Association between intrafollicular concentration of benzene and outcome of controlled ovarian stimulation in IVF/ICSI cycles: a pilot study.

BACKGROUND: Several studies have shown that exposure to benzene is associated to menstrual disorders, miscarriages and other disorders of the reproductive system. We performed an observational prospective pilot study to evaluate if levels of benzene in follicular fluid were correlated with response to controlled ovarian stimulation. METHOD: Thirty-four normogonadotrophic women undergoing IVF were enrolled. Intra-follicular benzene levels were evaluated by chromatography/mass spectrometry. Based on median benzene level, we divided the study population in two groups: Group A with a "low" intra-follicular benzene concentration (n=19, benzene <0.54 ng/mL) and Group B with a "high" intra-follicular benzene concentration (n=15, benzene ≥ 0.54 ng/mL). The ovarian response to gonadotrophins and the outcome of IVF were analyzed in the two groups. RESULTS: The two groups did not differ in terms of demographic or anthropometric characteristics. Group B had significantly higher basal FSH levels, lower estradiol peak concentration, and fewer oocytes retrieved and embryos transferred (p<0.05). Number of gonadotrophin vials, length of controlled ovarian stimulation and ongoing pregnancy rate were similar in the two groups. CONCLUSION: In conclusion, ovarian response to endogenous and exogenous gonadotrophins appeared to be influenced by intra-follicular benzene levels.

Epirubicin permeation of personal protective equipment can induce apoptosis in keratinocytes.

The present study investigated the epirubicin (EPI) permeability of various commercially available glove types, as well as toxicity mechanisms and effects on human keratinocyte cell line (HaCaT). Permeability experiments were carried out on various commercially available gloves, differing as regards material and thickness. Permeability was evaluated after different "contact times" and the influence of EPI solution's pH (acid and neutral) on permeability was also examined. Toxicity of EPI toward skin was tested by evaluating the effects of the drug on cell growth and apoptosis, by using an in vitro model based on cultured immortalized human keratinocytes. No permeation was detected in the case of EPI neutral solutions; in contrast, acid solutions were found to penetrate low thickness nitrile gloves. Obtained results also showed the induction of apoptosis in epithelial cells through the activation of intrinsic pathway p53-independent occurring even when cells are exposed at low drug concentration. EPI solution's pH influences the glove's permeability; once penetrated, EPI at concentrations lower than those able to penetrate the nitrile glove during the 8-h work-shift can cause apoptosis in epithelial cells. The findings reported here highly support the choice of either natural rubbers gloves or high thickness nitrile ones for preventing the occupational exposure to EPI.

Screening of several drugs of abuse in Italian workplace drug testing: performance comparisons of on-site screening tests and a fluorescence polarization immunoassay-based device.

According to the Italian laws, some categories of workers entrusted with duties possibly constituting a threat to security, physical safety, and health of third parties have to be screened to exclude the use/abuse of the following drugs of abuse: opiates, cocaine, cannabinoids, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methadone, and buprenorphine. Toxicological tests can be performed with urinary on-site rapid screening devices, provided that sensitivities up to specified cutoffs are ensured. The present study reports performances, in terms of sensitivity, specificity, and accuracy, of an automatic on-site test and of an FPIA-based device, using gas chromatography/mass spectrometry (GC/MS) as a reference methodology. Three levels of concentration were tested, corresponding to the cutoff and to 2 and 3 times the limits, respectively. In terms of sensitivities, neither the on-site nor the benchtop instrumentations gave positive results, since values of zero percentage were obtained for concentrations up to 2-fold the limits. Even if good results were obtained in terms of specificity and accuracy by both devices, none of them seem to be adequate for the current application to the toxicological screening at workplaces. In fact, a rapid screening device can be used for drug tests provided that it ensures sensitivity at the prescribed cutoffs. Data showed that such is completely rejected and a more sensitive instrumentation should be preferred.

Solid-phase microextraction-gas chromatography-mass spectrometry method validation for the determination of endogenous substances: urinary hexanal and heptanal as lung tumor biomarkers.

Hexanal and heptanal are endogenous aldehydes coming from membrane lipid oxidation, found in lung cancer patients' blood, and suggested as lung tumor biomarkers. Here the urinary matrix was investigated instead of blood and the difficulties related to the determination of endogenous substances in biological matrices were faced by developing an external calibration HS-SPME/GC/MS method. The methodology was validated according to international validation procedures and it was verified analyzing unknown biological samples from cancer patients and healthy subjects. Percentage accuracy and precision, ranging from -11.25 to 10.85% and from 0.45 to 4.46%, respectively, were obtained, together with limits of detection (LODs) and lower limits of quantification (LLOQs) of 0.11 and 0.23 pg µL(-1) for hexanal and of 0.10 and 0.21 pg µL(-1) for heptanal. Analytes percentage recoveries (66.3%, hexanal and 70.5%, heptanal) and stability were evaluated. No analytes degradation was found at room temperature, while the remarkable analytes loss found after 1 month storage suggests analyzing biological samples within a week from storage. Results coming from the analysis of unknown biological samples showed no evident differences of heptanal urinary excretion between lung cancer patients and healthy subjects (0.22-0.95 and 0.21-0.69 pg µL(-1), respectively), while hexanal urinary concentrations in cancer patients (0.24-4.36 pg µL(-1)) were slightly higher than those found in control group ones (0.23-1.26 pg µL(-1)). The obtained results highly suggest to do further investigations in order to collect statistically significant biological data to discriminate between the pathological state of lung cancer patients and physiological conditions of healthy subjects, using the simple, rapid and cheap method here reported for the quantification of urinary aldehydes.

Analytical and pharmacological aspects of therapeutic drug monitoring of mTOR inhibitors.

Mammalian Target Of Rapamycin (mTOR) inhibitors represent a new class of immunosuppressant drugs extensively used for the prevention and the treatment of graft rejection in organ transplant recipients. Their current use is due to referred low nephrotoxic effects, particularly important in kidney transplanted and/or patients with renal failure. The most representative drugs of such class are Sirolimus (Siro) and Everolimus (Rad). Both drugs show a narrow therapeutic window, therefore, monitoring of whole-blood drug levels is recommended in order to optimize the therapy. Among the available assays, Liquid Chromatography coupled with UltraViolet or Electrospray Tandem Mass Spectrometry methods (LC/UV or LC/ESI-MSMS) are the most accurate and specific ones. A reliable alternative is represented by immunoassays, which offer the opportunity to minimize sample pre-treatment, thus reducing the time between drawing blood sample and measuring the drug concentration, an important aspect in high-throughput analyses. Despite this, a limitation in the use of immunoassays for therapeutic drug monitoring is the lower specifity compared with the chromatographic methods when analysing structurally-related drugs. New insights to optimize mTOR inhibitors regimens seem to be offered by the evaluation of CYP450 3A activity by using the probe drug approach. To such purpose, there are a number of major probe drugs used for in vivo studies including: midazolam, cortisol, lidocaine, nifedipine, dextromethorphan, erythromycin, dapsone and alfentanil. The aim of the present paper is to report the most recent knowledge concerning this issue, supplying a critical and comprehensive review for whom are involved both in the clinical and analytical areas.

Biological monitoring of nurses exposed to doxorubicin and epirubicin by a validated liquid chromatography/fluorescence detection method.

OBJECTIVES: Occupational exposure to antineoplastic drugs can represent a potential health risk for hospital staff. Assessing exposure is the first step in providing a safe work environment; the present study aimed to perform a biological monitoring (BM) of nurses exposed to doxorubicin and epirubicin. In order to assure data accuracy and reproducibility, the high-performance liquid chromatography with fluorescence detection method was validated. METHODS: Validation experiments were carried out according to the Food and Drug Administration guidelines. A detailed questionnaire about workplace practices and work organization was administered to 56 nurses of oncology department of two hospitals (A and B) located in southern Italy. End-shift urine samples were collected. Amounts of drugs handled were registered. RESULTS: The quantification and detection limits were 1.1 and 0.6 pg microl(-1) (doxorubicin) and 2.0 and 1.2 pg microl(-1) (epirubicin); moreover, the analytical method fulfilled all guidelines requirements. Questionnaire information evidenced that vertical laminar flow hoods were present in both hospitals, surfaces were cleaned with inappropriate detergents, no antispilling devices were adopted, and gloves were not changed during the work shift. A lower percentage of positive samples was found in the hospital where higher amounts of anthracyclines were handled (3.4% in A and 14.8% in B), suggesting individual incorrect working/cleaning practices in hospital A and overall hygienic standards to be improved in hospital B, where 'critical practices' were carried out. CONCLUSIONS: Results showed the crucial role of adopting effective safety precautions and handling practices to reduce exposure. Environmental and BM should be performed to discriminate between incorrect personal working modalities and general hygienic standards.

Doxorubicin can penetrate nitrile gloves and induces apoptosis in keratinocytes cell lines.

Doxorubicin (DOXO) is an anthracycline antibiotic which is used in the treatment of human malignancies such as leukemia, lymphoma and a number of solid tumors, particularly breast cancer. Anthracyclines have been reported to contaminate chemotherapy workstation surfaces as well as other workplaces surfaces. The occupational exposure to these drugs could occur in hospitals, for nurses involved in anthracyclines preparation and administration, in chemical industries during the commercial formulate syntheses, and in analytical laboratories. Numerous studies investigated cutaneous effects related to DOXO administration, on the contrary few literature data are available about effects on the skin due to the direct contact with the drug. The present study investigated the DOXO permeability of three commercially available gloves' types used to protect skin in occupational contexts, as well as the effects of DOXO on human keratinocyte cell line (HaCaT). The results suggest that the DOXO permeability of gloves depends not only on glove material but also on DOXO solutions' pH, in fact nitrile gloves can be penetrated by acid solutions, while neither natural rubbers nor nitrile gloves are permeable to neutral solutions. Moreover, DOXO solutions, even at low concentration, cause apoptosis in epithelial cells, through activation of intrinsic pathway p53-independent.

Study of the fragmentation pattern of ketamine-heptafluorobutyramide by gas chromatography/electron ionization mass spectrometry.

Ketamine is an anaesthetic compound used in human and veterinary medicine with hallucinogen properties that have resulted in its increased illicit use by teenagers at rave parties. Although several gas chromatography/mass spectrometry (GC/MS) methods have been reported for the quantification of the drug both in urine and in hair, its electron ionization (EI) fragmentation after derivatization with different reagents has been not yet fully investigated. The present work reports the study of the fragmentation of ketamine, derivatized with heptafluorobutyric anhydride (HFBA-Ket), using gas chromatography/electron ionization mass spectrometry (GC/EI-MS). The complete characterization of the fragmentation pattern represented an intriguing exercise and required tandem mass spectrometry (MS(n)) experiments, high-resolution accurate mass measurements and the use of deuterated d(4)-ketamine to corroborate the proposed structures and to characterize the fragment ions carrying the unchanged aromatic moiety. Extensive fragmentation was observed, mainly located at the cyclohexanone ring followed by rearrangement of the fragment ions, as confirmed by the mass spectra obtained from the deuterated molecule. The GC/EI-MS analysis of HFBA-Ket will represent a useful tool in forensic science since high-throughput analyses are enabled, preserving both the GC stationary phase and the cleanliness of the mass spectrometer ion optics.

Evaluation of occupational exposure to antiblastic drugs in an Italian hospital oncological department.

The determination of the current antiblastic drug contamination levels in an Italian hospital oncology ward was carried out. Statistical evaluation of data aiming to identify potential exposure causes was performed. Cyclophosphamide (CP), ifosfamide (IF) and 5-fluorouracil (5-FU) were determined by wipe tests, extracted with diatomaceous earths and quantified by GC/MSMS or HPLC/UV. Data were analysed with respect to the potential contamination levels of sampled surfaces, and various amounts of handled analyte. chi(2) tests and Spearman correlation coefficients were calculated. Median concentration levels of 0.086, 0.071, 2.363 microg/dm(2) were obtained, (CP, IF, 5-FU, respectively). 3.8 and 13.5% of investigated surfaces showed CP and IF concentrations higher than 1 microg/dm(2) (up to 26.96 microg/dm(2)) and 13.4% of samples contained 5-FU concentrations in the range 20-208.9 microg/dm(2). Analytes' concentration levels were dependent on sampling sites, with significant correlations showing a progressive contamination decrement going from workbenches, floor, hood planes and other surfaces. A diffuse contamination (traces of all the three analytes) was found on all investigated surfaces, even when analytes had not been used during the sampling days. A significant correlation (rho s=0.303, p=0.001) between the measured analyte concentration and the analyte handled amount was found only in the case of IF. The risk management strategy should be improved, as suggested by the measured and widespread levels of contamination. Since contamination also depends on other factors attributable to working modalities and cleaning procedures, the obtained results suggest that performance of specific training courses as well as scheduling environmental monitoring plans to achieve an actual decrement of the observed contamination levels should be implemented.

Analytical method validation for the evaluation of cutaneous occupational exposure to different chemical classes of pesticides.

In occupational exposure to pesticides, validated methodologies are available only in regards to homogeneous chemical classes of substances and the inhaling exposure, neglecting the cutaneous one that, especially in agriculture, represents an important route of absorption. An analytical methodology for the simultaneous quantification of different chemical classes of pesticides by using pads as environmental matrix and GC-MS/SIM as detection method was developed and validated. The extraction step of analytes from pads was optimized by comparing analytes recovery percentages obtained with different extraction solvents. High recoveries were obtained with ether and, above all, with acetonitrile. Validation experiments following the Food and Drug Administration Guidelines were carried out.

Immunoassay determination of rapamycin: reliability of the method with respect to liquid chromatography mass spectrometric quantification.

Immunochemical assays represent a promising tool for quantification of immunosuppressants in organ transplanted patients, because they require small sample volumes and minimum sample pre-treatment; nevertheless considerations about method specificity, sensitivity and reproducibility cannot be overlooked. The present paper investigates the reliability of using the immunoparticle enzyme immunoassay (MEIA) for the quantification of blood rapamycin (RAPA) levels in therapeutic drug monitoring of renal transplanted patients with respect to a validated liquid chromatography tandem mass spectrometric (LC/ESI-MSMS) method, used as reference. Linearity of MEIA was tested over the range 0.0-30.0 ng/mL, with accuracy and precision within acceptable limits. Fifty-two blood samples were collected from 42 renal transplanted patients and analyzed simultaneously by both methods. The Pearson's regression analysis gave the following parameters: correlation equation [RAPA](MEIA) = 1.330 + 0.776 [RAPA](LC/ESI-MSMS), r = 0.8526, SD = 1.778, p < 0.0001. The obtained average rapamycin concentration was 8.8 +/- 3.4 ng/mL using MEIA and 9.6 +/- 3.7 ng/mL for LC/ESI-MSMS, with an overall underestimation of about 6% of the immunoenzymatic test. Accuracy of MEIA ranged from -33% to 36% with respect to the reference mass spectrometric method. Although immunoenzymatic test represents a fast and sufficiently accurate method for its use in clinical practice, specificity of the assay is still not sufficiently investigated and reference methods and/or Proficiency Testing Scheme should be used as external control.

Determination of rapamycin: quantification of the sodiated species by an ion trap mass spectrometer as an alternative to the ammoniated complex analysis by triple quadrupole.

Rapamycin is a potent immunosuppressive drug capable of significantly reducing acute graft rejection in kidney, liver and heart transplant patients. Its immunosuppressive activity and adverse effects have been related to rapamycin concentration, and therapeutic drug monitoring of the drug is deemed appropriate. This work was aimed at developing a new quantification method based on the isolation of the [M+Na]+ ion as precursor and its further fragmentation through an ion trap mass spectrometer equipped with an electrospray ionization source. A limit of detection (LOD) of 0.7 ng/mL was obtained, while the lower limit of quantification (LLOQ) was 2.4 ng/mL. The accuracy and reproducibility of the responses were evaluated and compared with results obtained when the [M+NH4]+ ion was chosen as the precursor in a triple quadrupole mass spectrometer. In this case the LOD was 0.5 ng/mL and the LLOQ 1.7 ng/mL. Data showed that it would be possible to use the quantification of the sodiated species for the routine determination of rapamycin, as an alternative to the commonly adopted method based on the ammoniated complex.

A case study: surface contamination of cyclophosphamide due to working practices and cleaning procedures in two Italian hospitals.

The efficacy of preventive and organisational measures implemented in Italy to prevent the contamination of cytotoxic drug preparation rooms has been investigated, and oncologic wards of two Italian hospitals were examined. The sampling strategy was based not only on potential sources of contamination but also on responses to detailed questionnaires on workplace practices and work organisation. Wipe samples were taken from different surfaces of preparation rooms, before and after the work shift, over a span of a month. Cyclophosphamide was taken as the marker drug that reflects exposure to cytotoxic drugs, being measurable by GC/MS. In one of the two hospitals (Hospital A), a large amount of cyclophosphamide was found, both before and after shift, on the workbench (median value, 2.55 microg dm(-2), before shift), on the floor between the operator working position and the waste bin (>10 microg dm(-2), after shift), as also on door handles and storage shelves. No quantifiable levels of cytotoxic drug were detected in the second hospital investigated (Hospital B). These results could be attributed to the efficacy of cleaning procedures and working practices. In fact, both hospitals were provided with vertical-laminar airflow hoods and the (male) nurses had attended special training courses; but in Hospital A, cleaning procedures were carried out without substances used specifically for the cleaning of surfaces contaminated by cytotoxic drugs such as sodium hypochlorite. Working practices did not include Luer Lock devices. Cyclophosphamide concentrations found in both hospitals, compared with the quantities of drug handled, gave evidence of the importance of the correct handling of cytotoxic agents as a major tool in reducing contamination levels. The results reveal the insufficiency of the risk management measures which do not take into account working practices that are prevailing, and stress the necessity for periodic environmental monitoring, indispensable for evolving effective procedures to prevent antineoplastic drug exposure.

Application of the standard addition approach for the quantification of urinary benzene.

Urinary benzene is used as biomarker of exposure to evaluate the uptake of this solvent both in non-occupationally exposed population and in benzene-exposed workers. The quantitative determination of benzene in urine is carried out in a three steps procedure: urine collection, sample analysis by head space/solid phase microextraction/gas chromatography/mass spectrometry and analyte quantification. The adopted quantification method influences the initial step, hence the whole procedure. Two quantification approaches were compared as regards precision and accuracy: the calibration curves and the standard addition method. Even if calibration curves obtained by using urine samples from different subjects were always linear, their slopes and intercepts showed noteworthy variations, attributable to the influence of the biological matrix on benzene recovery. The standard addition method showed to be more suitable for compensating matrix effects, and a three-point standard addition protocol was used to quantify benzene in urine samples of 11 benzene-exposed workers (smokers and non-smokers). Urine from occupationally exposed workers was collected before and after work-shift. Besides urinary benzene, the applicability of the method was verified by measuring the urinary concentration of the S-phenylmercapturic acid, a specific benzene metabolite, generally adopted as biomarker in biological monitoring procedures. A similar trend of concentration levels of both analytes measured in urine samples collected before work-shift with respect to the after work-shift ones was found, showing the actual applicability of the standard addition method for biological monitoring purposes.

Styrene-7,8-oxide activates a complex apoptotic response in neuronal PC12 cell line.

Styrene-7,8-oxide (SO), the major in vivo metabolite of styrene, one of the most important plastic monomers worldwide, is classified as carcinogenic in humans and animals. Although the toxic effects of SO have been extensively documented in human lymphocytes, the molecular mechanisms responsible for SO-induced cell damage are still unknown. In the present study, we evaluated the effect of SO on growth and apoptosis, assessed by FACS and gel ladder analysis, in neuronal PC12 cell line. Our results demonstrate that SO triggered PC12 cell apoptosis in a dose- and time-dependent manner. PC12 apoptosis was associated with caspase-3 activation and modulation of the Bcl-2 family proteins. In addition, examination of the cytoskeleton showed that SO induced F-actin depolymerization and a rapid cell rounding before caspase-3 activation, suggesting that the changes in cell shape involving cytoskeletal structure are an early step in the apoptotic pathway. Therefore, SO triggers a complex apoptotic response consisting of a loss of cytoskeletal organization that precedes caspase-3 activation. These mechanisms may represent the molecular basis of the different SO sensitivity to tumor promotion among species and organs.

Ion trap mass spectrometry in the structural analysis of haemoglobin peptides modified by epichlorohydrin and diepoxybutane.

Ion trap mass spectrometry has been shown to be particularly suitable for the structural analysis of high molecular weight peptides directly fragmented in the mass analyser without needing further sub-digestion reactions. Here we report the advantages of using multi-stage ion trap mass spectrometry in the structural characterisation of haemoglobin alkylated with epichlorohydrin and diepoxybutane. Alkylated globins were digested with trypsin and the peptide mixtures were analysed by MS(3). This technique allows the sequential fragmentation of peptides under analysis, giving rise to MS(3) product ion spectra with additional information with respect to MS(2) mass spectra. The results obtained complete the previously reported structural characterisation of alkylated haemoglobin, demonstrating the potential of ion trap mass spectrometry.

Structural analysis of styrene oxide/haemoglobin adducts by mass spectrometry: identification of suitable biomarkers for human exposure evaluation.

The structural characterisation of adducts formed by the in vitro reaction of haemoglobin (Hb) with styrene oxide (SO), the most reactive metabolite of the industrial reagent styrene, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human Hb chains. The reactive sites of human Hb towards SO were identified through characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation with tandem mass spectrometry (MALDI-MS/MS). A procedure was set up based on this characterisation, allowing Hb modification to be assessed by monitoring SO/Hb adducts using HPLC with selected ion recording (SIR) mass spectrometry. By this methodology it was also possible to compare advantages and disadvantages of presently available strategies for the measurement of Hb adducts with SO. The results obtained could most plausibly lead to the optimisation of molecular dosimetry of SO adducts, and the analytical procedure described herein could be applied to the biological monitoring of styrene exposure in the workplace.