Quantification of modified nucleotides and nucleosides by isotope dilution mass spectrometry.

Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5-methyl-2'-deoxycycytidine. Furthermore, 5-mdC can be oxidized to form three new modifications, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxy-2'-deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean-up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations.

Evaluation of different isotope dilution mass spectrometry strategies for the characterization of naturally abundant and isotopically labelled peptide standards.

Natural abundance and isotopically labelled tryptic peptides are routinely employed as standards in quantitative proteomics. The certification of the peptide content is usually carried out by amino acid analysis using isotope dilution mass spectrometry (IDMS) after the acid hydrolysis of the peptide. For the validation and traceability of the amino acid analysis procedure, expensive certified peptides must be employed. In this work we evaluate different IDMS alternatives which will reduce the amount of certified peptide required for validation of the amino acid analysis procedure. In this context, the characterization of both natural and isotopically labelled synthetic angiotensin I peptides was carried out. First, we applied a fast procedure for peptide hydrolysis based on microwave-assisted digestion and employed two certified peptide reference materials SRM 998 angiotensin I and CRM 6901-b C-peptide for validation of the hydrolysis procedure. The amino acids proline, leucine, isoleucine, valine, tyrosine, arginine and phenylalanine were evaluated for their suitability for peptide certification by IDMS by both liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography with mass spectrometry (GC)-MS/MS. Then, natural angiotensin I and 13C1-labelled angiotensin I were synthesized in-house and purified by preparative liquid chromatography. The concentration of the 13C1-labelled angiotensin I peptide was established by reverse IDMS in its native form using SRM 998 angiotensin I as reference. The concentration of the natural synthesized peptide was determined by IDMS both using the 13C1-labelled peptide in its native form and by amino acid analysis showing comparable results. Finally, the synthetic naturally abundant angiotensin I peptide was employed as "in-house" standard for the validation of subsequent peptide characterization procedures. Therefore, the novelty of this work relies on, first, the development of a faster hydrolysis procedure assisted by focused microwaves, providing complete hydrolysis in 150 min, and secondly, a validation strategy combining GC-MS and LC-MS/MS that allowed us to certify the purity of an in-house-synthesized peptide standard that can be employed as quality control in further experiments.

Comparison between one and two-dimensional liquid chromatographic approaches for the determination of plasmatic stroke biomarkers by isotope dilution and tandem mass spectrometry.

This work presents the evaluation of one- and two-dimensional liquid chromatography for the quantification of three stroke outcome predictors in plasma. Isotopically labelled analogues of L-arginine (L-Arg), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are used to quantify the three analytes by isotope dilution and tandem mass spectrometry. Chromatographic isotope effects were not observed between natural L-Arg and its 15N-labelled analogue but they were observed between natural ADMA and SDMA and their multiple deuterated analogues. Under these conditions, bidimensional chromatography through the use of an automated multiple heart cutting mode provided unsatisfactory results for ADMA and SDMA due to the different amounts of natural and labelled compounds transferred from the first to the second chromatographic dimension. In contrast, using one dimensional liquid chromatography after a derivatization step to esterify carboxylic groups, chromatographic isotope effects did not alter the initial mass balance as full coelution of natural and labelled analogues or baseline resolution between the analytes was not required. This method was successfully validated following the Clinical & Laboratory Standards Institute guidelines and applied to the analysis of plasma samples from patients who had suffered from an intraparenchymal haemorrhagic stroke.

Determination of methylmercury and inorganic mercury in human hair samples of individuals from Colombian gold mining regions by double spiking isotope dilution and GC-ICP-MS.

With the aim to distinguish between routes of exposition to mercury (Hg) in artisanal and small-scale gold mining (ASGM) communities and to distinguish between Hg contamination sources, Hg species composition should be performed in human biomarkers. In this work, Hg species-specific determination were determined in human hair samples (N = 96), mostly non-directly occupied in ASGM tasks, from the six most relevant gold mining Colombian regions. Therefore, MeHg, Hg(II) and THg concentrations were simultaneously determined by double spiking species-specific isotope dilution mass spectrometry (IDMS) and GC-ICP-MS. Only 16.67% of participants were involved at some point in AGSM works and fish consumption ranged from 3 to 7 times/week, which is between medium and high intake levels. The median concentration of THg obtained from all samples is higher than the reference dose weekly acceptable of MeHg intake established by the EPA (1 ppm), whereas a 25% were more than 4 times higher than the WHO level (2.2 µg Hg g-1). Median THg value of individuals consuming fish 5-7 times per week was significantly higher (p < 0.05) than those of the other consuming groups (12.5 µg Hg g-1). Most of the samples presented a % of MeHg relative to THg higher than 80%. The average % of Hg(II)/THg was 11% and only 10 individuals presented a Hg(II) content over 30%. No significant differences (p > 0.05) were found when the amount of Hg(II) was compared between people involved in AGSM task and people not involved. Interestingly, significant differences among the evaluated groups where found when the percentage of the Hg(II)/THg ratio of these groups were compared. In fact, people involved in AGSM tasks showed 1.7 times higher Hg(II)/THg vs. inhabitants uninvolved. This suggest that Hg(II) determination by IDMS-GC-ICP-MS could be a good proxy for evaluating Hg(II) adsorption by direct exposure to mercury vapors onto hair.

Impaired condensin complex and Aurora B kinase underlie mitotic and chromosomal defects in hyperdiploid B-cell ALL.

B-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD being an initiating oncogenic event affiliated with childhood B-ALL, the mitotic and chromosomal defects associated with HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated with chromosome-alignment defects at the metaphase plate leading to robust chromosome-segregation defects and nonmodal karyotypes. Mechanistically, biochemical, functional, and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness, and mislocalization of the chromosome passenger complex proteins Aurora B kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and an impaired spindle assembly checkpoint (SAC), thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated with a defective condensin complex, AURKB, and SAC.

Determination of 3-monoiodotyrosine and 3,5-diiodotyrosine in newborn urine and dried urine spots by isotope dilution tandem mass spectrometry.

High levels of 3-mono- and 3,5-diiodotyrosine (MIT and DIT, respectively) in urine have been related to iodotyrosine dehalogenase 1 deficiency, a type of congenital hypothyroidism. However, the determination of MIT and DIT in urine is not included in newborn screening programs performed in clinical laboratories to detect inborn errors of metabolism. We report here on the development of an analytical method for the determination of MIT and DIT in newborn urine and dried urine spots (DUS) by Liquid Chromatography Isotope Dilution tandem Mass Spectrometry (LC-IDMSMS). The development included the synthesis of 15N-monoiodotyrosine and 13C2-diiodotyrosine through the iodination of 15N-tyrosine and 13C2-tyrosine, respectively, using bis(pyridine)iodonium(I) tetrafluoroborate (IPy2BF4). Both labelled analogues were added at the beginning of the sample preparation procedure and used to develop both single- and double-spike LC-IDMS methods for the determination of MIT and DIT. The developed double spike methodology was able to quantify and correct possible MIT ↔ DIT interconversions throughout the sample preparation, which was observed for concentrated urine samples but not for DUS. Suppression matrix effects on the absolute signals of MIT and DIT were observed in urine samples but did not affect the IDMS results as recoveries on urine samples at different dilution factors could be considered quantitative. Method detection limits were 0.018 and 0.046 ng g-1 (limits of quantification 0.06 and 0.15 ng g-1) by single-spike IDMS, for MIT and DIT, respectively, in the analysis of urine samples and 0.07 and 0.05 ng g-1 (limits of quantification 0.23 and 0.17 ng g-1) for MIT and DIT, respectively, in the analysis of DUS. No significant differences were obtained for MIT concentrations in the analysis of the same newborn samples stored as liquid urine or DUS when the results were corrected for the creatinine content. Finally, 36 DUS samples from healthy newborns were analyzed and MIT was detected in all samples at low ng mg-1creatinine levels.

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.

Development of a Common Procedure for the Determination of Methylmercury, Ethylmercury, and Inorganic Mercury in Human Whole Blood, Hair, and Urine by Triple Spike Species-Specific Isotope Dilution Mass Spectrometry.

We report the first common methodology for the simultaneous determination of methylmercury (MeHg), ethylmercury (EtHg), and inorganic mercury (Hg(II)) in human blood hair and urine. With the exception of the initial sample mass (0.15 g for blood, 0.5 g for urine, and 0.1 g for hair), the same sample preparation and gas chromatography-inductively coupled plasma mass spectrometry (GC-ICPMS) measurement conditions are employed for the three matrixes providing experimental values in agreement with the certified values in the analysis of NIST SRM 955c (Caprine Blood) Level 3 and the certified human hairs IAEA 085 and IAEA 086. Also, the method provides quantitative recoveries for the three Hg species in the analysis of fortified human urine samples at 1, 2, and 5 ng Hg g-1. Mercury species concentrations for levels 2 and 4 of SRM 955c are reported here for the first time. A systematic interconversion of EtHg into Hg(II) was obtained for all matrixes reaching values up to 95% in blood, 29% in hair, and 11% in urine. MeHg dealkylation was also observed in a lesser extent in blood and hair analyses, but it was not observed when analyzing urine samples. Hg methylation was not observed in any matrix. The amount of NaBPr4 added for derivatization has been found to be the main factor responsible for Hg species interconversion. This work demonstrates for the first time that experimental conditions optimized for SRM 955c (caprine blood) are not valid for human blood samples as the optimum initial sample amount for a real sample is more than 3 times lower than that for SRM 955c.

Melatonin Decreases Glucose Metabolism in Prostate Cancer Cells: A 13C Stable Isotope-Resolved Metabolomic Study.

The pineal neuroindole melatonin exerts an exceptional variety of systemic functions. Some of them are exerted through its specific membrane receptors type 1 and type 2 (MT1 and MT2) while others are mediated by receptor-independent mechanisms. A potential transport of melatonin through facilitative glucose transporters (GLUT/SLC2A) was proposed in prostate cancer cells. The prostate cells have a particular metabolism that changes during tumor progression. During the first steps of carcinogenesis, oxidative phosphorylation is reactivated while the switch to the "Warburg effect" only occurs in advanced tumors and in the metastatic stage. Here, we investigated whether melatonin might change prostate cancer cell metabolism. To do so, 13C stable isotope-resolved metabolomics in androgen sensitive LNCaP and insensitive PC-3 prostate cancer cells were employed. In addition to metabolite 13C-labeling, ATP/AMP levels, and lactate dehydrogenase or pentose phosphate pathway activity were measured. Melatonin reduces lactate labeling in androgen-sensitive cells and it also lowers 13C-labeling of tricarboxylic acid cycle metabolites and ATP production. In addition, melatonin reduces lactate 13C-labeling in androgen insensitive prostate cancer cells. Results demonstrated that melatonin limits glycolysis as well as the tricarboxylic acid cycle and pentose phosphate pathway in prostate cancer cells, suggesting that the reduction of glucose uptake is a major target of the indole in this tumor type.

Determination of Polychlorinated Biphenyls in Solid Samples by Isotope Dilution Mass Spectrometry Using ³7Cl-Labeled Analogues.

This work describes the first application of (37)Cl-labeled compounds to isotope dilution mass spectrometry (IDMS). The synthesis of 12 (37)Cl-labeled polychlorinated biphenyls (PCBs) was carried out by the chlorination of biphenyl with isotopically enriched chlorine gas, generated by the direct oxidation of Na(37)Cl with potassium peroxymonosulfate. After an exhaustive purification due to the presence of other congeners, the concentration and the isotopic enrichment of all (37)Cl-labeled PCBs in the mixture was determined. The proposed procedure allows the simultaneous quantification of every isotope diluted PCB congener in a single gas chromatography-tandem mass spectrometry (GC-MS/MS) injection without resorting to a methodological calibration graph. The results obtained here demonstrate that the use of (37)Cl-labeled analogues provides results in agreement with the certified values of three different Certified Reference Materials (marine sediment SRM 1944, fish tissue 1947, and loamy soil CRM 962-50) and analytical figures of merit comparable to those obtained using regular IDMS procedures based on the use of commercially available (13)C-labeled analogues.

Simultaneous determination of creatinine and creatine in human serum by double-spike isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).

This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors derived from the sample preparation step.

Correction of creatine-creatinine conversion during serum creatinine quantification by two-dimensional liquid chromatography and double-spike isotope dilution tandem mass spectrometry.

BACKGROUND AND AIMS: Development of a candidate reference method based on bidimensional liquid chromatography coupled to ESI-MS/MS and double spike isotope dilution for serum creatinine quantification capable of correcting for creatinine-creatine interconversion during sample pretreatment. Study of the impact of the creatine-creatinine interconversion during the analysis of human serum samples. MATERIALS AND METHODS: 13C1-creatinine and 13C2-creatine are added to the serum sample. Separation carried out by bidimensional liquid chromatography combining reversed phase and a strong cation exchange chromatography. The heart cut, containing creatine and creatinine, is automatically transferred to the second dimension. Quantification carried out by double spike isotope dilution tandem MS/MS. RESULTS: Minimization of spectral interferences and ion suppression due to matrix effects while increasing sample throughput compared to the direct coupling of cation exchange chromatography to the ESI source. Trueness of the method studied with the satisfactory analysis of two certified reference materials. Satisfactory intra- and inter-day precisions obtained analysing a serum pool and control sera. Analysis of 93 serum samples revealed negligible interconversions with no correlation with creatine levels. CONCLUSIONS: The method provides adequate analytical figures of merit for serum creatinine determination according to CSLI guidelines. Negligible creatine-creatinine interconversion is promoted with the applied sample preparation procedure.

Species-specific stable isotope analysis by the hyphenation of chromatographic techniques with MC-ICPMS.

This work reviews the basis and all the existing publications on the hyphenation of chromatography-based techniques to MC-ICPMS for isotopic studies that were published until the end of 2010. A brief historical retrospective of the measurement of isotope ratios from transient signals by ICPMS with different sample introduction techniques is also included. The most important experimental parameters and data reduction strategies affecting the accurate and precise measurement of compound-specific isotope ratios by either HPLC or GC coupled to MC-ICPMS are discussed. All the applications are reported and critically reviewed in terms of analytical characteristics, performances, optimization, advantages and disadvantages and future applicability to the environmental, geochemical, or bioinorganic studies.

Effectiveness of interventions using apps to improve physical activity, sedentary behavior and diet: An umbrella review.

Technology has been recently found to be an effective tool to deliver public health interventions [1]. More specifically, the effects of interventions using apps to improve health have been targeted lately [2]. The goal of the present study was to conduct a systematic review of systematic reviews to summarize the scientific evidence. Three research questions were formulated to guide the research: RQ1. Are interventions using apps effective to improve PA? RQ2. Are interventions using apps effective to improve sedentary behavior? RQ3. Are interventions using apps effective to improve diet? This review of reviews was registered with PROSPERO (CRD42022345909). Systematic reviews were included following the PICOTS framework (population, intervention, comparator, outcomes, time and setting). In addition, reviews with several research objectives were included only when they comprised more than two-thirds of the studies analyzing one or more of the objectives of this review. As a result, 12 systematic reviews were selected for data extraction. Findings uncovered that apps could be effective to improve individuals' PA, sedentary behavior and diet. However, elements like the intervention components, the context/environment/setting, the length of the intervention or the population targeted should be carefully considered in future studies.

Influence of Post-Processing on the Properties of Multi-Material Parts Obtained by Material Projection AM.

The great geometric complexity that additive manufacturing allows in parts, together with the possibility of combining several materials in the same part, establishes a new design and manufacturing paradigm. Despite the interest of many leading sectors, the lack of standardization still makes it necessary to carry out characterization work to enjoy these advantages in functional parts. In many of these techniques, the process does not end with the end of the machine cycle, but different post-processing must be carried out to consider the part finished. It has been found that the type of post process applied can have a similar effect on part quality as other further studied process parameters. In this work, the material projection technique was used to manufacture multi-material parts combining resins with different mechanical properties. The influence of different post-processing on the tensile behavior of these parts was analyzed. The results show the detrimental effect of ultrasonic treatment with isopropyl alcohol in the case of the more flexible resin mixtures, being advisable to use ultrasonic with mineral oil or furnace treatment. For more rigid mixtures, the furnace is the best option, although the other post-processing techniques do not significantly deteriorate their performance.

How class cohesion and teachers' relatedness supportive/thwarting style relate to students' relatedness, motivation, and positive and negative outcomes in physical education.

The main goal of this study was to examine the links between class cohesion and teachers' relatedness teaching style with students' relatedness needs, motivation, and positive and negative outcomes in Physical Education. A total of 1294 students 10-18 years old (M = 14.40, SD = 1.99), 613 males (M = 14.48, SD = 1.95) and 681 females (M = 14.33, SD = 2.02), agreed to participate. They were enrolled in 88 classes belonging to 13 different primary and secondary schools in southwestern Spain. The study followed a correlational research design. Results of the multilevel path model showed a positive relationship between teachers' relatedness support and class cohesion and behavioral and emotional engagement through relatedness need satisfaction and autonomous and controlled motivation. Results also showed a positive relationship between teachers' relatedness thwarting and disruptive behaviors and problematic relationships through relatedness need frustration and amotivation. In conclusion, teachers' relatedness behaviors and class cohesion can significantly impact the students' relatedness and motivation, which in turn will affect their engagement and behaviors. A whole cascade of consequences begins with the way teachers teach and the cohesion generated in class. These first steps cannot be overlooked.

Development and evaluation of an electrochemical biosensor for creatinine quantification in a drop of whole human blood.

An electrochemical biosensor for creatinine determination in a drop of whole human blood was developed and applied to the determination of creatinine in real clinical samples. It is based on the modification of a dual carbon working electrode with a combination of three enzymes: creatinine amidohydrolase (CNN), creatine amidinohydrolase (CRN) and sarcosine oxidase (SOX). Electrochemical transduction is performed using horseradish peroxidase (HRP) and potassium hexacyanoferrate(II) as mediator. A drop of human blood is enough to carry out the measurements by differential chronoamperometry where one carbon electrode detects creatine and the other both creatine and creatinine. The integrated differential signal obtained in the biosensor is linear with the concentration of creatinine in blood in the range 0.5-15 mg/dL and the enzyme-modified electrodes are stable for at least 3 months at 4 °C. The biosensor was lined to a reference method based on Isotope Dilution Mass Spectrometry (IDMS) with 50 real human blood samples and the results compared with those obtained by alternative routine techniques based on Jaffé method and an enzymatic method (Cobas 8000 Roche®, Crep2 Roche®). There were no significant differences between the creatinine concentrations found by the routine techniques and the developed biosensor.

Fast and accurate procedure for the determination of Cr(VI) in solid samples by isotope dilution mass spectrometry.

We present here a new environmental measurement method for the rapid extraction and accurate quantification of Cr(VI) in solid samples. The quantitative extraction of Cr(VI) is achieved in 10 minutes by means of focused microwave assisted extraction using 50 mmol/L Ethylendiamintetraacetic acid (EDTA) at pH 10 as extractant. In addition, it enables the separation of Cr species by anion exchange chromatography using a mobile phase which is a 1:10 dilution of the extracting solution. Thus, neutralization or acidification steps which are prone to cause interconversion of Cr species are not needed. Another benefit of using EDTA is that it allows to measure Cr(III)-EDTA complex and Cr(VI) simultaneously in an alkaline extraction solution. The application of a 10 minutes focused microwave assisted extraction (5 min at 90 °C plus 5 min at 110 °C) has been shown to quantitatively extract all forms of hexavalent chromium from the standard reference materials (SRM) candidate NIST 2700 and NIST 2701. A double spike isotope dilution mass spectrometry (IDMS) procedure was employed to study chromium interconversion reactions. It was observed that the formation of a Cr(III)-EDTA complex avoided Cr(III) oxidation for these two reference materials. Thus, the use of a double spiking strategy for quantification is not required and a single spike IDMS procedure using isotopically enriched Cr(VI) provided accurate results.

Development of a routine method for the simultaneous confirmation and determination of clenbuterol in urine by minimal labeling isotope pattern deconvolution and GC-EI-MS.

A novel and fast routine method for the simultaneous determination and confirmation of clenbuterol in bovine and human urine samples by gas chromatography electron ionization mass spectrometry (GC-EI-MS) has been developed. The method employs isotope dilution mass spectrometry (IDMS) and is based on a combination of minimal labeling (a single (13)C label in the molecule) and isotope pattern deconvolution (IPD). This new methodology does not require the construction of a methodological calibration graph, and was compared with the classical IDMS procedure employed in clenbuterol analysis based on the use of a deuterated compound as internal standard (d(9)-clenbuterol) and a calibration curve. The sample preparation consists of simple extraction with dichloromethane, which was dried and derivatized with chloro(chloromethyl)dimethylsilane, generating a cyclic dimethylsilamorpholine (DMS) derivative suitable for GC(EI)MS detection and identification. This compound produces five intense ions in the electron ionization source, which allow the presence of clenbuterol to be confirmed in just one analysis, as demanded by European Union directives. The accuracy of the method was studied by performing recovery experiments at different concentration levels (from 0.3 to 5 ng g(-1)) in 5 mL bovine urine samples using two labeled compounds: an in-house-synthesized (13)C(1)-clenbuterol and a commercially available d(9)-clenbuterol. The detection limit of the method in human urine was 0.050 ng g(-1) with a sample volume of 10 mL, and is thus suitable for antidoping control purposes. Finally, the (13)C(1)-clenbuterol standard was employed for the determination of clenbuterol in two reference materials, BCR-503 and BCR-504 (lyophilized bovine urine). The concentrations obtained were in agreement with the certified values, with a reproducibility of below 1% RSD.

Effects of Family-Based Interventions Using Mobile Apps on Youth's Physical Activity: A Systematic Review.

Objective. This review synthesized the currently available literature on the effects of family-based interventions using smartphone apps on youth physical activity. Design. Systematic review. Data Sources. 1037 studies from eight databases were retrieved. Eligibility Criteria for Selecting Studies. The seven articles included in this review met the following inclusion criteria: (1) experimental studies, (2) using smartphone apps, and (3) involving families with healthy children/adolescents. Results. Studies were stratified according to whether they used smartphone apps only or the combination of sports wearables and their associated companion app. The smartphone app interventions showed significant improvements in youth's PA levels. All but one of the studies reported no significant improvement in PA levels after the intervention. However, positive PA-related outcomes were found, and the combination of sports wearables and their associated companion app showed inconclusive results due to the small number of studies. A trend of the relevance of families in improving the PA levels of youths was found. Conclusions. The findings of this review indicate that more research is needed on the effects of family-based interventions using mobile apps on youth's physical activity. Mixed results were found for variables related to the PA of the youth involved in these programs. Although strong evidence was found that youth's physical activity levels do not always improve with the implementation of these programs, promising results were found for a positive impact on different variables related to physical activity. Therefore, more experimental studies using only a mobile app to promote PA as the main outcome are needed to understand the real effect of mobile apps on youth's PA levels. Future studies need to further explore this topic by developing programs based on designs of high methodological quality.