Sensitive Analysis of Idarubicin in Human Urine and Plasma by Liquid Chromatography with Fluorescence Detection: An Application in Drug Monitoring.

A new approach for the sensitive, robust and rapid determination of idarubicin (IDA) in human plasma and urine samples based on liquid chromatography with fluorescence detection (LC-FL) was developed. Satisfactory chromatographic separation of the analyte after solid-phase extraction (SPE) was performed on a Discovery HS C18 analytical column using a mixture of acetonitrile and 0.1% formic acid in water as the mobile phase in isocratic mode. IDA and daunorubicin hydrochloride used as an internal standard (I.S.) were monitored at the excitation and emission wavelengths of 487 and 547 nm, respectively. The method was validated according to the FDA and ICH guidelines. The linearity was confirmed in the range of 0.1-50 ng/mL and 0.25-200 ng/mL, while the limit of detection (LOD) was 0.05 and 0.125 ng/mL in plasma and urine samples, respectively. The developed LC-FL method was successfully applied for drug determinations in human plasma and urine after oral administration of IDA at a dose of 10 mg to a patient with highly advanced alveolar rhabdomyosarcoma (RMA). Moreover, the potential exposure to IDA present in both fluids for healthcare workers and the caregivers of patients has been evaluated. The present LC-FL method can be a useful tool in pharmacokinetic and clinical investigations, in the monitoring of chemotherapy containing IDA, as well as for sensitive and reliable IDA quantitation in biological fluids.

A 96-Monolithic inorganic hollow fiber array as a new geometry for high throughput solid-phase microextraction of doxorubicin in water and human urine samples coupled with liquid chromatography-tandem mass spectrometry.

Innovations in extraction phases, extraction modes and hyphenated instrument configurations, are the most important issues to address for progress in the solid phase microextraction (SPME) methodology. In this regard, we have embarked on the development of a novel biocompatible 96-monolithic inorganic hollow fiber (96-MIHF) array as a new configuration for high-throughput SPME on a 96-well plate system. An arrangement of highly ordered 96 titania/Hydroxyapatite (TiO2/HAP) nanocomposite hollow fibers and corresponding stainless-steel needles on a Teflon plate holder were used as the extraction module. The inorganic hollow fibers were prepared via a rapid and reproducible template approach (Polypropylene hollow fiber) in combination with a sol-gel method in the presence of polyvinyl alcohol (PVA), as a network maker. The hollow fiber-shape sorbents were obtained with excellent precision by weight (RSD% = 4.98, n = 10) and length (RSD% = 1.08, n = 10) criteria. The proposed design can overcome a number of geometrically dependent drawbacks of conventional high-throughput SPME methods, mainly the ones related to sorbent amount and surface area due to possessing inner/outer surfaces without additional internal supports. The SPME platform, for the first time, was successfully applied for the extraction and preconcentration of doxorubicin from urine and water media without requiring sample preparation and free from significant matrix effect. The extracted analyte was analyzed by liquid chromatography-ion trap tandem mass spectrometry (LC-MS/MS). Highly satisfactory analytical figures of merit were obtained under optimized conditions. The limit of detection (LOD), limit of quantification (LOQ) and linearity of determination were 0.1 ng mL-1, 0.25 ng mL-1 and 0.25 to 4000 ng mL-1, respectively. The interday, intraday and inter sorbent precisions for three concentration levels ranged from 2.01 to 8.09 % (n = 3), 1.02 to 8.65 % (n = 5) and 0.99 to 1.02% (n = 15), respectively. The mean intra-well RSD value for 96 individual wells in 96-MIHF-SPME-LC-MS/MS (n = 3) at the medium concentration level was 7.81%.

Pharmacokinetics, drug metabolism, and tissue distribution of CPX-351 in animals.

CPX-351, a liposomal encapsulation of cytarabine and daunorubicin at a synergistic 5:1 molar ratio, is indicated for adults with newly diagnosed, therapy-related acute myeloid leukemia or acute myeloid leukemia with myelodysplasia-related changes. In preclinical species, this article demonstrated (1) similar release of cytarabine and daunorubicin by CPX-351 in plasma; (2) similar patterns of metabolism of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal cytarabine/daunorubicin combination; (3) prolonged tissue exposure to CPX-351; (4) dramatically different tissue distribution of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination (tissue:plasma ratios generally <1 versus >1, respectively); and (5) dramatically lower unbound plasma and tissue concentrations of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination. Together, these results provide insight into the safety profile of CPX-351, as well as mechanisms that drive the improved efficacy observed for CPX-351 versus the conventional 7 + 3 cytarabine/daunorubicin regimen in clinical studies.

Ag-4-ATP-MWCNT electrode modified with dsDNA as label-free electrochemical sensor for the detection of daunorubicin anticancer drug.

This paper describes the synthesis of Ag-4-ATP-MWCNT nanocomposite and its use as a modifier of working electrode. The surface of the electrochemical Ag-4-ATP-MWCNT electrode was modified with a double-stranded DNA (dsDNA) to detect daunorubicin-DNA interactions. Differential pulse voltammetry was applied to develop an electroanalytical procedure for the determination of daunorubicin and evaluate its interaction with dsDNA immobilized on the electrode surface. After the optimization of operational parameters, the linear dependence of the peak current on the daunorubicin concentration was observed in the range of 0.10×10-8 to 1.00×10-5molL-1, with the detection and quantification limits of 3.00×10-10 and 1.00×10-9molL-1, respectively. The proposed biosensor was successfully applied to validate its capability for the determination of daunorubicin in human serum and urine samples.

Detection of daunomycin using phosphatidylserine and aptamer co-immobilized on Au nanoparticles deposited conducting polymer.

A highly sensitive and selective sensor for daunomycin was developed using phosphatidylserine (PS) and aptamer as bioreceptors. The PS and aptamer were co-immobilized onto gold nanoparticles modified/functionalized [2,2':5',2″-terthiophene-3'-(p-benzoic acid)] (polyTTBA) conducting polymer. Direct electrochemistry of daunomycin was used to fabricate a label free sensor that monitors current at -0.61 V. The formation of each layer was confirmed with XPS, SEM, and QCM. Response of the sensor was compared with and without PS in terms of sensitivity and selectivity. Interaction between the sensor probe and daunomycin was determined with DPV. The experimental parameters affecting sensor performance were optimized in terms of concentration of immobilized aptamer, PS:aptamer ratio, temperature, pH, and reaction times. The dynamic range for daunomycin analysis ranged between 0.1 and 60.0 nM with a detection limit of 52.3 ± 2.1 pM. Sensor was also examined for interference effect of other drugs. The present sensor exhibited long term stability and successfully detected daunomycin in a real human urine spiked with daunomycin.

Simultaneous determination of cyclophosphamide, ifosfamide, doxorubicin, epirubicin and daunorubicin in human urine using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry: bioanalytical method validation.

A reversed-phase high-performance liquid chromatography (rp-HPLC) system interfaced with an electrospray ionization (ESI) source coupled to tandem mass spectrometry (MS/MS) was developed and validated for the determination of cyclophosphamide (CP), ifosfamide (IF), daunorubicin (DNR), doxorubicin (DXR), and epirubicin (EPI) in human urine. The analysis of samples containing multiple analytes with a dissimilar range of polarities was carried out using a conventional reversed-phase chromatographic BDS Hypersil C8 column. The analytical run was 15 min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 0.2 to 4.0 microg.L(-1) for CP, IF, DXR, EPI and 0.15-2.0 microg.L(-1) for DNR in human urine. The lower limit of quantification (LLOQ) was 0.2 microg.L(-1) for CP, IF, EPI and was set at 0.3 and 0.15 microg.L(-1) for DXR and DNR, respectively. The relative standard deviations (RSD%) were <11.2% for inter- and intra-day precisions. The overall accuracy was also within 114.7% for all analytes at the concentrations of the quality control samples. The potential of ionization suppression resulting from the endogenous biological material on the rp-HPLC/MS/MS method was evaluated and measured. The feasibility of the proposed HPLC/ESI-MS/MS procedure was demonstrated by analyzing urine samples from pharmacy technicians and nurses working in hospitals or personnel employed in drug-manufacturing plants.

Determination of daunomycin in human plasma and urine by using an interference-free analysis of excitation-emission matrix fluorescence data with second-order calibration.

Daunorubicin (DNR) is a significant antineoplastic antibiotic, which is usually applied to a chemotherapy of acute lymphatic and myelogenous leukaemia. Unfortunately, cardiotoxicity research in animals has indicated that DNR is cardiotoxic. Therefore, it is important to quantify DNR in biological fluids. A new algorithm, the alternating fitting residue (AFR) method, and the traditional parallel factor analysis (PARAFAC) have been utilized to directly determine DNR in human plasma and urine. These methodologies fully exploit the second-order advantage of the employed three-way fluorescence data, allowing the analyte concentrations to be quantified even in the presence of unknown fluorescent interferents. Furthermore, in contrast to PARAFAC, more satisfactory results were gained with AFR.

Liposomal daunorubicin plasmatic and renal disposition in patients with acute leukemia.

Liposomal formulations of anthracyclines have been developed to increase their delivery to solid tumors while reducing toxicity in normal tissues. DaunoXome (DNX, NeXstar) is a liposomal-encapsulated preparation of daunorubicin registered for treatment of Kaposi's sarcoma that during prior in vitro studies showed a toxicity to leukemic cells at least comparable to that of free daunorubicin. The aim of our study was to determine DNX pharmacokinetics in 11 poor-risk patients with acute leukemia treated with DNX 60 mg/m2 IV on days 1, 3, and 5. Blood and urine samples were collected at appropriate intervals after each of the three DNX administrations. The total amount of daunorubicin (free and entrapped) (t-DNR) and of its metabolite daunorubicinol (DNRol) was assayed by HPLC. The main pharmacokinetic parameters (t1/2alpha 4.54 +/- 0.87 h; VdSS 2.88 +/- 0.93 l/m2; Cl 0.47 +/- 0.26 l/h/m2) showed that in patients with acute leukemia liposomal-entrapped daunorubicin pharmacokinetics greatly differed from that observed for the conventional formulation. In fact, DNX produced mean plasma AUC levels (t-DNR AUC0-infinity 456.27 +/- 182.64 microg/ml/h) about 100- to 200-fold greater than those reported for the free drug at comparable doses due to a very much lower total body clearance. Volume of distribution at steady state was 200-to 500-fold lower than for the free drug. Plasma AUC of DNRol (17.62 +/- 7.13 microg/ml x h) was similar to or even greater than that observed with free daunorubicin for comparable doses. Cumulative urinary excretion showed that about 6% and 12% of the total dose of DNX administered was excreted in urine as daunorubicin and daunorubicinol, respectively. No major toxicity was encountered. Therefore, pharmacokinetic characteristics suggest that DNX may be more convenient than free daunorubicin in the treatment of acute leukemia. In fact, liposomal formulation may allow a reduction of daunorubicin captation in normal tissues. thus minimizing toxicity at least for the parent drug, and guarantee an unimpeded access to leukemic cells in the bloodstream and bone marrow, thus theoretically improving efficacy.

High-performance liquid chromatographic analysis of idarubicin and fluorescent metabolites in biological fluids.

A specific, sensitive, and reliable high-performance liquid chromatographic (HPLC) method for the determination of idarubicin (IDA) and its known fluorescent metabolites idarubicinol (IDAol) and 4-demethoxy-daunomycinone (AG1) in biological fluids (human plasma and urine) was developed and tested. Plasma samples were solid-phase-extracted (C18 bonded silica cartridges). Complete separation of unchanged drugs and metabolites was achieved on a Cyanopropyl chromatographic column (25 cm x 4.6 mm inside diameter; particle size, 5 microns) using fluorescence detection (excitation wavelength, 470 nm; emission wavelength, 580 nm). Sensitivity was better than 0.2 ng/ml for all analytes; rates of recovery of unchanged drug and metabolites were better than 84.5% (IDA), 80.3% (IDAol), and 83.9% (AG1). The interassay coefficient of variation was 6.5% for IDA, 5.8% for IDAol, and 9.8% for AG1. Mean intra-assay precision was 4.6% for IDA, 5.9% for IDAol, and 5.0% for AG1 at sample concentrations of above 1 ng/ml and 12.1% for IDA, 10.8% for IDAol, and 14.1% for AG1 at sample concentrations of below 1 ng/ml.

Hepatobiliary metabolism and urinary excretion of 4-demethoxydaunorubicin as compared to daunorubicin in rats.

The hepatic metabolism and biliary excretion of 4-demethoxydaunorubicin (4DDM) was studied in Crl: CD(SD) BR rats by the liver perfusion technique. In the same strains of rats urinary excretion was investigated in vivo. Daunorubicin (DM) was always included for comparison. The drugs and their metabolites were determined in the perfusion medium, in the bile and liver and in the urine by high-performance liquid chromatography with fluorimetric detection. Compared to its analogue DM, 4DDM markedly differed in the metabolic and excretory profile. The cumulative biliary and urinary excretion of 4DDM and the metabolites was quantitatively lower than that of DM (18% vs 36% of the dose) and was consistent with prolonged persistence of 4DDM in plasma in vivo. The extensive carbonyl reduction of 4DDM and DM observed in previous in vivo pharmacokinetic studies was also evident in this study. 13-hydroxy metabolites, daunorubicinol (DMol) and 4-demethoxydaunorubicinol (4DDMol), either as such or after glycosidic cleavage, i.e. 4DDMol aglycone, were present in appreciable amounts in the perfusion medium, bile, liver and urine. In the hepatobiliary system, however, the 13-hydroxy derivative of DM amounted to a much lower fraction than the DM aglycone (17% vs 50% of the total dose), 80% of the total 4DDM dose was accounted for by 4DDMol aglycone. In urine uncleaved DMol or 4DDMol represented more than 75% of the total amount excreted for both drugs. Conjugation, a major step in the excretion of aglycones, seems to play a minor role in the biliary and urinary excretion of 4DDM and 4DDMol.

High-speed countercurrent chromatography.

Support-free high-speed countercurrent liquid chromatography provides a rich domain of applications, some beyond reach of conventional liquid chromatography.

Method for the determination of 4-demethoxydaunorubicin, its quinone and hydroquinone metabolites in human plasma and urine by high-performance liquid chromatography.

4-Demethoxydaunorubicin (4-DMDNR) is a new orally active analogue of daunorubicin (DNR). We have developed a high-performance liquid chromatography (HPLC) method capable of separating and identifying 4-DMDNR, five possible fluorescent quinone metabolites and three possible non-fluorescent hydroquinone metabolites. Methods are described for high-yield synthesis of reference metabolites. The limit of detection of the fluorescence assay was less than 1 ng/ml after extraction of 1 ml plasma or urine with chloroform/propan-2-ol (2:1), with coefficients of variation in k' (HPLC column capacity factors) of less than 3% throughout the day. Efficiency of the extraction method described exceeded 80% in control experiments. Blood and urine samples were analysed from four cancer patients who had received 50 mg/m2 orally as three divided doses every 8 h. A typical urinary profile of the drug and its metabolites was: parent drug, 13%; 4-demethoxydaunorubicinol (4-DMDNOL), 80%; 4-DMDNR 7-hydroxyaglycone, 4% and 4-DMDNOL 7-hydroxyaglycone, 3%. 4-DMDNOL was the major metabolite detected in plasma. A further metabolite identified as the 7-deoxyaglycone of 4-DMDNOL was detected in plasma of two patients at concentrations equal to or greater than the parent drug. In the other two patients no trace of the metabolite was detected.

Limitations of the salmonella/mammalian microsome assay (Ames test) to determine occupational exposure to cytostatic drugs.

Urine samples of nursing personnel working in medical oncology divisions of several Swiss hospitals were examined for mutagenic activity. Urine samples were concentrated 100 times following XAD-2 chromatography and mutagenicity was determined using the Salmonella/mammalian microsome assay (Ames test). Apart from the urine samples of patients treated with cytostatic drugs and urine samples of nurses who are cigarette smokers, no mutagenic activity could be found. Also following exposure to an increased and defined quantity of cytostatic drugs no mutagenicity could be recovered from the urine. Four different nurses worked with cyclophosphamide, methotrexate, 5-fluorouracil, adriamycin and cis-platinum for 3-4 hr without using any protection such as gloves, masks or a vertical laminar airflow hood. Aqueous extracts of filters, through which air was pumped during the whole experiment (a personal air-sampler was fixed near the face of the test persons), were non-mutagenic. Parallel to the mutagenicity test chemical analyses were also done. The methotrexate content was determined in serum samples and the aqueous filter extracts and urine samples were examined for cis-platinum. All chemical determinations were negative. With the aid of urine concentrates of a patient treated with sub-therapeutic doses of cyclophosphamide as well as with normal urine to which single small amounts of different cytostatics (adriamycin, cyclophosphamide, cis-platinum) were added, the detection limits for the corresponding cytostatic drugs were determined and found to be in the range of 2-10 mg for cyclophosphamide and approx. 10 micrograms for adriamycin. Cis-platinum was lost during the passage through the XAD-2 columns. With these results at hand the sensitivity of the hitherto preferably used method (Ames test) for the monitoring of exposure to cytostatic drugs must be seriously questioned.

Doxorubicin and daunorubicin plasmatic, hepatic and renal disposition in the rabbit with or without enterohepatic circulation.

The pharmacokinetics, metabolism and disposition of doxorubicin and daunorubicin were studied for periods up to 100 hr in rabbits with (group II) or without a biliary fistula (groups I and III) and with (group I) or without (groups II and III) ligatured ureters using high-performance liquid chromatography to separate parent drug and metabolites. The plasma decay of doxorubicin and daunorubicin was triexponential. Metabolites appearing in the plasma after doxorubicin and daunorubicin bolus i.v. injection were respectively doxorubicinol and daunorubicinol, the latter being the major compound after daunorubicin injection. The elimination of daunorubicin was faster than that of doxorubicin. No differences in the elimination were observed between the 3 groups. In bile, 21% of the injected dose of doxorubicin were excreted mainly as the parent drug and 60% of the injected dose of daunorubicin were excreted, mainly as daunorubicinol. Enterohepatic circulation did not affect the biliary excretion of both doxorubicin and daunorubicin. Ligature of ureters increased slightly the biliary excretion of doxorubicin. The hepatic clearance of daunorubicin was greater than that of doxorubicin. The total urinary excretion was not different between the II and III groups and amounted to 11.6 and 12.8% of the injected dose of doxorubicin and daunorubicin, respectively. Metabolic ratios of doxorubicinol/doxorubicin and daunorubicinol/daunorubicin were similar in bile and urine.

Pharmacokinetics of daunorubicinol in the rabbit: comparison with daunorubicin.

The pharmacokinetics of daunorubicinol (DOL), the main metabolite of daunorubicin (DNR), was studied in rabbits and compared to that of daunorubicin after an 8 mg/kg dose. High-performance liquid chromatography was used to separate parent drug and metabolites. The plasma disappearance of DNR and DOL was triexponential. DOL was the major species detected in plasma and urine. Both drugs had large volumes of distribution. About 70% of DNR or DOL were bound to plasma proteins and mainly to albumin. Pharmacokinetic parameters of DOL obtained after injection of DOL were different from those calculated for DNR and those calculated for DOL after injection of DNR. The total urinary excretions of DNR or DOL were similar and amounted to 25% of the dose. No conjugates were identified in urine after enzymatic treatment. No fluorescent drug was identified in the feces. Anthracyclines were degraded in vitro in rabbit feces. The rabbit seems to be a good model for the study of anthracycline pharmacokinetics as our results in rabbits after DNR injection were similar to those in human studies.

A rapid chromatographic procedure for the determination of adriamycin, daunomycin and their 13-OH metabolites adriamycinol and daunomycinol.

A rapid chromatographic procedure for the quantitative determination of the anthracycline antibiotics adriamycin and daunorubicin and their chief metabolites adriamycinol and daunorubicinol in plasma and urine is described. The extraction is performed using SEP-PAK silica cartridges. After filtration the eluate is chromatographed on a reversed-phase column.

Disposition and metabolism of [14-14C] 4-demethoxydaunorubicin HCl (idarubicin) and [14-14C]daunorubicin HCl in the rat. A comparative study.

The disposition of [14-14C]4-demethoxydaunorubicin HCl ([14-14C]idarubicin HCl, [14C]IDR) and of [14-14C]daunorubicin HCl ([14C]DNR) was studied in male Sprague Dawley rats. [14C]IDR was administered either IV at 0.25 mg/kg body weight or PO at 1 mg/kg body weight, whereas [14C]DNR was dosed IV at 1 mg/kg body weight. The main elimination route for both compounds was the bile, fecal excretion representing 0.75-0.8 times the total dose at 72 h. Radioactivity due to [14C]IDR-derived species is released by the tissues at a slower rate than activity derived from [14C]DNR. After IV treatment comparable plasma levels are obtained, but tissue radioactivity is markedly lower with [14C]IDR, in keeping with the lower dosage. The ratio of plasma to tissue radioactivity is even higher in animals treated PO with [14C]IDR, because of the more extensive metabolism after this route of administration. The 13-dihydro derivatives of both [14C]IDR and [14C]DNR are the main metabolites in tissues, but in the case of the former, products of phase II reactions become more important at later times in liver and kidney and in excreta.

Continuous extraction of urinary anthracycline antitumor antibiotics with the horizontal flow-through coil planet centrifuge.

Extraction of doxorubicin (adriamycin) and daunorubicin and their metabolites from human urine was attempted utilizing the horizontal flow-through coil planet centrifuge. Partition coefficients of the drugs for various combinations of non-aqueous phases and aqueous salt solutions were determined. Optimal coefficients for adriamycin and daunorubicin were achieved with n-butanol-0.3 M disodium hydrogen phosphate. Extraction efficiencies of the drugs from human urine comparable to those obtained by standard resin column techniques could be realized by employing the n-butanol-urine (containing 0.3 M disodium hydrogen phosphate) system in the coil planet centrifuge, at flow-rates of 500-600 ml/h, and at 650 rpm revolutional speed. Small quantities of drugs and metabolites could be continuously concentrated into small volumes of the n-butanol phase from large volumes of salted urine. The versatility of the technique was demonstrated by its application to extraction of aclacinomycin A, a novel anthracycline antitumor agent, and its metabolites from human urine.