Chemical signature of colorectal cancer: case-control study for profiling the breath print.

BACKGROUND: Effective screening for colorectal cancer can reduce mortality by early detection of tumours and colonic polyps. An altered pattern of volatile organic compounds (VOCs) in exhaled breath has been proposed as a potential non-invasive diagnostic tool for detection of cancer. The aim of this study was to evaluate the reliability of breath-testing for colorectal cancer screening and early diagnosis using an advanced breath sampler. METHODS: The exhaled breath of patients with colorectal cancer and non-cancer controls with negative findings on colonoscopy was collected using the ReCIVA® Breath Sampler. This portable device is able to capture the alveolar breath fraction without environmental contamination. VOCs were desorbed thermally and analysed by gas chromatography-mass spectrometry. The discriminatory ability of VOCs in detecting colorectal cancer was evaluated by receiver operating characteristic (ROC) curve analysis for each VOC, followed by cross-validation by the leave-one-out method, and by applying stepwise logistic regression analysis. RESULTS: The study included 83 patients with colorectal cancer and 90 non-cancer controls. Fourteen VOCs were found to have significant discriminatory ability in detecting patients with colorectal cancer. The model with the diagnosis of cancer versus no cancer resulted in a statistically significant likelihood of discrimination of 173·45 (P < 0·001), with an area under the ROC curve of 0·979. Cross-validation of the model resulted in a true predictive value for colorectal cancer of 93 per cent overall. Reliability of the breath analysis was maintained irrespective of cancer stage. CONCLUSION: This study demonstrated that analysis of exhaled VOCs can discriminate patients with colorectal cancer from those without. This finding may eventually lead to the creation of a smart online sensory device, capable of providing a binary answer (cancer/no cancer) and directing to further screening.

ANTECEDENTES: Un cribaje efectivo del cáncer colorrectal (colorectal cáncer, CRC) puede reducir la mortalidad mediante la detección precoz de cáncer/pólipos del colon. La identificación de un patrón de compuestos volátiles orgánicos (volatile organic compounds, VOCs) en el aire espirado se ha propuesto como un procedimiento potencial de diagnóstico no invasivo para la detección del cáncer. El objetivo de este estudio fue evaluar la factibilidad del test de la respiración para el cribaje del CRC y diagnóstico precoz empleando un equipo avanzado de muestreo del aliento. MÉTODOS: Se recogieron muestras de aire espirado de 83 pacientes con CRC y de 90 controles sin cáncer con colonoscopia negativa empleando el ReCIVA Breath Sampler©. Este equipo portátil es capaz de capturar la fracción de aire alveolar espirada ausente de contaminación ambiental. Los VOCs fueron aislados térmicamente y analizados mediante cromatografía de gases acoplada a espectrometría de masas. La capacidad discriminatoria de los VOCs para detectar pacientes con CCR se evaluó mediante un análisis de la curva ROC para cada VOC seguida de validación cruzada mediante el método ir eliminando paso a paso cada uno de los VOCs en un modelo de regresión logística. RESULTADOS: Se observó que 14 VOCs tenían habilidad discriminatoria significativa para la detección de pacientes con CRC. El modelo con el diagnóstico de cáncer versus no cáncer mostró una probabilidad estadísticamente significativa de 151,03 (P < 0,0001) con un área bajo la curva (area under the curve, AUC) de 0,963. En la validación cruzada del modelo se obtuvo un valor global predictivo verdadero para el CRC del 92,5%. La fiabilidad del análisis del aire espirado se mantuvo con independencia del estadio del cáncer. CONCLUSIÓN: Este estudio ha demostrado que el análisis de los VOCs en el aire espirado puede discriminar pacientes con CRC de pacientes sin cáncer. Este hallazgo podría ser de ayuda para diceñar un dispositivo sensorial inteligente en línea, capaz de proporcionar una respuesta binaria (cáncer/NO cáncer) y asimismo contribuir a la indicación de una futura colonoscopia.

Solid phase microextraction--liquid chromatography (SPME-LC) determination of chloramphenicol in urine and environmental water samples.

A solid phase microextraction--liquid chromatography with ultraviolet detection (SPME-LC-UV) method for the determination of the antimicrobial agent chloramphenicol was developed. The performances of three commercially available fibers were compared; the Carbowax/TPR-100 was found to provide the most efficient extraction. All the aspects influencing the fiber adsorption (extraction time, temperature, pH, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte were investigated. The method was eventually applied to the determination of the drug in both biological (urine) and environmental (tap and sea water) samples. The optimized procedure required a simple sample pretreatment, isocratic elution, and provided enough sensitivity for the analyte determination in the considered samples. The investigated linear ranges were 37-1000 ng/ml (urine), 0.1-10 ng/ml (tap water), 0.3-30 ng/ml (sea water). Within-day and between-days RSD% ranged between 5.5-6.2 and 8.7-9.0 (urine), 5.1-6.0 and 8.4-8.8 (tap water), 5.4-5.7 and 8.6-8.9 (sea water). Estimated LOD and LOQ were 37 and 95 ng/ml (urine), 0.1 and 0.3 ng/ml (tap water), 0.3 and 0.7 ng/ml (sea water).

Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles.

Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples.

Automated high-throughput method using solid-phase microextraction-liquid chromatography-tandem mass spectrometry for the determination of ochratoxin A in human urine.

A new automated, high-throughput method for the determination of ochratoxin A (OTA) in human urine samples has been optimized and validated using solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS). High-throughput was achieved by simultaneous preparation of up to 96 samples using multi-fiber SPME device and multi-well plates. A carbon-tape coating was chosen for the first time as the best extracting phase for this contaminant. The proposed method required only minimal sample pre-treatment to adjust sample pH to 3.0 using a dilution (1:1) with 0.5M phosphate-buffered saline. A simple gradient guaranteed a good chromatographic separation from matrix interferences in only 8min. Relative recovery (%), precision and linearity validation results met Food and Drug Administration acceptance criteria at three concentration levels (1, 10, and 50ng/mL), indicating excellent performance of the proposed method. Limits of detection and quantitation were 0.3 and 0.7ng/mL in urine, respectively. OTA determination in urine is a good marker for human exposure to this mycotoxin. It is also less invasive than blood analysis. This method is fully automated and the SPME technique is simpler, less time-consuming and cheaper compared with most widely adopted clean-up procedures for OTA extraction from urine.

Determination of clenbuterol in human urine and serum by solid-phase microextraction coupled to liquid chromatography.

A solid-phase microextraction (SPME)-LC-UV method for the determination of the beta-adrenergic drug clenbuterol in human urine and serum samples was developed for the first time using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated fiber. The procedure required very simple sample pretreatments, isocratic elution, and provided highly selective extractions. All the aspects influencing fiber adsorption (extraction time, temperature, pH, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte have been investigated. The linear ranges investigated in urine and serum were 10-500 and 5-500 ng/ml, respectively (that covers the typical clenbuterol concentration observed in biological fluids). Within-day and between-days R.S.D.% in urine ranged between 5.0-5.3 and 8.5-8.7, respectively, while in serum ranged between 5.5-5.9 and 8.7-9.1, respectively. Estimated LOD and LOQ were 9 and 32 ng/ml (spiked urine), respectively, and 5 and 24 ng/ml (spiked serum), respectively, well below the usual clenbuterol urinary and serum level.

Solid-phase microextraction coupled to gas chromatography-mass spectrometry for the study of soil adsorption coefficients of organophosphorus pesticides.

A solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) method for the simultaneous determination of the organophosphorus pesticides (OPPs), phorate, diazinon, methyl-parathion, fenitrothion, malathion, fenthion, ethyl-parathion and methidathion, has been developed to study their soil/water distribution. The method was used in conjunction with a conventional 'batch equilibrium method' to assess the soil adsorption coefficients (Koc) of the target compounds in different soil samples with known organic carbon content. Contrary to traditional techniques, the present method is fast, solvent-free and highly sensitive, thus permitting the assessment of the Koc values of the target compounds even at low soil concentration levels, close to those encountered in real field contamination, where the Freudlich adsorption isotherms can be considered to be linear. The estimated Koc values were found to be in good agreement with those reported in the literature.

Amino-bonded silica as stationary phase for liquid chromatographic determination of cyclopiazonic acid in fungal extracts.

A new high-performance liquid (HPLC) chromatographic method is described for cyclopiazonic acid (CPA) determination in fungal cultures on a propylamino-bonded stationary phase with a CH3CN/CH3COONH4 buffer as mobile phase. Retention of CPA on propylamino modified silica under acidic conditions (protonated amino groups and deprotonated CPA) is governed by a mixed ion-exchange-reversed-phase mechanism. In addition to non-polar (hydrophobic) interactions, polar interactions with the surface silanols are also possible and become important as the polarity of the mobile phase decreases. A detection limit of 25 pg of CPA standard is obtained that represents an improvement of more than two orders of magnitude compared to existing HPLC procedures. UV-detector response was linear to 200 ng of CPA. Fungal extracts can be analysed after a simple dilution step with UV diode array detection that provides peak identity/purity assessment. The suitability of the proposed method as a rapid confirmatory test to assess the toxigenic potential of different Aspergillus and Penicillium strains is demonstrated by the analysis of 54 fungal extracts.

Solid-phase microextraction and gas chromatography-mass spectrometry for the rapid screening of triazole residues in wine and strawberries.

A solid-phase microextraction gas chromatography-mass spectrometry method has been developed for the determination of triazole residues, such as triadimefon, propiconazole, myclobutanil and penconazole. The method has been successfully applied to the analysis of strawberries and wine samples. The procedure is solvent-free, simple and highly sensitive. Within-day and day-to-day RSDs ranged between 2-11% and 7-28%, respectively. Detection limits estimated at a signal-to-noise ratio of 3 ranged between 30 (propiconazole) and 100 ng/kg (triadimefon). Since the detection limits achieved by this method are well below the maximum residue levels for wine (or grapes) and strawberries recommended by the European legislation, it can be conveniently used as a low-cost rapid screening method for the contamination of the considered samples.

Determination of triazines in soil leachates by solid-phase microextraction coupled to gas chromatography-mass spectrometry.

A solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) method was developed for the evaluation of the leachability order of selected triazines (propazine, terbuthylazine, sebuthylazine, ametryn, prometryn and terbutryn) in soil/sediment samples (organic carbon content ranging from 0.19 to 0.42%), analysing fractions collected from a soil packed microcolumn elution experiments. The procedure is fast, simple, highly sensitive and solvent free. SPME-GC-MS was also employed for the quantitative determination of triazines in the soil leachate, since the method showed good recovery yield. Detection limits were always better than 1 ng ml(-1). The method was tested on a contaminated landfill top soil. Prometryn and ametryn were identified through their MS spectra and then quantified. Terbuthylazine was used to assess recovery. Results compared well with those obtained by solvent extraction followed by HPLC-UV detection.

Needle-type glucose microbiosensor based on glucose oxidase immobilised in an overoxidised polypyrrole film (an in-vitro study).

A fast response, needle-type glucose microbiosensor has been fabricated by a one-step electrochemical immobilisation of glucose oxidase in a polypyrrole film. The sensor shows a remarkable rejection of electroactive interferences, especially paracetamol. The maximum bias observed in the worst situation never exceeded the value of 6%. The fabrication procedure delivered very reproducible devices and the sensitivity of a newly prepared biosensor was typically 650 nA mM(-1) cm(-2). The kinetic parameters, obtained from an existing model, permitted to understand the sensor behaviour.

Degradation of chlortoluron in water disinfection processes: a kinetic study.

The kinetics of the reaction between chlortoluron, a phenylurea herbicide [N'-(2-hydroxy-4-methyl-5-chlorophenyl)-N,N-dimethylurea], and hypochlorite, the active species in water disinfection processes involving chlorine, were investigated by HPLC-UV and HPLC-electrospray ionisation mass spectrometry (HPLC-ESI-MS). In particular, the concentrations of the main chlortoluron by-products were monitored as a function of time by HPLC-ESI-MS and a kinetic model was developed to fit the relevant curves. The results showed that chlortoluron degradation starts with two parallel pathways, namely, chlorination and hydroxylation of the aromatic ring, which are then followed by consecutive chlorination reactions, and after almost 2 weeks by ring opening and partial mineralisation, as confirmed by head-space solid-phase microextraction gas chromatography-MS (SPME-GC-MS) and total organic carbon (TOC) measurements. Kinetic constants for the first reactions of the overall process, under pseudo-first-order conditions (hypochlorite excess), were estimated by a fitting procedure.

Detection of collagen synthesis by human osteoblasts on a tricalcium phosphate hydroxyapatite: an X-ray photoelectron spectroscopy investigation.

The effect of a hydroxyapatite-tricalcium phosphate (HA) material on collagen synthesis by human osteoblasts was investigated using X-ray photoelectron spectroscopy (XPS). To this aim, thin HA slices were exposed to osteoblasts harvested from three different patients, for 20 days and then analyzed by XPS. Platinum plates were also exposed to the cells for comparison, and control tests were performed on both materials using cell-free media. XPS analysis supported by standard spectra of some polyaminoacids and of collagen deposited on HA suggested that a deposition of collagen occurred on the HA slices in the presence of osteoblasts. On the other hand, only an aspecific deposition of proteins was observed on platinum and when cell-free media were used. These data were confirmed evaluating collagen synthesis by [(3)H]proline incorporation of osteoblasts exposed to HA.

An electrospray ionization ion trap mass spectrometric (ESI-MS-MSn) study of dehydroascorbic acid hydrolysis at neutral pH.

The hydrolysis of dehydroascorbic acid (DAAH) at neutral pH and 27 degrees C was investigated by direct infusion electrospray ionisation ion trap mass spectrometry (ESI-MS). This approach permitted derivatisation and elution procedures to be avoided, reducing to the minimum extent sample manipulation and allowing a rapid and direct observation of the species involved in the reaction. Six main peaks, related to hydrated dehydroascorbate (HyDAA-) and diketogulonate (HyDKG-) anions, were observed in the mass spectra of DAAH solutions at different times of incubation and were characterised by MSn experiments. The relevant signal intensities changed with time and a model, based on the irreversible pseudo-first order HyDAA(-)-->HyDKG- conversion, fitted successfully the data obtained for dehydroascorbate. The kinetic constant of the process was (3.2 +/- 0.5) x 10(-2) min-1. The influence of metal ion traces on the hydrolysis rate was also checked, performing experiments in the presence of EDTA, and was found to be negligible.

Electrospray ionization mass spectrometry of 5-methyl-2'-deoxycytidine and its determination in urine by liquid chromatography/electrospray ionization tandem mass spectrometry.

The behaviour of 5-methyl-2'-deoxycytidine (m(5)dCyd, claimed to be a potential marker for leukaemia) during the electrospray process was studied. In particular, considerations concerning the effect of solution chemistry (e.g. analyte concentration, pH, etc.) on electrospray ionization mass spectra were drawn. Furthermore, a procedure using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for the determination of urinary m(5)dCyd is described. The method is simple, sensitive and highly specific. The pre-treatment procedure gave an average recovery of 79% (relative standard deviation (RSD) of 3%). Method performance was evaluated on spiked urine samples covering the concentration range from 50 ng/mL to 10 microgram/mL, the same as that of an existing inhibition ELISA method. Contrary to findings based on this immunoassay technique, urinary m(5)dCyd in healthy individuals was not detectable and did not increase in the presence of the malignant disease.

Liquid chromatographic determination of urinary 5-methyl-2'-deoxycytidine and pseudouridine as potential biological markers for leukaemia.

A simple reversed-phase liquid chromatographic (LC) method for the determination of urinary 5-methyl-2'-deoxycytidine (m5dCyd), recently claimed (on the basis of an imuno-technique) to be a potential marker for leukaemia, has been developed. Sample pre-treatment is based on a microcolumn clean-up step with an average recovery of 79% and a RSD of 3%. Detection limit was 0.2 microg/ml which is about tenfold lower than levels previously measured by an ELISA method in urine of healthy individuals. The creatinine (Cre) excretion, necessary for normalising the m5dCyd excretion, was evaluated by ion-pair liquid chromatography which permitted the simultaneous determination of pseudouridine (psi), a modified nucleoside also potentially useful as a marker for leukaemia. The described LC procedures were applied to the analysis of urine samples from healthy individuals and leukaemia patients. While the urinary psi/Cre ratio was found significantly increased for leukaemia patients, the urinary m5dCyd levels in healthy individuals were below the detection limits and did not increase in presence of the malignant disease.

In vitro toxicity of N3-methyl-5'-deoxy-5-fluorouridine, a novel metabolite of doxifluridine: a bioanalytical investigation.

The cytotoxicity of N3-methyl-5'-deoxy-5-fluorouridine (N3-Me-5'-dFUR), a novel metabolite of the anticancer pro-drug 5'-deoxy-5-fluorouridine (5'-dFUR), has been evaluated by in vitro experiments with cultures of different cancer cell lines. The new metabolic product was found to be non-toxic in all the cell growth experiments performed. The absence of cytotoxicity could be explained by the observation that the metabolite was not recognized as a substrate by thymidine phosphorilase, the enzyme responsible for 5-fluorouracil (5-FU) release from doxifluridine, as ascertained by high-performance liquid chromatography/ultraviolet (HPLC-UV) analysis of the incubation mixture. The biomethylation process leading to N3-Me-5'-dFUR could be considered as a possible detoxification pathway, altering the drug bioavailability, in competition with 5'-dFUR cleavage to the active 5-FU.

Determination of N3-methyl-5'-deoxy-5-fluorouridine, a novel metabolite of doxifluridine, in body fluids by high performance liquid chromatography.

A high performance liquid chromatography method for the determination of N3-methyl-5'-deoxy-5-fluorouridine, a possible metabolic product of the anticancer pro-drug 5'-deoxy-5-fluorouridine, in human serum and urine is described. Sample treatment involved addition of internal standard (5-bromouracil) and protein precipitation with ammonium sulphate (serum samples) followed by liquid-liquid extraction with ethyl acetate-isopropanol (90:10, v/v). The average recovery at 0.5 mg ml-1 level was (80 +/- 4%). A linear response extending over two decades of concentration was observed. Detection limits of 50 and 100 ng ml-1 were obtained in serum and urine, respectively.

Gas chromatography-mass spectrometry identification of a novel N3-methylated metabolite of 5'-deoxy-5-fluorouridine in plasma of cancer patients undergoing chemotherapy.

Evidence of in vivo biomethylation of the anticancer pro-drug 5'-deoxy-5-fluorouridine (5'-dFUR) is presented for the first time. Biomethylation seems to occur specifically at the N3 site on the pyrimidine ring. This novel metabolic product was detected by gas chromatography-mass spectrometry of the trimethylsilylated extract of plasma samples from cancer patients undergoing doxifluridine chemotherapy. Considering the observed electron impact fragmentation pattern, the metabolic product was tentatively identified as N3-Me-5'-dFUR. Definite confirmation of the proposed structure was achieved by comparison of the mass spectra and chromatographic characteristics of the suspected metabolite with those of a synthetically prepared reference standard.

Simultaneous determination of pseudouridine, neopterine and creatinine in urine by ion-pair high-performance liquid chromatography with in-series ultraviolet and fluorescence detection.

A simple, fast and precise method for the simultaneous determination of neopterine (Npt), pseudouridine (Psu) and creatinine (Cre) in urine by using ion-pair HPLC has been developed. The urine specimen is subjected to a microcolumn clean-up step, providing a ten-fold sample dilution, and is then injected into the column. The eluate is forced through the respective cells of a fluorescence and then a UV detector, which are connected in series. Neopterine is monitored by fluorescence emission at 438 nm (excitation wavelength 353 nm) while Psu and Cre are monitored by UV absorption at 235 nm. This allows the determination of Npt/Cre and Psu/Cre concentration ratios in a single run on the same sample, and the use of urine specimens collected randomly instead of 24 hourly collections. The method has been applied to urine specimens from 19 healthy, male donors (mean Npt/Cre (mumol/mol) and Psu/Cre (mumol/mmol) values of 41.7 and 16.8, respectively) and 23 healthy, female donors (mean Npt/Cre (mumol/mol) and Psu/Cre (mumol/mol) values of 67.4 and 18.5, respectively). The within-run S(r) for Npt/Cre and Psu/Cre concentration ratios ranged between 3.3 and 4.6%.