Plasma free and total carnitine measured in children by tandem mass spectrometry

Free and total carnitine quantification is important as a complementary test for the diagnosis of unusual metabolic diseases, including fatty acid degradation disorders. The present study reports a new method for the quantification of free and total carnitine in dried plasma specimens by isotope dilution electrospray tandem mass spectrometry with sample derivatization. Carnitine is determined by looking for the precursor of ions of m/z = 103 of N-butylester derivative, and the method is validated by comparison with radioenzymatic assay. We obtained an inter- and intra-day assay coefficient of variation of 4.3 and 2.3, respectively. Free and total carnitine was analyzed in 309 dried plasma spot samples from children ranging in age from newborn to 14 years using the new method, which was found to be suitable for calculating reference age-related values for free and total carnitine (less than one month: 19.3 ± 2.4 and 23.5 ± 2.9; one to twelve months: 28.8 ± 10.2 and 35.9 ± 11.4; one to seven years: 30.7 ± 10.3 and 38.1 ± 11.9; seven to 14 years: 33.7 ± 11.6, and 43.1 ± 13.8 µM, respectively). No difference was found between males and females. A significant difference was observed between neonates and the other age groups. We compare our data with reference values in the literature, most of them obtained by radioenzymatic assay. However, this method is laborious and time consuming. The electrospray tandem mass spectrometry method presented here is a reliable, rapid and automated procedure for carnitine quantitation.

A clinical gamma camera-based pinhole collimated system for high resolution small animal SPECT imaging

The main objective of the present study was to upgrade a clinical gamma camera to obtain high resolution tomographic images of small animal organs. The system is based on a clinical gamma camera to which we have adapted a special-purpose pinhole collimator and a device for positioning and rotating the target based on a computer-controlled step motor. We developed a software tool to reconstruct the target’s three-dimensional distribution of emission from a set of planar projections, based on the maximum likelihood algorithm. We present details on the hardware and software implementation. We imaged phantoms and heart and kidneys of rats. When using pinhole collimators, the spatial resolution and sensitivity of the imaging system depend on parameters such as the detector-to-collimator and detector-to-target distances and pinhole diameter. In this study, we reached an object voxel size of 0.6 mm and spatial resolution better than 2.4 and 1.7 mm full width at half maximum when 1.5- and 1.0-mm diameter pinholes were used, respectively. Appropriate sensitivity to study the target of interest was attained in both cases. Additionally, we show that as few as 12 projections are sufficient to attain good quality reconstructions, a result that implies a significant reduction of acquisition time and opens the possibility for radiotracer dynamic studies. In conclusion, a high resolution single photon emission computed tomography (SPECT) system was developed using a commercial clinical gamma camera, allowing the acquisition of detailed volumetric images of small animal organs. This type of system has important implications for research areas such as Cardiology, Neurology or Oncology.

Classification of brain tumor extracts by high resolution ¹H MRS using partial least squares discriminant analysis

High resolution proton nuclear magnetic resonance spectroscopy (¹H MRS) can be used to detect biochemical changes in vitro caused by distinct pathologies. It can reveal distinct metabolic profiles of brain tumors although the accurate analysis and classification of different spectra remains a challenge. In this study, the pattern recognition method partial least squares discriminant analysis (PLS-DA) was used to classify 11.7 T ¹H MRS spectra of brain tissue extracts from patients with brain tumors into four classes (high-grade neuroglial, low-grade neuroglial, non-neuroglial, and metastasis) and a group of control brain tissue. PLS-DA revealed 9 metabolites as the most important in group differentiation: γ-aminobutyric acid, acetoacetate, alanine, creatine, glutamate/glutamine, glycine, myo-inositol, N-acetylaspartate, and choline compounds. Leave-one-out cross-validation showed that PLS-DA was efficient in group characterization. The metabolic patterns detected can be explained on the basis of previous multimodal studies of tumor metabolism and are consistent with neoplastic cell abnormalities possibly related to high turnover, resistance to apoptosis, osmotic stress and tumor tendency to use alternative energetic pathways such as glycolysis and ketogenesis.

Serum metabolic profiling of human gastric cancer based on gas chromatography/mass spectrometry

Research on molecular mechanisms of carcinogenesis plays an important role in diagnosing and treating gastric cancer. Metabolic profiling may offer the opportunity to understand the molecular mechanism of carcinogenesis and help to non-invasively identify the potential biomarkers for the early diagnosis of human gastric cancer. The aims of this study were to explore the underlying metabolic mechanisms of gastric cancer and to identify biomarkers associated with morbidity. Gas chromatography/mass spectrometry (GC/MS) was used to analyze the serum metabolites of 30 Chinese gastric cancer patients and 30 healthy controls. Diagnostic models for gastric cancer were constructed using orthogonal partial least squares discriminant analysis (OPLS-DA). Acquired metabolomic data were analyzed by the nonparametric Wilcoxon test to find serum metabolic biomarkers for gastric cancer. The OPLS-DA model showed adequate discrimination between cancer and non-cancer cohorts while the model failed to discriminate different pathological stages (I-IV) of gastric cancer patients. A total of 44 endogenous metabolites such as amino acids, organic acids, carbohydrates, fatty acids, and steroids were detected, of which 18 differential metabolites were identified with significant differences. A total of 13 variables were obtained for their greatest contribution in the discriminating OPLS-DA model [variable importance in the projection (VIP) value >1.0], among which 11 metabolites were identified using both VIP values (VIP >1) and the Wilcoxon test. These metabolites potentially revealed perturbations of glycolysis and of amino acid, fatty acid, cholesterol, and nucleotide metabolism of gastric cancer patients. These results suggest that gastric cancer serum metabolic profiling has great potential in detecting this disease and helping to understand its metabolic mechanisms.

Striatal and extrastriatal dopamine D2/3 receptors studied with [11C]raclopride and high-resolution PET

The human striatum is a heterogeneous structure representing a major part of the dopamine (DA) system’s basal ganglia input and output. Positron emission tomography (PET) is a powerful tool for imaging DA neurotransmission. However, PET measurements suffer from bias caused by the low spatial resolution, especially when imaging small, D2/3 -rich structures such as the ventral striatum (VST). The brain dedicated high-resolution PET scanner, ECAT HRRT (Siemens Medical Solutions, Knoxville, TN, USA) has superior resolution capabilities than its predecessors. In the quantification of striatal D2/3 binding, the in vivo highly selective D2/3 antagonist [11C] raclopride is recognized as a well-validated tracer. The aim of this thesis was to use a traditional test-retest setting to evaluate the feasibility of utilizing the HRRT scanner for exploring not only small brain regions such as the VST but also low density D2/3 areas such as cortex. It was demonstrated that the measurement of striatal D2/3 binding was very reliable, even when studying small brain structures or prolonging the scanning interval. Furthermore, the cortical test-retest parameters displayed good to moderate reproducibility. For the first time in vivo, it was revealed that there are significant divergent rostrocaudal gradients of [11C]raclopride binding in striatal subregions. These results indicate that high-resolution [11C]raclopride PET is very reliable and its improved sensitivity means that it should be possible to detect the often very subtle changes occurring in DA transmission. Another major advantage is the possibility to measure simultaneously striatal and cortical areas. The divergent gradients of D2/3 binding may have functional significance and the average distribution binding could serve as the basis for a future database. Key words: dopamine, PET, HRRT, [11C]raclopride, striatum, VST, gradients, test-retest.

Evaluation of noni (Morinda citrifolia) volatile profile by dynamic headspace and gas chromatography-mass spectrometry

Noni is a fruit that has interested the scientific community due to its medicinal and functional activities. Different products that contain noni are already in the market, but their consumption could be impaired by their distinctive unpleasant aroma and flavor. The aim of this work was to evaluate the noni pulp volatile profile by dynamic headspace and gas chromatography-mass spectrometry. Thirty seven volatile compounds were detected, mainly alcohols (63.3%), esters (26.9%), cetones (7.4%), and acids (1.2%).

(E)-2-Nonenal determination in brazilian beers using headspace solid-phase microextraction and gas chromatographic coupled mass spectrometry (HS-SPME-GC-MS)

(E)-2-nonenal is considered an important off-flavor of beer, related to the flavor of beer staling. In this study, a new method for determination of (E)-2-nonenal in beer using headspace solid-phase microextraction and gas chromatographic coupled mass spectrometry (HS-SPME-GC-MS) was developed and applied in Brazilian beer samples. The extractions were carried out in CAR-PDMS (carboxen-polydimethylsiloxane) fiber and the best results were found with 15 minutes of equilibrium and 90 minutes of extraction at 50 °C. The method was linear in the range from 0.02 to 4.0 μg.L-1 with correlation coefficient of 0.9994. The limits of detection and quantification were 0.01 and 0.02 μg.L-1, respectively. 96.5% of recovery and 4% precision (RSD) were obtained in the fortification of beer samples with 2.0 μg.L-1 of (E)-2-nonenal. The developed method proved to be simple, efficient and highly sensitive to the determination of this analyte being easily applied in the quality control of the brewery. (E)-2-nonenal was found in all beer samples analyzed with levels between 0.17 and 0.42 μg.L-1.

Monitoring of wine aging process by electrospray ionization mass spectrometry

The characterization of wine samples by direct insertion electrospray ionization mass spectrometry (ESI-MS), without pre-treatment or chromatographic separation, in a process denominated fingerprinting, has been applied to several samples of wine produced with grapes of the Pinot noir, Merlot and Cabernet Sauvignon varieties from the state o Rio Grande do Sul, in Brazil. The ESI-MS fingerprints of the samples detected changes which occurred during the aging process in the three grape varieties. Principal Component Analysis (PCA) of the negative ion mode fingerprints was used to group the samples, pinpoint the main changes in their composition, and indicate marker ions for each group of samples.

Development and validation of a method for detection and quantification of ochratoxin A in green coffee using liquid chromatography coupled to mass spectrometry

A method using Liquid Chromatography Tanden Mass Spectrometry (LC-MS/MS) with matrix-matched calibration curve was developed and validated for determining ochratoxin A (OTA) in green coffee. Linearity was found between 3.0 and 23.0 ng.g-1. Mean recoveries ranged between 90.45% and 108.81%; the relative standard deviation under repeatability and intermediate precision conditions ranged from 5.39% to 9.94% and from 2.20% to 14.34%, respectively. The limits of detection and quantification were 1.2 ng.g-1 and 3.0 ng.g-¹, respectively. The method developed was suitable and contributed to the field of mycotoxin analysis, and it will be used for future production of the Certified Reference Material (CRM) for OTA in coffee.

Characterization and classification of pequi trees (Caryocar brasiliense Camb.) based on the profile of volatile constituents using headspace solid-phase microextraction - gas chromatography - mass spectrometry and multivariate analysis

In order to determine the variability of pequi tree (Caryocar brasiliense Camb.) populations, volatile compounds from fruits of eighteen trees representing five populations were extracted by headspace solid-phase microextraction and analyzed by gas chromatography-mass spectrometry. Seventy-seven compounds were identified, including esters, hydrocarbons, terpenoids, ketones, lactones, and alcohols. Several compounds had not been previously reported in the pequi fruit. The amount of total volatile compounds and the individual compound contents varied between plants. The volatile profile enabled the differentiation of all of the eighteen plants, indicating that there is a characteristic profile in terms of their origin. The use of Principal Component Analysis and Cluster Analysis enabled the establishment of markers (dendrolasin, ethyl octanoate, ethyl 2-octenoate and β-cis-ocimene) that discriminated among the pequi trees. According to the Cluster Analysis, the plants were classified into three main clusters, and four other plants showed a tendency to isolation. The results from multivariate analysis did not always group plants from the same population together, indicating that there is greater variability within the populations than between pequi tree populations.

Developing correlative Optical-Photoacustic microscopy for high-resolution in vivo imaging

PhotoAcoustic Imaging (PAI) is a branch in clinical and pre-clinical imaging, that refers to the techniques mapping acoustic signals caused by the absorption of the short laser pulse. This conversion of electromagnetic energy of the light to the mechanical (acoustic) energy is usually called photoacoustic effect. PAI, by combining optical excitation with acoustical detection, is able to preserve the diffraction limited spatial resolution. At the same time, the penetration depth is extended beyond the diffusive limit. The Laser-Scanning PhotoAcoustic Microscope system (LS-PAM) has been developed, that offers the axial resolution of 7.75 µm with the lateral resolution better than 10 µm. The first in vivo imaging experiments were carried out. Thus, in vivo label-free imaging of the mouse ear was performed. The principle possibility to image vessels located in deep layers of the mouse skin was shown. As well as that, a gold printing sample, vasculature of the Chick Chorioallantoic Membrane Assay, Drosophila larvae were imaged by PAI. During the experimental work, a totally new application of PAM was found, in which the acoustic waves, generated by incident light can be used for further imaging of another sample. In order to enhance the performance of the presented system two main recommendation can be offered. First, the current system should be transformed into reflection-mode setup system. Second, a more powerful source of light with the sufficient repetition rate should be introduced into the system.

A fast atom bombardment mass spectrometry study of tris (3, 6-dioxaheptyl) amine-alkali metal halide complexes and hydrogen bonded complexes

The objective of this thesis was to demonstrate the potential of fast atom bombardment mass spectrometry (FABMS) as a probe of condensed phase systems and its possible uses for the study of hydrogen bonding. FABMS was used to study three different systems. The first study was aimed at investigating the selectivity of the ligand tris(3,6-dioxaheptyl) amine (tdoha) for the alkali metal cations. FABMS results correlated well with infrared and nmr data. Systems where a crown ether competed with tdoha for a given alkali metal cation were also investigated by fast atom bombardment. The results were found to correlate with the cation affinity of tdoha and the ability of the crown ether to bind the cation. In the second and third studies, H-bonded systems were investigated. The imidazole-electron donor complexes were investigated and FABMS results showed the expected H-bond strength of the respective complexes. The effects of concentration, liquid matrix, water content, deuterium exchange, and pre-ionization of the complex were also investigated. In the third system investigated, the abundance of the diphenyl sulfone-ammonium salt complexes (presumably H-bonded) in the FABMS spectrum were found to correlate with qualitative considerations such as steric hindrance and strength of ion pairs.

Fast atom bombardment and electron impact mass spectrometry studies of some aryltin compounds and ferrocenes

Both El MS and FAB MS behavior of two groups of compounds, aryltin and ferrocene compounds, have been studied. For the aryltin compounds, the effect of substituent group position, substituent group type and ligand type on the El spectra have been explored in the El MS studies. The fragmentation mechanism has been investigated under El with linked scans, such as fragment ion scans(BJE), parent ion scans(B2JE) and constant neutral radical loss scans(B2(1-E)JE2). In the FAB MS studies, matrix optimization experiments have been carried out. The positive ion FAB MS studies focused on the effect of substituent group position, substituent group type and ligand type on the spectra. The fragmentation mechanisms of all the samples under positive ion FAB have been studied by means of the linked scans. The CA positive ion FAB fragmentation studies were also carried out for a typical sample. Negative ion FAB experiments of all the compounds have been done. And finally, the comparison of the El MS and FAB MS has been made. For ferrocenes, the studies concentrated on the fragmentation mechanism of each compound under El with linked scan techniques in the first field-free region and the applicability of positive/negative ion FAB MS to this group of compounds. The fragmentation mechanisms under positive ion FAB of those ferrocenes which can give positive ion FAB MS spectra were studied with the linked scan techniques. The CA +ve F AB fragmentation studies were carried out for a typical sample. Comparison of the E1 MS and FAB MS has been made.