Analytical Methods for the Determination of Perfume-related Substances.

Las fragancias son productos químicos que desprenden un olor agradable y que se utilizan en multitud de productos de consumo a los que confieren su característico aroma. Así, se pueden encontrar en productos cosméticos de todo tipo (tales como perfumes, champús, desodorantes o jabones) y también en productos para el hogar (como detergentes, ambientadores o velas). El uso de productos cosméticos se ha incrementado progresivamente con el paso del tiempo, considerándose un indicador del progreso y bienestar de un país. El reciente Reglamento Europeo sobre productos cosméticos armoniza íntegramente las normas comunitarias a fin de lograr la libre circulación de los productos y garantizar un elevado nivel de protección de la salud humana. Este Reglamento regula aspectos importantes, como el etiquetado y la composición, indicando las sustancias prohibidas, así como la cantidad máxima permitida de algunos ingredientes cosméticos y su aplicación. En particular, los perfumes requieren una cuidadosa elaboración que asegure que, además de que se cumple el Reglamento, el producto acabado posee las características olfativas que se pretendía al diseñar su formulación. Para realizar los controles de calidad correspondientes se requieren métodos analíticos adecuados. Además, el Reglamento Europeo sobre productos cosméticos prohíbe o restringe la concentración en el producto acabado de más de 50 fragancias, debido a sus posibles efectos secundarios para la salud del usuario. Además, otras sustancias, que no son fragancias propiamente dichas, pero que pueden aparecer en el producto acabado a través del proceso de fabricación o envasado también están prohibidas o restringidas debido a sus efectos secundarios. Por tanto, se requieren métodos analíticos capaces de detectar la posible presencia de trazas de ingredientes prohibidos y restringidos, en las materias primas y en los productos acabados. Por otra parte, todas estas sustancias relacionadas con las fragancias pueden llegar indirectamente al medio ambiente si su eliminación en las plantas de tratamiento de aguas no es suficientemente efectiva y, también adicionalmente, debido a su presencia en productos cosméticos, pueden alcanzar directamente el medio ambiente debido a actividades como el baño en mares o ríos. Por lo tanto, es también necesario desarrollar métodos analíticos para estimar su potencial de bioacumulación en estos ecosistemas. En la Sección I se describen algunos conceptos fundamentales sobre los perfumes y su legislación, los antecedentes del tema y las metodologías analíticas utilizadas en la presente Tesis Doctoral. El objetivo de la presente Tesis Doctoral es desarrollar métodos analíticos para la detección y determinación de fragancias y sustancias relacionadas con los perfumes que permitan un adecuado control de calidad de los perfumes y una evaluación del impacto medioambiental de las fragancias. En base a este objetivo, se han desarrollado diversos métodos analíticos: un método para la determinación de ftalatos en perfumes. Estos analitos pueden presentarse en el producto de forma intencionada (utilizados como fijadores, disolventes de algunas fragancias, desnaturalizantes de los alcoholes usados en su fabricación…) o de forma no intencionada (debido a su migración desde los envases de plástico al producto). Se han determinado los ftalatos prohibidos en el Reglamento Europeo utilizando cromatografía de gases acoplada a espectrometría de masas (GC-MS) como técnica analítica. un método para la determinación, en perfumes, de dos sustancias procedentes de extractos vegetales naturales utilizados como perfumantes, denominados atranol y chloroatranol, de conocido potencial alérgeno. Estas sustancias están en vía de inclusión en el listado de sustancias prohibidas del Reglamento Europeo. El método está basado en GC-MS como técnica analítica. Como etapa de tratamiento de la muestra se utiliza la extracción liquido-líquido (LLE) seguida de extracción liquido-líquido dispersiva (DLLME) y derivatización simultanea de los compuestos. un método que permite estudiar y seleccionar el tiempo de maceración implicado en la elaboración de los perfumes. El método está basado en GC-FID (ionización de llama) como técnica analítica. Los datos se tratan posteriormente mediante análisis discriminante lineal (LDA). un método para la determinación de nitro almizcles en aguas medioambientales y en aguas provenientes de plantas de tratamiento basado en DLLME como etapa de tratamiento de la muestra seguido de CG-MS como técnica analítica. Estos compuestos han sido ampliamente utilizados como fragancias debido a su agradable olor. Sin embargo, debido a sus efectos nocivos el uso de musk ambrette, musk tibetene y musk moskene está prohibido en productos cosméticos y el de musk xylene y musk ketone está restringido a unas concentraciones máximas por el Reglamento Europeo. Además, estos compuestos están considerados contaminantes persistentes con una fuerte tendencia a la bioacumulación. un método para la determinación de nitro almizcles en aguas medioambientales y en aguas provenientes de plantas de tratamiento basado en extracción en fase sólida (SPE) utilizando sorbentes molecularmente impresos como tratamiento de la muestra seguido de CG-MS como técnica analítica. La metodología de trabajo utilizada en la realización de los trabajos que forman parte de esta Tesis Doctoral ha sido: 1. Desarrollo y optimización de la etapa de detección/determinación empleando patrones de las sustancias a estudiar. Debido a la volatilidad de los analitos a estudiar se ha empleado la cromatografía de gases. En general, se ha utilizado la espectrometría de masas como detector, con el que se obtienen bajos límites de detección, aptos para el análisis de trazas. 2. Desarrollo y optimización de estrategias de limpieza y concentración de los analitos. Para llevar a cabo la determinación de compuestos a nivel de trazas es de vital importancia incluir un paso previo al análisis, que implique una concentración de los analitos. Además esta etapa también debería eliminar las sustancias interferentes que pueda contener la matriz de la muestra. En este sentido, se han desarrollado nuevos métodos que utilizan como etapa de tratamiento de la muestra la DLLME o la SPE utilizando sorbentes molecularmente impresos. 3. Validación de los métodos propuestos. Se han evaluado los métodos desarrollados en base a sus características analíticas, tales como límites de detección y cuantificación, exactitud, precisión o factor de enriquecimiento. Los métodos propuestos se han aplicado al análisis de muestras reales y se ha evaluado la influencia de la matriz mediante estudios de recuperación. 4. Elaboración de conclusiones y difusión de los resultados Los resultados y conclusiones se han difundido mediante los medios científicos habituales (presentación en congresos, redacción de artículos, etc.) Los trabajos realizados en la presente Tesis Doctoral han dado lugar a 6 publicaciones que se incluyen en el Anexo presentado. Las conclusiones detalladas de cada uno de los trabajos realizados, se muestran en los resúmenes elaborados en la Sección II. Finalmente, se presentan las conclusiones generales de la Tesis Doctoral presentada.Fragrances are chemical compounds with a pleasant odour. They are used in many consumer products, providing them with their characteristic aroma. In this way, they can be found in all kinds of cosmetic products (such as perfumes, shampoos, deodorants or soaps) and household products (such as detergents, air fresheners or candles). The use of cosmetic products has progressively increased over time and is now considered an indicator of the progress and prosperity of a country. The recent European Union (EU) Cosmetic Regulation harmonises the guidelines in order to achieve free movement of products and ensure a high level of protection of human health. This Regulation controls important aspects such as labelling and composition, indicating the prohibited substances and the maximum permitted quantity of certain cosmetic ingredients. Concerning perfumes, these products require careful preparation to ensure that, in addition to the compliance of the Regulation, the finished product has the originally designed olfactory characteristics. Suitable analytical methods, that enable the appropriate quality controls, are required. Furthermore, the EU Cosmetic Regulation prohibits or restricts the concentration of over 50 fragrances, due to possible side effects. Additionally, other substances, which are not fragrances themselves, but could appear in the finished product due to the manufacturing process or the product packaging, are also prohibited or restricted due to their side effects. Therefore, new analytical methods that enable the detection of prohibited or restricted ingredients in the raw materials and the finished products are required. Moreover, all these fragrance-related substances could indirectly reach the environment if wastewater treatment plants are not sufficiently effective. In addition, because of their presence in cosmetics, they can also reach the environment due to activities such as bathing in seas or rivers. Therefore, it is also necessary to develop new analytical methods to estimate the potential for bioaccumulation in these ecosystems. Some fundamental concepts about perfumes and legislation, the background of the topic, and the methodologies used in this PhD thesis are described in Section I. The aim of this PhD thesis is the development of analytical methods for the detection and determination of fragrance compounds and perfume-related compounds that enable the adequate quality control of perfumes and the environmental impact assessment of fragrance substances. Based on this aim, various analytical methods have been developed: a method for the determination of phthalates in perfumes. These analytes may be present in the product intentionally (used as fixatives, solvents of some fragrances, denaturing of the alcohols used in manufacturing...) or unintentionally (due to migration from plastic packaging to the product). The phthalates banned in the EU Cosmetic Regulation are determined using gas chromatography coupled to mass spectrometry (GC-MS) as analytical technique. a method to determine atranol and chloroatranol in perfumes. These two substances are found in natural plant extracts usually used as fragrances. However, they have an important allergenic potential and are in the process of inclusion in the list of prohibited substances of the EU Cosmetic Regulation. The method is based on GC-MS as analytical technique. As sample treatment liquid-liquid extraction (LLE) followed by dispersive liquid-liquid microextraction (DLLME) and simultaneous derivatization of the compounds is performed. a method to study and select the maceration time involved in the preparation of perfumes. The method is based on GC-FID (flame ionization detector) as analytical technique. The data are further processed by linear discriminant analysis (LDA). a method for the determination of nitro musks in environmental water and wastewater based on DLLME as sample treatment step followed by GC-MS as analytical technique. These compounds have been widely used as fragrances due to its pleasant odour. However, due to its harmful effects the use of musk ambrette, musk tibetene and musk moskene is prohibited in cosmetics and the use of musk xylene and musk ketone is restricted to a maximum concentration by the EU Cosmetic Regulation. Furthermore, these compounds are considered persistent pollutants with a strong tendency to bioaccumulate. a method for the determination of nitro musks in environmental water and wastewater based on solid phase extraction (SPE) using molecularly imprinted sorbents as sample treatment followed by GC-MS as analytical technique.   The work methodology followed to carry out the work of this doctoral thesis is: 1. Development and optimization of the detection/determination step using standards of the substances studied. Due to the volatility of the analytes gas chromatography has been used. In general, a mass spectrometer detector has also been used, with which low limits of detection are obtained, suitable for trace analysis. 2. Development and optimization strategies for cleaning and concentrating the analytes To carry out the determination of compounds at trace levels it is essential to include a step prior to the analysis, involving a concentration of the analytes. Besides, this stage should also remove interfering substances that may contain the sample matrix. In this sense, new methods that include DLLME or SPE using molecularly imprinted sorbents as sample treatment steps have been developed. 3. Validation of the proposed methods. The developed methods have been evaluated based on their analytical characteristics, such as limits of detection and quantification, accuracy, precision or enrichment factors. The proposed methods have been applied to the analysis of real samples and the influence of the matrix has been evaluated by recovery studies. 4. Conclusions and diffusion of results The results and conclusions have been diffused through the usual scientific means (presentation at conferences, published articles, etc.). The work performed in this PhD thesis has led to the six publications included in the Annex. The detailed conclusions of each of the works are shown in the summaries written in Section II. Finally, the general conclusions of the doctoral thesis are shown.

Isoprostanoids Levels in Cerebrospinal Fluid Do Not Reflect Alzheimer’s Disease

Previous studies showed a relationship between lipid oxidation biomarkers from plasma samples and Alzheimer’s Disease (AD), constituting a promising diagnostic tool. In this work we analyzed whether these plasma biomarkers could reflect specific brain oxidation in AD. In this work lipid peroxidation compounds were determined in plasma and cerebrospinal fluid (CSF) samples from AD and non-AD (including other neurological pathologies) participants, by means of an analytical method based on liquid chromatography coupled with mass spectrometry. Statistical analysis evaluated correlations between biological matrices. The results did not show satisfactory correlations between plasma and CSF samples for any of the studied lipid peroxidation biomarkers (isoprostanes, neuroprostanes, prostaglandines, dihomo-isoprostanes). However, some of the analytes showed correlations with specific CSF biomarkers for AD and with neuropsychological tests (Mini-Mental State Examination (MMSE), Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)). In conclusion, lipid peroxidation biomarkers in CSF samples do not reflect their levels in plasma samples, and no significant differences were observed between participant groups. However, some of the analytes could be useful as cognitive decline biomarkers

Lipid Peroxidation Assessment in Preclinical Alzheimer Disease Diagnosis

Alzheimer disease (AD) is an increasingly common neurodegenerative disease, especially in countries with aging populations. Its diagnosis is complex and is usually carried out in advanced stages of the disease. In addition, lipids and oxidative stress have been related to AD since the earliest stages. A diagnosis in the initial or preclinical stages of the disease could help in a more effective action of the treatments. Isoprostanoid biomarkers were determined in plasma samples from preclinical AD participants (n = 12) and healthy controls (n = 31) by chromatography and mass spectrometry (UPLC-MS/MS). Participants were accurately classified according to cerebrospinal fluid (CSF) biomarkers and neuropsychological examination. Isoprostanoid levels did not show differences between groups. However, some of them correlated with CSF biomarkers (t-tau, p-tau) and with cognitive decline. In addition, a panel including 10 biomarkers showed an area under curve (AUC) of 0.96 (0.903–1) and a validation AUC of 0.90 in preclinical AD prediction. Plasma isoprostanoids could be useful biomarkers in preclinical diagnosis for AD. However, these results would require a further validation with an external cohort

Validated analytical method to determine new salivary lipid peroxidation compounds as potential neurodegenerative biomarkers

International audienceLipid peroxidation is closely related to neurodegenerative diseases since brain shows high lipid composition and oxygen consumption. The determination of lipid peroxidation compounds in non-invasive biological samples would help to monitor the patients' oxidative stress status. A new analytical method based on ultrasound-assisted liquid-liquid semi-microextraction (UA-LLsME) followed by Ultra Performance Liquid Chromatography coupled to tandem Mass Spectrometry was developed to determine 18 lipid peroxidation biomarkers in saliva samples. The variables affecting the UA-LLsME efficiency were systematically studied. Under the optimum conditions, the methodology was validated and showed high-throughput, high sensitivity (limits of detection 0.02-2 nmol L −1), and satisfactory precision (coef-ficients of variation 2-11% (intra-day) and 5-12% (inter-day)). The reliability of the described method was assessed analysing spiked saliva samples, and the recoveries were between 80% and 120% for most of the analytes. Then, the method suitability was demonstrated by analysing saliva samples (n = 30) from elderly people with neurodegenerative diseases. To conclude, the new developed analytical method is a useful tool to determine salivary lipid peroxidation compounds as potential biomarkers in further clinical studies in which oxidative stress plays an important role

Plasma lipid peroxidation biomarkers for early and non-invasive Alzheimer Disease detection

International audienceIntroduction: Alzheimer Disease (AD) standard diagnosis is based on evaluations and biomarkers that are non-specific, expensive, or requires invasive sampling. Therefore, an early, and non-invasive diagnosis is required. As regards molecular mechanisms, recent research has shown that lipid peroxidation plays an important role.Methods: Well-defined participants groups were recruited. Lipid peroxidation compounds were determined in plasma using a validated analytical method. Statistical studies consisted of an elastic-net-penalized logistic regression adjustment.Results: The regression model fitted to the data included six variables (lipid peroxidation biomarkers) as potential predictors of early AD. This model achieved an apparent area under the receiver operating characteristics (AUC-ROCs) of 0.883 and a bootstrap-validated AUC-ROC of 0.817. Calibration of the model showed very low deviations from real probabilities.Conclusion: A satisfactory early diagnostic model has been obtained from plasma levels of 6 lipid peroxidation compounds, indicating the individual probability of suffering from early AD

New screening approach for Alzheimer's disease risk assessment from urine lipid peroxidation compounds

International audienceAlzheimer Disease (AD) standard biological diagnosis is based on expensive or invasive procedures. Recent research has focused on some molecular mechanisms involved since early AD stages, such as lipid peroxidation. therefore, a non-invasive screening approach based on new lipid peroxidation compounds determination would be very useful. Well-defined early AD patients and healthy participants were recruited. Lipid peroxidation compounds were determined in urine using a validated analytical method based on liquid chromatography coupled to tandem mass spectrometry. Statistical studies consisted of the evaluation of two different linear (Elastic Net) and non-linear (Random Forest) regression models to discriminate between groups of participants. The regression models fitted to the data from some lipid peroxidation biomarkers (isoprostanes, neuroprostanes, prostaglandines, dihomo-isoprostanes) in urine as potential predictors of early AD. these prediction models achieved fair validated area under the receiver operating characteristics (AUc-Rocs > 0.68) and their results corroborated each other since they are based on different analytical principles. A satisfactory early screening approach, using two complementary regression models, has been obtained from urine levels of some lipid peroxidation compounds, indicating the individual probability of suffering from early AD

Calorie restriction improves metabolic state independently of gut microbiome composition: a randomized dietary intervention trial

BACKGROUND: The gut microbiota has been suggested to play a significant role in the development of overweight and obesity. However, the effects of calorie restriction on gut microbiota of overweight and obese adults, especially over longer durations, are largely unexplored. METHODS: Here, we longitudinally analyzed the effects of intermittent calorie restriction (ICR) operationalized as the 5:2 diet versus continuous calorie restriction (CCR) on fecal microbiota of 147 overweight or obese adults in a 50-week parallel-arm randomized controlled trial, the HELENA Trial. The primary outcome of the trial was the differential effects of ICR versus CCR on gene expression in subcutaneous adipose tissue. Changes in the gut microbiome, which are the focus of this publication, were defined as exploratory endpoint of the trial. The trial comprised a 12-week intervention period, a 12-week maintenance period, and a final follow-up period of 26 weeks. RESULTS: Both diets resulted in ~5% weight loss. However, except for Lactobacillales being enriched after ICR, post-intervention microbiome composition did not significantly differ between groups. Overall weight loss was associated with significant metabolic improvements, but not with changes in the gut microbiome. Nonetheless, the abundance of the Dorea genus at baseline was moderately predictive of subsequent weight loss (AUROC of 0.74 for distinguishing the highest versus lowest weight loss quartiles). Despite the lack of consistent intervention effects on microbiome composition, significant study group-independent co-variation between gut bacterial families and metabolic biomarkers, anthropometric measures, and dietary composition was detectable. Our analysis in particular revealed associations between insulin sensitivity (HOMA-IR) and Akkermansiaceae, Christensenellaceae, and Tanerellaceae. It also suggests the possibility of a beneficial modulation of the latter two intestinal taxa by a diet high in vegetables and fiber, and low in processed meat. CONCLUSIONS: Overall, our results suggest that the gut microbiome remains stable and highly individual-specific under dietary calorie restriction. TRIAL REGISTRATION: The trial, including the present microbiome component, was prospectively registered at ClinicalTrials.govNCT02449148 on May 20, 2015. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13073-022-01030-0

Pre-diagnostic plasma bile acid levels and colon cancer risk: A prospective study

BACKGROUND: Bile acids have been proposed to promote colon carcinogenesis. However, there are limited prospective data on circulating bile acid levels and colon cancer risk in humans. METHODS: Associations between prediagnostic plasma levels of 17 primary, secondary, and tertiary bile acid metabolites (conjugated and unconjugated) and colon cancer risk were evaluated in a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Bile acid levels were quantified by tandem mass spectrometry in samples from 569 incident colon cancer cases and 569 matched controls. Multivariable logistic regression analyses were used to estimate odds ratios (ORs) for colon cancer risk across quartiles of bile acid concentrations. RESULTS: Positive associations were observed between colon cancer risk and plasma levels of seven conjugated bile acid metabolites: the primary bile acids glycocholic acid (ORquartile 4 vs quartile 1= 2.22, 95% confidence interval [CI] = 1.52 to 3.26), taurocholic acid (OR = 1.78, 95% CI = 1.23 to 2.58), glycochenodeoxycholic acid (OR = 1.68, 95% CI = 1.13 to 2.48), taurochenodeoxycholic acid (OR = 1.62, 95% CI = 1.11 to 2.36), and glycohyocholic acid (OR = 1.65, 95% CI = 1.13 to 2.40), and the secondary bile acids glycodeoxycholic acid (OR = 1.68, 95% CI = 1.12 to 2.54) and taurodeoxycholic acid (OR = 1.54, 95% CI = 1.02 to 2.31). By contrast, unconjugated bile acids and tertiary bile acids were not associated with risk. CONCLUSIONS: This prospective study showed that prediagnostic levels of certain conjugated primary and secondary bile acids were positively associated with risk of colon cancer. Our findings support experimental data to suggest that a high bile acid load is colon cancer promotive

Pre-diagnostic plasma bile acid levels and colon cancer risk : A prospective study

BACKGROUND: Bile acids have been proposed to promote colon carcinogenesis. However, there are limited prospective data on circulating bile acid levels and colon cancer risk in humans. METHODS: Associations between prediagnostic plasma levels of 17 primary, secondary, and tertiary bile acid metabolites (conjugated and unconjugated) and colon cancer risk were evaluated in a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Bile acid levels were quantified by tandem mass spectrometry in samples from 569 incident colon cancer cases and 569 matched controls. Multivariable logistic regression analyses were used to estimate odds ratios (ORs) for colon cancer risk across quartiles of bile acid concentrations. RESULTS: Positive associations were observed between colon cancer risk and plasma levels of seven conjugated bile acid metabolites: the primary bile acids glycocholic acid (ORquartile 4 vs quartile 1= 2.22, 95% confidence interval [CI] = 1.52 to 3.26), taurocholic acid (OR = 1.78, 95% CI = 1.23 to 2.58), glycochenodeoxycholic acid (OR = 1.68, 95% CI = 1.13 to 2.48), taurochenodeoxycholic acid (OR = 1.62, 95% CI = 1.11 to 2.36), and glycohyocholic acid (OR = 1.65, 95% CI = 1.13 to 2.40), and the secondary bile acids glycodeoxycholic acid (OR = 1.68, 95% CI = 1.12 to 2.54) and taurodeoxycholic acid (OR = 1.54, 95% CI = 1.02 to 2.31). By contrast, unconjugated bile acids and tertiary bile acids were not associated with risk. CONCLUSIONS: This prospective study showed that prediagnostic levels of certain conjugated primary and secondary bile acids were positively associated with risk of colon cancer. Our findings support experimental data to suggest that a high bile acid load is colon cancer promotive