Comparative evaluation of the My5-FU™ immunoassay and LC-MS/MS in monitoring the 5-fluorouracil plasma levels in cancer patients.

BACKGROUND: Chemotherapies of solid tumors commonly include 5-fluorouracil (5-FU). With standard doses of 5-FU, substantial inter-patient variability has been observed in exposure levels and treatment response. Recently, improved outcomes in colorectal cancer patients due to pharmacokinetically guided 5-FU dosing were reported. We aimed at establishing a rapid and sensitive method for monitoring 5-FU plasma levels in cancer patients in our routine clinical practice. METHODS: Performance of the Saladax My5-FU™ immunoassay was evaluated on the Roche Cobas® Integra 800 analyzer. Subsequently, 5-FU concentrations of 247 clinical plasma samples obtained with this assay were compared to the results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and other commonly used clinical analyzers (Olympus AU400, Roche Cobas c6000, and Thermo Fisher CDx90). RESULTS: The My-FU assay was successfully validated on the Cobas Integra 800 analyzer in terms of linearity, precision, accuracy, recovery, interference, sample carryover, and dilution integrity. Method comparison between the Cobas Integra 800 and LC-MS/MS revealed a proportional bias of 7% towards higher values measured with the My5-FU assay. However, when the Cobas Integra 800 was compared to three other clinical analyzers in addition to LC-MS/MS including 50 samples representing the typical clinical range of 5-FU plasma concentrations, only a small proportional bias (≤1.6%) and a constant bias below the limit of detection was observed. CONCLUSIONS: The My5-FU assay demonstrated robust and highly comparable performance on different analyzers. Therefore, the assay is suitable for monitoring 5-FU plasma levels in routine clinical practice and may contribute to improved efficacy and safety of commonly used 5-FU-based chemotherapies.

Inverse relationship between leukaemic cell burden and plasma concentrations of daunorubicin in patients with acute myeloid leukaemia.

AIMS: It has been shown that the cellular uptake and cytotoxicity of anthracyclines decrease with increasing cell density in vitro, an event termed 'the inocculum effect'. It is not known whether such an effect occurs in vivo. In this study the relationships between white blood cell (WBC) count, plasma and cellular concentrations of daunorubicin (DNR) in patients with acute myeloid leukaemia were investigated. METHODS: Plasma and mononuclear blood cells were isolated from peripheral blood from 40 patients with acute myeloid leukaemia at end of infusion (time 1 h), 5 and 24 h following the first DNR infusion. DNR concentrations were determined by high-pressure liquid chromatography and related to the WBC count at diagnosis. A population pharmacokinetic model was used to estimate the correlations between baseline WBC count, volume of distribution and clearance of DNR. RESULTS: A clear but weak inverse relationship between the baseline WBC count and plasma concentrations of DNR (r(2)=0.11, P<0.05) at time 1 was found. Furthermore, a clear relationship between baseline WBC count and DNR central volume of distribution using population pharmacokinetic modelling (dOFV 4.77, P<0.05) was also noted. Analysis of plasma DNR and the metabolite daunorubicinol (DOL) concentrations in patients with a high WBC count support that the low DNR/DOL concentrations are due a distribution effect. CONCLUSION: This study shows that the leukaemic cell burden influences the plasma concentrations of anthracyclines. Further studies are needed to explore if patients with high a WBC count may require higher doses of anthracyclines.

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 x 4 mm)) and an analytical column (Zorbax XDB C18, 150 x 4.6 mm, 5 microm) with a 23 min total cycle time. The method is linear (r(2)>0.998) over the range of 0.5-25 microg/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 microg/mL. Intra- and inter-day precision were acceptable (< or = 15% RSD) at and above the LOQ. The method was applied to the analysis of serum samples from patients undergoing chemotherapy with these agents.

Simple and sensitive HPLC method for determination of amrubicin and amrubicinol in human plasma: application to a clinical pharmacokinetic study.

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for determination of amrubicin and its metabolite amrubicinol in human plasma. After protein precipitation with methanol without evaporation procedure, large volume samples were injected and separated by two monolithic columns with a guard column. The mobile phase consisted of tetrahydrofuran-dioxane-water (containing 2.3 mM acetic acid and 4 mM sodium 1-octanesulfonate; 2:6:15, v/v/v). Wavelengths of fluorescence detection were set at 480 nm for excitation and 550 nm for detection. Under these conditions, linearity was confirmed in the 2.5-5000 ng/mL concentration range of both compounds. The intra- and inter-day precision and intra- and inter-day accuracy for both compounds were less than 10%. The method was successfully applied to a clinical pharmacokinetic study of amrubicin and amrubicinol in cancer patients.

CE separation approaches for combinations of anthracyclines and taxanes.

For the first time the versatility of CE is demonstrated for the separation of different types of anticancer drugs - anthracyclines and taxanes simultaneously. The use of these drugs in combination therapy for cancer has sparked interest in the development of methods for potential application. The simultaneous analysis of anthracyclines and taxanes can significantly increase a sample throughput of a clinical laboratory. The study shows the potential of CE for such a challenge: anthracyclines and taxanes were separated by CZE, MEKC and MEEKC. The MEEKC method was successfully applied to these compounds for the first time and was characterised by very short separation time, high efficiencies of peaks and was proven to be generic for the separation of different combinations of anthracyclines and taxanes. This separation approach could be highly beneficial for clinical analysis if applied with a sensitive detection system. MEKC and high-speed MEEKC methods were proven to show good potential in their application to plasma samples.

Plasma concentration of amrubicinol in plateau phase in patients treated for 3 days with amrubicin is correlated with hematological toxicities.

Amrubicinol (AMR-OH) is an active metabolite of amrubicin (AMR), a novel synthetic 9-aminoanthracycline derivative. The time-concentration profile of AMR-OH exhibits a continuous long plateau slope in the terminal phase. To determine the relationships between the steady-state plasma concentration of AMR-OH and treatment effects and toxicities associated with AMR therapy, we carried out a pharmacokinetic/pharmacodynamic study in patients treated with AMR alone or the combination of AMR+cisplatin (CDDP). AMR was given at a dose of 30 or 40 mg/m(2) on days 1-3. Plasma samples were collected 24 h after the third injection (day 4). Plasma concentrations of AMR-OH or total CDDP were determined by a high-performance liquid chromatography or an atomic absorption spectrometry. Percent change in neutrophil count (dANC) and the plasma concentration of AMR-OH were evaluated using a sigmoid E(max) model. A total of 35 patients were enrolled. Significant relationships were observed between AMR-OH on day 4 and the toxicity grades of leukopenia, neutropenia, and anemia (P=0.018, P=0.012, and P=0.025, respectively). Thrombocytopenia grade exhibited a tendency toward relationship with AMR-OH on day 4 (P=0.081). The plasma concentration of AMR-OH on day 4 was positively correlated with dANC in the group of all patients, as well as in patients treated with AMR alone and in patients coadministered with CDDP. In conclusion, the plasma concentration of AMR-OH on day 4 was correlated with hematological toxicities in patients treated with AMR. The assessment of plasma concentration of AMR-OH at one timepoint might enable the prediction of hematological toxicities.

Uptake of anthracyclines in vitro and in vivo in acute myeloid leukemia cells in relation to apoptosis and clinical response.

AIMS: To study anthracycline-induced apoptosis in leukemic cells isolated from patients with acute myelogenous leukemia (AML) in vitro and to compare intracellular anthracycline concentrations causing apoptosis in vitro with those obtained in vivo during anthracycline treatment. METHODS: Mononuclear blood cells from AML patients were isolated before (n = 20) and after anthracycline infusion (n = 24). The pre-treated cells were incubated in vitro with daunorubicin (DNR) and/or idarubicin (IDA). Anthracycline concentrations were determined by high-performance liquid chromatography, and apoptosis was detected by propidium iodine staining using a flow cytometer. RESULTS: There was a clear concentration-response relationship between intracellular anthracycline levels and apoptosis albeit with a large interindividual variation. Intracellular levels >1200 muM always led to high apoptosis development (>60%) in vitro. The intracellular concentrations of DNR in vivo (n = 24) were more than tenfold lower than the concentrations needed to induce effective apoptosis in vitro, although a significant relation between in vivo concentrations and clinical remission was found. We also found a significant relation between apoptosis induction in leukemic cells by IDA in vitro and clinical remission. CONCLUSIONS: Our results indicate that intracellular anthracycline levels in vivo are suboptimal and that protocols should be used that increase intracellular anthracycline levels.

Pulse gradient, large-volume injection, high-throughput ultra-performance liquid chromatographic/tandem mass spectrometry bioanalysis for measurement of plasma amrubicin and its metabolite amrubicinol.

In this communication, we report the development of a new ultra-performance liquid chromatographic/tandem mass spectrometry (UPLC-MS-MS) assay for measurement of amrubicin (an anthracycline anti-cancer agent) and its active metabolite, amrubicinol, in plasma. The enhanced electrospray ionization signal intensity of the analytes achieved by modifying the mobile phase with formic acid was associated with improvement in the lower limit of quantification. These favorable effects were electrolyte concentration-dependent. In order to maximize assay throughput, we used methanol protein precipitation to prepare the plasma samples, and simplified sample preparation by injecting 40 microL of the supernatant containing methanol at 87.5% (v/v) directly onto the UPLC column without any intermediary solvent evaporation step. The large-volume injection of highly organic supernatant sample increased matrix and elutropic effects, but these drawbacks were respectively overcome by using a 5mM formic acid-modified mobile phase and a new pulse gradient method. To our knowledge, this is the first report successfully using large-volume injection of strong organic samples with UPLC-MS-MS bioanalysis. The pulse gradient elution also resulted in band compression and enhanced the robustness of the chromatography. The promising new approach illustrated herein is extremely straightforward to optimize, and may be used for UPLC-MS-MS bioanalytical assay of other compounds.

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%.

[A case of advanced small cell lung cancer complicated by chronic renal failure treated with amrubicin].

A 64-year old man first visited our clinic approximately 10 years ago because of diabetic nephropathy that had developed into chronic renal failure. He was hospitalized to examine a left S10 tumor shadow. Based on the results of these examinations, a primary left S10 T2N0M1, ED small cell lung cancer, was diagnosed. During his outpatient visits nephropathy was found. Following admission, he began dialysis (HD). During the detailed examinations, chemotherapy with amrubicin (AMR)was performed and the blood concentration of the drug was measured. The results showed no significant variations in blood concentration before and after the dialysis. While PR was achieved in this patient, a reduction in grade 4 eosinophils was observed as an adverse reaction.

[A case of small cell lung cancer under hemodialysis with chronic renal failure treated with amrubicin while measuring blood concentration of the drug].

We reported a case of small cell lung cancer treated with amrubicin while receiving hemodialysis. An 83-year-old man with chronic renal failure being treated by hemodialysis was admitted because of a left hilar mass. Small cell lung cancer with liver metastasis (cT2NOM1) was diagnosed. Two courses of chemotherapy with amrubicin resulted in partial response. Toxicity was relatively mild. We measured blood concentration of amrubicin during the first course of chemotherapy. There was no significant difference in blood cell and plasma concentration of amrubicin hydrochloride and amrubicinol between days when he received hemodialysis and when he did not receive hemodialysis. Thus, we considered that amrubicin hydrochloride may be a good candidate for the treatment of small cell lung cancer patients with chronic renal failure under hemodialysis.

pH gradient loading of anthracyclines into cholesterol-free liposomes: enhancing drug loading rates through use of ethanol.

Application of cholesterol-free liposomes as carriers for anticancer drugs is hampered, in part, because of standard pH gradient based loading methods that rely on incubation temperatures above the phase transition temperature (Tc) of the bulk phospholipid to promote drug loading. In the absence of cholesterol, liposome permeability is enhanced at these temperatures which, in turn, can result in the collapse of the pH gradient and/or unstable loading. Doxorubicin loading studies, for example, indicate that the drug could not be loaded efficiently into cholesterol-free DSPC liposomes. We demonstrated that this problem could be circumvented by the addition of ethanol as a permeability enhancer. Doxorubicin loading rates in cholesterol-free DSPC liposomes were 6.6-fold higher in the presence of ethanol. In addition, greater than 90% of the added doxorubicin was encapsulated within 2 h at 37 degrees C, an efficiency that was 2.3-fold greater than that observed in the absence of ethanol. Optimal ethanol concentrations ranged from 10% to 15% (v/v) and these concentrations did not significantly affect liposome size, retention of an aqueous trap marker (lactose) or, most importantly, the stability of the imposed pH gradient. Cryo-transmission electron micrographs of liposomes exposed to increasing concentrations of ethanol indicated that at 30% (v/v) perturbations to the lipid bilayer were present as evidenced by the appearance of open liposomes and bilayer sheets. Ethanol-induced increased drug loading was temperature-, lipid composition- and lipid concentration-dependent. Collectively, these results suggest that ethanol addition to preformed liposomes is an effective method to achieve efficient pH gradient-dependent loading of cholesterol-free liposomes at temperatures below the Tc of the bulk phospholipid.

Equilibrium binding of anthracycline cytostatics to serum albumin and small unilamellar phospholipid vesicles as measured by gel filtration.

A Sephadex G-200 gel filtration method was used to measure directly the equilibrium binding of five important anthracycline analogs to serum albumin. The order of the overall binding constant (K) in a 150 mM NaCl, 20 mM Hepes buffer (pH 7.45) was doxorubicin < daunorubicin < 4-demethoxydaunorubicin approximately 13-dihydro-4'-deoxy-4'-iododoxorubicin < 4'-deoxy-4'-iododoxorubicin for human serum albumin (K = 2.67 +/- 0.07 mM(-1) to 24.5 +/- 3.1 mM[-1]) and bovine serum albumin (K = 1.36 +/- 0.25 mM(-1) to 48.4 +/- 5.2 mM[-1]). Data were given on the pH-dependence of K. The anthracycline-albumin association reaction was compared with measurements of drug partitioning into unilamellar phospholipid membranes and octanol. The results provide important new data required for a systematic kinetic analysis of anthracycline transport in tumor cells under serum conditions in a biological system.

Prediction of anthracycline cardiotoxicity after chemotherapy by biomarkers kinetic analysis.

Anthracyclines are active drugs against breast cancer, but can exert cardiotoxic effects. We analyzed the association between the kinetics of various biomarkers during chemotherapy, and the risk of subsequent cardiac toxicity. 50 patients (49 women) with early breast cancer surgically treated and eligible to anthracycline-based adjuvant chemotherapy were analyzed. The left ventricular ejection fraction (LVEF) together with the plasma concentration of several blood markers was measured at the beginning of anthracycline chemotherapy (t (0)), 5 months (t (1)), 16 months (t (2)), 28 months (t (3)), and 40 months later (t (4)). A single measured LVEF value less than 50% or a clinically overt congestive heart failure (CHF) was considered cardiotoxic effects. We tested whether the kinetics of LVEF and blood biomarkers measured during chemotherapy was predictive of subsequent cardiotoxicity and overall cardiac fitness. The left ventricular ejection fraction measured at the end of treatment as well as the rate of change of hemoglobin concentration during anthracycline-based chemotherapy predicted cardiotoxicity in a 3-year follow-up period. When LVEF at the end of chemotherapy was lower than 53% or hemoglobin blood concentration declined more than 0.33 g/dL/month during chemotherapy, the odds ratio of subsequent cardiotoxicity was 37.3 and 18, respectively. The specificity of these two tests was 93.3% and 80%, whereas the sensitivity was 90.9 and 81.2%, respectively. Testing the rate of change of hemoglobin concentration during anthracycline-based chemotherapy, as well as the left ventricular ejection fraction at the end of treatment, seems a powerful method to assess the effects of anthracyclines on cardiac fitness and identify patients at high risk of CHF. Further validation of these tests on a large cohort of patients and cost-benefit analysis should be encouraged.

Pharmacokinetic and pharmacodynamic study on amrubicin and amrubicinol in Japanese patients with lung cancer.

PURPOSE: The pharmacokinetic (PK)-pharmacodynamic (PD) relationship of amrubicin and its active metabolite, amrubicinol, has only been evaluated using trough levels of these agents since the full PK profiles not yet been clarified so far. This study was performed to analyze the full PK profiles of amrubicin and amrubicinol and to evaluate their toxicity-PK relationships in Japanese patients. METHODS: Amrubicin (35-40 mg/m(2)) was administered to 21 lung cancer patients on days 1-3 every 3-4 weeks. Fourteen blood samples were obtained per patient over the course of 3 administration days. The plasma concentrations of amrubicin and amrubicinol were quantitated by HPLC, and the relationships between PK parameters of these compounds and hematological toxicities were evaluated. RESULTS: The overall PK profiles of amrubicin and amrubicinol were well characterized using a 3-compartment model and a 1-compartment model with a first-order metabolic process, respectively. The major toxicities were hematological. The clearance of amrubicinol was significantly correlated with grade 4 neutropenia (P = 0.01). The percentage decreases in the neutrophil count, hemoglobin level and platelet count were well correlated with the amrubicinol AUC. CONCLUSION: The pharmacokinetic profiles of amrubicin and amrubicinol were clarified, and the subsequent PK-PD analyses indicate that the clearance of amrubicinol is the major determinant of neutropenia.

Quantitative liquid chromatographic analysis of anthracyclines in biological fluids.

Anthracyclines are amongst the most widely used drugs in oncology, being part of the treatment regimen in most patients receiving systemic chemotherapy. This review provides a comprehensive summary of the sample preparation techniques and chromatographic methods that have been developed during the last two decades for the analysis of the 4 most administered anthracyclines, doxorubicin, epirubicin, daunorubicin and idarubicin in plasma, serum, saliva or urine, within the context of clinical and pharmacokinetic studies or for assessing occupational exposure. Following deproteinization, liquid-liquid extraction, solid phase extraction or a combination of these techniques, the vast majority of methods utilizes reversed-phase C18 stationary phases for liquid chromatographic separation, followed by fluorescence detection, or, more recently, tandem mass spectrometric detection. Some pros and cons of the different techniques are addressed, in addition to potential pitfalls that may be encountered in the analysis of this class of compounds.

Co-administration of irinotecan decreases the plasma concentration of an active metabolite of amrubicin, amrubicinol in rats.

PURPOSE: This study examined the pharmacokinetics of irinotecan (CPT-11), active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), SN-38 glucuronide (SN-38G) amrubicin (AMR), and active metabolite amrubicinol (AMR-OH) after intravenous administration of this combination therapy in rats. METHODS: Male Sprague-Dawley rats were treated with 10 mg/kg CPT-11 with 10 mg/kg AMR. AMR, AMR-OH, CPT-11, SN-38 and SN-38G were measured in plasma, bile, and tissues using high-performance liquid chromatography. RESULTS: Co-administration of CPT-11 resulted in a significant decrease in plasma concentrations and area under the curves (AUC) of AMR-OH compared with treatment with AMR alone. On the other hand, co-administration of AMR resulted in a slight increase in the initial plasma concentration of SN-38; however, there were no differences in AUC values in CPT-11 and SN-38. The cumulative biliary excretion curves of AMR, CPT-11, and their active metabolites were not changed. CPT-11 inhibited the conversion of AMR to AMR-OH in rat cytosolic fractions. CONCLUSIONS: CPT-11 did not affect the pharmacokinetic of AMR but decreased the plasma concentration of AMR-OH and might affect the formation of AMR-OH from AMR in hepatocytes.

Development and validation of a liquid chromatographic method for the simultaneous determination of four anthracyclines and their respective 13-S-dihydro metabolites in plasma and saliva.

A quantitative HPLC method with fluorescence detection has been developed for the simultaneous determination of four anthracyclines (doxorubicin, epirubicin, daunorubicin and idarubicin) and their respective 13-S-dihydro metabolites (doxorubicinol, epirubicinol, daunorubicinol and idarubicinol) in plasma and saliva, using epidaunorubicin as internal standard. A progressive optimization matrix led to a two-step extraction based on a protein precipitation with ethanol followed by a liquid-liquid extraction with dichloromethane after pH adjustment to 8.5. The chromatographic separation was performed in 14min on a C18 column, applying gradient elution with a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The analytes were detected and quantified at an excitation and emission wavelength of 480 and 555nm, respectively. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.3 or 0.75, and 1 or 2.5ng/mL, respectively. Linearity by means of weighted (1/x) regression was obtained from the LLOQ up to 1000 or 2500ng/mL for the parent drugs and up to 400 or 1000ng/mL for the metabolites. Intra-assay and inter-assay relative standard deviation values were all less than 14% at low, medium and high levels, and below 17% at the LLOQ. Accuracy ranged between 91 and 113% at low, medium and high concentrations and between 83 and 118% at the LLOQ. Absolute recoveries were between 78 and 88% in plasma, and between 70 and 79% in saliva, respectively. Autosampler, benchtop, freeze-thaw and long-term stability samples fulfilled acceptance criteria. This selective method was applied successfully to the analysis of plasma and saliva samples from patients administered epirubicin intravenously.

Analysis of antibiotics by capillary electrophoresis.

Recent developments in the characterization of antibiotics are reviewed. Many capillary electrophoretic techniques have been utilized in their analyses, addressing various aspects of quantifying, profiling, and monitoring. Laser-induced fluorescence detection systems demonstrated their usefulness in clinical settings and in the monitoring of residue levels in food matrices. Different sample introduction methods have been explored, enhancing detection sensitivity, or reducing or eliminating sample manipulation prior to injection.