Extraction and determination of trace amounts of three anticancer pharmaceuticals in urine by three-phase hollow fiber liquid-phase microextraction based on two immiscible organic solvents followed by high-performance liquid chromatography.

An automated three-phase hollow fiber liquid-phase microextraction based on two immiscible organic solvents followed by high-performance liquid chromatography with UV-Vis detection method was applied for the extraction and determination of exemestane, letrozole, and paclitaxel in water and urine samples. n-Dodecane was selected as the supported liquid membrane and its polarity was justified by trioctylphosphine oxide. Acetonitrile was used as an organic acceptor phase with desirable immiscibility having n-dodecane. All the effective parameters of the microextraction procedure such as type of the organic acceptor phase, the supported liquid membrane composition, extraction time, pH of the donor phase, hollow fiber length, stirring rate, and ionic strength were evaluated and optimized separately by a one variable at-a-time method. Under the optimal conditions, the linear dynamic ranges were 1.8-200 (R2  = 0.9991), 0.9-200 (R2  = 0.9987) and 1.2-200 µg/L (R2  = 0.9983), and the limits of detection were 0.6, 0.3, and 0.4 µg/L for exemestane, letrozole, and paclitaxel, respectively. To evaluate the capability of the proposed method in the analysis of biological samples, three different urinary samples were analyzed under the optimal conditions. The relative recoveries of the three pharmaceuticals were in the range of 91-107.3% for these three analytes.

Combined quantification of paclitaxel, docetaxel and ritonavir in human feces and urine using LC-MS/MS.

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human feces and urine is described. The drugs were extracted from 200 µL urine or 50 mg feces followed by high-performance liquid chromatography analysis coupled with positive ionization electrospray tandem mass spectrometry. The validation program included calibration model, accuracy and precision, carry-over, dilution test, specificity and selectivity, matrix effect, recovery and stability. Acceptance criteria were according to US Food and Drug Administration guidelines on bioanalytical method validation. The validated range was 0.5-500 ng/mL for paclitaxel and docetaxel, 2-2000 ng/mL for ritonavir in urine, 2-2000 ng/mg for paclitaxel and docetaxel, and 8-8000 ng/mg for ritonavir in feces. Inter-assay accuracy and precision were tested for all analytes at four concentration levels and were within 8.5% and <10.2%, respectively, in both matrices. Recovery at three concentration levels was between 77 and 94% in feces samples and between 69 and 85% in urine samples. Method development, including feces homogenization and spiking blank urine samples, are discussed. We demonstrated that each of the applied drugs could be quantified successfully in urine and feces using the described assay. The method was successfully applied for quantification of the analytes in feces and urine samples of patients.

Simultaneous quantification of reparixin and paclitaxel in plasma and urine using ultra performance liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS): Application to a preclinical pharmacokinetic study in rats.

A liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay was developed and validated to simultaneously quantify anticancer drugs reparixin and paclitaxel in this study. The compounds were extracted from plasma and urine samples by protein precipitation with acetone (supplemented with 0.1% formic acid). Chromatographic separation was achieved using a C18 column, and drug molecules were ionized using dual ion source electrospray and atmospheric pressure chemical ionization (DUIS: ESI-APCI). Reparixin and paclitaxel were quantified using negative and positive multiple reaction monitoring (MRM) mode, respectively. Stable isotope palcitaxel-D5 was used as the internal standard (IS). The assay was validated for specificity, recovery, carryover and sample stability under various storage conditions; it was also successfully applied to measure drug concentrations collected from a pharmacokinetic study in rats. The results confirmed that the assay was accurate and simple in quantifying both reparixin and paclitaxel in plasma and urine with minimal sample pretreatment.

The development of a quantitative and qualitative method based on UHPLC-QTOF MS/MS for evaluation paclitaxel-tetrandrine interaction and its application to a pharmacokinetic study.

Paclitaxel is a broad-spectrum anti-cancer drug by targeting microtubulin. However, multidrug resistant (MDR) makes its clinical application more difficult and results in failure of chemotherapy. Tetrandrine as a potential multidrug resistant modulator could be combined with other anti-cancer drugs. In this study, ultra-performance liquid chromatography (UHPLC) combined with quadrupole time-of-flight mass spectrometry (QTOF) was applied to simultaneously qualitative and quantitative analysis of paclitaxel for the pharmacokinetic studies while combined with tetrandrine. This method was developed based on non-target screening mode IDA (Information Dependent Acquisition). As a result, the validated range was 0.25-64ng/ml (30µl plasma) for paclitaxel. Totally 33 metabolites of paclitaxel and tetrandine were identified in vivo and in vitro. The main metabolites of PTX were dose-dependent decreased with different amounts of tetrandine co-administration no matter in vivo and in vitro, the exposure of PTX increased in pharmacokinetic study. The verified method is sensitive accurate and effective for the simultaneous determination of paclitaxel and its metabolites in blood, urine and live microsome incubation samples and it was successfully applied to evaluate the pharmacokinetics and drug-drug interaction between paclitaxel and tetrandine. Furthermore, a biosensor technology, surface plasmon resonance (SPR) analysis was applied to preliminary evaluate the competitive protein binding of multiple components. The SPR analysis indicated that the affinity between 6-hydroxy-paclitaxel and micotubulin is similar to that between paclitaxel and micotubulin, and tetrandrine also does not form a competitive combination with paclitaxel. For human, 6-hydroxy-paclitaxel is the one of main metabolites of paclitaxel, so the results suggested that tetrandine has an influence on the metabolite of paclitaxel, but tetrandine and the main metabolites of PTX probably do not affect PTX's biological targeting, the effect of its pharmacological action needs to be further studied.

Effect of dimethyl sulfoxide on bladder tissue penetration of intravesical paclitaxel.

Our laboratory has shown that the efficacy of bladder cancer intravesical therapy is in part limited by the poor penetration of drugs into the urothelium. We further found that paclitaxel, because of its lipophilicity, shows a higher penetration than other commonly used drugs such as mitomycin C and doxorubicin. However, the commercial formulation of paclitaxel (i.e., Taxol) contains Cremophor, which forms micelles that entrap the drug and reduce its free fraction. The present study evaluated the effect of DMSO on paclitaxel release from Cremophor micelles and paclitaxel penetration in bladders of dogs given an intravesical dose of paclitaxel (500 microg/20 ml in 0.22% Cremophor, 0.21% ethanol, and 50% DMSO). Cremophor produced a concentration-dependent reduction of the free fraction of paclitaxel (reduced to 23% at 0.25% Cremophor). This Cremophor effect was reversed by DMSO in a concentration-dependent manner, resulting in a 92% free fraction at 50% DMSO. DMSO also increased the average size of Cremophor micelles from 13 nm to 230 nm at 50% DMSO. A comparison of the tissue penetration data in the presence of Cremophor and/or DMSO indicates the following effects of DMSO: (a). increase in urine production rate and, consequently, a 36% reduction of the final urine concentration; (b). 2-fold increase in paclitaxel penetration across bladder urothelium; (c). increase in drug removal from bladder tissues (30% more rapidly); and (d). a 60% increase of the amount of drug in bladder tissue. These results indicate that DMSO caused rearrangement of Cremophor micelles, reversed the entrapment of paclitaxel in Cremophor micelles and thereby increased the free fraction of paclitaxel in solution, enhanced the urine production rate and enhanced drug removal by the perfusing capillaries, with an overall effect of increasing the bladder tissue delivery of paclitaxel formulated in Cremophor.

Phase I and pharmacokinetic study of the new taxane analog BMS-184476 given weekly in patients with advanced malignancies.

PURPOSE: The study was designed to establish the maximum administered dose and maximum tolerated dose (MTD) of BMS-184476, an analogue of paclitaxel, given weekly for 3 consecutive weeks every 28 days, later amended to a regimen of weekly administration for 2 consecutive weeks every 21 days. EXPERIMENTAL DESIGN: Adult patients with solid tumors received BMS-184476 i.v. on days 1, 8, and 15 without premedication. The trial followed a modified accelerated titration design. Doses of 7, 14, 28, 40, 50, and 60 mg/m(2)/wk were investigated. Pharmacokinetics of BMS-184476 in plasma and urine were investigated by high-performance liquid chromatography assay. RESULTS: Fifty-three patients were treated; the maximum administered dose was 60 mg/m(2)/wk, and the MTD was 50 mg/m(2)/wk. Dose-limiting neutropenia was the main toxicity. Neutropenia at the higher dose levels frequently prevented administration of the day 15 dose, and a modified schedule at MTD dosing on days 1 and 8 every 21 days was evaluated and found more feasible for Phase II studies. Diarrhea was the main nonhematological toxicity; other toxicities were vomiting, cumulative fatigue, and loss of appetite. Two patients died of neutropenia-related complications. Antitumor activity was observed in patients with breast and non-small cell lung cancer, with confirmed partial responses in 22% of patients. BMS-184476 was the main species found in the plasma with <5% present as paclitaxel or sulfoxide metabolites. The PKs of BMS-184476 appeared to be linear in the dose range of 7-60 mg/m(2). CONCLUSION: The recommended dose and schedule of weekly BMS-184476 is 50 mg/m(2) on days 1 and 8 every 21 days.

Electrochemical determination of the anticancer drug taxol at a ds-DNA modified pencil-graphite electrode and its application as a label-free electrochemical biosensor.

In this study a novel biosensor for determination of taxol is described. The interaction of taxol with salmon-sperm double-stranded DNA (ds-DNA) based on the decreasing of the oxidation signals of guanine and adenine bases was studied electrochemically with a pencil-graphite electrode (PGE) using a differential pulse voltammetry (DPV) method. The decreases in the intensity of the guanine and adenine oxidation signals after interaction with taxol were used as indicator signals for the sensitive determination of taxol. DPV exhibits a linear dynamic range of 2.0×10(-7)-1.0×10(-5) M for taxol with a detection limit of 8.0×10(-8) M. Finally, this modified electrode was used for determination of taxol in some real samples.

Cremophor reduces paclitaxel penetration into bladder wall during intravesical treatment.

PURPOSE: We have previously shown that paclitaxel, when dissolved in water and instilled into the bladder, readily penetrates the urothelium. The FDA-approved formulation uses Cremophor and ethanol to dissolve paclitaxel. In the present study, the effects of this solvent system on the urine, bladder tissue, and plasma pharmacokinetics of intravesical paclitaxel were evaluated. METHODS: Plasma, urine, and tissue pharmacokinetics were determined in five dogs treated for 120 min with paclitaxel (500 microg per 20 ml of 0.22% w/v Cremophor and 0.21% v/v ethanol) by intravesical instillation. Equilibrium dialysis was used to determine the free fraction of paclitaxel and the presence of Cremophor micelles was verified using a fluorescent probe method. RESULTS: The average bladder tissue concentration was > 1600-fold higher than the plasma concentration. Comparison of the results for paclitaxel dissolved in Cremophor/ethanol with our previous results of paclitaxel dissolved in water (500 microg per 20 ml) indicates that Cremophor/ethanol decreased the paclitaxel partition across the urothelium and reduced the average bladder tissue concentration by 75%, but did not alter the rate of paclitaxel penetration across the bladder wall, the urine pharmacokinetics or the plasma pharmacokinetics of paclitaxel. For Cremophor, the urine concentrations during the 120-min treatment ranged from 0.12% to 0.22%, and the concentration in bladder tissue from 0.00004% to 0.0009%. The threshold Cremophor concentration for micelle formation was 0.008%. We found that ethanol at concentrations up to 1% and Cremophor at concentrations below 0.01% did not alter the free fraction of paclitaxel, whereas Cremophor at higher concentrations, i.e. 0.065% and 0.25%, significantly reduced the free fraction by two- to six-fold, respectively. These results indicate that during intravesical instillation of the FDA-approved paclitaxel formulation, the concentration of Cremophor in urine was sufficient to form micelles, resulting in sequestration of paclitaxel into micelles, reduction in the free fraction of paclitaxel and consequently a reduction in paclitaxel penetration across the urothelium. In contrast, the Cremophor concentrations in bladder tissue were inadequate to form micelles and thus did not alter the drug penetration through the bladder tissue. CONCLUSIONS: We conclude that intravesical paclitaxel treatment using the FDA-approved formulation provides a significant bladder tissue targeting advantage, although the advantage is lower than when paclitaxel is dissolved in water.

Determination of a new polymer-bound paclitaxel derivative (PNU 166945), free paclitaxel and 7-epipaclitaxel in dog plasma and urine by reversed-phase high-performance liquid chromatography with UV detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of PNU 166945, a new polymer-bound paclitaxel derivative, free paclitaxel and 7-epipaclitaxel in dog plasma and urine has been developed. The method involves a solid-phase extraction of free paclitaxel and its possible degradation product 7-epipaclitaxel from plasma and urine, previously buffered with an equal volume of 0.05 M or 1 M KH2PO4 respectively, on 1-ml cyanopropyl columns. Cartridges elution was performed with the mobile phase, 0.05 M (pH 4.6) monobasic potassium phosphate-acetonitrile mixture (45:55, v/v). The samples were chromatographed on a reversed-phase octyl 4-microns column with UV detection at 229 nm. The retention times of paclitaxel and 7-epipaclitaxel were about 14 and 22 min, respectively. Determination of total paclitaxel (free + polymer-bound) was performed after release of paclitaxel from the polymeric carrier by chemical hydrolysis at room temperature (22 degrees C) for 20 h. After addition of 0.5 ml of methanol-0.1 M KH2PO4 mixture (50:50, v/v, pH = 7.5) to 0.5 ml of plasma or urine, paclitaxel was analysed as described above. PNU 166945 concentration was then determined by subtraction of free from total paclitaxel. The linearity, precision, accuracy and recovery of the method were evaluated. The limit of quantitation of the method was 5 ng/ml for biological fluid for paclitaxel and 7-epipaclitaxel and 20 ng/ml for PNU 166945 (as paclitaxel equivalent).

[Determination of new anticancer drug, paclitaxel, in biological fluids by high performance liquid chromatography].

A simple and reproducible high performance liquid chromatographic (HPLC) method using an internal standard (I.S.) was developed for the determination of an anticancer drug, paclitaxel, in biological fluids. The sample preparation involves a solid-phase extraction step. HPLC was performed on an ODS column using acetonitrile-2 mM phosphoric acid (45:55) as a mobile phase with detection at 227 nm. A linear relationship was obtained in the range of 0.02-2.0 micrograms/ml in the rat plasma and urine. Since the recovery of the drug was as high as that of I.S. (> 96%), a standard curve generated from the solution of the drug with I.S. in the mobile phase could be used for determination. The method could be applicable for human and dog plasma as well as rat plasma, and the intra- and interday coefficients of variation at 0.05, 2.0 and 10.0 micrograms/ml were less than 7%. This method is useful for pharmacokinetic studies of paclitaxel.

The elimination of MTC-220, a novel anti-tumor agent of conjugate of paclitaxel and muramyl dipeptide analogue, in rats.

PURPOSE: MTC-220, a conjugate of paclitaxel and muramyl dipeptide analogue, was reported to exhibit anti-tumor ability and anti-metastatic effect. The aim of present study was to investigate the elimination of MTC-220 and the related mechanisms in rats. METHODS: The excretion of MTC-220 and its metabolites in bile and urine were determined in rats after intravenous administration at 4 mg/kg. Caco-2 cell monolayer, in situ liver perfusion model and in vivo pharmacokinetics with selected inhibitors in rats were used to confirm the involvement of hepatic transporters in the elimination of MTC-220. The metabolic stability of MTC-220 was assessed by the incubation with rat liver microsomes and plasma. RESULTS: Approximately 72 % of MTC-220 was excreted into bile and less than 0.02 % into urine after administration in rats. The Caco-2 cell monolayer was impermeable to MTC-220. In in situ liver perfusion model, the hepatic extraction ratio of MTC-220 was reduced to 40 % of control in the presence of rifampicin, an Oatps inhibitor, and the cumulative biliary excretion rates of MTC-220 were reduced to 52.9, 71.5 and 62.9 % of control when concomitant perfusion with probenecid, novobiocin and verapamil, the inhibitors of Mrp2, Bcrp and P-gp, respectively. Co-administration of rifampicin, probenecid, novobiocin and verapamil with MTC-220 increased the AUC0-t and decreased the CL of MTC-220 in certain extents in rats. MTC-220 remained metabolically intact in rat liver microsomes, but less stable in plasma incubation. CONCLUSIONS: In summary, the elimination of MTC-220 was mainly through the biliary excretion in unchanged form in rats. Liver transporters including Oatps, Mrp2, Bcrp and P-gp might be all involved in the hepatic elimination of MTC-220. MTC-220 exhibited the high metabolic stability in liver microsomes, but less stable in plasma. The esterases might involve in the metabolism of MTC-220 in plasma.

Direct and fast determination of paclitaxel, morphine and codeine in urine by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was developed for the determination of paclitaxel, morphine and codeine in human urine from patients under cancer treatment. The background electrolyte consisted of a borate buffer (pH 9.2; 20 mM) with sodium dodecyl sulfate (60 mM) and 5% MeOH. The applied voltage was 25 kV, temperature was 20 °C and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in a fused silica capillary with an internal diameter of 75 µm and a total length of 57 cm. The detection of target compounds was performed at 212 nm. Under these conditions, a complete separation of paclitaxel, morphine and codeine was achieved in less than 15 min. According to the validation study, the developed method was proved to be accurate, precise, sensitive, specific, rugged and robust. This method was applied to the analysis of six urines samples from different cancer patients undergoing treatment with paclitaxel or/and codeine. In all the urine paclitaxel determination were done.

High-performance liquid chromatographic procedures for the quantitative determination of paclitaxel (Taxol) in human urine.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the quantitative determination of paclitaxel in human urine. A comparison is made between solid-phase extraction (SPE) and liquid-liquid extraction (LLE) as sample pretreatment. The HPLC system consists of an APEX octyl analytical column and acetonitrile-methanol-0.2 microM ammonium acetate buffer pH 5 (4:1:5, v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The SPE procedure involves extraction on Cyano Bond Elut columns. n-Butylchloride is the organic extraction fluid used for the LLE. The recoveries of paclitaxel in human urine are 79 and 75% for SPE and LLE, respectively. The accuracy for the LLE and SPE sample pretreatment procedures is 100.4 and 104.9%, respectively, at a 5 micrograms/ml drug concentration. The lower limit of quantitation is 0.01 microgram/ml for SPE and 0.25 microgram/ml for LLE. Stability data of paclitaxel in human urine are also presented.

Determination of Taxotere in human plasma by a semi-automated high-performance liquid chromatographic method.

A rapid, selective and reproducible high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed for the determination of the anti-cancer agent Taxotere in biological fluids. The method involves a solid-phase extraction step (C2 ethyl microcolumns) using a Varian Advanced Automated Sample Processor (AASP) followed by reversed-phase HPLC. The validated quantitation range of the method is 10-2500 ng/ml in plasma with coefficients of variation < or = 11%. The method is also suitable for the determination of Taxotere in urine samples under the same conditions. The method was applied in a phase I tolerance study of Taxotere in cancer patients, allowing the pharmacokinetic profile of Taxotere to be established.

Determination of paclitaxel and metabolites in mouse plasma, tissues, urine and faeces by semi-automated reversed-phase high-performance liquid chromatography.

We have developed and validated a sensitive and selective assay for the quantification of paclitaxel and its metabolites 6 alpha, 3'-p-dihydroxypaclitaxel, 3'-p-hydroxypaclitaxel and 6 alpha-hydroxypaclitaxel in plasma, tissue, urine and faeces specimens of mice. Tissue and faeces were homogenized (approximately 0.1-0.2 g/ml) in bovine serum albumin (40 g/l) in water, and urine was diluted (1:5, v/v) in blank human plasma. Sample pretreatment involved liquid-liquid extraction of 200-1000 microliters of sample with diethyl ether followed by automated solid-phase extraction using cyano Bond Elut columns. 2'-Methylpaclitaxel was used as internal standard. The overall recovery of the sample pretreatment procedure ranged from 76 to 85%. In plasma, the lower limit of detection (LOD) and the lower limit of quantitation (LLQ) are 15 and 25 ng/ml, respectively, using 200 microliters of sample. In tissues, faeces and urine the LLQs are 25-100 ng/g, 125 ng/g and 25 ng/ml, respectively, using 1000 microliters (faeces: 200 microliters) of homogenized or diluted sample. The concentrations in the various biological matrices, for validation procedures spiked with known amounts of the test compounds, are read from calibration curves constructed in blank human plasma in the range 25-100,000 ng/ml for paclitaxel and 25-500 ng/ml for the metabolites. The accuracy and precision of the assay fall within the generally accepted criteria for bio-analytical assays.

Taxol metabolism and disposition in cancer patients.

The objective of this study was to determine the metabolic fate and disposition of taxol in cancer patients. Five patients received 225 or 250 mg/m2 of taxol together with 100 microCi of [3H]taxol as a 3-hr infusion, followed by cisplatin and 5-fluorouracil. Urine, feces, and blood samples were collected for 120 hr and analyzed for total radioactivity, taxol, and metabolites by reversed-phase HPLC and tandem MS. Total urinary excretion was 14.3 +/- 1.4% (SE) of the dose, with unchanged taxol and an unknown polar metabolite as the main excretion products. Total fecal excretion was 71.1 +/- 8.2%, with 6 alpha-hydroxytaxol being the largest component by far. Unchanged taxol and four other metabolites could also be identified from fecal extracts. The plasma area under the curve for unchanged taxol was 20.5 +/- 2.3 microM.hr and that for total taxol metabolites was 14.2 +/- 4.5 microM.hr. The half-life of total metabolites (5.6 +/- 0.4 hr), however, greatly exceeded that of unchanged taxol (2.9 +/- 0.3 hr). Thus, at 5-hr posttaxol infusion, the plasma concentrations of the five metabolites together exceeded the taxol concentration by 2.4-fold. The findings from this study should be of importance as a guide to further therapeutic evaluation of this drug.

Paclitaxel metabolites in human plasma and urine: identification of 6 alpha-hydroxytaxol, 7-epitaxol and taxol hydrolysis products using liquid chromatography/atmospheric-pressure chemical ionization mass spectrometry.

Reversed-phase high-performance liquid chromatography/mass spectrometry (LC/MS), with an atmospheric-pressure chemical ionization (APCI) interface, has been applied to the identification of metabolites and derivatives of paclitaxel (taxol) in plasma and urine of patients treated with this new anticancer drug. Protonated molecules with substantial fragmentation were obtained using this ionization technique. The three ion series observed are characteristic of the intact molecule, the taxane ring, and the side chain at C13. Their analysis gives information about chemical modifications of the taxane structure at different positions of the molecule. Urine and plasma extracts were evaluated using the capacity to perform MS analysis directly on the entire effluent from conventional LC columns. Excellent spectra were obtained with 50 pmol of separated compounds in full scan mode. This technique allowed highly sensitive identification of 6 alpha-hydroxytaxol, the major human biliary metabolite, and of 7-epitaxol in extracts of plasma and urine from patients. Taxol hydrolysis derivatives were observed for the first time in urine 24 hours after the end of the infusion period. Sensitivity could be increased further using single ion monitoring (SIM) mode, once a target derivative was identified. These results demonstrate that LC/MS with an APCI interface is useful for the characterization and pharmacokinetic analysis of taxoids in biological matrices.

Assay of paclitaxel (Taxol) in plasma and urine by high-performance liquid chromatography.

A new, rapid and sensitive high-performance liquid chromatographic method for the analysis of paclitaxel (Taxol) in human plasma and urine was developed and validated. After addition of an internal standard, paclitaxel was extracted from plasma or urine by a liquid-liquid extraction using diethyl ether. Extraction efficiency averaged 90%. Chromatography was performed isocratically on a reversed-phase column monitored at 227 nm. Retention times were 7.7 and 6.7 min for paclitaxel and docetaxel, respectively, and the assay was linear in the range 25-1000 ng/ml. The limits of quantification for paclitaxel were 25 and 40 ng/ml in plasma and urine, respectively. The assay was shown to be suitable for pharmacokinetic studies of children involved in a phase I clinical trial.

Tissue distribution, metabolism and excretion of paclitaxel in mice.

So far, all animal pharmacokinetic studies of paclitaxel, which used analytical procedures based on HPLC, have not been sensitive enough to quantify drug levels below 500 ng/ml. Consequently, the interpretation of the results is restricted because drug levels of paclitaxel as low as at least 50 nM (43 ng/ml) are relevant for the pharmacology of this drug. We recently described an accurate and very sensitive method based on HPLC for the determination of paclitaxel and the metabolites 3'-p-hydroxypaclitaxel (I), 6 alpha-hydroxypaclitaxel (II) and 6 alpha,3'-p-dihydroxypaclitaxel (III) in a wide variety of biological matrices. We have now implemented this methodology in a comprehensive pharmacokinetic study in female FVB mice. Previous pharmacokinetic studies in humans demonstrated a large steady-state volume of distribution, indicating that the drug is widely distributed into tissues. Comprehensive tissue distribution studies may, therefore, be helpful in providing more insight into possible relationships between plasma levels, drug levels in tissues and toxicity. Paclitaxel, formulated in Cremophor EL and ethanol (1:1, v/v), was given as a single i.v. bolus dose of 2, 10 and 20 mg/kg to female FVB mice. Except for the brain, the distribution of paclitaxel to all other tissues in the female mice was substantial and maximum drug levels were achieved within 0.5 or 1 h. A marked non-linear increase in the area under the concentration-time curve (AUC) in plasma was observed, which was not paralleled by a proportional increase in the tissue AUC levels. It is postulated that this effect may be related to the substantial amounts of Cremophor EL administered concurrently. The recovery of paclitaxel in the feces (0-96 h) was reduced from 58% at the 2 mg/kg dose level to 44% at the 20 mg/kg dose level. Small amounts of metabolites I and II were detected in the gut, liver and gall bladder, but not in the systemic circulation or any other tissue. Metabolite III was not detected. Metabolites I and II are likely excreted directly into the bile, and since their recovery in the feces accounts for about 25% of the administered dose, their formation thus represents an important pathway of detoxification.

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine.

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5-1000 ng/ml with RSDs < or = 10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.