UHPLC-MS/MS method for the simultaneous determination of nicotine and tobacco-specific nitrosamines NNN and NNK for use in preclinical studies.

A new method was developed and validated for the simultaneous determination of nicotine and tobacco-specific nitrosamines (TSNAs) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) in two different tests matrices: porcine buccal epithelium tissue and phosphate buffered saline (PBS) extracts of smokeless tobacco products. The novelty of this work is in the development of a liquid chromatography tandem mass spectrometry method that can provide simultaneous quantification of trace levels of TSNAs and high concentrations of nicotine in biological media. Precision, accuracy, and stability were evaluated during method validation to ensure the method was fit for purpose. Several sample preparation and extraction methods were evaluated to minimize matrix effects and maximize analyte recoveries. The method was accurate in the range of 81.1% - 117%; repeatability was estimated in the range of 1.5% - 13.6% across multiple concentrations. The linear regression correlation coefficient (R2) was greater than 0.9959 for all analytes, and the limit of detection (LOD) was determined for nicotine, NNK, and NNN at 1 ng/mL 0.005 ng/mL, and 0.006 ng/ mL, respectively. Our method was found to be appropriate for the analysis of nicotine, NNN, and NNK in the porcine buccal epithelium and PBS extracts of smokeless tobacco products.

Hepatic Metabolic Profiling of Lifelong Exercise Training Rats.

Regular physical exercise has been investigated as a primary preventive measure of several chronic diseases and premature death. Moreover, it has been shown to synchronize responses across multiple organs. In particular, hepatic tissue has proven to be a descriptive matrix to monitor the effect of physical activity. In this study, we performed an untargeted metabolomics-based analysis of hepatic tissue extracts from rats that have undergone either lifelong or chronic exercise training. For this purpose, 56 hepatic samples were collected and were analyzed by UHPLC-TOF-MS in negative ionization mode. This approach involved untargeted metabolite detection on hepatic tissue extracts accompanied by an in-house retention time/accurate mass library enabling confident metabolite identification. Unsupervised (PCA) and supervised (OPLS-DA) multivariate analysis showed significant metabolic perturbation on a panel of 28 metabolites, including amino acids, vitamins, nucleotides, and sugars. The training regime employed in this study resulted in a probable acceleration of the bioenergetic processes (glycolysis, glycogen metabolism), promoted catabolism of purines, and supplied biosynthetic precursors via the pentose phosphate pathway and pentose and glucuronate interconversions. Overall, the applied methodology was able to discriminate the different training schedules based on the rat liver metabolome.

Investigation of salivary biomarkers as indicators of skeletal and dental maturity in children.

OBJECTIVE: Estimation of patient's skeletal maturity in orthodontics is essential for the diagnosis and treatment planning. The aim of the study was to investigate the potential use of metabolic fingerprint of saliva for bone growth and tooth development estimation. MATERIALS AND METHODS: Saliva samples from 54 young patients were analysed by an untargeted gas chromatography-mass spectrometry metabolomics-based method. The skeletal maturity was calculated with the cervical vertebrae maturation method, and the dental age was estimated with the Demirjian method. Multivariate analysis and univariate analysis were performed to investigate differences within skeletal, dental and chronological age groups. RESULTS: Metabolomic analysis identified 61 endogenous compounds. Mannose, glucose, glycerol, glyceric acid and pyroglutamic acid levels differentiated significantly with skeletal age (P = .02 to .043), while mannose, lactic acid, glycolic acid, proline, norleucine, 3-aminoisobutyric acid, threonine, cadaverine and hydrocinnamic acid levels differed within the dental age groups (P = .018 to .04); according to the chronological age, only the levels of mannose and 3-hydroxyphenylacetic acid showed variation (P = .029 and .048). The principal component analysis did not manage to highlight differences between the groups of the studied parameters. CONCLUSION: Differentiated levels of mannose, glucose, glycerol, glyceric acid and pyroglutamic acid related to skeletal maturation were identified. According to dental development, the levels of mannose, lactic acid, glycolic acid, proline, norleucine, 3-aminoisobutyric acid, threonine, cadaverine and hydrocinnamic acid differed within the groups, while regarding chronological age, only the levels of mannose and 3-hydroxyphenylacetic acid showed variations. Further studies are required to prove their relation to skeletal and dental development pathway by applying complementary analytical techniques to wider cover the metabolome.

Impact of religious fasting on metabolic and hematological profile in both dyslipidemic and non-dyslipidemic fasters.

BACKGROUND/OBJECTIVES: Religious fasting (RF) is practiced annually by millions of Christian and Muslim followers worldwide. Scarce data exist on the impact of RF on the metabolic and hematological profile of individuals with or without dyslipidemia. SUBJECTS/METHODS: The present study included: (i) 60 Greek Orthodox participants, 30 with dyslipidemia and 30 without dyslipidemia, who abstained from meat, fish and dairy products for seven consecutive weeks, and (ii) 15 young, non-dyslipidemic Muslim participants abstaining totally from food and liquid from dawn till sunset during 30 days. Biochemical (iron, ferritin, vitamin B12, calcium, low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (TC), triglyceride and fasting glucose) and hematological (hemoglobin, hematocrit) serum blood test results of study participants were measured pre- and post- RF (at weeks 0 and 7 for Orthodox participants and at weeks 0 and 4 for Muslim participants). RESULTS: In dyslipidemic and non-dyslipidemic Orthodox participants, a significant reduction of fasting glucose, HDL, LDL and TC levels was found post-RF. Hemoglobin, hematocrit, iron and ferritin levels were significantly increased, while post-RF vitamin B12 and calcium levels were substantially decreased. Subanalysis between dyslipidemic and non-dyslipidemic Orthodox participants revealed a greater decrease of cholesterol levels in the former. In Muslim participants, triglyceride, LDL and total cholesterol levels were increased post-RF (all p values < 0.05). CONCLUSIONS: Our study adds to the existing literature evidence about the significant impact of RF on metabolic and hematological profiles of Orthodox and Muslim followers. The prevention of calcium and B12 deficiency during Orthodox RF by supplement consumption as well as the protection from dehydration and dysregulation of lipid metabolism during Ramadan RF should concern both clinicians and dietician nutritionists. Nevertheless, studies with larger sample size and/or long-term follow-up are warranted before reaching definite conclusions about the effects of RF on human health.

Headspace gas chromatography-mass spectrometry in the analysis of lavender's essential oil: Optimization by response surface methodology.

A static headspace gas chromatography - mass spectrometry (HS-GC/MS) method was developed and optimized with the aim to be applied in the analysis of lavender essential oil. To obtain a comprehensive profile of the essential oil, the optimum HS-GC/MS method parameters were selected based on a Design of Experiments (DοE) process. Plackett-Burman experimental design was applied by utilizing seven parameters of the HS injection system. Incubation equilibration temperature and time, agitator's vortex speed, post injection dwell time, inlet temperature, split ratio and injection flow rate were screened to select the optimum conditions on the basis of the number and the intensity of the identified compounds. Other parameters, such as sample volume and dilution solvent ratio, were also examined to achieve a comprehensive profile in a chromatographic run of 55 min. With the obtained optimum method, more than 40 volatile compounds were identified in lavender's essential oils from different geographical regions in Greece. The method can be utilized for the quality assessment of lavender's essential oil and provide information on its characteristic aroma and discrimination among species based on the acquired GC-MS profiles.

Development and validation of an UHPLC-qTOF-MS method for the quantification of cyclic polyesters oligomers in pasta by applying a modified QuEChERS clean-up.

An Ultra High-Performance Liquid chromatography method quadruple time-of-flight mass spectrometry has been developed for the analysis of 11 cyclic polyesters oligomers, following a modified QuEChERS clean-up with alumina/primary secondary amine, in pasta. Target analytes were polyethylene terephthalate (PET) 1st series cyclic dimer to heptamer, polybutylene terephthalate (PBT) dimer to pentamer and a polyurethane oligomer. Standard addition method was applied for the calibration, and the limits of quantification ranged from 3.2 to 17.2 ng g-1. Recoveries ranged from 86.4 to 109.8%, RSDs were lower than 12% for all analytes, and matrix effect never exceeded ± 2.5%. The method was successfully applied to real commercial pasta samples, where the PET 1st series cyclic trimer was the most abundant oligomer, being found in all tested samples. The 1st series PET cyclic dimer and tetramer, as well as 1,4,7-trioxacyclotridecane-8,13-dione, were found in considerable amounts. Traces of the 2nd and 3rd series PET cyclic dimers were also found.

Amniotic Fluid and Maternal Serum Metabolic Signatures in the Second Trimester Associated with Preterm Delivery.

Preterm delivery (PTD) represents a major health problem that occurs in 1 in 10 births. The hypothesis of the present study was that the metabolic profile of different biological fluids, obtained from pregnant women during the second trimester of gestation, could allow useful correlations with pregnancy outcome. Holistic and targeted metabolomics approaches were applied for the complementary assessment of the metabolic content of prospectively collected amniotic fluid (AF) and paired maternal blood serum samples from 35 women who delivered preterm (between 29 weeks + 0 days and 36 weeks +5 days gestation) and 35 women delivered at term. The results revealed trends relating the metabolic content of the analyzed samples with preterm delivery. Untargeted and targeted profiling showed differentiations in certain key metabolites in the biological fluids of the two study groups. In AF, intermediate metabolites involved in energy metabolism (pyruvic acid, glutamic acid, and glutamine) were found to contribute to the classification of the two groups. In maternal serum, increased levels of lipids and alterations of key end-point metabolites were observed in cases of preterm delivery. Overall, the metabolic content of second-trimester AF and maternal blood serum shows potential for the identification of biomarkers related to fetal growth and preterm delivery.

Sample preparation optimization in fecal metabolic profiling.

Metabolomic analysis of feces can provide useful insight on the metabolic status, the health/disease state of the human/animal and the symbiosis with the gut microbiome. As a result, recently there is increased interest on the application of holistic analysis of feces for biomarker discovery. For metabolomics applications, the sample preparation process used prior to the analysis of fecal samples is of high importance, as it greatly affects the obtained metabolic profile, especially since feces, as matrix are diversifying in their physicochemical characteristics and molecular content. However there is still little information in the literature and lack of a universal approach on sample treatment for fecal metabolic profiling. The scope of the present work was to study the conditions for sample preparation of rat feces with the ultimate goal of the acquisition of comprehensive metabolic profiles either untargeted by NMR spectroscopy and GC-MS or targeted by HILIC-MS/MS. A fecal sample pooled from male and female Wistar rats was extracted under various conditions by modifying the pH value, the nature of the organic solvent and the sample weight to solvent volume ratio. It was found that the 1/2 (wf/vs) ratio provided the highest number of metabolites under neutral and basic conditions in both untargeted profiling techniques. Concerning LC-MS profiles, neutral acetonitrile and propanol provided higher signals and wide metabolite coverage, though extraction efficiency is metabolite dependent.

Quantitative profiling of polar primary metabolites using hydrophilic interaction ultrahigh performance liquid chromatography-tandem mass spectrometry.

A hydrophilic interaction liquid chromatography (HILIC)-MS/MS profiling method was developed for the efficient separation and quantification of small polar molecules, mostly primary metabolites. The method was based on an ultrahigh performance liquid chromatography (UHPLC) separation system coupled with ESI mass spectrometry on a triple quadrupole mass spectrometer, operating in both positive and negative ionisation mode using rapid polarity switching. With the developed method quantitation of 135 compounds belonging in four major classes of polar compounds (sugars, aminoacids, organic acids and amines) was achieved in a single run of 30 min. The method was applied to grape extracts from different varieties and provided information on primary metabolite content. Multivariate statistical analysis was applied using the concentrations found, with the aim of investigating the differences in metabolite profiles. Classification of grapes according to their skin colour was carried out using principle component analysis based on the concentration variation of a number of the metabolites studied.

Sample preparation prior to the LC-MS-based metabolomics/metabonomics of blood-derived samples.

Blood represents a very important biological fluid and has been the target of continuous and extensive research for diagnostic, or health and drug monitoring reasons. Recently, metabonomics/metabolomics have emerged as a new and promising 'omics' platform that shows potential in biomarker discovery, especially in areas such as disease diagnosis, assessment of drug efficacy or toxicity. Blood is collected in various establishments in conditions that are not standardized. Next, the samples are prepared and analyzed using different methodologies or tools. When targeted analysis of key molecules (e.g., a drug or its metabolite[s]) is the aim, enforcement of certain measures or additional analyses may correct and harmonize these discrepancies. In omics fields such as those performed by holistic analytical approaches, no such rules or tools are available. As a result, comparison or correlation of results or data fusion becomes impractical. However, it becomes evident that such obstacles should be overcome in the near future to allow for large-scale studies that involve the assaying of samples from hundreds of individuals. In this case the effect of sample handling and preparation becomes very serious, in order to avoid wasting months of work from experts and expensive instrument time. The present review aims to cover the different methodologies applied to the pretreatment of blood prior to LC-MS metabolomic/metabonomic studies. The article tries to critically compare the methods and highlight issues that need to be addressed.

Determination of two COX-2 inhibitors in serum and synovial fluid of patients with inflammatory arthritis by ultra performance liquid chromatography-inductively coupled plasma mass spectroscopy and quadrupole time-of-flight mass spectrometry.

The determination of two sulphur-containing drugs, the COX-2 inhibitors celecoxib and etoricoxib, in the serum and synovial fluid of inflammatory arthritis patients, is described using a sensitive ultra performance liquid chromatography-inductively coupled plasma mass spectroscopy (UPLC/ICPMS) method. Confirmation of the identity of the analytes in the samples was also performed by electrospray quadruple time-of-flight mass spectrometry in positive electrospray ionisation mode. The two COX-2 inhibitors were extracted from serum and synovial fluid following dilution with acetate buffer (pH 5) and liquid-liquid extraction (LLE) into ethyl acetate. Extracted samples were then analysed using UPLC/ICPMS with sulphur-specific detection. The limit of detection by UPLC/ICPMS was 0.45 ng/ml of sulphur in both serum and synovial fluid. The UPLC/ICPMS method was applied to the analysis of samples from patients receiving either 200 mg/day of celecoxib (2x 100 mg), 90 mg/day etoricoxib or placebo. The range of concentrations detected in the samples for the two drugs was from 0.3 to 3.3 microg/ml.