Solid phase microextraction, mass spectrometry and metabolomic approaches for detection of potential urinary cancer biomarkers: a powerful strategy for breast cancer diagnosis

A sensitive assay to identify volatile organic metabolites (VOMs) as biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. Therefore the aim of this study was to establish the urinary metabolomic profile of breast cancer patients and healthy individuals (control group) and to explore the VOMs as potential biomarkers in breast cancer diagnosis at early stage. Solid-phase microextraction (SPME) using CAR/PDMS sorbent combined with gas chromatography–mass spectrometry was applied to obtain metabolomic information patterns of 26 breast cancer patients and 21 healthy individuals (controls). A total of seventy-nine VOMs, belonging to distinct chemical classes, were detected and identified in control and breast cancer groups. Ketones and sulfur compounds were the chemical classes with highest contribution for both groups. Results showed that excretion values of 6 VOMs among the total of 79 detected were found to be statistically different (p < 0.05). A significant increase in the peak area of (−)-4-carene, 3-heptanone, 1,2,4-trimethylbenzene, 2-methoxythiophene and phenol, in VOMs of cancer patients relatively to controls was observed. Statiscally significant lower abundances of dimethyl disulfide were found in cancer patients. Bioanalytical data were submitted to multivariate statistics [principal component analysis (PCA)], in order to visualize clusters of cases and to detect the VOMs that are able to differentiate cancer patients from healthy individuals. Very good discrimination within breast cancer and control groups was achieved. Nevertheless, a deep study using a larger number of patients must be carried out to confirm the results.

Determination of the highest no-effect dose (HNED) and of the elimination pattern for cocaine in horses

Cocaine is one of the most widespread illegal stimulants utilized by the human population throughout the world. The aim of this study was to establish the highest no-effect dose (HNED) of cocaine on the spontaneous locomotor activity (SLA) of horses in a behavior chamber, and thereby to determine the maximal acceptable threshold of the urinary drug concentration in horses. Twelve English thoroughbred mares received 0.02, 0.03, 0.04, 0.08 or 0.12 mg kg(-1) cocaine i.v. or saline solution (control). It was noted that doses above 0.04 mg kg(-1) induced a significant increase in SLA (P < 0.05, Tukey's test). No significant increase in SLA was seen in the mares that received 0.03 mg kg(-1), but the animals showed important behavioral changes that did not occur after the 0.02 mg kg(-1) dose. It was concluded that the HNED of cocaine for horses in a behavior chamber is 0.02 mg kg(-1). After injection of this dose in five horses, urine samples were collected at predetermined intervals through vesical catheterization. The concentrations of cocaine, norcocaine, benzoylecgonine and ecgonine methyl ester were quantified by liquid chromatography/electrospray ionization tandem mass spectrometry. Cocaine and norcocaine concentrations remained consistently below the level of detection. Benzoylecgonine reached a mean (+/- SEM) maximum concentration of 531.9 +/- 168.7 ng ml(-1) after 4 h, whereas ecgonine methyl ester peaked 2 h after injection at a concentration of 97.2 +/- 26.5 ng ml(-1). The maximum admissible concentration for cocaine and/or metabolites in the urine of horses is difficult to establish unequivocally because of the substantial individual variation in the drug elimination pattern observed in horses, which can be inferred by the large standard error of the means obtained. Copyright (C) 2002 John Wiley Sons, Ltd.

Chemical Composition of Lipids Present in Cat and Dog Oocyte by Matrix-Assisted Desorption Ionization Mass Spectrometry (MALDI- MS)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Determination of gatifloxacin in bulk and tablet preparations by high-performance liquid chromatography

A sensitive, precise, and specific high-performance liquid chromatographic (HPLC) method was developed for the assay of gatifloxacin (GATX) in raw material and tablets. The method validation parameters yielded good results and included the range, linearity, precision, accuracy, specificity, and recovery. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. The HPLC separation was carried out by reversed-phase chromatography on a C18 absorbosphere column (250 x 4.6 mm id, 5 pm particle size) with a mobile phase composed of acetic acid 50/o--acetonitrile-methanol (70 + 15 + 15, v/v/v) pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 287 nm. The calibration graph for GATX was linear from 4.0 to 14.0 mu g/mL. The interday and intraday precisions (relative standard deviation) were less than 1.05%.

Determination of lomefloxacin in tablet preparations by liquid chromatography

A sensitive, precise, and specific high-performance liquid chromatography (HPLC) method was developed for the assay of lomefloxacin (LFLX) in raw material and tablet preparations. The method validation parameters yielded good results and included the range, linearity, precision, accuracy, specificity, and recovery. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. The HPLC separation was performed on a reversed-phase Phenomenex C18 column (150 x 4.6 mm id, 5 pm particle size) with a mobile phase composed of 1% acetic acid-acetonitrile-methanol (70 + 15 + 15, v/v/v), pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 280 nm. The calibration graph for LFLX was linear from 2.0 to 7.0 mg/mL. The interday and intraday precisions (relative standard deviation) were less than 1.0%. The method was applied for the quality control of commercial LFLX tablets to quantitate the drug.

Study of RF-excited Diethylene Glycol Dimethyl Ether Plasmas by Mass Spectrometry

This paper deals with the study of the fragmentation process of diethylene glycol dimethyl ether (CH3O(CH2CH2O)(2)CH3) (diglyme here in) molecule in low pressure RF excited plasma discharges. The study was carried out using mass spectrometry. The results showed that for a fixed pressure, the increase of the RF power coupled to the plasma chamber from 1 to 35 W produced a plasma environment much more reactive which increases the population of the ionized species like CH3+ (15 amu), C2H4+ (28 amu), CH3O+ (31 amu), C2H4O+ (44 amu), CH3OCH2CH2+ (59 amu) and CH3OCH2CH2O+ (75 amu). This fact may be attributed to the increase of the electronic temperature that makes predominant the occurrence of inelastic processes that promotes molecular fragmentation. For a fixed value of RF power the increase of pressure from 50 mTorr to 100 mTorr produces the decreasing of the above mentioned chemical species due the lower electronic mean free path. These results suggest that if one wants to keep the monomer's functionality within the plasma deposited films resulting from such kind of discharges one must operate in low power conditions.

Quantitation of malondialdehyde in gingival crevicular fluid by a high-performance liquid chromatography-based method

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development and application of methods for determination of residual monomer in dental acrylic resins using high performance liquid chromatography

Two high-performance liquid chromatographic methods for determination of residual monomer in dental acrylic resins are described. Monomers were detected by their UV absorbance at 230 nm, on a Nucleosil((R)) C-18 (5 mu m particle size, 100 angstrom pore size, 15 x 0.46 cm i.d.) column. The separation was performed using acetonitrile-water (55:45 v/v) containing 0.01% triethylamine (TEA) for methyl methacrylate and butyl methacrylate, and acetonitrile-water (60:40 v/v) containing 0.01% TEA for isobutyl methacrylate and 1,6-hexanediol dimethacrylate as mobile phases, at a flow rate of 0.8 mL/min. Good linear relationships were obtained in the concentration range 5.0-80.0 mu g/mL for methyl methacrylate, 10.0-160.0 mu g/mL for butyl methacrylate, 50.0-500.0 mu g/mL for isobutyl methacrylate and 2.5-180.0 mu g/mL for 1,6-hexanediol dimethacrylate. Adequate assay for intra- and inter-day precision and accuracy was observed during the validation process. An extraction procedure to remove residual monomer from the acrylic resins was also established. Residual monomer was obtained from broken specimens of acrylic disks using methanol as extraction solvent for 2 h in an ice-bath. The developed methods and the extraction procedure were applied to dental acrylic resins, tested with or without post-polymerization treatments, and proved to be accurate and precise for the determination of residual monomer content of the materials evaluated. Copyright (c) 2005 John Wiley & Sons, Ltd.

Phenylalanine ammonia-lyase activity in soybean roots extract measured by reverse-phase high performance liquid chromatography

The high performance liquid chromatography (HPLC) technique was applied to measure phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity in soybean (Glycine max L. Merril cv. BR16) roots. t-Cinnamate, the catalytic product of the PAL reaction was quantified at 275 nm by isocratic elution with methanol:water through an ODS(M) column. Comparative experiments were carried out with 1.0 mM ferulic acid, an inducer of PAL activity. The results suggest that liquid chromatography is a rapid and sensitive method to analyze PAL activity in non-purified extract.

Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Monitoring pharmaceuticals photo-Fenton degradation process by using Solid Phase Extraction and liquid chromatography

The recovery of the pharmaceuticals bezafibrate and tetracycline from water was evaluated, using Solid Phase Extraction (SPE) with the aim of applying this technique to interrupt the pharmaceuticals' photodegradation by photo-Fenton process for further analysis. Sep-Pack C-18, Strata X, and Oasis HLB cartridges were evaluated. Oasis HLB showed the most satisfactory recovery and repeatability results: 98% (CV - 1%) for bezafibrate (20.0 mg L-1) and 76% (CV = 1%) for tetracycline (25.0 mg L-1). There was not a significant decrease in recovery at lower concentrations of the pharmaceuticals, and neither when present in Sewage Treatment Plant (STP) effluent matrix.

Effect of Ionic Liquid on the Determination of Aromatic Amines as Contaminants in Hair Dyes by Liquid Chromatography Coupled to Electrochemical Detection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatography

A rapid, sensitive and reliable reverse-phase HPLC method was used for the quantitative determination of the anti-fungal and insecticide amides, dihydropiplartine (1), piplartine (2), Delta(alpha,beta)-dihydropiperine (3) and pellitorine (4) in plants in natura, in plantlets in vitro and ex vitro, and in callus of Piper tuberculatum. Well-resolved peaks were obtained with good detection response and linearity in the range of 15.0-3000 mug/mL. The plants in natura contained compounds 1-4, the plantlets ex vitro and in vitro accumulated compounds 1-2 and 1-4, respectively, while only amide 4 was found in callus. Copyright (C) 2003 John Wiley Sons, Ltd.

Quantitative determination of maitenin and 22 beta-hydroxymaitenin in callus of Maytenus aquifolium (Celastraceae) by reverse phase high performance liquid chromatography

Explants of Maytenus aquifolium were induced to form callus and, subsequently, suspension cultures. The isolation of natural products from callus led to the identification of the cytotoxic triterpene quinonemethides, maitenin (1) and 22 beta-hydroxymaitenin (2), A rapid, sensitive and reliable reversed-phase high-performance liquid chromatography method was developed using a Cls column and isocratic elution for the determination of 1 and 2, the elaborated method gave well-resolved peaks for these compounds with good detection response and linearity in the range of 0.08-72.0 mu g. The quantification of 1 and 2 was performed by an external standard method. (C) 1998 John Wiley & Sons, Ltd.

Determination of the betaine content of feed ingredients using high-performance liquid chromatography

A series of studies was conducted to establish a methodology for the accurate and efficient determination of betaine in different feed ingredients. The final methodology involves an extraction step in which the feed sample is heated for 3h in a methanolic KOH solution using a Goldfisch apparatus. Impurities are removed by the addition of activated charcoal and concentrated (36%) HCl. After centrifugation the extractant is passed through a strong cation exchange resin (Dowex 50W-X12, H+). The betaine retained in the column is eluted with 1.5 N HCl. A 2 nil aliquot of the elute is air dried and reconstituted with 1 ml of deionised water. HPLC separation with a cation exchange column (Partisil SCX-10) is used for the separation of betaine from other compounds. The mobile phase is kept constant at 50mm KH2PO4 in water, and eluted compounds are detected by UV absorbance (200nm). The flow rate is maintained at 1.5ml min(-1). This assay is very accurate over the range of betaine concentrations from 15 to 650 mug ml(-1), with a lower detection limit in feeds of approximately 500 mug g(-1) when 4g of sample is extracted. Recovery assays done with standard betaine hydrochloride and hard red wheat resulted in a consistent recovery of 80%. Betaine content was quantified in several feed ingredients, including alfalfa (1.77 mg kg(-1)), wheat (3.96 mg kg(-1)), wheat middlings (4.98 mg kg(-1)) and poultry meal (0.77 mg kg(-1)). Betaine in corn and soybean meal was not detectable by this method, even when 16g of sample was used (<125 mg kg(-1)). Betaine present in several feed ingredients should influence choline supplementation to animal feeds and may have implications for human health. (C) 2002 Society of Chemical Industry.