Determination of selected endogenous anabolic androgenic steroids and ratios in urine by ultra high performance liquid chromatography tandem mass spectrometry and isotope pattern deconvolution

An isotope dilution mass spectrometry (IDMS) method for the determination of selected endogenous anabolic androgenic steroids (EAAS) in urine by UHPLC–MS/MS has been developed using the isotope pattern deconvolution (IPD) mathematical tool. The method has been successfully validated for testosterone, epitestosterone, androsterone and etiocholanolone, employing their respective deuterated analogs using two certified reference materials (CRM). Accuracy was evaluated as recovery of the certified values and ranged from 75% to 108%. Precision was assessed in intraday (n = 5) and interday (n = 4) experiments, with RSDs below 5% and 10% respectively. The method was also found suitable for real urine samples, with limits of detection (LOD) and quantification (LOQ) below the normal urinary levels. The developed method meets the requirements established by the World Anti-Doping Agency for the selected steroids for Athlete Biological Passport (ABP) measurements, except in the case of androsterone, which is currently under study

Climate change and impact on renewable energies in the Azores strategic visions for sustainability

The energy sector is the largest contributor to global greenhouse gas emissions, but could also be seriously affected by climate change, calling into question society’s current consumption patterns. In this communication, climate projections based on a set of numerical models of global circulation are used to simulate the climate until the end of the century and keep in mind the alternative scenarios of pollutant emissions. Apart from solar energy, the results for the Azores region show a negative impact on the production and consumption of renewable energies. In the regional context, this issue assumes special relevance, given the geographical constraints, such as territorial discontinuity and insularity. Based on these assumptions, measures and recommendations are pointed out for the sectors that most penalize greenhouse gas emissions, considering the energy sustainability in the Azores and the commitments and goals assumed under international agreements.info:eu-repo/semantics/publishedVersio

Distribution of GABAergic Neurons and VGluT1 and VGAT Immunoreactive Boutons in the Ferret (Mustela putorius) Piriform Cortex and Endopiriform Nucleus. Comparison With Visual Areas 17, 18 and 19

We studied the cellular organization of the piriform network [comprising the piriform cortex (PC) and endopiriform nucleus (EP)] of the ferret (Mustela putorius)—a highly excitable region prone to seizures—and, more specifically, the distribution and morphology of different types of gamma-aminobutyric acid (GABA)ergic neurons, and the distribution and ratio of glutamatergic and GABAergic boutons, and we compared our findings to those in primary visual area 17, and secondary areas 18 and 19. We accomplished this by using cytochrome oxidase and immunohistochemistry for mature neuronal nuclei (NeuN), GABAergic neurons [glutamic acid decarboxylase-67 (GAD67), calretinin (CR) and parvalbumin (PV)], and for excitatory (vesicular glutamate transporter 1; VGluT1) and inhibitory (vesicular GABA transporter; VGAT) boutons. In the ferret, the cellular organization of the piriform network is similar to that described in other species such as cats, rats and opossums although some differences also exist. GABAergic immunolabeling showed similarities between cortical layers I–III of the PC and visual areas, such as the relative distribution of GABAergic neurons and the density and area of VGluT1- and VGAT-immunoreactive boutons. However, multiple differences between the piriform network and visual areas (layers I–VI) were found, such as the percentage of GABAergic neurons with respect to the total number of neurons and the ratio of VGluT1- and VGAT-immunoreactive boutons. These findings are relevant to better understand the high excitability of the piriform network

2-Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy

Despite recent advances in the development of new cancer therapies, the treatment options for glioma remain limited, and the survival rate of patients has changed little over the past three decades. Here, we show that 2-hydroxyoleic acid (2OHOA) induces differentiation and autophagy of human glioma cells. Compared to the current reference drug for this condition, temozolomide (TMZ), 2OHOA combated glioma more efficiently and, unlike TMZ, tumor relapse was not observed following 2OHOA treatment. The novel mechanism of action of 2OHOA is associated with important changes in membrane-lipid composition, primarily a recovery of sphingomyelin (SM) levels, which is markedly low in glioma cells before treatment. Parallel to membrane-lipid regulation, treatment with 2OHOA induced a dramatic translocation of Ras from the membrane to the cytoplasm, which inhibited the MAP kinase pathway, reduced activity of the PI3K/Akt pathway, and downregulated Cyclin D-CDK4/6 proteins followed by hypophosphorylation of the retinoblastoma protein (RB). These regulatory effects were associated with induction of glioma cell differentiation into mature glial cells followed by autophagic cell death. Given its high efficacy, low toxicity, ease of oral administration, and good distribution to the brain, 2OHOA constitutes a new and potentially valuable therapeutic tool for glioma patients

Post-traumatic stress disorder associated with life-threatening motor vehicle collisions in the WHO World Mental Health Surveys

BACKGROUND: Motor vehicle collisions (MVCs) are a substantial contributor to the global burden of disease and lead to subsequent post-traumatic stress disorder (PTSD). However, the relevant literature originates in only a few countries, and much remains unknown about MVC-related PTSD prevalence and predictors. METHODS: Data come from the World Mental Health Survey Initiative, a coordinated series of community epidemiological surveys of mental disorders throughout the world. The subset of 13 surveys (5 in high income countries, 8 in middle or low income countries) with respondents reporting PTSD after life-threatening MVCs are considered here. Six classes of predictors were assessed: socio-demographics, characteristics of the MVC, childhood family adversities, MVCs, other traumatic experiences, and respondent history of prior mental disorders. Logistic regression was used to examine predictors of PTSD. Mental disorders were assessed with the fully-structured Composite International Diagnostic Interview using DSM-IV criteria. RESULTS: Prevalence of PTSD associated with MVCs perceived to be life-threatening was 2.5 % overall and did not vary significantly across countries. PTSD was significantly associated with low respondent education, someone dying in the MVC, the respondent or someone else being seriously injured, childhood family adversities, prior MVCs (but not other traumatic experiences), and number of prior anxiety disorders. The final model was significantly predictive of PTSD, with 32 % of all PTSD occurring among the 5 % of respondents classified by the model as having highest PTSD risk. CONCLUSION: Although PTSD is a relatively rare outcome of life-threatening MVCs, a substantial minority of PTSD cases occur among the relatively small proportion of people with highest predicted risk. This raises the question whether MVC-related PTSD could be reduced with preventive interventions targeted to high-risk survivors using models based on predictors assessed in the immediate aftermath of the MVCs

A Novel Clinical and Stress Cardiac Magnetic Resonance (C-CMR-10) Score to Predict Long-Term All-Cause Mortality in Patients with Known or Suspected Chronic Coronary Syndrome

[EN] Vasodilator stress cardiac magnetic resonance (stressCMR) has shown robust diagnostic and prognostic value in patients with known or suspected chronic coronary syndrome (CCS). However, it is unknown whether integration of stressCMR with clinical variables in a simple clinical-imaging score can straightforwardly predict all-cause mortality in this population. We included 6187 patients in a large registry that underwent stressCMR for known or suspected CCS. Several clinical and stressCMR variables were collected, such as left ventricular ejection fraction (LVEF) and ischemic burden (number of segments with stress-induced perfusion defects (PD)). During a median follow-up of 5.56 years, we registered 682 (11%) all-cause deaths. The only independent predictors of all-cause mortality in multivariable analysis were age, male sex, diabetes mellitus (DM), LVEF and ischemic burden. Based on the weight of the chi-square increase at each step of the multivariable analysis, we created a simple clinical-stressCMR (C-CMR-10) score that included these variables (age >= 65 years = 3 points, LVEF 5 segments = 1 point). This 0 to 10 points C-CMR-10 score showed good performance to predict all-cause annualized mortality rate ranging from 0.29%/year (score = 0) to >4.6%/year (score >= 7). The goodness of the model and of the C-CMR-10 score was separately confirmed in 2 internal cohorts (n> 3000 each). We conclude that a novel and simple clinical-stressCMR score, which includes clinical and stressCMR variables, can provide robust prediction of the risk of long-term all-cause mortality in a population of patients with known or suspected CCS.This work was supported by the Instituto de Salud Carlos III and co-funded by Fondo Europeo de Desarrollo Regional (FEDER) (grant numbers PI17/01836 and CIBERCV16/11/00486).Marcos-Garces, V.; Gavara-Doñate, J.; Monmeneu-Menadas, JV.; Lopez-Lereu, MP.; Pérez, N.; Rios-Navarro, C.; De Dios, E.... (2020). A Novel Clinical and Stress Cardiac Magnetic Resonance (C-CMR-10) Score to Predict Long-Term All-Cause Mortality in Patients with Known or Suspected Chronic Coronary Syndrome. Journal of Clinical Medicine. 9(6):1-13. https://doi.org/10.3390/jcm9061957S11396Hendel, R. C., Friedrich, M. G., Schulz-Menger, J., Zemmrich, C., Bengel, F., Berman, D. S., … Nagel, E. (2016). CMR First-Pass Perfusion for Suspected Inducible Myocardial Ischemia. JACC: Cardiovascular Imaging, 9(11), 1338-1348. doi:10.1016/j.jcmg.2016.09.010Chang, S.-A., & Kim, R. J. (2016). The Use of Cardiac Magnetic Resonance in Patients with Suspected Coronary Artery Disease: A Clinical Practice Perspective. Journal of Cardiovascular Ultrasound, 24(2), 96. doi:10.4250/jcu.2016.24.2.96Kiaos, A., Tziatzios, I., Hadjimiltiades, S., Karvounis, C., & Karamitsos, T. D. (2018). Diagnostic performance of stress perfusion cardiac magnetic resonance for the detection of coronary artery disease. International Journal of Cardiology, 252, 229-233. doi:10.1016/j.ijcard.2017.11.066Li, M., Zhou, T., Yang, L., Peng, Z., Ding, J., & Sun, G. (2014). Diagnostic Accuracy of Myocardial Magnetic Resonance Perfusion to Diagnose Ischemic Stenosis With Fractional Flow Reserve as Reference. JACC: Cardiovascular Imaging, 7(11), 1098-1105. doi:10.1016/j.jcmg.2014.07.011Siontis, G. C., Mavridis, D., Greenwood, J. P., Coles, B., Nikolakopoulou, A., Jüni, P., … Windecker, S. (2018). Outcomes of non-invasive diagnostic modalities for the detection of coronary artery disease: network meta-analysis of diagnostic randomised controlled trials. BMJ, k504. doi:10.1136/bmj.k504Nagel, E., Greenwood, J. P., McCann, G. P., Bettencourt, N., Shah, A. M., Hussain, S. T., … Berry, C. (2019). Magnetic Resonance Perfusion or Fractional Flow Reserve in Coronary Disease. New England Journal of Medicine, 380(25), 2418-2428. doi:10.1056/nejmoa1716734Siontis, G. C., Branca, M., Serruys, P., Silber, S., Räber, L., Pilgrim, T., … Hunziker, L. (2019). Impact of left ventricular function on clinical outcomes among patients with coronary artery disease. European Journal of Preventive Cardiology, 26(12), 1273-1284. doi:10.1177/2047487319841939Buckert, D., Kelle, S., Buss, S., Korosoglou, G., Gebker, R., Birkemeyer, R., … Bernhardt, P. (2016). Left ventricular ejection fraction and presence of myocardial necrosis assessed by cardiac magnetic resonance imaging correctly risk stratify patients with stable coronary artery disease: a multi-center all-comers trial. Clinical Research in Cardiology, 106(3), 219-229. doi:10.1007/s00392-016-1042-5Catalano, O., Moro, G., Perotti, M., Frascaroli, M., Ceresa, M., Antonaci, S., … Priori, S. G. (2012). Late gadolinium enhancement by cardiovascular magnetic resonance is complementary to left ventricle ejection fraction in predicting prognosis of patients with stable coronary artery disease. Journal of Cardiovascular Magnetic Resonance, 14(1). doi:10.1186/1532-429x-14-29Lipinski, M. J., McVey, C. M., Berger, J. S., Kramer, C. M., & Salerno, M. (2013). Prognostic Value of Stress Cardiac Magnetic Resonance Imaging in Patients With Known or Suspected Coronary Artery Disease. Journal of the American College of Cardiology, 62(9), 826-838. doi:10.1016/j.jacc.2013.03.080Gargiulo, P., Dellegrottaglie, S., Bruzzese, D., Savarese, G., Scala, O., Ruggiero, D., … Filardi, P. P. (2013). The Prognostic Value of Normal Stress Cardiac Magnetic Resonance in Patients With Known or Suspected Coronary Artery Disease. Circulation: Cardiovascular Imaging, 6(4), 574-582. doi:10.1161/circimaging.113.000035Kwong, R. Y., Ge, Y., Steel, K., Bingham, S., Abdullah, S., Fujikura, K., … Simonetti, O. P. (2019). Cardiac Magnetic Resonance Stress Perfusion Imaging for Evaluation of Patients With Chest Pain. Journal of the American College of Cardiology, 74(14), 1741-1755. doi:10.1016/j.jacc.2019.07.074Marcos-Garces, V., Gavara, J., Monmeneu, J. V., Lopez-Lereu, M. P., Bosch, M. J., Merlos, P., … Bodi, V. (2020). Vasodilator Stress CMR and All-Cause Mortality in Stable Ischemic Heart Disease. JACC: Cardiovascular Imaging, 13(8), 1674-1686. doi:10.1016/j.jcmg.2020.02.027Heitner, J. F., Kim, R. J., Kim, H. W., Klem, I., Shah, D. J., Debs, D., … Judd, R. M. (2019). Prognostic Value of Vasodilator Stress Cardiac Magnetic Resonance Imaging. JAMA Cardiology, 4(3), 256. doi:10.1001/jamacardio.2019.0035Bodi, V., Sanchis, J., Lopez-Lereu, M. P., Nunez, J., Mainar, L., Monmeneu, J. V., … Llacer, A. (2007). Prognostic Value of Dipyridamole Stress Cardiovascular Magnetic Resonance Imaging in Patients With Known or Suspected Coronary Artery Disease. Journal of the American College of Cardiology, 50(12), 1174-1179. doi:10.1016/j.jacc.2007.06.016Bodi, V., Husser, O., Sanchis, J., Núñez, J., Monmeneu, J. V., López-Lereu, M. P., … Llacer, Á. (2012). Prognostic Implications of Dipyridamole Cardiac MR Imaging: A Prospective Multicenter Registry. Radiology, 262(1), 91-100. doi:10.1148/radiol.11110134Ponikowski, P., Voors, A. A., Anker, S. D., Bueno, H., Cleland, J. G. F., Coats, A. J. S., … van der Meer, P. (2016). 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. European Heart Journal, 37(27), 2129-2200. doi:10.1093/eurheartj/ehw128Marschner, I. C., Colquhoun, D., Simes, R. J., Glasziou, P., Harris, P., Singh, B. B., … Tonkin, A. (2001). Long-term risk stratification for survivors of acute coronary syndromes. Journal of the American College of Cardiology, 38(1), 56-63. doi:10.1016/s0735-1097(01)01360-2Knuuti, J., Wijns, W., Saraste, A., Capodanno, D., Barbato, E., Funck-Brentano, C., … Cuisset, T. (2019). 2019 ESC Guidelines for the diagnosis and management of chronic coronary syndromes. European Heart Journal, 41(3), 407-477. doi:10.1093/eurheartj/ehz425Klem, I., Shah, D. J., White, R. D., Pennell, D. J., van Rossum, A. C., Regenfus, M., … Kim, R. J. (2011). Prognostic Value of Routine Cardiac Magnetic Resonance Assessment of Left Ventricular Ejection Fraction and Myocardial Damage. Circulation: Cardiovascular Imaging, 4(6), 610-619. doi:10.1161/circimaging.111.964965Grothues, F., Smith, G. C., Moon, J. C. ., Bellenger, N. G., Collins, P., Klein, H. U., & Pennell, D. J. (2002). Comparison of interstudy reproducibility of cardiovascular magnetic resonance with two-dimensional echocardiography in normal subjects and in patients with heart failure or left ventricular hypertrophy. The American Journal of Cardiology, 90(1), 29-34. doi:10.1016/s0002-9149(02)02381-0Timmis, A., Raharja, A., Archbold, R. A., & Mathur, A. (2018). Validity of inducible ischaemia as a surrogate for adverse outcomes in stable coronary artery disease. Heart, 104(21), 1733-1738. doi:10.1136/heartjnl-2018-313230Pontone, G., Andreini, D., Bertella, E., Loguercio, M., Guglielmo, M., Baggiano, A., … Masci, P. G. (2015). Prognostic value of dipyridamole stress cardiac magnetic resonance in patients with known or suspected coronary artery disease: a mid-term follow-up study. European Radiology, 26(7), 2155-2165. doi:10.1007/s00330-015-4064-xHeydari, B., Juan, Y.-H., Liu, H., Abbasi, S., Shah, R., Blankstein, R., … Kwong, R. Y. (2016). Stress Perfusion Cardiac Magnetic Resonance Imaging Effectively Risk Stratifies Diabetic Patients With Suspected Myocardial Ischemia. Circulation: Cardiovascular Imaging, 9(4). doi:10.1161/circimaging.115.004136Vincenti, G., Masci, P. G., Monney, P., Rutz, T., Hugelshofer, S., Gaxherri, M., … Schwitter, J. (2017). Stress Perfusion CMR in Patients With Known and Suspected CAD. JACC: Cardiovascular Imaging, 10(5), 526-537. doi:10.1016/j.jcmg.2017.02.006Buckert, D., Cieslik, M., Tibi, R., Radermacher, M., Rottbauer, W., & Bernhardt, P. (2017). Cardiac magnetic resonance imaging derived quantification of myocardial ischemia and scar improves risk stratification and patient management in stable coronary artery disease. Cardiology Journal, 24(3), 293-304. doi:10.5603/cj.a2017.0036Zemrak, F., & Petersen, S. E. (2011). Late Gadolinium Enhancement CMR Predicts Adverse Cardiovascular Outcomes and Mortality in Patients With Coronary Artery Disease: Systematic Review and Meta-Analysis. Progress in Cardiovascular Diseases, 54(3), 215-229. doi:10.1016/j.pcad.2011.07.003El Aidi, H., Adams, A., Moons, K. G. M., Den Ruijter, H. M., Mali, W. P. T. M., Doevendans, P. A., … Leiner, T. (2014). Cardiac Magnetic Resonance Imaging Findings and the Risk of Cardiovascular Events in Patients With Recent Myocardial Infarction or Suspected or Known Coronary Artery Disease. Journal of the American College of Cardiology, 63(11), 1031-1045. doi:10.1016/j.jacc.2013.11.048Fox, K. A. A., Metra, M., Morais, J., & Atar, D. (2019). The myth of ‘stable’ coronary artery disease. Nature Reviews Cardiology, 17(1), 9-21. doi:10.1038/s41569-019-0233-ySchiele, F., Ecarnot, F., & Chopard, R. (2017). Coronary artery disease: Risk stratification and patient selection for more aggressive secondary prevention. 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Normalization of sphingomyelin levels by 2-hydroxyoleic acid induces autophagic cell death of SF767 cancer cells

The very high mortality rate of gliomas reflects the unmet therapeutic need associated with this type of brain tumor. We have discovered that the plasma membrane fulfills a critical role in the propagation of tumorigenic signals, whereby changes in membrane lipid content can either activate or silence relevant pathways. We have designed a synthetic fatty acid, 2-hydroxyoleic acid (2OHOA), that specifically activates sphingomyelin synthase (SGMS), thereby modifying the lipid content of cancer cell membranes and restoring lipid levels to those found in normal cells. In reverting, the structure of the membrane by activating SGMS, 2OHOA inhibits the RAS-MAPK pathway, which in turn fails to activate the CCND (Cyclin D)-CDK4/CDK6 and PI3K-AKT1 pathways. The overall result in SF767 cancer cells, a line that is resistant to apoptosis, is the sequential induction of cell cycle arrest, cell differentiation and autophagy. Such effects are not observed in normal cells (MRC-5) and thus, this specific activation of programmed cell death infers greater efficacy and lower toxicity to 2OHOA than that associated with temozolomide (TMZ), the reference drug for the treatment of glioma

Risk score for early risk prediction by cardiac magnetic resonance after acute myocardial infarction

[EN] Background: Cardiac magnetic resonance (CMR) performed early after ST-segment elevation myocardial infarction (STEMI) can improve major adverse cardiac event (MACE) risk prediction. We aimed to create a simple clinical-CMR risk score for early MACE risk stratification in STEMI patients. Methods: We performed a multicenter prospective registry of reperfused STEMI patients (n = 1118) in whom early (1-week) CMR-derived left ventricular ejection fraction (LVEF), infarct size and microvascular obstruction (MVO) were quantified. MACE was defined as a combined clinical endpoint of cardiovascular (CV) death, nonfatal myocardial infarction (NF-MI) or re-admission for acute decompensated heart failure (HF). Results: During a median follow-up of 5.52 [2.63-7.44] years, 216 first MACE (58 CV deaths, 71 NF-MI and 87 HF) were registered. Mean age was 59.3 +/- 12.3 years and most patients (82.8%) were male. Based on the four variables independently associated with MACE, we computed an 8-point risk score: time to reperfusion >4.15 h (1 point), GRACE risk score > 155 (3 points), CMR-LVEF 1.5 segments (1 point). This score permitted MACE risk stratification: MACE per 100 person-years was 1.96 in the low-risk category (0-2 points), 5.44 in the intermediate-risk category (3-5 points), and 19.7 in the high-risk category (6-8 points): p 4.15 h and GRACE risk score > 155) and CMR (LVEF 1.5 segments) variables allows for simple and straightforward MACE risk stratification early after STEMI. External validation should confirm the applicability of the risk score.This work was supported by the Instituto de Salud Carlos III and cofunded by Fondo Europeo de Desarrollo Regional (FEDER) (grants PI20/00637 and CIBERCV16/11/00486), "Marató TV3" [grant number 20153030-31-32], the Catalonian Society of Cardiology 2015, La Caixa Foundation [HR17-00527], and by Sociedad Española de Cardiología (grant SEC/FECINV-CLI 21/024). D.M. acknowledges financial support from the Conselleria d'Educació, Investigació, Cultura i Esport, Generalitat Valenciana (grants AEST/ 2019/037 and AEST/2020/029). J. G. acknowledges financial support from the Agencia Estatal de Investigación (grant FJC2020-043981-I / AEI/10.13039/501100011033).Marcos-Garcés, V.; Perez, N.; Gavara-Doñate, J.; Lopez-Lereu, MP.; Monmeneu, JV.; Rios-Navarro, C.; De Dios, E.... (2022). Risk score for early risk prediction by cardiac magnetic resonance after acute myocardial infarction. International Journal of Cardiology. 349:150-154. https://doi.org/10.1016/j.ijcard.2021.11.05015015434

Oxidative stress mediates epigenetic modifications and the expression of miRNAs and genes related to apoptosis in diabetic retinopathy patients

Producción CientíficaKnowledge on the underlying mechanisms and molecular targets for managing the ocular complications of type 2 diabetes mellitus (T2DM) remains incomplete. Diabetic retinopathy (DR) is a major cause of irreversible visual disability worldwide. By using ophthalmological and molecular-genetic approaches, we gathered specific information to build a data network for deciphering the crosslink of oxidative stress (OS) and apoptosis (AP) processes, as well as to identify potential epigenetic modifications related to noncoding RNAs in the eyes of patients with T2DM. A total of 120 participants were recruited, being classified into two groups: individuals with T2MD (T2MDG, n = 67), divided into a group of individuals with (+DR, n = 49) and without (−DR, n = 18) DR, and a control group (CG, n = 53). Analyses of compiled data reflected significantly higher plasma levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and significantly lower total antioxidant capacity (TAC) in the +DR patients compared with the −DR and the CG groups. Furthermore, the plasma caspase-3 (CAS3), highly involved in apoptosis (AP), showed significantly higher values in the +DR group than in the −DR patients. The microRNAs (miR) hsa-miR 10a-5p and hsa-miR 15b-5p, as well as the genes BCL2L2 and TP53 involved in these pathways, were identified in relation to DR clinical changes. Our data suggest an interaction between OS and the above players in DR pathogenesis. Furthermore, potential miRNA-regulated target genes were identified in relation to DR. In this concern, we may raise new diagnostic and therapeutic challenges that hold the potential to significantly improve managing the diabetic eye.Instituto de Salud Carlos III, Red de Enfermedades Inflamatorias (REI), Fondo Europeo de Desarrollo Regional (FEDER), NextGenerationEU - (grant RD21/0002/0032)Comunidad Valenciana, Fundación para el Fomento de la Salud y la Investigación Biomédica (FISABIO) - (Project MACBIO/2022-2023: UGP-21-216

Evaluation of uncertainty sources in the determination of testosterone in urine by calibration-based and isotope dilution quantification using ultra high performance liquid chromatography tandem mass spectrometry

Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been used for the determination of testosterone in urine by LC-MS/MS. Uncertainty has been calculated and compared for the three methodologies through intra- and inter-laboratory reproducibility assays. IPD showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant and clearly worse at the lowest concentration assayed (2 ng/mL) reaching RSD values up to 16%. The inter-laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for the three procedures revealed that intercept and slope were the most important factors contributing to uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard measured.The authors acknowledge financial support from the Generalitat Valenciana (Research group of excellence Prometeo II 2014/023 and Collaborative Research on Environment and Food Safety ISIC/2012/016), as well as University Jaume I for project PB1-1B2013-55. Finally, the authors are grateful to the Serveis Centrals d'Instrumentació Científica (SCIC) of University Jaume I for using Acquity and TQD instruments