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ABSTRACT Objective: To develop a rapid method for analysing polyphenols, which are potentially active antioxidants against neonatal oxidative stress, from small human milk (HM) volumes. Methods: Acid and alkaline extractions were compared using two dyes: Folin-Ciocalteu and Fast Blue BB. Linearity, sensitivity, recovery percentage, polyphenol content, precision, and stability were assessed in 14 HM samples and compared using the Kruskal-Wallis H test (p<0.05). The best technique was applied to 284 HM samples to determine their polyphenolic content and its association with maternal diet by multifactorial linear regression. Results: Acidic extraction successfully recovered the gallic acid reference standard, whereas alkaline extraction overestimated it. Calibration curves for all methods were linear (R2>0.96) up to 500 mg/L. All bicarbonate-based Folin-Ciocalteu methods assayed were stable and repeatable, whereas Fast Blue BB-based variants were not. HM polyphenols (mean=94.68 mg/L) positively correlated to the dietary intake of hydroxycinnamic acids, the most consumed polyphenolic family in this population. Conclusions: A bicarbonate-based Folin-Ciocalteu micromethod allowed the accurate determination of polyphenols in HM, which might be useful for translational research settings and HM banks.
RESUMO Objetivo: Desenvolver um método rápido para analisar polifenóis, que são antioxidantes potencialmente ativos contra o estresse oxidativo neonatal, em pequenos volumes de leite humano (LH). Métodos: Foram comparadas extrações ácidas e alcalinas usando dois corantes: Folin-Ciocalteu e Fast Blue BB. Foram avaliadas variáveis como linearidade, sensibilidade, percentagem de recuperação, teor de polifenóis, precisão e estabilidade em 14 amostras de LH, comparadas usando o teste de Kruskal-Wallis H (p<0,05). A melhor técnica foi aplicada a 284 amostras de LH para determinar seu teor polifenólico e sua associação com a dieta materna por regressão linear multifatorial. Resultados: A extração ácida recuperou com sucesso o padrão de referência do ácido gálico, enquanto a extração alcalina o superestimou. As curvas de calibração para todos os métodos foram lineares (R2>0,96) até os 500 mg/L. Todos os métodos testados baseados em Folin-Ciocalteu com bicarbonato foram estáveis e repetíveis, enquanto as variantes baseadas em Fast Blue BB não. Os polifenóis do HM (média=94,68 mg/L) correlacionaram-se positivamente com a ingestão dietética de ácidos hidroxicinâmicos, a família de polifenóis mais consumida nesta população. Conclusões: Um micrométodo baseado em bicarbonato de Folin-Ciocalteu permitiu a determinação precisa de polifenóis no HM, o que pode ser útil para configurações de pesquisa translacional e bancos de HM.
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Benzodiazepines(BDZs)are used in clinics for anxiolysis,anticonvulsants,sedative hypnosis,and muscle relaxation.They have high consumptions worldwide because of their easy availability and potential addiction.They are often used for suicide or criminal practices such as abduction and drug-facilitated sexual assault.The pharmacological effects of using small doses of BDZs and their detections from complex biological matrices are challenging.Efficient pretreatment methods followed by accurate and sensitive detections are necessary.Herein,pretreatment methods for the extraction,enrichment,and preconcentration of BDZs as well as the strategies for their screening,identification,and quantitation developed in the past five years have been reviewed.Moreover,recent advances in various methods are summarized.Characteristics and advantages of each method are encompassed.Future directions of the pretreatment and detection methods for BDZs are also reviewed.
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Improved analytical methods for the metabolomic profiling of tissue samples are constantly needed.Currently,conventional sample preparation methods often involve tissue biopsy and/or homogenization,which disrupts the endogenous metabolome.In this study,solid-phase microextraction(SPME)fibers were used to monitor changes in endogenous compounds in homogenized and intact ovine lung tissue.Following SPME,a Biocrates AbsoluteIDQ assay was applied to make a downstream targeted metab-olomics analysis and confirm the advantages of in vivo SPME metabolomics.The AbsoluteIDQ kit enabled the targeted analysis of over 100 metabolites via solid-liquid extraction and SPME.Statistical analysis revealed significant differences between conventional liquid extractions from homogenized tissue and SPME results for both homogenized and intact tissue samples.In addition,principal component analysis revealed separated clustering among all the three sample groups,indicating changes in the metabolome due to tissue homogenization and the chosen sample preparation method.Furthermore,clear differences in free metabolites were observed when extractions were performed on the intact and homogenized tissue using identical SPME procedures.Specifically,a direct comparison showed that 47 statistically distinct metabolites were detected between the homogenized and intact lung tissue samples(P＜0.05)using mixed-mode SPME fibers.These changes were probably due to the disruptive homogenization of the tissue.This study's findings highlight both the importance of sample preparation in tissue-based metabolomics studies and SPME's unique ability to perform minimally invasive extractions without tissue biopsy or homogenization while providing broad metabolite coverage.
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Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a new imaging technique with label-free, rapid, and high throughput features. It has bloomed in the analysis on the spatial distribution of biomolecules such as drugs, metabolites, peptides and proteins on the tissue surface in virtue of providing high data throughput from non-targeted full analysis and high accuracy from targeted analysis. The acquisition of MSI signal response with high sensitivity, high spatial resolution, and good stability is directly depended on the appropriate sample preparation approaches, and flexible and various data processing tools will help the non-target data mining to meet the demands of visualization, spatial distribution and multiple index applications so as to reveal the scientific rules beneath the data. This review briefly summarizes the key advances in MALDI-MSI from aspects of sample preparation procedures, data processing and visualization. It also illustrates the characteristics, difficulties and probable solutions derived from these key techniques.
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Bile acids (BAs) are a group of endogenous steroid molecules that regulate lipid, glucose and energy metabolism. They play an important role in maintaining body homeostasis and physiological functions as key signaling molecules for host and gut microbial metabolism. The accurate characterization and quantification of BAs in vivo is of great importance in basic and clinical research. Over the past decades, enzymatic assay, enzyme-linked immunoassay, nuclear magnetic resonance (NMR), chromatography, and other related techniques have been developed and applied to the detection of BAs. The diverse structures of BAs, the existence of isomers and the complex matrix of biological samples pose great challenges for the detection of endogenous BAs. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is a robust analytical technique that combines the rapid separation capacities of UPLC with the powerful structural identification capabilities of MS/MS, facilitating the more rapid separation, characterization and accurate quantitative of target analytes in biological samples. UPLC-MS/MS has been widely used in the quantitative analysis of BAs in recent years for its high selectivity, high sensitivity, and high accuracy. This paper summarized the biosynthetic pathways of BAs, sample pretreatment methods, common analytical detection techniques, and highlights the current status of the application of UPLC-MS/MS technology in the analysis of endogenous BAs over the past five years, to provide a reference for the accurate detection of endogenous BAs and further research development and application.
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Sample preparation is considered as the bottleneck step in bioanalysis because each biological matrix has its own unique challenges and complexity.Competent sample preparation to extract the desired analytes and remove redundant components is a crucial step in each bioanalytical approach.The matrix effect is a key hurdle in bioanalytical sample preparation,which has gained extensive consideration.Novel sample preparation techniques have advantages over classical techniques in terms of accuracy,automation,ease of sample preparation,storage,and shipment and have become increasingly popular over the past decade.Our objective is to provide a broad outline of current developments in various bioanalytical sample preparation techniques in chromatographic and spectroscopic examinations.In addition,how these techniques have gained considerable attention over the past decade in bioanalytical research is mentioned with preferred examples.Modern trends in bioanalytical sample preparation techniques,including sorbent-based microextraction techniques,are primarily emphasized.
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For identifying and quantifying prohibited substances,solid-phase microextraction(SPME)continues to arouse interest as a sample preparation method.However,the practical implementation of this method in routine laboratory testing is currently hindered by the limited number of coatings compatible with the ubiquitous high-performance liquid chromatography(HPLC)systems.Only octadecyl(C18)and poly-dimethylsiloxane/divinylbenzene ligands are currently marketed for this purpose.To address this situ-ation,the present study evaluated 12 HPLC-compatible coatings,including several chemistries not currently used in this application.The stationary phases of SPME devices in the geometry of thin film-coated blades were prepared by applying silica particles bonded with various functional ligands(C18,octyl,phenyl-hexyl,3-cyanopropyl,benzenesulfonic acid,and selected combinations of these),as well as unbonded silica,to a metal support.Most of these chemistries have not been previously used as microextraction coatings.The 48 most commonly misused substances were selected to assess the extraction efficacy of each coating,and eight desorption solvent compositions were used to optimize the desorption conditions.All samples were analyzed using an HPLC system coupled with triple quadrupole tandem mass spectrometry.This evaluation enables selection of the best-performing coatings for quantifying prohibited substances and investigates the relationship between extraction efficacy and the physicochemical characteristics of the analytes.Ultimately,using the most suitable coatings is essential for trace-level analysis of chemically diverse prohibited substances.
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Abstract The objective of the study was to develop an easy, cheap, effective, and safe, small-scale method for sample preparation suitable for the simultaneous high-performance liquid chromatography (HPLC)-ultraviolet (UV) assay of capecitabine and its 5′-deoxy-5-fluorocytidine (5′-DFCR) metabolite in mouse blood plasma. The suitability of the proposed method of sample preparation was verified by the optimal effectiveness and efficiency achieved in the overall analytical workflow. The chromatographic separation of capecitabine and its first metabolite was performed on a Hypersil GOLD aQ column with a mobile phase consisting of 1% formic acid, methanol, and water, and run in a gradient elution mode. The absence of interfering endogenous components at the retention times of each analyte was confirmed by the chromatographic analysis of blank matrices and matrices spiked with the corresponding standards. The absence of any tactile matrix effect was also recorded. For the first time, the effect of the vacutainer's anticoagulant on the extraction efficiency of both analytes was evaluated. The method was found to be accurate, precise, and specific. The estimated mean "extraction" efficiencies were ≥90% for each analyte. The lower limit of quantitation for both capecitabine and 5′-DFCR was 0.05 μg/mL.
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Mass spectrometry imaging （MSI） is a new imaging technology that can simultaneously detect and record the spatial distribution information of multiple molecules on the sample surface without labeling. The main principle of MSI is to combine mass spectrometry with imaging technology and irradiate the sample slice with ion beam or laser to ionize the molecules on its surface, obtain the mass spectrometry signal through the detector, convert the obtained data into pixel points by the imaging software, and then construct the spatial distribution image of the target compound on the tissue surface. The sample preparation for MSI include： sample collection and storage, tissue section, tissue pretreatment, selection and application of matrix. At present, this technology has been widely used in the fields of biomedicine, new drug development and proteomics, and its application in the field of forensic toxicology has also gradually attracted attention. This article reviews the principles and sample preparation process of MSI, describes the application of MSI in abused substances and metabolites of various material matrices, herbal mixtures, latent fingerprints, hair and animal and plant tissues, and discusses the prospects of the application of this technology in forensic toxicology, in order to provide ideas and references for the application of MSI technology in forensic toxicology.
Subject(s)
Animals , Humans , Diagnostic Imaging , Forensic Toxicology , Mass Spectrometry , Plants , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Single-cell proteome analysis can perform in-depth research on cell heterogeneity and further promote the development of precision medicine and tumor research. In recent years, mass spectrometry-based proteomics has made substantial progress, but proteomics analysis at the single-cell level still faces sensitivity challenges. At present, liquid chromatography tandem mass spectrometry has become the main analysis method of proteomics. Because of its high sensitivity, high throughput, and high stability, it has been widely used in the field of single cells. In recent years, the representative single-cell proteomics methods can be divided into three types: single-tube technology, microfluidic platform, and integrated processing platform according to different sample processing methods. Different kinds of data collection and analysis methods have their own advantages and disadvantages. If single-cell proteomics, still a laboratory study, can be applied in diagnostic practice as soon as possible, it will definitely promote the progress of precision medicine and oncology research.
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A sample preparation method was developed to simulate the process of intracellular metabolites metabonomics analysis of Escherichia coli. The Escherichia coli cell was firstly quenched with cold sodium chloride solution ( 0. 85 %，precooled at -80℃ for 15 min).The quenched bacterial cell was treated by using the technique of vacuum freeze-drying and liquid nitrogen freezing combined with ultrasonic processing to increase cell membrane penetrability. Finally，a cold aqueous solution of methanol (MeOH:H2O, 1:1，V/ V, 4 ℃) was used as extraction solvent to extract metabolites. In the present research，flow cytometry and OD value recovery were performed to evaluate the degree of cell damage caused by quenching at single cell level and at integral level respectively. The tested results indicated that the degree of damage to cells caused by cold sodium chloride solution was less than 5%. The peak quantity and the total ion intensity detected by LC-TOF in low collision energy were used to evaluate extraction effects. Three different cell membrane penetrability modes and 4 kinds of extraction solvents were investigated and compared.The results showed that the technique of liquid nitrogen freezing combined with ultrasonic processing for cell membrane penetrability and a cold aqueous solution of methanol (MeOH/H2O，1:1，V/V, 4℃) for extraction of metabolites had the best extraction effect(peak quantity was greater than or equal to 105，and total ion intensity was in the range of 106-107).Therefore，in this work，the freeze drying，grinding with liquid nitrogen and ultrasonic extraction were combined to extract metabolites. In this way，it effectively promoted cell lysis and improved the efficiency of extraction. The result of synthetic analysis showed that the method proposed here could meet the requirements of the metabonomics analysis of Escherachaa coli.
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Objective To prepare the trueness verification materials of C-reactive protein (CRP) and evaluate its homogeneity, stability and commutability. Methods The high and low CRP concentrations trueness verification materials were from patient leftover sera which were pooled, mixed thoroughly, filtered and aliquoted. The homogeneity, stability and commutability of these materials were evaluated according to CNAS(China National Accreditation Service for Conformity Assessment, CNAS)-GL29:2010 "Reference materials-General and statistical principles for certification (ISO Guide35:2006)"and the Clinical and Laboratory Standards Institute (CLSI) EP30A. The trueness verification materials were used to evaluate the commutability in 10 clinical CRP detection systems, using forty-five patients' leftover sera with different CRP concentration evaluated by Deming regression in EP30A of CLSI. Meanwhile, the commutability of dilution series of ERM DA-474/IFCC were evaluated using the same method. Results A total of two CRP concentration level trueness verification materials were prepared, with high and low concentration levels of 754 and 743 vials, 1 ml each, respectively. The preparation showed good homogeneity (F<F0.05(14,30);On the condition of room temperature, 2-8 ℃ and -80 ℃, these materials were stable for 7 days and 44 months respectively, the slope of the linear equation of | b1 | less than t0.95,n-2 · s(b1), there was no statistically significant difference between the slope and zero, the stability is satisfied. The materials and the dilution series of ERM-DA 474/IFCC also showed good commutability among patient sera in 10 systems. Conclusions The trueness verification materials of C-reactive protein (CRP) showed good homogeneity, stability and commutability. The dilution series ERM DA-474/IFCC also have good commutability. These provided experimental support for the value transfer and application of the trueness verification materials .
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Due to the diversity and complexity, the change of chemical components in medicinal plant according to the time, cultivated varieties or ecological condition is difficult to recognize using traditional phytochemistry method. In order to analyze the pharmacodynamics material basis in Uighur medicinal plant Artemisia rupestris L. in an effective and comprehensive way, a plant metabolomics approach was established based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study firstly focused on the effect of extraction solvents,redissolve solvents and ultrasonic time on the untargeted metabolomics, then the optimal preparation condition was selected according to metabolites coverage. After methodology validation, the approach was applied to acquire metabolic information in root, stem, branchlet, leaf and flower of Artemisia rupestris L. The results showed that the metabolome in flower was obviously different with the other organs. Coupling with multivariate statistical analysis, a batch of differential metabolites were picked out, in which 61 flavonoids, 97 rupestonic acid derivatives, 7 chlorogenic acids and 15 other compounds were primarily recognized according to the characteristic fragmentation rules of specific structure type and database retrieval. Additionally,the distribution characteristics of the above 180 differential metabolites was illustrated by cluster heat map. In conclusion,this study provided important information about the rational utilization of effective parts from Artemisia rupestris L.,and offered a novel strategy for quality control,variety improvement and reasonable development of medicinal plants.
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Objetivo: O presente estudo teve como objetivo determinar e comparar o limiar de positividade e a sensibilidade dos métodos de centrífugo-flutuação em sulfato de zinco (Faust et al.) e sedimentação espontânea (Lutz) para o diagnóstico de cistos de Giardia duodenalis. Métodos: Para obtenção de amostras fecais com quantidades conhecidas de cistos de G. duodenalis, amostras positivas para o parasito foram purificadas e quantificadas, e posteriormente alíquotas com diferentes quantidades foram adicionadas a amostras fecais negativas para parasitos. Após a contaminação de oito amostras negativas com quantidades variando entre 1.000 e 200.000 cistos por grama de fezes (c/g/f), as mesmas foram submetidas aos métodos de Faust et al. e Lutz, onde o primeiro se mostrou mais sensível para a detecção de cistos de G. duodenalis. Resultados: O limiar de positividade do método de Faust et al. foi de 11.000 c/g/f, e do método de Lutz foi de 100.000 c/g/f, portanto, cargas parasitárias inferiores a esses limiares levariam a resultados falso-negativos. Conclusão: O método de Lutz não é adequado para o diagnóstico de giardíase, portanto deve ser sempre utilizado em conjunto com o método de Faust et al.
Objective: The present study aimed to determine and compare the positivity threshold and sensitivity of the methods of zinc sulfate centrifugal flotation (Faust et al.) and spontaneous sedimentation (Lutz) for the diagnosis of Giardia duodenalis. Methods: To obtain fecal samples containing known amounts of G. duodenalis cysts, the samples with the parasite were purified and quantified, then aliquots with different amounts were added to fecal samples negative for parasites. After the contamination of eight negative samples with amounts ranging between 1.000 and 200.000 cysts per gram of feces, they were subjected to methods of Faust et al. and Lutz, where the first was more sensitive for the detection of G. duodenalis cysts. Results: The positivity threshold of the method of Faust et al. was 11.000 c/g/f, and the method of Lutz was 100.000 c/g/f, so parasitic loads below those thresholds would lead to false-negative results. Conclusion: The method of Lutz is not suitable for the diagnosis of giardiasis, therefore must always be associated with the method Faust et al.
Subject(s)
Humans , Parasitic Diseases/diagnosis , Sensitivity and Specificity , Giardia lamblia , Analytic Sample Preparation Methods , GiardiasisABSTRACT
O aprimoramento das técnicas analíticas viabilizou a metabolômica, uma área da ciência que busca compreender, de forma comparativa, os metabólitos envolvidos nas vias bioquímicas. A metabolômica está inserida no contexto das "ômicas", que teve início na "Era Genômica", a qual permitiu a identificação de diversos genes. Em seguida, o interesse dos pesquisadores centrou no estudo dos metabólitos (metabolômica) mostrando ser uma ferramenta valiosa na pesquisa do transplante renal, que exige um tratamento medicamentoso por meio de imunossupressores. A combinação destes imunossupressores pode minimizar a rejeição do órgão transplantado, reforçando a necessidade de um estudo metabolômico, a fim de avaliar e comparar as mudanças ocorridas após o transplante em nível molecular, melhorando o conhecimento sobre a influência destes regimes e dando subsídios sobre prognósticos possíveis na área de transplante renal. Nesta tese foram avaliadas 2 terapias: Everolimo/ Prednisona/Tacrolimo (grupo 1) e Micofenolato mofetil/Prednisona/Tacrolimo (grupo 2) a partir de uma abordagem untargeted. No presente trabalho foram coletadas amostras de urina de pacientes ao longo de 6 meses. Foi necessário determinar a melhor condição para análise das amostras de urina dos pacientes. Desta forma, foram realizados estudos sobre alguns parâmetros que impactam no preparo de amostra abordando a influência da urease, tipos e proporção de solventes para precipitação de proteína, seleção do melhor agente derivatizante e tratamento de dados. A avaliação da medida de qualidade dos tratamentos com urease foi feita a partir do desvio padrão relativo (RSD) dos valores de intensidade de pico. A concentração de 10 mg mL-1 apresentou o melhor resultado. O estudo mostrou também que o teor de ureia na urina pode influenciar na identificação dos compostos. O número de compostos identificados foi menor quando a urina não foi tratada com urease, com aproximadamente 10 compostos a menos em relação à amostra tratada com a enzima, na mesma concentração de ureia adicionada. Dos solventes orgânicos testados para precipitação de proteínas nas amostras de urina, o isopropanol mostrou ser o solvente mais adequado na proporção 1:6 urina:solvente (v/v), utilizando-se 100 µL de urina. Foram testados dois protocolos de derivatização para análises por GC-MS: metoximação e sililação utilizando BSTFA e cloroformiato de metila. A comparação mostrou que o procedimento por BSTFA, com 40 metabólitos identificados, foi superior ao cloroformiato de metila, com 13 compostos identificados. No tratamento de dados com o software XCMS, os seguintes parâmetros foram avaliados: largura a meia altura do pico (fwhm), largura da banda (bw) e threshold (sntresh). Para avaliar a melhor combinação destes parâmetros, foi feita uma variação univariada destes valores. A qualidade do resultado de cada combinação foi monitorada pelos valores gerados de número de missing values, quantidade de picos com RSD <15% e número de valores duplicados. Os valores ótimos foram obtidos para a combinação: fwhm=4, bw=2 e threshold=5. A abordagem do estudo dos dois grupos de pacientes baseou-se inicialmente na comparação entre o dia 7 da terapia com os demais períodos (dia 14, mês 1, mês 3 e mês 6) e posteriormente avaliou-se a evolução temporal. A partir do mês 3 os valores de correlação e predição dos modelos de PLS-DA são melhores e já é eficaz na diferenciação entre os dois grupos. Foram observadas perturbações no metabolismo de carboidratos em ambos os grupos, como açúcares, glicerol e N-acetil-D-manosamina. No grupo 1, foram observados metabólitos discriminantes da classe dos poliois e das vias do ciclo do ácido cítrico e degradação de xenobióticos, enquanto que, no grupo 2, foi observada alteração do hidroxibutirato, um corpo cetônico. Neste grupo, foi observado também um aumento do ácido hipúrico, ácido acetamido butírico, ácido benzoico, entre outros. Nesta tese foi possível demonstrar que a metabolômica é uma ferramenta importante para comparar metabólitos discriminantes entre dois regimes imunossupressores, sendo um estudo piloto que visa fornecer subsídios para avaliação da influência destas terapias no prognóstico de transplante renal
The improvement of analytical techniques enabled the emergence of metabolomics, which aims to compare the metabolites involved in biochemical pathways, in certain biological conditions. Metabolomics is inserted in the "omics" context, which began in the "Genomic Age", and allowed the identification of several genes. After that, the researchers focused on the study of metabolites. Among several applications, metabolomics can be a valuable tool in renal transplant research, which requires a drug treatment through immunosuppressants. The combination of these immunosuppressants can minimize toxicity and rejection of the transplanted organ, reinforcing the need for a metabolomic study, in order to evaluate and compare changes after transplantation at the molecular level, improving knowledge about the influence of these regimens and giving subsidies on prognosis in the area of renal transplantation. In this thesis two immunosuppressive therapies were evaluated by an untargeted approach: Everolimus/Prednisone/Tacrolimus (group 1) and Mycophenolate mofetil/Prednisone/Tacrolimus (group 2). In this study, urine samples were collected from patients over 6 months. It was necessary to determine the best condition for analysis of patients' urine samples. Thus, studies were carried out on some parameters that impact on sample preparation, evaluating the influence of urease, types and proportion of solvents for protein precipitation, selection of the best derivatizing agent, and data treatment. The evaluation of the quality measure of the urease treatments was made from the relative standard deviation (RSD) of the peak intensity values. The concentration of 10 mg mL-1 presented the best result. The study also showed that urine urea content may influence the identification of the compounds. The number of identified compounds was lower when urine was not treated with urease, with approximately 10 compounds less than the enzyme-treated sample, at the same concentration of urea added. In the evaluation of the organic solvents tested for protein precipitation in the urine samples, isopropanol was the most suitable solvent in the ratio 1: 6 urine:solvent (v/v), using 100 µL of urine. Two derivatization protocols were tested for GC-MS analysis: metoximation and silylation with BSTFA and methyl chloroformate. The comparison between the two derivatization protocols showed that the BSTFA procedure, with 40 identified metabolites, was superior to methyl chloroformate with 13 compounds identified. In data processing with the XCMS software, the following parameters were evaluated: full width at half maximum of the peak (fwhm), bandwidth (bw) and threshold (sntresh). To evaluate the best combination of these parameters, a univariate variation of these values was made. The quality of the result of each combination was monitored by the number of missing values, number of peaks with RSD <15%, and number of duplicate values. The optimal values were obtained for the combination: fwhm=4, bw=2 and threshold =5. The study of the two groups of patients was initially based on the comparison between day 7 of the therapy with the other periods (day 14, month 1, month 3 and month 6) and later the temporal evolution was evaluated. From month 3 the values of correlation and prediction of the PLS-DA models are better and already effective in the differentiation between the two groups. Disorders in carbohydrate metabolism were observed in both groups with sugars and glycerol and N-acetyl-D-mannosamine as discriminant metabolites. In group 1, discriminant metabolites of the class of polyols and citric acid cycle pathways and degradation of xenobiotics were observed, and in group 2 alteration of hydroxybutyrate, a ketone body, was observed. In this group an increase of hippuric acid, acetamido butyric acid, benzoic acid, among others, was also observed. In this thesis it was possible to demonstrate that metabolomics is an important tool to compare discriminant metabolites between two immunosuppressive regimens, being a pilot study that aims to provide future subsidies to evaluate the influence of these therapies on the renal transplant prognosis
Subject(s)
Humans , Male , Female , Therapeutics/instrumentation , Kidney Transplantation , Metabolomics/classification , Preservation of Water Samples/analysis , Multivariate Analysis , Immunosuppressive AgentsABSTRACT
O aprimoramento das técnicas analíticas viabilizou a metabolômica, uma área da ciência que busca compreender, de forma comparativa, os metabólitos envolvidos nas vias bioquímicas. A metabolômica está inserida no contexto das "ômicas", que teve início na "Era Genômica", a qual permitiu a identificação de diversos genes. Em seguida, o interesse dos pesquisadores centrou no estudo dos metabólitos (metabolômica) mostrando ser uma ferramenta valiosa na pesquisa do transplante renal, que exige um tratamento medicamentoso por meio de imunossupressores. A combinação destes imunossupressores pode minimizar a rejeição do órgão transplantado, reforçando a necessidade de um estudo metabolômico, a fim de avaliar e comparar as mudanças ocorridas após o transplante em nível molecular, melhorando o conhecimento sobre a influência destes regimes e dando subsídios sobre prognósticos possíveis na área de transplante renal. Nesta tese foram avaliadas 2 terapias: Everolimo/ Prednisona/Tacrolimo (grupo 1) e Micofenolato mofetil/Prednisona/Tacrolimo (grupo 2) a partir de uma abordagem untargeted. No presente trabalho foram coletadas amostras de urina de pacientes ao longo de 6 meses. Foi necessário determinar a melhor condição para análise das amostras de urina dos pacientes. Desta forma, foram realizados estudos sobre alguns parâmetros que impactam no preparo de amostra abordando a influência da urease, tipos e proporção de solventes para precipitação de proteína, seleção do melhor agente derivatizante e tratamento de dados. A avaliação da medida de qualidade dos tratamentos com urease foi feita a partir do desvio padrão relativo (RSD) dos valores de intensidade de pico. A concentração de 10 mg mL-1 apresentou o melhor resultado. O estudo mostrou também que o teor de ureia na urina pode influenciar na identificação dos compostos. O número de compostos identificados foi menor quando a urina não foi tratada com urease, com aproximadamente 10 compostos a menos em relação à amostra tratada com a enzima, na mesma concentração de ureia adicionada. Dos solventes orgânicos testados para precipitação de proteínas nas amostras de urina, o isopropanol mostrou ser o solvente mais adequado na proporção 1:6 urina:solvente (v/v), utilizando-se 100 µL de urina. Foram testados dois protocolos de derivatização para análises por GC-MS: metoximação e sililação utilizando BSTFA e cloroformiato de metila. A comparação mostrou que o procedimento por BSTFA, com 40 metabólitos identificados, foi superior ao cloroformiato de metila, com 13 compostos identificados. No tratamento de dados com o software XCMS, os seguintes parâmetros foram avaliados: largura a meia altura do pico (fwhm), largura da banda (bw) e threshold (sntresh). Para avaliar a melhor combinação destes parâmetros, foi feita uma variação univariada destes valores. A qualidade do resultado de cada combinação foi monitorada pelos valores gerados de número de missing values, quantidade de picos com RSD <15% e número de valores duplicados. Os valores ótimos foram obtidos para a combinação: fwhm=4, bw=2 e threshold=5. A abordagem do estudo dos dois grupos de pacientes baseou-se inicialmente na comparação entre o dia 7 da terapia com os demais períodos (dia 14, mês 1, mês 3 e mês 6) e posteriormente avaliou-se a evolução temporal. A partir do mês 3 os valores de correlação e predição dos modelos de PLS-DA são melhores e já é eficaz na diferenciação entre os dois grupos. Foram observadas perturbações no metabolismo de carboidratos em ambos os grupos, como açúcares, glicerol e N-acetil-D-manosamina. No grupo 1, foram observados metabólitos discriminantes da classe dos poliois e das vias do ciclo do ácido cítrico e degradação de xenobióticos, enquanto que, no grupo 2, foi observada alteração do hidroxibutirato, um corpo cetônico. Neste grupo, foi observado também um aumento do ácido hipúrico, ácido acetamido butírico, ácido benzoico, entre outros. Nesta tese foi possível demonstrar que a metabolômica é uma ferramenta importante para comparar metabólitos discriminantes entre dois regimes imunossupressores, sendo um estudo piloto que visa fornecer subsídios para avaliação da influência destas terapias no prognóstico de transplante renal.
The improvement of analytical techniques enabled the emergence of metabolomics, which aims to compare the metabolites involved in biochemical pathways, in certain biological conditions. Metabolomics is inserted in the "omics" context, which began in the "Genomic Age", and allowed the identification of several genes. After that, the researchers focused on the study of metabolites. Among several applications, metabolomics can be a valuable tool in renal transplant research, which requires a drug treatment through immunosuppressants. The combination of these immunosuppressants can minimize toxicity and rejection of the transplanted organ, reinforcing the need for a metabolomic study, in order to evaluate and compare changes after transplantation at the molecular level, improving knowledge about the influence of these regimens and giving subsidies on prognosis in the area of renal transplantation. In this thesis two immunosuppressive therapies were evaluated by an untargeted approach: Everolimus/Prednisone/Tacrolimus (group 1) and Mycophenolate mofetil/Prednisone/Tacrolimus (group 2). In this study, urine samples were collected from patients over 6 months. It was necessary to determine the best condition for analysis of patients' urine samples. Thus, studies were carried out on some parameters that impact on sample preparation, evaluating the influence of urease, types and proportion of solvents for protein precipitation, selection of the best derivatizing agent, and data treatment. The evaluation of the quality measure of the urease treatments was made from the relative standard deviation (RSD) of the peak intensity values. The concentration of 10 mg mL-1 presented the best result. The study also showed that urine urea content may influence the identification of the compounds. The number of identified compounds was lower when urine was not treated with urease, with approximately 10 compounds less than the enzyme-treated sample, at the same concentration of urea added. In the evaluation of the organic solvents tested for protein precipitation in the urine samples, isopropanol was the most suitable solvent in the ratio 1: 6 urine:solvent (v/v), using 100 µL of urine. Two derivatization protocols were tested for GC-MS analysis: metoximation and silylation with BSTFA and methyl chloroformate. The comparison between the two derivatization protocols showed that the BSTFA procedure, with 40 identified metabolites, was superior to methyl chloroformate with 13 compounds identified. In data processing with the XCMS software, the following parameters were evaluated: full width at half maximum of the peak (fwhm), bandwidth (bw) and threshold (sntresh). To evaluate the best combination of these parameters, a univariate variation of these values was made. The quality of the result of each combination was monitored by the number of missing values, number of peaks with RSD <15%, and number of duplicate values. The optimal values were obtained for the combination: fwhm=4, bw=2 and threshold =5. The study of the two groups of patients was initially based on the comparison between day 7 of the therapy with the other periods (day 14, month 1, month 3 and month 6) and later the temporal evolution was evaluated. From month 3 the values of correlation and prediction of the PLS-DA models are better and already effective in the differentiation between the two groups. Disorders in carbohydrate metabolism were observed in both groups with sugars and glycerol and N-acetyl-D-mannosamine as discriminant metabolites. In group 1, discriminant metabolites of the class of polyols and citric acid cycle pathways and degradation of xenobiotics were observed, and in group 2 alteration of hydroxybutyrate, a ketone body, was observed. In this group an increase of hippuric acid, acetamido butyric acid, benzoic acid, among others, was also observed. In this thesis it was possible to demonstrate that metabolomics is an important tool to compare discriminant metabolites between two immunosuppressive regimens, being a pilot study that aims to provide future subsidies to evaluate the influence of these therapies on the renal transplant prognosis
Subject(s)
Humans , Male , Female , Kidney Transplantation , Metabolomics , Immunosuppressive Agents/therapeutic use , Multivariate AnalysisABSTRACT
Phosphopeptide enrichment is of great significance in phosphoproteomics related research. Pre-analysis enrichment helps concentrating phosphopeptides of low abundance, and other interferences of high abundance, such as non-phosphorylated peptides, will be eliminated at the same time. By this way, the enhanced sensitivity of mass-spectrometry analysis of phosphopeptides can be achieved, as well as better detection and identification performance. The key for phosphopeptide enrichment is enrichment materials with specific affinity towards phosphopeptides. Enrichment materials with different phosphopeptide adsorption mechanisms have been developed, while various improvements on material morphologies, enrichment operation, and enrichment specificity are also reported. In the review, we summarized recent progresses in phosphopeptide enrichment from perspectives of both enrichment materials with different enrichment mechanisms, and improvement on methodologies of phosphopeptide enrichment based on various enrichment materials.
ABSTRACT
Objective To establish UV spectrophotometry and HPLC methods for content determinations of total flavonoids and puerarin from Puerariae Lobatae Radix and Puerariae Thomsonii Radix; To compare the ultrasonic method at room temperature, conventional refluxing method and ultrasonic method at heating conditions at the aspect of content determinations. Methods The content determinations of total flavonoids was determined by UV spectrophotometry at 250 nm; the content of puerarin was determined by HPLC with octadecylsilane-bonded silica gel as the stationary phase, a mixture of methanol and water (25:75) as the mobile phase, 256 nm as the detection wavelength, 1.0 mL/min as the flow rate. Results Contents of total flavonoids in Puerariae Lobatae Radix by ultrasonic method at room temperature, conventional refluxing method and ultrasonic method were15.09%, 14.48%, and 12.71% (n=3), respectively. The contents of puerarin were 4.37%, 4.09%, and 3.80% (n=3), respectively. Contents of total flavonoids in Puerariae Thomsonii Radix were 2.09%, 2.23%, and 2.17% (n=3), respectively. The contents of puerarin were 0.50%, 0.53%, and 0.52% (n=3), respectively. Conclusion Ultrasonic method at room temperature can replace conventional refluxing method for content determinations of total flavonoids and puerarin from Puerariae Lobatae Radix, and ultrasonic method at heating conditions also can replace conventional refluxing method for content determinations of total flavonoids puerarin from Puerariae Thomsonii Radix. Puerarin contents from Puerariae Lobatae Radix and Puerariae Thomsonii Radix in South Anhui Province are all in line with the Pharmacopoeia standards.
ABSTRACT
Objective To establish UV spectrophotometry and HPLC methods for content determinations of total flavonoids and puerarin from Puerariae Lobatae Radix and Puerariae Thomsonii Radix; To compare the ultrasonic method at room temperature, conventional refluxing method and ultrasonic method at heating conditions at the aspect of content determinations. Methods The content determinations of total flavonoids was determined by UV spectrophotometry at 250 nm; the content of puerarin was determined by HPLC with octadecylsilane-bonded silica gel as the stationary phase, a mixture of methanol and water (25:75) as the mobile phase, 256 nm as the detection wavelength, 1.0 mL/min as the flow rate. Results Contents of total flavonoids in Puerariae Lobatae Radix by ultrasonic method at room temperature, conventional refluxing method and ultrasonic method were15.09%, 14.48%, and 12.71% (n=3), respectively. The contents of puerarin were 4.37%, 4.09%, and 3.80% (n=3), respectively. Contents of total flavonoids in Puerariae Thomsonii Radix were 2.09%, 2.23%, and 2.17% (n=3), respectively. The contents of puerarin were 0.50%, 0.53%, and 0.52% (n=3), respectively. Conclusion Ultrasonic method at room temperature can replace conventional refluxing method for content determinations of total flavonoids and puerarin from Puerariae Lobatae Radix, and ultrasonic method at heating conditions also can replace conventional refluxing method for content determinations of total flavonoids puerarin from Puerariae Thomsonii Radix. Puerarin contents from Puerariae Lobatae Radix and Puerariae Thomsonii Radix in South Anhui Province are all in line with the Pharmacopoeia standards.
ABSTRACT
Electromembrane extraction (EME) is an analytical microextraction technique, where charged analytes (such as drug substances) are extracted from an aqueous sample (such as a biological fluid), through a supported liquid membrane (SLM) comprising a water immiscible organic solvent, and into an aqueous acceptor solution. The driving force for the extraction is an electrical potential (dc) applied across the SLM. In this paper, EME is reviewed. First, the principle for EME is explained with focus on extraction of cationic and anionic analytes, and typical performance data are presented. Second, papers published in 2016 are reviewed and discussed with focus on (a) new SLMs, (b) new support materials for the SLM, (c) new sample additives improving extraction,(d) new technical configurations, (e) improved theoretical understanding, and (f) pharmaceutical new applications. Finally, important future research objectives and directions are defined for further development of EME, with the aim of establishing EME in the toolbox of future analytical laboratories.