High-Throughput Method for the Simultaneous Determination of Doxorubicin Metabolites in Rat Urine after Treatment with Different Drug Nanoformulations.

Doxorubicin (DOX) is one of the most effective cytotoxic agents against malignant diseases. However, the clinical application of DOX is limited, due to dose-related toxicity. The development of DOX nanoformulations that significantly reduce its toxicity and affect the metabolic pathway of the drug requires improved methods for the quantitative determination of DOX metabolites with high specificity and sensitivity. This study aimed to develop a high-throughput method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for the quantification of DOX and its metabolites in the urine of laboratory animals after treatment with different DOX nanoformulations. The developed method was validated by examining its specificity and selectivity, linearity, accuracy, precision, limit of detection, and limit of quantification. The DOX and its metabolites, doxorubicinol (DOXol) and doxorubicinone (DOXon), were successfully separated and quantified using idarubicin (IDA) as an internal standard (IS). The linearity was obtained over a concentration range of 0.05-1.6 µg/mL. The lowest limit of detection and limit of quantitation were obtained for DOXon at 5.0 ng/mL and 15.0 ng/mL, respectively. For each level of quality control (QC) samples, the inter- and intra-assay precision was less than 5%. The accuracy was in the range of 95.08-104.69%, indicating acceptable accuracy and precision of the developed method. The method was applied to the quantitative determination of DOX and its metabolites in the urine of rats treated by novel nanoformulated poly(lactic-co-glycolic acid) (DOX-PLGA), and compared with a commercially available DOX solution for injection (DOX-IN) and liposomal-DOX (DOX-MY).

A sensitive procedure for the quantitation of free and N-(2-hydroxypropyl) methacrylamide polymer-bound doxorubicin (PK1) and some of its metabolites, 13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in human plasma and urine by reversed-phase HPLC with fluorimetric detection.

A high-performance liquid chromatographic assay has been developed and validated for the determination in plasma and urine of doxorubicin (DXR) and some of its metabolites released in vivo from an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer containing DXR linked through its aminosugar moiety to the polymer via an oligopeptide spacer (PK1). The method also allows measurement of the DXR still bound to the polymer. Following addition of two internal standards, the free compounds were extracted twice with isopropanol-chloroform (25:75, v/v). The first extraction was performed at physiological pH and the second after buffering at pH 8.4, in order to extract the aglycones and the glycosides, respectively. Determination of total DXR (polymer-bound plus free DXR) was performed, after quantitative acid hydrolysis to release doxorubicinone from free or polymer-bound DXR, by extraction with the same solvent mixture at pH 7.4. In both cases the organic phase was evaporated to dryness; the compounds were then separated by reversed-phase high-performance liquid chromatography (HPLC) under isocratic conditions and quantitated by fluorimetric detection. In the chromatograms all the analytes appeared to be separated at the baseline and no interference from blank human plasma and urine was observed. The suitability of the method for in vivo samples was checked by the analysis of plasma and urine samples obtained from a cancer patient who had received a single intravenous dose of the test compound.

Determination of anthracycline purity in patient samples and identification of in vitro chemical reduction products by application of a multi-diode array high-speed spectrophotometric detector.

We describe the application of a high-speed spectrophotometric detector and high-performance liquid chromatography to the determination of anthracycline purity in extracted patient specimens and to the identification of chemical reduction products. Blood contained pure anthracyclines whilst in urine, tissue and tumour there was evidence of co-eluting endogenous peaks and complexation. Aerobic reduction yielded two main products: a C13 alcohol and a fully reduced, non-fluorescent, yellow hydroquinone. Anaerobic reduction in the presence of DNA yielded a 7-deoxyaglycone metabolite end product instead of the fully reduced hydroquinone. Eight other separate chromatographic species were identified, all of which showed unique absorbance characteristics, having a visible lambda max at 530 nm and being coloured purple/blue.

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.

Rapid quantitative determination of seven anthracyclines and their hydroxy metabolites in body fluids.

A reversed-phase high-performance liquid chromatographic method is described for the determination of seven anthracycline analogues and their hydroxy metabolites. The method is suitable for analysis of 30 plasma samples a day. The detection limit is 0.5 ng/ml for all compounds. Examples of the pharmacokinetics of adriamycin and carminomycin in man are shown.

Purification and characterization of aclacinomycin A and its metabolites from human urine.

Eleven metabolites of the anthracycline antineoplastic antibiotic aclacinomycin A (Acm) were isolated and purified from the urine of four patients treated with 100-120 mg/m2 of Acm. In an initial step, pooled urine was extracted and Acm and its metabolites were concentrated in n-butanol with a horizontal flow-through coil planet centrifuge. Individual metabolites were subsequently isolated and purified by thin-layer chromatography (TLC). Purified urinary species were characterized and compared to standards of Acm and its known metabolites by TLC, high performance liquid chromatography (HPLC), acid and enzymatic hydrolysis, and mass spectrometry. Nine of the urinary species were identified as either Acm or metabolites previously described in fermentation broths of Streptomyces galileus, in vitro reactions, animal plasma, or human plasma. Another urinary species corresponded to the major and previously unidentified Acm metabolite observed in human plasma. Sufficient quantitites of this material were isolated to allow its characterization and identification as bisanhydroaklavinic acid. The most polar urinary species isolated was identified as a beta-glucuronide conjugate of bisanhydroaklavinic acid. As such, these studies demonstrate the ability of humans to produce all of the previously proposed metabolites of Acm and establish the identity of the major Acm metabolite observed in human plasma.

[Studies on the absorption, excretion and distribution of aclacinomycin A: absorption, excretion and distribution of 14C- or 3H-aclacinomycin A in mice, rats and rabbits (author's transl)].

A new anthracycline antitumor antibiotic, aclacinomycin A, was labeled with 3H uniformly or with 14C simultaneously at the anthracycline nucleus and L-rhodosamine. These labeled drugs were administered intravenously to normal dd mice, solid type Sarcoma 180 tumor-bearing ICR mice, normal or pregnant Wistar rats and normal rabbits, respectively. 14C-Aclacinomycin A given to rabbits (5 mg/kg) was rapidly cleared from the blood and transferred to tissues. But low level of radioactivity (equivalent to about 0.5 mcg/ml) was remained in the blood even 8 approximately 10 hours after administration. About 45% of the radioactivity were recovered from the urine and 20% from the feces by 72 hours after administration. Tissue levels of 3H-14C-aclacinomycin A given to normal and tumor-bearing mice were highest in the lungs and spleen. Higher distribution was observed also in the liver and kidneys 2 hours after administration. Bioassay revealed that the drug was present in the lungs and spleen in biologically active form and in the liver and kidneys in inactive form, respectively. In the tumor tissue the radioactivity was low but it persisted for 48 hours. Autoradiography with 14C-aclacinomycin A in rats demonstrated that radioactivity due to the drug distributed in the lungs, spleen, kidneys, thymus, intestine, lymph nodes, bone marrow, salivary gland, hypophysis and pineal body but it was rapidly cleared. About 0.2% of radioactivity given to a pregnant rat were transferred to a fetus when 14C-aclacinomycin A was administered intravenously on the 18 approximately 19th day of pregnancy.

[Studies on the absorption, excretion and distribution of aclacinomycin A: absorption, excretion and distribution of aclacinomycin A in mice, rabbits and dogs by photometric assay (author's transl)].

An anthracycline antitumor antibiotic, aclacinomycin A, was given to mice, rabbits or dogs intravenously to study the pharmacokinetics by photometric assay based on the absorption of anthracycline ring. The drug was rapidly eliminated from the blood in these animals. Drug levels were much higher in the blood cells than in the plasma. Tissue levels in dogs were 50 approximately 100 times higher than the blood levels, which showed the drug was rapidly transferred from the blood to tissues after administration. Higher levels were observed in the lungs, spleen and lymph nodes, where the drug was present as aclacinomycin A itself and the glycoside-type metabolites that were biologically active. The active form was also detected in the pancreas, heart, thymus, bone marrow and gastrointestinal tract. In the liver and kidneys, biologically inactive aglycone-type metabolites were observed. About 2 approximately 4% of the drug given to rabbits or dogs was recovered in the urine by 72 hours after administration, in which only 10% of the excreted drug was active form in rabbits but about 65% in dogs. The rest was inactive aglycone-type metabolites that were excreted almost in the conjugated form. Biliary excretion also contributed to the total clearance of the drug. Aclacinomycin A was absorbed even by oral administration in rabbits and dogs. Tissue distribution of the drug orally given to dogs was similar to that in intravenous administration, except that higher levels of active form were detected in the gastrointestinal tract and of inactive form in the liver.