs, EAad(I) – EAad(VI) and EAad(I) – EAad(XI), were consistently predicted to be about 8–9 and 17–18 kcal/mol, respectively, by the DF, B3LYP and AM1-HE models. The performance of the PM3 and SAM1 models was not as good as the AM1 model. Of all the methods tested, the B3LYP/6-311G(d,p) model is concluded to give the most accurate quantitative trend (I(42.6) > VI(33.1) > XI(23.7)) in EAad. The predicted trend in EA can satisfactorily be rationalized by the calculated LUMO orbital energies, atomic charges and spin density distributions. Analysis of the spin density data predicts that phenoxyl- and anilino-type radical anions predominate in the p-benzosemiquinones of I and XI, respectively, while both phenoxyl- and anilino-type radicals contribute to the structure of the p-benzosemiquinone of VI, with the anilino-type predominating.     

Anthracycline-dependent heat-induced transition from positive to negative supercoiled DNA

The conformational stability of individual DNA topoisomers depends on the concentration of DNA intercalating drugs. To study the DNA-drug interaction, we used ethidium bromide (EtBr) and negative supercoiled pUC19 as a model system. The effects of two anthracyclines widely used in cancer therapy, daunorubicin (Dau) and doxorubicin (Doxo), and EtBr were compared. In spite of their different chemical structures and intercalation mode, all intercalating agents show similar effects on the conformational stability of supercoiled DNA. Our observations show that the studied intercalators have at least two main effects on the supercoiled DNA: (i) they decrease the level of negative supercoiling and, at certain concentrations, they may induce positive supercoiling in DNA; (ii) a temperature increase can cause a recovery of negative supercoiling in DNA. The conformational stability of plasmid DNA-drug complexes has been investigated by temperature gradient gel electrophoresis (TGGE). We demonstrate the suitability of TGGE for this purpose, because it offers a global view on DNA-drug complexes over a continuous range of temperature. Images of DNA plasmids adsorbed onto a substrate at different temperatures and drug concentrations were acquired by atomic force microscopy (AFM), allowing us to distinguish directly the conformation chirality assumed by the plasmid under different conditions confirming TGGE results. Our detection system allows to characterize unknown drugs and to determine their intercalating properties.     

Anticancer Drug Modified GCFE,for the Study of Its In‐Vivo Interaction Mechanism

An anticancer drug (Adriamycin) modified Glassy Carbon Fiber Electrode (GCFE) has been prepared to study its interaction with ds‐DNA. The redox reaction of Adriamycin molecules at the chemically modified GCFE helps in understanding the in‐vivo mechanism of action of this anti cancer drug. The modified electrode has been fabricated by the adsorption of Adriamycin on GCFE surface. The results of Differential Pulse Voltammetric (DPV) analysis in acetate buffer of pH 4.5 ± 0.1, showed that the interaction between DNA guanine and adenine bases and electrode surface, is easily detected. A suitable mechanism for the oxidation and reduction of Adriamycin in‐situ intercalated in ds‐DNA immobilized on to the GCFE surface has been explained. The drug‐DNA complex formation at GCFE surface has also been studied. The prepared modified electrode is of utmost relevance because the mechanism of interaction of DNA‐Adriamycin at charged interfaces is parallel to the in‐vivo DNA‐Adriamycin complex reaction, where the nucleic acid is in close contact with charged phospholipid membranes and proteins. The interaction studies of Adriamycin at modified GCFE using DPV method help in understanding the DNA‐Adriamycin reaction mechanism.     

CE-LIF method for the separation of anthracyclines: application to protein binding analysis in plasma using ultrafiltration

Anthracyclines are chemotherapeutic drugs that are widely used in the treatment of cancers such as lung and ovarian cancers. The simultaneous determination of the anthracyclines, daunorubicin, doxorubicin and epirubicin, was achieved using CE coupled to LIF, with an excitation and emission wavelength of 488 and 560 nm, respectively. Using a borate buffer (105 mM, pH 9.0) and 30% MeOH, a stable and reproducible separation of the three anthracyclines was obtained. The method developed was shown to be capable of monitoring the therapeutic concentrations (50-50 000 ng/mL) of anthracyclines. LODs of 10 ng/mL, calculated at an S/N = 3, were achieved. Using the CE method developed, the in vitro protein binding to plasma was measured by ultrafiltration, and from this investigation the estimated protein binding was determined to be in the range of 77-94%.     

Lipophilicity profiling of anthracycline antibiotics by microemulsion electrokinetic chromatography–effects on cardiotoxicity and endotheliotoxicity

Accurate profiling of the lipophilicity of amphoteric compounds might be complex and laborious. In the present work the lipophilicity of 12 anthracycline antibiotics–four parent drugs: doxorubicin, daunorubicin, epidoxorubicin, and epidaunorubicin and eight novel formamidyne derivatives with attached morpholine, hexamethylenoimine or piperidine rings–was determined based on novel approach using MEEKC. In the second stage, lipophilicity was correlated with anthracycline toxicity towards two cell lines. In rat cardiomyoblast cell line (h9c2) a significant correlation between the logP and toxicity was found. The anthracycline lipophilicity was not correlated with toxicity towards the endothelial hybrid cell line (EAhy.926). In conclusion, the lipophilicity of anthracyclines seems to determine their toxicity towards cardiomyoblasts but not on endothelial cells, suggesting a different mechanism of anthracyclines intercellular transport or extrusion in cardiomyoblast and endothelial cells.