Surviving adversity: Exploring the presence of Lunularia cruciata (L.) Dum. on metal-polluted mining waste.

The tailings dump of Barraxiutta (Sardinia, Italy) contains considerable concentrations of heavy metals and, consequently, is scarcely colonized by plants. However, wild populations of the liverwort Lunularia cruciata (L.) Dum. form dense and healthy-looking carpets on this tailing dump. L. cruciata colonizing the tailing dump was compared with a control population growing in a pristine environment in terms of: (i) pollutant content, (ii) photochemical efficiency, and (iii) volatile secondary metabolites in thalli extracts. L. cruciata maintained optimal photosynthesis despite containing considerable amounts of soil pollutants in its thalli and had higher sesquiterpene content compared to control plants. Sesquiterpenes have a role in plant stress resistance and adaptation to adverse environments. In the present study, we propose enhanced sesquiterpenes featuring Contaminated L. cruciata as a defence strategy implemented in the post-mining environment.

Effectiveness of a combined UV-C and ozone treatment in reducing healthcare-associated infections in hospital facilities.

BACKGROUND: Hospital-acquired infections pose an ongoing threat to patient safety due to the presence of multi-drug-resistant organisms (MDROs) and other pathogens such as Clostridioides difficile which are dependent on thorough and effective cleaning and disinfection by personnel. METHODS: This study evaluated the influence of UV-C air treatment: the air in the room was sanitized by UV-C and redirected into the room. In addition, ozone was released into the room to treat actual surfaces in low-risk areas such as hospital gyms, and high- to medium-risk areas such as hospital rooms. To this aim, a portable device designed for treating the environment air was tested against nine bacterial strains including Aspergillus spp. and Clostridioides spp. RESULTS: The use of UV-C air treatment during daily operations and ozone treatment achieved at least a 2-log10 pathogen reduction except for Clostridioides spp. CONCLUSION: Effective prevention of C. difficile normally requires the use of combined approaches that include chemical compounds and disinfection agents whose toxicity can be harmful not only to patients but also to healthcare personnel. Thus, the proposed no-touch device may be evaluated in future research to assess the needed requirements for its possible and full implementation in hospitals.

[Antineoplastic profile assessment for the evaluation of occupational exposure in hospital personnel involved in handling cytotoxic agents]. / Definizione del profile di chemioterapici antiblastici nella valutazione dell'esposizione professionale in ambito sanitario.

Environmental and biological monitoring performed in health care settings during a 14-year period allowed numerous data to be obtained by using the HPLC coupled with tandem mass spectrometry (HPLC-MS/MS). Data was stored in a specific data-base for the assessment of risk exposure to antineoplastic agents (CA). The strategy of analysis was based on the simultaneous determination of different active substances with a lower limit of quantification (LOQ) optimized in the range of sub-units of microg/L. In the present study, the MRM-ESI-MS/MS profiles of seven antineoplastic agents in both environmental and biological matrices are reported. These methods validated according to FDA guidelines allowed our lab to define a profile of antineoplastic agents that was representative of the four IARC classes, such as cyclophosphamide (group 1), cisplatin and doxorubicin (group 2A), daunorubicin (group 2B), 5-fluorouracil, ifosfamide (group 3), taxol, and gemcitabine (group 4). Moreover, contamination levels on a number of work surfaces and trends over a 14-year period are presented. The evaluation of occupational exposure to CA has been based on ALARA principle for most of the past decades but this principle is nowadays overwhelmed by the fast development of technology. The assessment of a possible in-take of CA in hospital personnel, when the sources of environmental contamination are identified, has become possible by the defining of a limit of exposure close to the limit of detection of the analytical method.

OEM-TACE: a new therapeutic approach in unresectable intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a rare life-threatening disease, whose only treatment with potential for cure is surgical resection. However, only 27% of patients at most are suitable for surgery when first diagnosed. For patients with unresectable disease, therapeutic options are chemotherapy or chemoradiation. We evaluated the feasibility and safety of oxaliplatin-eluting microspheres transarterial chemoembolization (OEM-TACE) associated with chemotherapy (ChT) in patients affected by unresectable ICC. Between December 2005 and May 2008 we treated nine patients (six female and three male) with unresectable ICC. All patients had undergone OEM-TACE associated with chemotherapy with oxaliplatin and gemcitabine. A retrospective comparison was carried out with a historical group of 11 patients treated with ChT only, estimating the prevalence of adverse effects and the median survival of the two groups. A total of 30 TACEs were performed during the observational time (ranging from one to seven procedures per patient). OEM-TACEs were followed by few adverse effects (AEs), without G4 AEs, according to CTACAE 3.0. According to RECIST criteria, 44% (4/9) of patients achieved partial responses and 56% (5/9) stabilization of disease. Overall survival analysis in the two groups showed a significantly increased survival in patients treated with ChT and OEM-TACE, with respect to those treated with ChT (30 vs. 12.7 months; p=0.004). In conclusion, in our experience OEM-TACE associated with ChT in the treatment of advanced unresectable ICC is a safe and feasible treatment causing no major adverse events. Although RECIST criteria can underestimate the rate of responses in patients treated with locoregional therapies, we achieved very encouraging results. A randomized multicentric trial is warranted to assess the actual superiority of OEM-TACE associated with ChT compared to conventional chemotherapy.

[Headache as "sentinel" symptom in personnel involved in the preparation and administration of antineoplastic drugs]. / La Cefalea sintomo "sentinella" nel personale addetto alla preparazione e somministrazione di Chemioterapici Antiblastici.

The increased prevalence of neoplastic diseases observed over the last years has resulted for in more frequent operations of preparation and administration of antiblastic drugs performed by the medical personnel. In this study, we examined a group of subjects involved in the preparation of ACNU, Methotrexate, Novantrone, Vincristine, Cyclophosphamide, Carboplatinum, Mythoxantrone, in order to elucidate whether headache may represent an early symptom of exposure to these products. At the same time, we measured the degree of pollution in the air surrounding the vertical laminar flow aspiration cabinet used for drug preparation. The 66.6% of the 12 subjects studied, 9 females and 3 males, complained of headache. However, the environmental detection using high performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC/MS/MS) gave negative results with respect to a possible environmental damage. These findings suggest that headache is the onset symptom of the toxic effect of antiblastic chemiotherapics in the medical personnel involved in drug preparation and administration.

High-performance liquid chromatography/tandem mass spectrometry for the quantitative analysis of a novel taxane derivative (BAY59-8862) in biological samples and characterisation of its metabolic profile in rat bile samples.

A sensitive, specific, accurate and reproducible high-performance liquid chromatography (HPLC) analytical method was developed and validated for the quantification of the novel oral taxane analogue BAY59-8862 in mouse plasma and tissue samples. A fully automated solid-phase extraction procedure was applied to the plasma after internal standard (IS) addition, with only 0.2 mL volume of the sample loaded on a CN-Sep-pak cartridge. In the case of the tissues a very simple acetonitrile extraction was used to recover BAY59-8862 and its internal standard from liver. The procedure for the quantification of BAY59-8862 and its IS (IDN5127) is based on high-performance liquid chromatography/ion spray-tandem mass spectrometry, operating in selected ion monitoring mode. The retention times of BAY and IS were 7.21 and 10.36 min, respectively. In both plasma and tissue specimens the assay was linear in the range 50-5000 ng/mL (ng/g). The overall precision and accuracy were assessed on three different days. The results for plasma were within 6.1% (precision) and between 99 and 112% (accuracy), and for the liver samples within 7.3% and between 104 and 118%, respectively. The LOD was 5 ng/mL and 20 ng/g in the plasma and liver, respectively. In addition, the biliary excretion of the compound in rats was studied. The study showed that an oxidative chemical reaction was the preferred metabolic pathway for biliary excretion, and two sets of mono- and dihydroxylated metabolites were detected by LC/ISP-MS/MS experiments. With this method, BAY59-8862 pharmacokinetic was determined in mice. The combined results demonstrate that the methodology can be considered a valid approach to conduct pharmacokinetic and metabolic studies during preclinical and clinical investigations.

Distribution of paclitaxel within the nervous system of the rat after repeated intravenous administration.

The distribution of paclitaxel (Taxol) within the central and peripheral nervous system after repeated administration of this antineoplastic agent is still largely unknown. In this study we determined for the first time paclitaxel tissue concentration in the brain, spinal cord, dorsal root ganglia (DRG) and sciatic nerve using an experimental paradigm in the rat which reproduces the features of paclitaxel peripheral neurotoxicity in humans. Pathological confirmation of the onset of paclitaxel-induced peripheral neurotoxicity was performed. In order to achieve reliable results even with low concentrations of paclitaxel, a newly reported analytical method (high-performance liquid chromatography with tandem mass spectrometry) was used. We demonstrated that paclitaxel has easy access to the DRG, where it accumulates, while the lowest concentrations of the drug were measured in the brain. The intermediate concentrations of paclitaxel observed in the sciatic nerve and spinal cord may be due to paclitaxel transport along the centrifugal and centripetal branches of the DRG neuron axons.

Rapid and sensitive determination of paclitaxel (Taxol(R)) in environmental samples by high-performance liquid chromatography tandem mass spectrometry

A new, rapid and sensitive method for the quantitative determination of paclitaxel (Taxol(R)) in environmental samples is developed and validated. This highly sensitive and specific method was successfully applied to an environmental monitoring study for identifying occupational exposure situations to anticancer drugs. After addition of an internal standard, 2'-methylpaclitaxel, aqueous extraction of wipe samples or air filters followed by a single-step liquid-liquid extraction with ethyl acetate was performed. Extraction efficiency averaged 90%. The method is based on analysis by liquid chromatography with tandem mass spectrometry in selected reaction monitoring mode of environmental samples from the preparation and administration of cytostatic agents. The assay is linear (r = 0.998) in the range 2-64 ng on filters (air samples), and 20-320 ng on wipe samples. The accuracy of the assay is always

Determination of methotrexate in human urine at trace levels by solid phase extraction and high-performance liquid chromatography/tandem mass spectrometry.

For biological monitoring of hospital personnel occupationally exposed to antineoplastic agents, highly sensitive and specific methods are required. In order to detect trace MTX urinary concentrations, a precise and accurate high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedure, incorporating solid phase extraction, has been developed. Urine samples were purified by solid phase extraction (SPE) on octadecyl bonded, endcapped silica SPE columns. After eluting with methanol, the solvent was evaporated obtaining a 25-fold concentration of the analyte. This procedure was validated by using 7-OHMTX as internal standard. Calibration curves had correlation coefficients always higher than 0.999, and the limit of detection was assessed at 0.2 microg L(-1). High specificity of the HPLC-MS/MS technique assures that no interfering substances are detected rather than the analyte of interest.

[Risk assessment concerning hospital personnel participating in the preparation and administration of antineoplastic drugs]. / La valutazione del rischio nel personale ospedaliero addetto alla preparazione e alla somministrazione di farmaci antitumorali.

24 workers (10 involved in the preparation and 14 in administration) exposed to cyclophosphamide (CP) and ifosfamide (IF) in two Italian hospitals were monitored. The extent of exposure was assessed by the analysis of air samples, wipe samples, pads and gloves. Urinary excretion at the beginning and at the end of the work shift was also measured by liquid-liquid extraction and analysis by HPLC-MS/MS. 3 out of 24 air samples resulted to be positive for CP or IF. In wipe samples, CP concentrations ranging from < 0.001 to 82.4 micrograms/dm2 in Hospital A (32 samples) and from 0.2 to 383.3 micrograms/dm2 in Hospital B (17 samples), were found. IF concentrations varied from < 0.001 to 90.9 micrograms/dm2 in Hospital A and from 0.01 to 141.5 micrograms/dm2 in Hospital B. Pads (from 11 to 13 for each operator) were contaminated with CP and IF especially on arms, legs and chest. The use of a plastic-backed liner on the working tray in the laminar flow hoods was demonstrated to compromise the containment properties of the hood. Urine samples were positive for CP in 50% of the workers (range: 0.1-2.1 micrograms/L), whereas IF was detected in 2 subjects only (range: 0.1-0.8 microgram/L). The results from this investigation demonstrated that vertical laminar airflow hoods, when incorrectly used, might represent a source of contamination and that higher risk may depend on lack of educational programmes and observance of preventive guidelines.

Human IL-1 receptor antagonist from Escherichia coli: large-scale microbial growth and protein purification.

Interleukin-1 receptor antagonist (IL-1ra) is a recently discovered cytokine which specifically inhibits IL-1 pro-inflammatory activities in various experimental conditions. In this work, the growth conditions of a recombinant E. coli strain which in laboratory studies expressed human IL-1ra mostly in insoluble form, have been optimized at the level of 6-1 bioreactors and then scaled up to a 50-1 process. As a result, a high amount (0.43 g l-1 of microbial culture) of soluble, active IL-1ra has been directly obtained in the large-scale cell lysate with no need for protein solubilization. Also, an efficient purification procedure has been developed for the soluble protein, based on cation exchange expanded bed adsorption directly followed by anion exchange chromatography. This process, which does not include any intermediate dialysis step or gradient elutions, can be easily scaled up to larger production volumes and is therefore well-suited for manufacturing. As a result of the overall optimization study, more than 12 g of pure IL-1ra have been obtained from a single 50-1 fermentation run, without any denaturation/renaturation process. The final product, whose identity and purity have been checked also by MALDI-TOF and ESI-MS, shows full biological activity both in cellular assays and in in vivo experiments with Cynomolgus monkeys.

Application of high performance liquid chromatography/tandem mass spectrometry in the environmental and biological monitoring of health care personnel occupationally exposed to cyclophosphamide and ifosfamide.

Twenty four workers (10 involved in the preparation and 14 in administration) exposed to cyclophosphamide (CP) and ifosfamide (IF) in two Italian hospitals were monitored. The extent of exposure was assessed by the analysis of air samples, wipe samples, pads and gloves. Urinary excretion at the beginning and at the end of the work shift was also measured by liquid-liquid extraction and analysis by high performance liquid chromatography/tandem mass spectrometry. Three out of 24 air samples were positive for CP or IF. In wipe samples, CP concentrations ranging from < 0.001 to 82.4 micrograms/dm2 in Hospital A (32 samples) and from 0.2 to 383.3 micrograms/dm2 in Hospital B (17 samples), were found. IF concentrations varied from < 0.001 to 90.9 micrograms/dm2 in Hospital A and from 0.01 to 141.5 micrograms/dm2 in Hospital B. Pads (from 11 to 13 for each operator) were contaminated with CP and IF especially on arms, legs and chest. The use of a plastic-backed liner on the working tray in the laminar flow hoods was demonstrated to compromise the containment properties of the hood. Urine samples were positive for CP in 50% of the workers (range: 0.1-2.1 micrograms/L), whereas IF was detected in 2 subjects only (range: 0.1-0.8 microgram/L). The results of this investigation demonstrate that vertical laminar airflow hoods, when incorrectly used, might represent a source of contamination and that higher risk may depend on lack of educational programmes and observance of preventive guidelines.

Liquid-liquid extraction procedure for trace determination of cyclophosphamide in human urine by high-performance liquid chromatography tandem mass spectrometry.

A sensitive, specific and accurate high performance liquid chromatography/ionspray-tandem mass spectrometry procedure (HPLC/MS/MS) has been developed to quantify cyclophosphamide in human urine from hospital personnel involved in drug preparation and administration of antineoplastic alkylating agents. This methodology, which includes liquid-liquid extraction with ethylacetate, requires no derivatization procedures, preventing cyclophosphamide (CP) from possible thermal and chemical decomposition reactions. We detected the excretion of this unmetabolized alkylating drug in 50% of all the study participants. The amount of CP ranged from 0.1 ng microL-1 to 1.9 ng microL-1 urine. This methodology was validated by the use of ifosfamide as internal standard. The assay was linear over the range 0 to 3.2 ng microL-1 urine, with a lower limit of quantification of 0.2 microL-1. The limit of detection was assessed at 0.05 ng microL-1 urine. This method is characterized by a coefficient of variation < 10%. Standard calibration curves, obtained on three different days, had correlation coefficients always greater than 0.998. The intra and interday precision were within 11%, and accuracy was in the range 99-103%. The mean extracted recovery assessed at three different concentrations (0.5, 0.8, 3.2 ng microL-1) was always more than 85%. The extraction efficiency of cyclophosphamide from urine samples was also studied at six different pH values (pH 4, 5, 6, 7, 8, 10). The maximum extraction efficiency was obtained when the pH of urine solutions was adjusted to 7.0

High-performance liquid chromatography tandem mass spectrometry procedure with automated solid phase extraction sample preparation for the quantitative determination of paclitaxel (Taxol) in human plasma.

A sensitive, specific, accurate and reproducible analytical method was developed and validated for the quantitation of the anticancer agent paclitaxel in human plasma. This procedure is based on high performance liquid chromatography/ion spray-tandem mass spectrometry. This methodology is highly specific because a MS/MS technique (multiple reactant-ion monitoring, MRM) was used for both paclitaxel and its internal standard. The use of a fully automated solid phase extraction procedure, using a CN Sep-pak cartridge, to improve the detection limit and quantification limit of paclitaxel in human plasma samples, was evaluated. The method involves the addition of methyl-paclitaxel as internal standard (i.s.). The retention times of paclitaxel and the I.S. were 2.8 and 4.0 min., respectively. The assay was linear over the range 5 to 500 ng/mL, with a quantification limit of 5 ng/mL having a coefficient of variation (c.v.) < 10%. Standard calibration curves, performed on three different days, had correlation coefficients always greater than 0.998. The intra and inter-day precision were within 12%, and accuracy was included in the range 102-110%. Paclitaxel recovery assessed at 15,250 and 500 ng/mL, was determined to be greater than 85%. The assay is applicable to clinical pharmacokinetic studies.

[The determination of urinary cyclophosphamide at low concentration levels by liquid-liquid extraction and HPLC/MS/MS analysis]. / Determinazione di ciclofosfamide urinaria a bassi livelli di concentrazione mediante estrazione liquido-liquido e analisi in HPLC/MS/MS.

A sensitive, specific and accurate high-performance liquid chromatography-ion spray-tandem mass spectrometry procedure (HPLC/MS/MS) has been developed to quantify cyclophosphamide in human urine. This methodology, which includes the liquid-liquid extraction with ethyl acetate, requires no derivatization procedures, preventing cyclophosphamide from possible thermal and chemical decomposition reactions. This methodology was validated by the use of ifosfamide as internal standard (I.S.). The assay was linear over the range 0 to 3.2 ng mL-1 urine, having a low limit of quantification of 0.2 ng mL-1. The low limit of detection was assessed at 0.05 ng mL-1 urine. This method is characterized by a coefficient of variation < 10%. Standard calibration curves, performed on three different days, had correlation coefficient always greater than 0.998. The intra and inter-day precision were within 11%, and accuracy was included in the range 99-103%. The mean extracted recovery assessed at three different concentrations (0.5, 0.8, 3.2 ng mL-1) was always more than 85%. The extraction efficiency of cyclophosphamide from urine samples was also studied at six different pH values (pH 4, 5, 6, 7, 8, 10). CP gave the maximum extraction efficiency when pH urine solutions was adjusted to 7.0 and somewhat lower at the other considered values.

Matrix performance in matrix-assisted laser desorption/ionization for molecular weight determination in sialyl and non-sialyl oligosaccharide proteins.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry relies on the formation of intact molecular ions to determine molecular weight. In biochemical research, conventional methods of protein analysis at picomol to fentomol sensitivity, such as sodium dodecyl sulphate polyacrylamide gel electrophoresis, have been replaced by this new ionization method. Unfortunately, problems caused by the mass accuracy and low resolution restrict the use of this ionization technique, particularly when a high mass accuracy in a high mass range is required. In this paper it is shown that the appropriate choice of parameters which determine the desorption/ionization of glycoproteins can improve the quality of MALDI mass spectra as well as mass reproducibility and resolution. The study of sample-matrix solution composition, pH and instrumental conditions allow the molecular weight determination of highly glycosylated proteins with a high percentage of sialic acid, e.g. erythropoietin. The glycosylation of this molecule which interferes with the production of multiply charged ions in electrospray ionization does not affect the desorption/ionization in MALDI analysis. We report the best operating conditions used to establish the degree of heterogeneity of erythropoietin.

Structural characterization of mono- and dihydroxylated metabolites of paclitaxel in rat bile using liquid chromatography/ion spray tandem mass spectrometry.

The capability of high performance liquid chromatography/ion spray mass spectrometry (HPLC/ISP-MS) and HPLC/ISP-tandem mass spectrometry (HPLC/ISP-MS/MS) were investigated to achieve mass separation as well as structural characterization of taxol metabolites directly in rat bile, without their previous isolation. HPLC/ISP-MS yielded information on the molecular weights of several hydroxylated derivatives while HPLC/ISP-MS/MS allowed the on-line structural characterization of all metabolites present in different ratios in rat bile. The approach led to the extraction of nine metabolites and their distinction from the other endogenous contaminants. These metabolites were recognized as three dihydroxytaxols, four monohydroxytaxols, one deacetyltaxol and one containing the taxane ring. Among the derivatives, we were able to identify four new metabolites of paclitaxel belonging to the dihydroxy and monohydroxy series, never previously detected. HPLC/ISP-MS/MS enabled the classification of all di- and monohydroxy isomers. These results demonstrate that the high sensitivity of this method, based on the combined use of tandem mass spectrometry with chromatographic separation, can be considered as offering a valid approach to the detection of new taxol derivatives directly in biological fluids.

Inhibitors of myo-inositol monophosphatase, ATCC 20928 factors A and C. Isolation, physico-chemical characterization and biological properties.

During the course of a screening program for inhibitors of myo-inositol monophosphatase we fermented the strain ATCC 20928, a known producer of L-671,776. We now show that this strain produces a complex of at least three sesquiterpenic compounds, L-671,776 (termed factor B) and two structurally related substances, termed factors A and C. Both factors A and C, like L-671,776, exhibited inhibitory activity against myo-inositol monophosphatase. Six other fungi producing the above mentioned compounds were also isolated and taxonomically characterized.