Cristina Sottani

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.

Occupational exposure to antineoplastic drugs in four Italian health care settings.

Exposure assessment of health care workers to antineoplastic drugs (ADs) is still an open issue since new, critical, and emerging factors may put pharmacists who prepare hazardous drugs or nurses who administer anti cancer agents to an increased risk of developing adverse health effects. Overall, eight pharmacies and nine patient areas have been surveyed in this study. Wipe and pad samples were experienced during the surveillance program in four Italian health care settings. Urine samples were collected from workers handling ADs. Cyclophosphamide (CP), ifosfamide (IF), and gemcitabine (GEM) were detected in all the work environments by using a LC-MS/MS method-based capable of analysing all the three drugs simultaneously. In total, 54% of wipe samples were positive for at least one drug and 19% of pad samples were shown to be contaminated by cyclophosphamide. Pharmacies were generally more contaminated than patient areas with the exception of one site where a nurse had an acute exposure during the cleaning-up of an hazardous drug solution spill. In total, 22 urine samples collected from pharmacists and 78 urine samples from nurses had no detectable concentrations of any antineoplastic drugs. Despite the adherence to the recommended safety practices residue contamination on surfaces and floors has continued to be assessed in all the investigated sites.

Occupational exposure to antineoplastic drugs in seven Italian hospitals: the effect of quality assurance and adherence to guidelines

In health care facilities, dermal contact and inhalation are considered to be the main routes of exposure to cytotoxic antineoplastic drugs (ADs). Hand-to-mouth contamination or accidental needle sticks as well as events due to inadequate disposal may also contribute to exposure. In order to measure the extent of contamination, biological and environmental monitoring are essential tools for routine testing. Moreover, reliable sampling and analytical procedures are required. During the last decade, several methods have been developed and validated. The appropriate analytical techniques were used to quantify even very low levels of some of the more commonly used ADs, such as cyclophosphamide, 5-fluoruracil, taxol, anthracyclines, and platinum-compounds. The main objective of this study is to assess the adherence to existing standards of practice through an effective monitoring program, including environmental and biological measurements. In seven hospitals located in Northern-Central Italy, periodic surveys were scheduled to verify continuing compliance with guidelines over a 5-year period. All biological samples were found to be below detection limits and a progressive, significant decrease in workplace contamination was observed. Our results confirm that a cost-effective monitoring regime, including fast and simple sample pre-treatment procedures, simultaneous determination of the analytes and their metabolites, validated procedures including uncertainty evaluation, and periodic surveys, is the adequate approach for the collection of reliable exposure data and hence for effective intervention.

Validation of an LC–MS/MS method for the determination of epirubicin in human serum of patients undergoing Drug Eluting Microsphere-Transarterial Chemoembolization (DEM-TACE)

Drug Eluting Microsphere-Transarterial Chemoembolization (DEM-TACE) is a new delivery system to administrate drugs in a controlled manner useful for application in the chemoembolization of colorectal cancer metastases to the liver. DEM-TACE is focused to obtain higher concentrations of the drug to the tumor with lower systemic concentrations than traditional cancer chemotherapy. Therefore a specific, precise and sensitive LC-ESI-MS/MS assay procedure was properly designed to detect and quantify epirubicin at the concentrations expected from a transarterial chemoembolization with microspheres. Serum samples were kept acidic (pH approximately of 3.5) and sample preparation consisted of a solid phase extraction (SPE) procedure with HLB OASIS cartridges using a methylene chloride/2-propanol/methanol mixture solution to recover epirubicin. The analyses consisted of reversed-phase high-performance liquid chromatography (rp-HPLC) coupled with tandem mass spectrometry (MS/MS). Accuracy, precision and matrix effect of this procedure were carried out by analyzing four quality control samples (QCs) on five separate days. The validation parameters were assessed by recovery studies of spiked serum samples. Recoveries were found to vary between 92 and 98% at the QC levels (5, 40, 80 and 150 microg/L) with relative standard deviation (RSD) always less than 3.7%. The limit of detection (LOD) was set at 1 microg/L. The developed procedure has been also applied to investigate the different capability of two types of commercially available microspheres to release epirubicin into the human circulatory system.

Simultaneous measurement of doxorubicin and reduced metabolite doxorubicinol by UHPLC–MS/MS in human plasma of HCC patients treated with TACE

A sensitive, selective, accurate and precise method for simultaneous quantification of doxorubicin (DOX) and doxorubicinol (DOXol) in human plasma of patients diagnosed as having intermediate stage unresectable hepatocellular carcinoma (HCC) was developed. The method was based on electrospray tandem mass spectrometry in selected reaction monitoring mode. DOX, DOXol and trofosfamide, an internal standard, were extracted from plasma by using a simple solid phase extraction (SPE) procedure after the addition of 0.1 M hydrochloric acid. A 200-μL aliquot of the extracted sample reconstituted in mobile phase was analyzed on a Zorbax SB-C18 UHPLC column (50 mm × 2.1 mm, 1.8 μm particle size) in 8 min. The mobile phase consisted of acetonitrile and 0.1% formic acid pH 4.5 (95:05 v/v). Good accuracy and precision of this method were demonstrated by determination of spiked plasma QC samples in four consecutive days. The SPE extraction recoveries ranged from 72.3 to 77.3% and 75.5 to 98.4% for doxorubicin and doxorubicinol, respectively. The intra-day and inter-day precisions were less than 11.4%. The limit of quantitation was 1.0 ng/mL for both compounds. The calibration curves of DOX and DOXol were analyzed by weighted linear regression with 1/x as a weighting factor. They were linear over the concentration range of 1.0-100.0 ng/mL with R(2) greater than 0.99. This developed method was successfully applied to study plasma pharmacokinetics in patients affected by HCC and treated with transarterial chemoemolization practices (TACEs) using HepaSphere™ pre-loaded with DOX in a standardized procedure.

Monitoring Surface Contamination by Antineoplastic Drugs in Italian Hospitals: Performance-Based Hygienic Guidance Values (HGVs) Project

Antineoplastic drugs (ADs) will continue to represent a potential risk for personnel involved in the handling of these compounds and great concerns have been raised by the presence of ADs in many surveyed workplaces. Eight hospitals were investigated by means of wipe sampling for surface residue determination. Each wipe sample was tested for five ADs considered suitable exposure markers. Cyclophosphamide (CP), gemcitabine (GEM), 5-fluorouracil (5-FU), platinum-containing drugs (Pt), and epi-doxorubicin (EPI) contamination levels were measured in 85 per cent of the studied pharmacies and 93 per cent of outpatient care units (OpCUs). This study showed that 83 out of 349 samples were positive in Pharmacies, this proportion being statistically significant (χ2 = 42.9, p < 0.001). The positive samples provided evidence of at least one substance with levels greater than the limit of detection (LOD). The two most frequently detected substances were Pt (42%) and CP (30%). These accounted for 72 per cent of the whole dataset, followed by 5-FU and GEM. Based on the 90th percentile of wipe sampling data distribution, we suggest hygienic guidance values (HGVs) of 3.6, 1.0, 0.9, and 0.5 ng cm-2 for CP, 5-FU, GEM and Pt, respectively, as the best target levels of the surface contamination load in Italian pharmacies. The approach of proposing guidance values at the 90th percentile of results obtained from workplaces with good hygiene practice was found to be a simple and practical way of controlling occupational exposure. HGVs were challenged in this study as technical threshold limits to benchmark AD residual surface contamination at workplaces.

A new, sensitive and versatile assay for quantitative determination of α-fluoro-β-alanine (AFBA) in human urine by using the reversed-phase ultrahigh performance-tandem mass spectrometry (rp-UHPLC–MS/MS) system

A method for the quantitation of α-fluoro-β-alanine (AFBA), the main metabolite of capecitabine (Cape) and 5-fluoruracil (5-FU), is described. Among antineoplastic drugs (ADs), 5-FU and Cape (the new oral prodrug) are the most commonly applied drugs in cancer therapy. The main objective of this study was to develop a reliable method that would be easy to run on a reversed-phase UHPLC system coupled to tandem mass spectrometry. AFBA was derivatized with Sangers reagent to ensure complete yield of a stable 2,4 dinitrophenil-α-fluoro-β-alanine derivative. This method was based on the use of a mixed-mode anion exchange solid phase extraction enabling urinary extracts to be clear of endogenous interferences affecting quantitative results. The assay was validated in human urine according to FDA criteria with the use of a labeled internal standard (β-alanine-d4) to minimize experimental error. Good accuracy and precision were demonstrated by determining spiked urine QC samples in four consecutive days. The recovery of AFBA was between 70.0 and 82.6%, with a matrix effect that was 12.8%-18.5%. The lower limit of quantitation (LOQ) was 0.5 ng/mL with a coefficient of variation of 5.3%. This assay was successfully applied to determine the levels of this metabolite in a large number of urine samples taken from personnel who were occupationally exposed to ADs.

Trattamento loco regionale dell’epatocarcinoma non resecabile mediante chemioembolizzazione con microparticelle caricate con epirubicina: valutazione di efficacia, tollerabilità e analisi farmacocinetica in uno studio clinico monocentrico

Image-guided transcatheter arterial chemoembolization (TACE) is the first-line treatment for patients with intermediate stage HCC not candidate for curable therapies. Recently, microembolizing particles have been introduced in clinical practise; they are able to be loaded with chemotherapic agents, in particular doxorubicin, cisplatin and epirubicin. By now, two types of microparticles are mostly used in clinical practise: DC Bead® and HepaSphere Microsphere®. Drug Eluting Beads (DEBs)-TACE reduces side effects and improve local efficacy because the microparticles have a double effect: they embolize the arteries feeding the neoplastic lesion and they gradually realise the chemotherapic agent in the tumoral bed, to obtain high intratumoral drug levels and low systemic drug concentrations. The purpose of this study is to determine the prevalence of complication in patients affected by intermediate stage HCC treated with DEBs-TACE. Between January 2007 and December 2009, 80 patients have been treated with DEBs-TACE for a total of 125 treatments. Everyone was affected by intermediate stage HCC, mostly BCLC B; they received Drug Eluting Beads (DEBs)-TACE with DC Bead or HepaSphere MicroSphere pre-loaded with epirubicin (50 mg/vial). Biochemical blood analysis were performed before, 4 and 24 hours after the procedure to monitor some hematologic parameters. Side effects were reported following the Common Toxicity Criteria in order to evaluate post-TACE tossicity. Tumor response was assessed after 40 days from the procedure with CT scans according to the modified Response Evaluation Criteria in Solid Tumors (mRECIST). This study shows that the use of drug-eluting microspheres did not increase the risk of post-procedural complication and the prevalence of major adverse events are similar to conventional TACE, according to data reported in literature. We, also, noticed no differences in the prevalence of adverse events between patients treated with DC Bead-TACE and HepaSphereTACE. Moreover, in a selected group of 20 patients, 12 treated with DC Bead and 8 with HepaSphere, we performed peripheral blood samples analysis at the end of the embolic solution delivery and at 5, 10, 20, 40, 60, 120, 180, 360, 1,440 minutes after the procedure to assess epirubicin serum pharmacokinetic. The pharmacokinetic study showed low peak serum epirubicin concentrations as median peak level was 73.5+/-24.5 ng/mL for the DC Bead group and 33.9+/-11.0 ng/mL for the HepaSphere cohort. The highest drug concentration was observed after microspheres injection at 5 minutes in all 20 patients. The PK profile never dropped to zero up until the end of the experiment. In the time-interval included between 1 and 24 hours, persisting levels of epirubicin were detected in patients’peripheral blood samples, ranging from 2.3 to 24.2 ng/mL for both embolics. This study suggests that, after an earlier release effect, occurring during the very first few minutes, both microspheres are capable of a sustained kinetic release.