RESUMO
Merging optical images of tissue sections with the spatial distributions of molecules seen by imaging mass spectrometry is a powerful approach to better understand the metabolic roles of the mapped molecules. Here, we use histologically friendly desorption electrospray ionization-mass spectrometry (DESI-MS) to map the lipid distribution in tissue sections of ovaries from cows (N = 8), sows (N = 3), and mice (N = 12). Morphologically friendly DESI-MS imaging allows the same sections to be examined for morphological information. Independent of the species, ovarian follicles, corpora lutea, and stroma could be differentiated by principal component analysis, showing that lipid profiles are well conserved among species. As examples of specific findings, arachidonic acid and the phosphatidylinositol PI(38:4), were both found concentrated in the follicles and corpora lutea, structures that promoted ovulation and implantation, respectively. Adrenic acid was spatially located in the corpora lutea, suggesting the importance of this fatty acid in the ovary luteal phase. In summary, lipid information captured by DESI-MS imaging could be related to ovarian structures and data were all conserved among cows, sows, and mice. Further application of DESI-MS imaging to either physiological or pathophysiological models of reproductive conditions will likely expand knowledge of the roles of specific lipids and pathways in ovarian activity and mammalian fertility. Graphical abstract Desorption electrospray ionization-mass spectrometry (DESI-MS) is performed directly from frozen ovarian tissue sections placed onto glass slides. Because the desorption and ionization process of small molecules is so gentle, the tissue architecture is preserved. The sample can then be stained and tissue morphology information can be overlaid with the chemical information obtained by DESI-MS.
Assuntos
Metabolismo dos Lipídeos , Ovário/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Bovinos , Feminino , Camundongos , SuínosRESUMO
Intraoperative desorption electrospray ionization-mass spectrometry (DESI-MS) is used to characterize tissue smears by comparison with a library of DESI mass spectra of pathologically determined tissue types. Measurements are performed in the operating room within 3 min. These mass spectra provide direct information on tumor infiltration into white or gray brain matter based on N-acetylaspartate (NAA) and on membrane-derived complex lipids. The mass spectra also indicate the isocitrate dehydrogenase mutation status of the tumor via detection of 2-hydroxyglutarate, currently assessed postoperatively on biopsied tissue using immunohistochemistry. Intraoperative DESI-MS measurements made at surgeon-defined positions enable assessment of relevant disease state of tissue within the tumor mass and examination of the resection cavity walls for residual tumor. Results for 73 biopsies from 10 surgical resection cases show that DESI-MS allows detection of glioma and estimation of high tumor cell percentage (TCP) at surgical margins with 93% sensitivity and 83% specificity. TCP measurements from NAA are corroborated by indirect measurements based on lipid profiles. Notably, high percentages (>50%) of unresected tumor were found in one-half of the margin biopsy smears, even in cases where postoperative MRI suggested gross total tumor resection. Unresected tumor causes recurrence and malignant progression, as observed within a year in one case examined in this study. These results corroborate the utility of DESI-MS in assessing surgical margins for maximal safe tumor resection. Intraoperative DESI-MS analysis of tissue smears, ex vivo, can be inserted into the current surgical workflow with no alterations. The data underscore the complexity of glioma infiltration.
Assuntos
Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Glioma/patologia , Glioma/cirurgia , Monitorização Intraoperatória/métodos , Espectrometria de Massas por Ionização por Electrospray , Adulto , Idoso , Feminino , Substância Cinzenta/patologia , Substância Cinzenta/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Substância Branca/patologia , Substância Branca/cirurgiaRESUMO
Simultaneous determination of 30 common drugs of abuse, including opioids, benzodiazepines, fentanyl derivatives, methamphetamines, cocaine, substituted methylenedioxyphenethylamines, cathinones, antidepressants, and antipsychotics, in neat oral fluid was carried out using touch spray mass spectrometry from volumetric absorptive microsampling swabs. A simple and rapid (<2 min per sample) method using multiple reaction monitoring was developed and fully validated, yielding satisfactory analytical performance. Minimal sample volumes (10 µL) were used, and neither sample transfer nor preparation steps were required. Most detection limits were below 5 ng/mL from the complex drug mixture in the biological matrix. Simulations of in vivo sampling were carried out and acceptable quantitative results were obtained even under these conditions, indicating the potential of this technique for fast and noninvasive point-of-care drug testing in clinical or forensic settings.
Assuntos
Líquidos Corporais/química , Drogas Ilícitas/análise , Detecção do Abuso de Substâncias , Humanos , Espectrometria de Massas , Estrutura MolecularRESUMO
Isocitrate dehydrogenase (IDH) I and II mutations in gliomas cause an abnormal accumulation of 2-hydroxyglutarate (2-HG) in these tumor cells. These mutations have potential prognostic value in that knowledge of the mutation status can lead to improved surgical resection. Information on mutation status obtained by immunohistochemistry or genomic analysis is not available during surgery. We report a rapid extraction nanoelectrospray ionization (nESI) method of determining 2-HG. This should allow the determination of IDH mutation status to be performed intraoperatively, within minutes, using a miniature mass spectrometer. This study demonstrates that the combination of tandem mass spectrometry with low-resolution mass spectrometry allows this analysis to be performed with confidence. Graphical Abstract.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Isocitrato Desidrogenase/genética , Mutação , Espectrometria de Massas em Tandem/instrumentação , Desenho de Equipamento , Humanos , Papel , Espectrometria de Massas por Ionização por Electrospray/economia , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/métodos , Fatores de TempoRESUMO
Examination of tissue sections using desorption electrospray ionization (DESI)-MS revealed phospholipid-derived signals that differ between gray matter, white matter, gliomas, meningiomas, and pituitary tumors, allowing their ready discrimination by multivariate statistics. A set of lower mass signals, some corresponding to oncometabolites, including 2-hydroxyglutaric acid and N-acetyl-aspartic acid, was also observed in the DESI mass spectra, and these data further assisted in discrimination between brain parenchyma and gliomas. The combined information from the lipid and metabolite MS profiles recorded by DESI-MS and explored using multivariate statistics allowed successful differentiation of gray matter (n = 223), white matter (n = 66), gliomas (n = 158), meningiomas (n = 111), and pituitary tumors (n = 154) from 58 patients. A linear discriminant model used to distinguish brain parenchyma and gliomas yielded an overall sensitivity of 97.4% and a specificity of 98.5%. Furthermore, a discriminant model was created for tumor types (i.e., glioma, meningioma, and pituitary), which were discriminated with an overall sensitivity of 99.4% and a specificity of 99.7%. Unsupervised multivariate statistics were used to explore the chemical differences between anatomical regions of brain parenchyma and secondary infiltration. Infiltration of gliomas into normal tissue can be detected by DESI-MS. One hurdle to implementation of DESI-MS intraoperatively is the need for tissue freezing and sectioning, which we address by analyzing smeared biopsy tissue. Tissue smears are shown to give the same chemical information as tissue sections, eliminating the need for sectioning before MS analysis. These results lay the foundation for implementation of intraoperative DESI-MS evaluation of tissue smears for rapid diagnosis.
Assuntos
Neoplasias Encefálicas/metabolismo , Metabolismo dos Lipídeos , Metabolômica , Espectrometria de Massas por Ionização por Electrospray/métodos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Encéfalo/patologia , Diferenciação Celular , Glioma/metabolismo , Substância Cinzenta/patologia , Humanos , Análise de Componente Principal , Substância Branca/patologiaRESUMO
Insulin is a key hormone with important functions in energy metabolism and is involved in the regulation of reproduction. Hyperinsulinaemia is known to impair fertility (for example, in obese mothers); therefore, we aimed to investigate the impact of elevated insulin concentrations during the sensitive period of oocyte maturation on gene expression and lipid profiles of the bovine Day-8 embryo. Two different insulin concentrations were used during in vitro oocyte maturation (INS10=10µgmL-1 and INS0.1=0.1µgmL-1) in order to observe possible dose-dependent effects or thresholds for hyperinsulinaemia in vitro. By investigating gene expression patterns by an mRNA microarray in combination with lipid profile analysis by desorption electrospray ionisation-mass spectrometry (DESI-MS) of embryos derived from insulin-treated oocytes, we gained further insights regarding molecular responses of embryos to insulin provocation during the first days of development. Lipid metabolism appeared to be influenced on multiple levels according to gene expression results but the profiles collected in positive-ion mode by DESI-MS (showing mostly ubiquinone, cholesteryl esters and triacylglycerols) did not differ significantly from controls. There are parallels in follicular development of ruminants and humans that make this bovine model relevant for comparative research on early human embryonic development during hyperinsulinaemia.
Assuntos
Blastocisto/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/análise , Animais , Blastocisto/metabolismo , Bovinos , Relação Dose-Resposta a Droga , Desenvolvimento Embrionário/fisiologia , Feminino , Expressão Gênica/efeitos dos fármacos , Técnicas de Maturação in Vitro de OócitosRESUMO
Touch spray mass spectrometry using medical swabs is an ambient ionization technique (ionization of unprocessed sample in the open air) that has potential intraoperative application in quickly identifying the disease state of tissue and in better characterizing the resection margin. To explore this potential, we studied 29 human brain tumor specimens and obtained evidence that this technique can provide diagnostic molecular information that is relevant to brain cancer. Touch spray using medical swabs involves the physical sampling of tissue using a medical swab on a spatial scale of a few mm2 with subsequent ionization occurring directly from the swab tip upon addition of solvent and application of a high voltage. Using a tertiary mixture of acetonitrile, N,N-dimethylformamide, and ethanol, membrane-derived phospholipids and oncometabolites are extracted from the tissue, incorporated into the sprayed microdroplets, vacuumed into the mass spectrometer, and characterized in the resulting mass spectra. The tumor cell load was assessed from the complex phospholipid pattern in the mass spectra and also separately by measurement of N-acetylaspartate. Mutation status of the isocitrate dehydrogenase gene was determined via detection of the oncometabolite 2-hydroxyglutarate. The lack of sample pretreatment makes touch spray mass spectrometry using medical swabs a feasible intraoperative strategy for rapid surgical assessment.
Assuntos
Neoplasias Encefálicas/cirurgia , Glioma/cirurgia , Margens de Excisão , Espectrometria de Massas , Fosfolipídeos/análise , HumanosRESUMO
A method is presented for the detection of circulating tumor cells (CTC) using mass spectrometry (MS), through reporter-ion amplification. Particles functionalized with short-chain peptides are bound to cells through antibody-antigen interactions. Selective release and MS detection of peptides is shown to detect as few as 690 cells isolated from a 10 mL blood sample. Here we present proof-of-concept results that pave the way for further investigations.
Assuntos
Separação Celular/métodos , Células Neoplásicas Circulantes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anticorpos/química , Anticorpos/imunologia , Contagem de Células , Linhagem Celular Tumoral , Humanos , Técnicas Imunológicas/métodos , Limite de Detecção , Camundongos , Peso Molecular , Nanopartículas/química , Células Neoplásicas Circulantes/imunologia , Peptídeos/química , Dióxido de Silício/químicaRESUMO
BACKGROUND: One driving motivation in the development of point-of-care (POC) diagnostics is to conveniently and immediately provide information upon which healthcare decisions can be based, while the patient is on site. Ambient ionization mass spectrometry (MS) allows direct chemical analysis of unmodified and complex biological samples. This suite of ionization techniques was introduced a decade ago and now includes a number of techniques, all seeking to minimize or eliminate sample preparation. Such approaches provide new opportunities for POC diagnostics and rapid measurements of exogenous and endogenous molecules (e.g., drugs, proteins, hormones) in small volumes of biological samples, especially when coupled with miniature mass spectrometers. CONTENT: Ambient MS-based techniques are applied in diverse fields such as forensics, pharmaceutical development, reaction monitoring, and food analysis. Clinical applications of ambient MS are at an early stage but show promise for POC diagnostics. This review provides a brief overview of various ambient ionization techniques providing background, examples of applications, and the current state of translation to clinical practice. The primary focus is on paper spray (PS) ionization, which allows quantification of analytes in complex biofluids. Current developments in the miniaturization of mass spectrometers are discussed. SUMMARY: Ambient ionization MS is an emerging technology in analytical and clinical chemistry. With appropriate MS instrumentation and user-friendly interfaces for automated analysis, ambient ionization techniques can provide quantitative POC measurements. Most significantly, the implementation of PS could improve the quality and lower the cost of POC testing in a variety of clinical settings.
Assuntos
Técnicas de Laboratório Clínico , Hormônios/análise , Preparações Farmacêuticas/análise , Testes Imediatos , Proteínas/análise , Espectrometria de Massas por Ionização por Electrospray , HumanosRESUMO
Touch spray-mass spectrometry (TS-MS) is an ambient ionization technique (ionization of unprocessed samples in the open air) that may find intraoperative applications in quickly identifying the disease state of cancerous tissues and in defining surgical margins. In this study, TS-MS was performed on fresh kidney tissue (â¼1-5 cm(3)), within 1 h of resection, from 21 human subjects afflicted by renal cell carcinoma (RCC). The preliminary diagnostic value of TS-MS data taken from freshly resected tissue was evaluated. Principal component analysis (PCA) of the negative ion mode (m/z 700-1000) data provided the separation between RCC (16 samples) and healthy renal tissue (13 samples). Linear discriminant analysis (LDA) on the PCA-compressed data estimated sensitivity (true positive rate) and specificity (true negative rate) of 98 and 95 %, respectively, based on histopathological evaluation. The results indicate that TS-MS might provide rapid diagnostic information in spite of the complexity of unprocessed kidney tissue and the presence of interferences such as urine and blood. Desorption electrospray ionization-MS imaging (DESI-MSI) in the negative ionization mode was performed on the tissue specimens after TS-MS analysis as a reference method. The DESI imaging experiments provided phospholipid profiles (m/z 700-1000) that also separated RCC and healthy tissue in the PCA space, with PCA-LDA sensitivity and specificity of 100 and 89 %, respectively. The TS and DESI loading plots indicated that different ions contributed most to the separation of RCC from healthy renal tissue (m/z 794 [PC 34:1 + Cl](-) and 844 [PC 38:4 + Cl](-) for TS vs. m/z 788 [PS 36:1 - H](-) and 810 [PS 38:4 - H](-) for DESI), while m/z 885 ([PI 38:4 - H](-)) was important in both TS and DESI. The prospect, remaining hurdles, and future work required for translating TS-MS into a method of intraoperative tissue diagnosis are discussed. Graphical abstract Touch spray-mass spectrometry used for lipid profiling of fresh human renal cell carcinoma. Left) Photograph of the touch spray probe pointed at the MS inlet. Right) Average mass spectra of healthy renal tissue (blue) and RCC (red).
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Renais/química , Carcinoma de Células Renais/patologia , Neoplasias Renais/química , Rim/química , Fosfolipídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Algoritmos , Carcinoma de Células Renais/cirurgia , Diagnóstico Diferencial , Humanos , Rim/patologia , Rim/cirurgia , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Canine non-Hodgkin's lymphoma (NHL) is a heterogeneous group of cancers representing approximately 15% of all canine cancers. Further, canine NHL mimics human disease in regards to histopathology and clinical behavior and could function as a comparative model. Diagnosis is currently performed by histopathological evaluation of surgical biopsy specimens and fine needle aspirate (FNA) cytology, an alternative and less invasive method for diagnosis. Desorption electrospray ionization - mass spectrometry (DESI-MS) imaging was performed on tissue sections of surgical biopsies and FNA smears. Mass spectra acquired from normal lymph nodes and NHL tumors were explored using multivariate statistics (e.g. principal component analysis). Tissue sections yielded a predicted sensitivity of 100% for normal and 93.1% for tumor. Further, preliminary results suggest B-cell and T-cell lymphoma can be discriminated (CV sensitivity of 95.5% and 85.7%, respectively). Normal and B-cell NHL FNA samples analyzed by DESI produced spectra that were similar to spectra obtained from surgical biopsies. FNA samples were evaluated using a PCA-LDA classification system built using tissue section data, exploring if the chemical information obtained from the different sample types is similar and whether DESI-MS performed on FNA samples is of diagnostic value. FNA prediction rate for normal (85.7%) and B-cell NHL (89.3%) indicated that DESI-MS analysis of FNA, not previously explored, could provide rapid preliminary diagnosis. Certainly, MS provides complementary molecular information to be used in conjunction with histopathology/cytology, potentially improving diagnostic confidence. The methodology outlined here is applicable to canine NHL, further supports canine models of human NHL, and translation to humans is envisioned.
Assuntos
Biópsia por Agulha Fina , Metabolismo dos Lipídeos , Linfoma não Hodgkin/patologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Cães , Humanos , Imagem MolecularRESUMO
BACKGROUND: Air pollution and tobacco smoke can induce negative effects on the human health and often leads to the formation of oxidative stress. OBJECTIVE: The purpose of this study was to clarify the role of the urbanization degree and of passive exposure to tobacco smoke in the formation of oxidative stress. Thus, a group of non-smoking adolescents was recruited among those who live and attend school in areas with three different population densities. To each subject a spot of urine was collected to quantify 15-F2t isoprostane as a marker of oxidative stress and cotinine as a marker of passive exposure to tobacco smoke. Furthermore, respiratory functionality was also measured. RESULTS: Multiple linear regression analysis results showed a direct correlation (p<0.0001) of 15-F2t isoprostane with both the urbanization and passive smoke. Lung function parameters proved significantly lower for the subjects living in the most populous city of Torino. CONCLUSION: This remarks the negative effect that urbanization has on the respiratory conditions. Lastly, lung functionality presented a low inverse correlation with 15-F2t isoprostane, suggesting an independent mechanism than that of the urban factor.
Assuntos
Cotinina/urina , Exposição Ambiental , Isoprostanos/urina , Pulmão/fisiopatologia , Poluição por Fumaça de Tabaco/efeitos adversos , Adolescente , Biomarcadores/urina , Criança , Dinoprosta/análogos & derivados , Monitoramento Ambiental , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Itália , Masculino , Estresse Oxidativo , Medição de Risco , População Rural , Espirometria , População UrbanaRESUMO
Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacylglycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural information of great interest. This paper describes the concept and presents the results of lipid profiling by mass spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids, cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism, particularly in early embryo development and cell differentiation research.
Assuntos
Blastocisto/metabolismo , Lipídeo A/análise , Metabolismo dos Lipídeos/fisiologia , Espectrometria de Massas/métodos , Oócitos/metabolismo , Células-Tronco/metabolismo , Animais , Desenvolvimento Embrionário/fisiologia , HumanosRESUMO
Nutritional and environmental conditions around conception and during early embryonic development may have significant effects on health and well-being in adult life. Here, a bovine heifer model was used to investigate the effects of rumen-protected fat supplementation on oocyte quality and embryo development. Holstein-Friesian heifers (n=84) received a dietary supplement consisting of rumen-protected conjugated linoleic acid (CLA) or stearic acid (SA), each on top of an isocaloric basic diet. Oocytes were collected via ultrasound-guided follicular aspiration and subjected to in vitro maturation followed by either desorption electrospray ionisation mass spectrometry (DESI-MS) for lipid profiling of individual oocytes or in vitro fertilisation and embryo culture. The type of supplement significantly affected lipid profiles of in vitro-matured oocytes. Palmitic acid and plasmalogen species were more abundant in the mass spectra of in vitro-matured oocytes after rumen-protected SA supplementation when compared with those collected from animals supplemented with CLA. Lipid concentrations in blood and follicular fluid were significantly affected by both supplements. Results show that rumen-protected fatty-acid supplementation affects oocyte lipid content and may pave the way for the establishment of a large-animal model for studies towards a better understanding of reproductive disorders associated with nutritional impairments.
RESUMO
Paper spray mass spectrometry ambient ionization is utilized for rapid discrimination of bacteria without sample preparation. Bacterial colonies were smeared onto filter paper precut to a sharp point, then wetted with solvent and held at a high potential. Charged droplets released by field emission were sucked into the mass spectrometer inlet and mass spectra were recorded. Sixteen different species representing eight different genera from Gram-positive and Gram-negative bacteria were investigated. Phospholipids were the predominant species observed in the mass spectra in both the negative and positive ion modes. Multivariate data analysis based on principal component analysis, followed by linear discriminant analysis, allowed bacterial discrimination. The lipid information in the negative ion mass spectra proved useful for species level differentiation of the investigated Gram-positive bacteria. Gram-negative bacteria were differentiated at the species level by using a numerical data fusion strategy of positive and negative ion mass spectra.
Assuntos
Bactérias Gram-Negativas/classificação , Bactérias Gram-Positivas/classificação , Espectrometria de Massas/métodos , Análise Multivariada , PapelRESUMO
The increasing role of hair analysis in forensic toxicological investigations principally owes to recent improvements of mass spectrometric instrumentation. Research achievements during the last 6 years in this distinctive application area of analytical toxicology are reviewed. The earlier state of the art of hair analysis was comprehensively covered by a dedicated book (Kintz, 2007a. Analytical and practical aspects of drug testing in hair. Boca Raton: CRC Press and Taylor & Francis, 382 p) that represents key reference of the present overview. Whereas the traditional organization of analytical methods in forensic toxicology divided target substances into quite homogeneous groups of drugs, with similar structures and chemical properties, the current approach often takes advantage of the rapid expansion of multiclass and multiresidue analytical procedures; the latter is made possible by the fast operation and extreme sensitivity of modern mass spectrometers. This change in the strategy of toxicological analysis is reflected in the presentation of the recent literature material, which is mostly based on a fit-for-purpose logic. Thus, general screening of unknown substances is applied in diverse forensic contexts than drugs of abuse testing, and different instrumentation (triple quadrupoles, time-of-flight analyzers, linear and orbital traps) is utilized to optimally cope with the scope. Other key issues of modern toxicology, such as cost reduction and high sample throughput, are discussed with reference to procedural and instrumental alternatives.
Assuntos
Toxicologia Forense/métodos , Cabelo/química , Espectrometria de Massas/métodos , Preparações Farmacêuticas/análise , Detecção do Abuso de Substâncias/métodos , Animais , Desenho de Equipamento , Toxicologia Forense/instrumentação , Humanos , Espectrometria de Massas/instrumentação , Detecção do Abuso de Substâncias/instrumentaçãoRESUMO
Strep throat causing Streptococcus pyogenes was detected in vitro and in simulated clinical samples by performing touch spray ionization-mass spectrometry. MS analysis took only seconds to reveal characteristic bacterial and human lipids. Medical swabs were used as the substrate for ambient ionization. This work constitutes the initial step in developing a non-invasive MS-based test for clinical diagnosis of strep throat. It is limited to the single species, S. pyogenes, which is responsible for the vast majority of cases. The method is complementary to and, with further testing, a potential alternative to current methods of point-of-care detection of S. pyogenes.
Assuntos
Técnicas Bacteriológicas/instrumentação , Técnicas Bacteriológicas/métodos , Faringite/diagnóstico , Espectrometria de Massas por Ionização por Electrospray , Infecções Estreptocócicas/diagnóstico , Streptococcus pyogenes/metabolismo , Humanos , Lipídeos/análise , Faringite/microbiologia , Sistemas Automatizados de Assistência Junto ao Leito , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/isolamento & purificaçãoRESUMO
BACKGROUND: Buprenorphine (BUP) is a psychoactive pharmaceutical drug largely used to treat opiate addiction. Short-term therapeutic monitoring is supported by toxicological analysis of blood and urine samples, whereas long-term monitoring by means of hair analysis is rarely used. Aim of this work was to develop and validate a highly sensitive ultrahigh-performance liquid chromatography tandem mass spectrometry method to detect BUP and norbuprenorphine (NBUP) in head hair. METHODS: Interindividual correlation between oral dosage of BUP and head hair concentration was investigated. Furthermore, an intra-individual study by means of segmental analysis was performed on subjects with variable maintenance dosage. Hair samples from a population of 79 patients in treatment for opiate addiction were analyzed. RESULTS: The validated ultrahigh-performance liquid chromatography tandem mass spectrometry protocol allowed to obtain limits of detection and quantification at 0.6 and 2.2 pg/mg for BUP and 5.0 and 17 pg/mg for NBUP, respectively. Validation criteria were satisfied, assuring selective analyte identification, high detection capability, and precise and accurate quantification. Significant positive correlation was found between constant oral BUP dosage (1-32 mg/d) and the summed up head hair concentrations of BUP and NBUP. Nevertheless, substantial interindividual variability limits the chance to predict the oral dosage taken by each subject from the measured concentrations in head hair. In contrast, strong correlation was observed in the results of intra-individual segmental analysis, which proved reliable to detect oral dosage variations during therapy. CONCLUSIONS: Remarkably, all hair samples yielded BUP concentrations higher than 10 pg/mg, even when the lowest dosage was administered. Thus, these results support the selection of 10 pg/mg as a cutoff value.
Assuntos
Buprenorfina/análise , Monitoramento de Medicamentos/métodos , Cabelo/química , Antagonistas de Entorpecentes/análise , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Síndrome de Abstinência a Substâncias , Adulto , Buprenorfina/administração & dosagem , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Antagonistas de Entorpecentes/administração & dosagem , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Fatores de Tempo , Adulto JovemRESUMO
Here, we present a targeted polar metabolomics protocol for the analysis of biofluids and frozen tissue biopsies using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We describe steps for sample pretreatment, liquid-liquid extraction, and isolation of polar metabolites. We then detail procedures for target LC-MS/MS analysis. In this protocol, we focus on the analysis of plasma and serum samples. We also provide brief instructions on how to process other biological matrices as supplemental information. For complete details on the use and execution of this protocol, please refer to Coskun et al. (2022).1.