Nadia Miraglia

Determination of cyclophosphamide and ifosphamide in urine at trace levels by gas chromatography/tandem mass spectrometry

A specific and sensitive method based on gas chromatography/tandem mass spectrometry with on-column injection was developed to quantify simultaneously cyclophosphamide and ifosphamide in urine by using trophosphamide as an internal standard. The urine samples were extracted with diethyl ether and derivatization was performed with heptafluorobutyric anhydride. The detection limits of cyclophosphamide and ifosphamide in urine samples were 0.1 and 0.5 ng ml(-1), respectively, with a signal-to-noise ratio of 3 : 1. The sensitivity, the specificity and the low cost of the instrumentation involved make this method suitable for economical analysis on a large scale, such as for the biological monitoring of occupational exposure to cyclophosphamide and ifosphamide in production plants and in hospitals during their pharmacological use.

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.

A mechanistic study on the cardiotoxicity of 5-fluorouracil in vitro and clinical and occupational perspectives

Fluoropyrimidines are key agents for the treatment of gastrointestinal tract adenocarcinomas. The possible cardiotoxic effects in patients and occupationally exposed workers are multifactorial and remain a puzzle to solve for investigators. In the present study, we study what cell death pathways and what doses can determine direct cardiotoxic effects of 5-fluorouracil (5-FU) and doxorubicin (DOXO) on rat cardiocytes (H9c2) and a human colon adenocarcinoma (HT-29) cell line, already reported to be sensitive to 5-FU. We have found that 5-FU induced 50% growth inhibition (IC:50) at 72 h with concentrations of 400 μM and 4 μM on H9c2 and HT-29, respectively. Moreover, we have found that the addition of Levofolinic Acid (LF) to 5-FU potentiated the growth inhibition induced by 5-FU. The growth inhibition induced by 5-FU alone or in combination with LF in cardiocytes was paralleled by an increase of thiobarbituric acid-reactive species (Tbars) and end products of nitric oxide (NO) suggesting the increase of the oxidative stress status in cardiocytes. Interestingly, these effects were strongly potentiated by the addition of LF, a biochemical modulator of 5-FU activity. Our data suggest that agents such as 5-FU different from anthracyclines, conventionally related to the induction of cardiotoxic effects, can also induce cardiocyte damage paralleled by oxidative stress. The strategies based upon the use of scavengers could be used in order to prevent this effect.

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.

Blood lead, manganese, and aluminum levels in a regional Italian cohort of ALS patients: does aluminum have an influence?

Objectives: To study aluminum (Al), manganese (Mn), and lead (Pb) influence on amyotrophic lateral sclerosis (ALS) development. Methods: A total of 34 patients (10% of the regional ALS population) and 25 controls of an Italian region were enrolled. Metal concentrations were determined by atomic absorption spectroscopy. Results: Serum Al concentrations in patients and controls were similar and lower than those provided by the Italian Society of Reference Values. No differences were observed in serum Mn concentrations, while, as expected, blood Pb levels were significantly higher in patients with ALS than those in controls. Conclusions: Results confirmed the association between high Pb blood levels and ALS; on the contrary, Al and Mn did not differ significantly in patients and controls, suggesting that Mn and especially Al may play a less important role in the ALS pathogenesis.

An Integrated Approach Based on Multiplexed Protein Array and iTRAQ Labeling for In-Depth Identification of Pathways Associated to IVF Outcome

The emergence of high-throughput protein quantification methodologies has enabled the comprehensive characterization by longitudinal and cross-sectional studies of biological fluids under physiological and pathological conditions. In particular, the simultaneous investigation of cytokines and growth factors signaling pathways and their associated downstream effectors by integrated multiplexed approaches offers a powerful strategy to gain insights into biological networks and processes in living systems. A growing body of research indicates that bioactive molecules of human reproductive fluids, including human follicular fluid (hFF), may affect oocyte quality, fertilization and embryo development, thus potentially influencing the physiopathology of pregnancy-related conditions. In this work, an iTRAQ labeling strategy has been complemented with a multiplexed protein array approach to analyze hFFs with the aim to investigate biological processes and pathways related to in vitro fertilization (IVF) outcome. The iTRAQ labeling strategy lead to the quantification of 89 proteins, 30 of which were differentially expressed in hFFs with successful compared to unsuccessful IVF outcome. The targeted study, based on multiplexed antibody protein arrays, allowed the simultaneous quantification of 27 low abundance proteins, including growth factors, chemokines and cytokines endowed with pro- and anti-inflammatory activity. A significant number of differentially regulated proteins were involved in biological functions related to blood coagulation, acute phase response signaling and complement system. Overall, the present results provide an integrated overview of protein changes in hFFs associated to IVF outcome, thus improving current knowledge in reproductive medicine and fertility research.

Rapid peptidomic profiling of peritoneal fluid by MALDI-TOF mass spectrometry for the identification of biomarkers of endometriosis

Abstract Peptidomic profiling of peritoneal fluid by Matrix Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF-MS) may represent a promising, suitable, rapid method for early diagnosis and staging of endometriosis. In a case-control study, peritoneal fluid was collected from 23 patients affected by endometriosis (eight minimal/mild endometriosis and 15 moderate/severe endometriosis) and six “endometriosis free” women undergoing laparoscopy. MALDI-TOF mass spectra of the peptide fraction extracted from peritoneal fluid samples lead to identify biomarkers potentially suitable for discriminating between peritoneal fluid samples from women affected by minimal/mild endometriosis and those from women affected by moderate/severe endometriosis. Peptidomic analysis of peritoneal fluid samples may define putative peptide biomarkers suitable for staging endometriosis and improve our understanding of the pathogenesis of endometriosis. Chinese abstract 通过基质辅助激光角吸附电离飞行时间质谱（MALDI-TOF-MS）进行腹膜液多肽组学测定也许可为子宫内膜异位症的早期诊断与分期提供一个可观、适用、快速的方法。在一项病例对照研究中，腹腔镜下采集了23位子宫内膜异位症患者的腹膜液（8位极轻/轻度患者，15位中度/重度患者）与6位非子宫内膜异位症女性的腹膜液。对提取自腹膜液样本的肽片段通过MALDI-TOF质谱分析测定生物标志物，可鉴别腹膜液样本是来自极轻/轻度子宫内膜异位症患者还是来自中度/重度患者。腹膜液样本的肽测定也许证实了肽生物标志物适用于子宫内膜异位症分期这一推断，且促进了我们对子宫内膜异位症发病机制的了解。

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

Abstract:  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.

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.

Occupational Exposure to Chromium of Assembly Workers in Aviation Industries

Aircraft are constructed by modules that are covered by a “primer” layer, which can often contain hexavalent chromium [Cr(VI)], known carcinogen to humans. While the occupational exposure to Cr(VI) during aircraft painting is ascertained, the exposure assessment of assembly workers (assemblers) requires investigations. Three biological monitoring campaigns (BM-I,II,III) were performed in an aviation industry, on homogeneous groups of assemblers (N = 43) and controls (N = 23), by measuring chromium concentrations in end-shift urine collected at the end of the working week and the chromium concentration difference between end- and before-shift urines. BM-I was conducted on full-time workers, BM-II was performed on workers after a 3–4 day absence from work, BM-III on workers using ecoprimers with lower Cr(VI) content. Samples were analyzed by atomic absorption spectroscopy and mean values were compared by T-test. Even if Cr concentrations measured during BM-I were lower than Biological Exposure Indices by ACGIH, statistically significant differences were found between urinary Cr concentrations of workers and controls. Despite 3–4 days of absence from work, urinary chromium concentrations measured during BM-II were still higher than references from nonoccupationally exposed populations. In the BM-III campaign, the obtained preliminary results suggested the efficacy of using ecoprimers. The healthcare of workers exposed to carcinogenic agents follows the principle of limiting the exposure to “the minimum technically possible”. The obtained results evidence that assemblers of aviation industries, whose task does not involve the direct use of primers containing Cr(VI), show an albeit slight occupational exposure to Cr(VI), that must be carefully taken into consideration in planning suitable prevention measures during risk assessment and management processes.