RESUMO
The present study investigated the encapsulation of ß-galactosidase in carrageenan, pectin and its hybrid hydrogels by using the ionotropic gelation method. The material obtained was characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG/DTG) and scanning electron microscopy (SEM). The effects of pH, temperature and storage time were evaluated in terms of the catalytic activity of the free and encapsulated enzyme. Addition studies were conducted evaluating the performance of catalytic activity in vitro conditions. Carrageenan, pectin and hybrid hydrogels presented encapsulation efficiency of 58 ± 1%, 72 ± 1% and 77 ± 2%, respectively. The pectin hydrogel showed the higher ß-galactosidase activity in pH and temperature tests. However, the carrageenan hydrogel exhibited best stability after been stored for three months. Carrageenan and pectin hydrogels were 2.0 and 2.4 times more efficiently than commercial tablet in the releasing ß-galactosidase under in vitro conditions, respectively. The results suggest that pectin and carrageenan hydrogels may be useful for the development of new formulation of ß-galactosidase.
Assuntos
Carragenina/química , Portadores de Fármacos/química , Pectinas/química , beta-Galactosidase/química , Varredura Diferencial de Calorimetria , Química Farmacêutica , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Tecnologia FarmacêuticaRESUMO
BACKGROUND: Chromobacterium violaceum (C. violaceum) occurs abundantly in a variety of ecosystems, including ecosystems that place the bacterium under stress. This study assessed the adaptability of C. violaceum by submitting it to nutritional and pH stresses and then analyzing protein expression using bi-dimensional electrophoresis (2-DE) and Maldi mass spectrometry. RESULTS: Chromobacterium violaceum grew best in pH neutral, nutrient-rich medium (reference conditions); however, the total protein mass recovered from stressed bacteria cultures was always higher than the total protein mass recovered from our reference culture. The diversity of proteins expressed (repressed by the number of identifiable 2-DE spots) was seen to be highest in the reference cultures, suggesting that stress reduces the overall range of proteins expressed by C. violaceum. Database comparisons allowed 43 of the 55 spots subjected to Maldi mass spectrometry to be characterized as containing a single identifiable protein. Stress-related expression changes were noted for C. violaceum proteins related to the previously characterized bacterial proteins: DnaK, GroEL-2, Rhs, EF-Tu, EF-P; MCP, homogentisate 1,2-dioxygenase, Arginine deiminase and the ATP synthase ß-subunit protein as well as for the ribosomal protein subunits L1, L3, L5 and L6. The ability of C. violaceum to adapt its cellular mechanics to sub-optimal growth and protein production conditions was well illustrated by its regulation of ribosomal protein subunits. With the exception of the ribosomal subunit L3, which plays a role in protein folding and maybe therefore be more useful in stressful conditions, all the other ribosomal subunit proteins were seen to have reduced expression in stressed cultures. Curiously, C. violeaceum cultures were also observed to lose their violet color under stress, which suggests that the violacein pigment biosynthetic pathway is affected by stress. CONCLUSIONS: Analysis of the proteomic signatures of stressed C. violaceum indicates that nutrient-starvation and pH stress can cause changes in the expression of the C. violaceum receptors, transporters, and proteins involved with biosynthetic pathways, molecule recycling, energy production. Our findings complement the recent publication of the C. violeaceum genome sequence and could help with the future commercial exploitation of C. violeaceum.
Assuntos
Proteínas de Bactérias/metabolismo , Chromobacterium/crescimento & desenvolvimento , Chromobacterium/metabolismo , Proteômica/métodos , Vias Biossintéticas , Meios de Cultura/química , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Proteínas Ribossômicas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Estresse FisiológicoRESUMO
Raw glycerol is a byproduct of biodiesel production that currently has low to negative value for biodiesel producers. One option for increasing the value of raw glycerol is to use it as a feedstock for microbial production. Bacillus subtilis LSFM 05 was used for the production of fengycin in a mineral medium containing raw glycerol as the sole carbon source. Fengycin was isolated by acid precipitation at pH 2 and purified by silica gel column chromatography and characterized using electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) with collision-induced dissociation (CID). The mass spectrum revealed the presence of the ions of m/z 1,435.7, 1,449.9, 1,463.8, 1,477.8, 1,491.8 and 1,505.8, which were further fragmented by ESI-MS/MS. The CID profile showed the presence of a series of ions (m/z 1,080 and 966) and (m/z 1,108 and 994) that represented the different fengycin homologues A and B, respectively. Fengycin homologues A and B are variants that differ at position 6 of the peptide moiety, having either Ala or Val residues, respectively. Mass spectrometry analyses identified four fengycin A and three fengycin B variants with fatty acid components containing 14-17 carbons. These results demonstrate that raw glycerol can be used as feedstock to produce fengycin, and additional work should focus on the optimization of process conditions to increase productivity.
Assuntos
Bacillus subtilis/metabolismo , Glicerol/metabolismo , Lipopeptídeos/química , Tensoativos/química , Lipopeptídeos/biossíntese , Lipopeptídeos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Espectroscopia de Infravermelho com Transformada de Fourier , Tensoativos/isolamento & purificação , Tensoativos/metabolismo , Espectrometria de Massas em TandemRESUMO
BACKGROUND: The aim of this study was to evaluate protein expression profile and quantify the proteins present in follicular fluid (FF) samples from women with endometriosis and pregnant women without endometriosis. METHODS: A prospective case-control study was carried out including women with Stage III or IV endometriosis (Group I) and pregnant women without endometriosis (Group II), both at the maximum age of 35 years. Women were submitted to controlled ovarian stimulation for in vitro fertilization, and FF was collected after ultrasound-guided ovarian aspiration. FF from both ovaries was pooled, and patient samples were pooled according to Group I or II. Pooled protein samples were separated and analyzed by MudPIT (multidimensional protein identification technology followed by Expression(E) and label-free quantification with ProteinLynxGlobalServer 2.4v, Identity(E) and Expression(E) software). RESULTS: A total of 416 proteins or randomic sequence were identified, 62 proteins differentially expressed between Groups I and II. One (1.6%) was expressed at a higher level and 36 (58.1%) were uniquely expressed in Group I, whereas 8 (12.9%) were expressed at a higher level and 17 (27.4%) were uniquely expressed in Group II. Of all these, 15 (24.2%) are related to binding, 1 (1.6%) to immune response, 8 (12.9%) to cell division, 3 (4.8%) to cellular metabolism, 16 (25.8%) to general function and 19 (30.6%) do not yet present an identified function. CONCLUSIONS: Protein expression profiles of patients with and without endometriosis identified at least 64 proteins differentially expressed, which may be related to the physiopathology of endometriosis. These proteins may additionally be useful in determining potential biomarkers for diagnostics, as well as for therapeutic intervention in women with infertility due to endometriosis.
Assuntos
Endometriose/metabolismo , Fertilização in vitro , Líquido Folicular/metabolismo , Indução da Ovulação , Proteínas/metabolismo , Adulto , Feminino , Perfilação da Expressão Gênica , Humanos , Estudos ProspectivosRESUMO
BACKGROUND: Lactose intolerance is characterized by the absence of the enzyme lactase (beta-galactosidase) and affects two thirds of the world adult population. Our aim was to evaluate a lactase gastro-resistant formulation to ensure increased activity in the action site of the enzyme (lumen of the small intestine). Simultaneously, we also evaluated the commercial product stability and enzyme activity, because the product containing beta-galactosidase is classified as food supplement according to the Food and Drug Administration (FDA), so it is free to pass quality testing, efficacy and stability. So, it is possible that contain some irregularities as to the content and enzymatic activity. METHODS: The dissolution assay was performed using a dissolution test system and commercial product and the gastro-resistant formulation were evaluated according to a method adapted to the conditions recommended by United States Pharmacopeia (US Pharmacopeia) for gastro-resistant formulations. For the assessment of enzymatic activity throughout the dissolution test was employed the official method of lactase assay described in US Pharmacopoeia. This method is based on a colorimetric reaction which the substrate reacts with the enzyme generate a colored product further analyzed by UVVisible spectrophotometry. RESULTS: When carrying out dissolution test in commercial product it is noted that the existing formulation is not able to protect the enzyme from degrading action of gastric environment (a loss of 86.0 ± 0.8% of lactase activity was observed). Our proposed gastro-resistant pharmaceutical form there was no loss of activity during the acid step and the end of the dissolution test the found activity was 95 ± 1.3%. CONCLUSION: The formulations proposed in this work using hypromellose capsules ensure the enzymatic activity of lactase, preventing its contact with the acid medium. For the other side, the results of commercial tablets for lactase release indicate a significant loss of enzyme activity due to the immediate release of the enzyme in the simulated gastric fluids.
Assuntos
Lactase/química , Cápsulas/química , Química Farmacêutica/métodos , Solubilidade , Comprimidos/química , Estados Unidos , United States Food and Drug AdministrationRESUMO
Metal-ion contamination (Cd, Cu, Pb, and Zn) on sunflower (Helianthus annuus L.) growth and total leaf protein expression were studied in the present work. The height, mass production, and metal distribution (Ca, K, Fe, Mg, Na, and P) in all plant fractions (roots, stems, and leaves) were evaluated. Sunflowers plants contaminated with four metal ions decreases height and mass by 35% and 40%, respectively, compared to control. Significant differences of total protein composition were noted after SDS-PAGE separation. Sunflower proteomics were more affected when 500 mg L(-1) of metal ion was added as contaminant of both zinc and mixed ions solution. In these cases, proteins having a molar mass of 14.5, 34.5, and 54.0 kDa were present at a lower level and alterations in enzymatic activities (SOD and GR) were found. Sunflowers plants contaminated with zinc and the mixed ions solution showed some degree of oxidative stress.
Assuntos
Helianthus/efeitos dos fármacos , Helianthus/crescimento & desenvolvimento , Metais Pesados/farmacologia , Biomassa , Regulação da Expressão Gênica de Plantas , Helianthus/metabolismo , Íons/química , Metais Pesados/química , Peso Molecular , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Pancreatin is a biotechnological product containing an enzyme complex, obtained from porcine pancreas, that is employed in treating pancreatic diseases. Experiments regarding the stability of the pharmaceutical formulation containing pancreatin were performed using standard binary mixtures with 6 excipients in a 1:1 ratio (m/m) and a commercial formulation. To accomplish these goals, samples were stored for 1, 3 and 6 months at 40 ± 1 °C and 75 ± 5 % relative humidity (RH) and 40 ± 1 °C and 0 % RH. Stress testing was also performed. All samples were analyzed to evaluate the α-amylase, lipase and protease activities through UV/Vis spectrophotometry. The results revealed that the excipient proprieties and the storage conditions affected enzyme stability. Humidity was a strong influencing factor in the reduction of α-amylase and protease activities. Stress testing indicated that pH 9.0 and UV light did not induce substantial alterations in enzyme activity.
Assuntos
Excipientes/química , Fármacos Gastrointestinais/metabolismo , Pancreatina/metabolismo , Animais , Brasil , Química Farmacêutica , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Estabilidade Enzimática , Fármacos Gastrointestinais/química , Guias como Assunto , Temperatura Alta/efeitos adversos , Umidade/efeitos adversos , Concentração de Íons de Hidrogênio , Lipase/química , Lipase/metabolismo , Oxirredução , alfa-Amilases Pancreáticas/química , alfa-Amilases Pancreáticas/metabolismo , Pancreatina/química , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Pós , Sus scrofa , Raios Ultravioleta/efeitos adversosRESUMO
OBJECTIVE: To evaluate protein expression profile and to quantify proteins present in seminal plasma from men with spinal cord injury (SCI) and healthy men without SCI. DESIGN: Experimental study. SETTING: University hospital. PATIENT(S): Twelve SCI patients divided into two groups, six who underwent electroejaculation (EEJ) and six who underwent penile vibratory stimulation (PVS); and ten control subjects presenting normal sperm motility and concentration. INTERVENTION(S): EEJ and PVS. MAIN OUTCOME MEASURE(S): The seminal plasma protein profile was analyzed by two proteomic strategies: data-independent label-free quantitative proteomics (MS(E)) and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). RESULT(S): A total of 638 different proteins were identified by MS(E) and 18 by 2D SDS-PAGE followed by tandem mass spectrometry. Interactome analysis showed key reproductive biologic processes-insemination, sperm and oocyte fusion, and acrosome reaction-related to all groups, as were triglyceride stimuli. Processes related to actin and muscle function and to iron oxidation, transportation, and homeostasis were found only in the EEJ and PVS groups; response to hydrogen peroxide and increased immune response was found only in the PVS group. CONCLUSION(S): This study was able to demonstrate differential protein expression among control, PVS, and EEJ groups; SCI is responsible for alterations in seminal plasma protein profile leading to a deviation from homeostasis; proteins reported in both PVS and EEJ groups correlate with the pathophysiology of SCI-related infertility.
Assuntos
Proteínas/análise , Proteômica , Análise do Sêmen/métodos , Sêmen/química , Recuperação Espermática , Traumatismos da Medula Espinal/metabolismo , Adulto , Biomarcadores/análise , Estudos de Casos e Controles , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Hospitais Universitários , Humanos , Masculino , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Contagem de Espermatozoides , Motilidade dos Espermatozoides , Traumatismos da Medula Espinal/fisiopatologia , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Recent developments and applications in the production of thermosprays directly into flame furnaces to improve the analytical sensitivity in atomic absorption spectrometry are reviewed in this manuscript. Principles, characteristics, instrumentation, and applications of this analytical technique for trace elements determination in several matrices are discussed. The use of preconcentration procedures to allow low detection limits for ultra-trace levels using TS-FF-AAS is presented and current perspectives and future trends of this technique are also discussed.
Assuntos
Espectrofotometria Atômica/métodos , Oligoelementos/análise , Temperatura Alta , Limite de Detecção , Espectrofotometria Atômica/instrumentação , Espectrofotometria Atômica/tendênciasRESUMO
O cloridrato de ziprasidona foi físico-quimicamente caracterizado pelas técnicas de Calorimetria Exploratória Diferencial (DSC), Termogravimetria (TG), Espectroscopia no Infravermelho com Transformada de Fourier (FT-IR), Difração de Raios X de Pó (DRX) e Microscopia Eletrônica de Varredura (MEV). O estudo de compatibilidade foi realizado com 5 excipientes farmacêuticos diferentes (amido pré-gelatinizado, estearato de magnésio, celulose microcristalina, manitol e polivinilpirrolidona PVP) . Amostras de misturas binárias fármaco: excipiente 1:1 m/m foram estocadas por 3 meses em câmara de estabilidade (75% ± 5% de umidade relativa e 40 ºC ± 1 ºC), e então analisadas por Cromatografia Líquida de Alta Eficiência (CLAE) para avaliar o efeito de cada excipiente na estabilidade química e, consequentemente, no teor do fármaco, em cada amostra . Os resultados de DRX e FT-IR identificaram a forma polimórfica F, correspondente ao cloridrato de ziprasidona monohidrato. A análise térmica demonstrou que o fármaco apresentou uma perda de massa de 4%, até 100ºC, correspondente à saída de uma molécula de água. A próxima perda de massa ocorreu a partir da temperatura de fusão (297ºC), e o fármaco foi totalmente degradado até 600ºC. Os resultados de CLAE demonstraram que o estearato de magnésio foi o único, entre os 5 excipientes testados, que provocou uma redução significativa de teor do fármaco na amostra (teor encontrado = 77% ± 3%). Dessa forma, o fármaco foi compatível com amido pré-gelatinizado, celulose microcristalina, manitol e PVP; e incompatível com estearato de magnésio nas condições estudadas.
Ziprasidone hydrochloride was fully characterized by Differential Scanning Calorimetry (DSC), Thermogravimetry (TG), Fourier Transform Infrared Spectroscopy (FT-IR), Powder X-ray Diffraction (PXRD) and Scanning Electron Microscopy (SEM). The stability study was carried out with 5 different pharmaceutical excipients (pregelatinized starch, magnesium stearate, microcrystalline cellulose, mannitol, polyvinylpyrrolidone PVP). Binary mixtures of the drug-excipient were prepared in a 1:1(w/w) ratio and stored for 3 months in stability chamber (75% ± 5% of relative humidity and temperature of 40 ºC ± 1 ºC), then these samples were analyzed by High Performance Liquid Chromatography (HPLC) to evaluate the effect of each excipient on chemical stability and, consequently, on amount of drug in each sample. Data obtained by FT-IR and PXRD shown the polymorphic form F, corresponding to monohydrate ziprasidone hydrochloride. The thermal analysis demonstrated a mass loss of 4% until 100ºC, corresponding to a water molecule. The following mass loss occurred from melting temperature (297ºC) to 600ºC, with total sample degradation. The HPLC results shown that, between 5 tested excipients, only magnesium stearate caused significant amount reduction of drug in the sample (amount found = 77% ± 3%). Then, the drug was compatible with pregelatinized starch, microcrystalline cellulose, mannitol and PVP; and incompatible with magnesium stearate, in these work conditions.
Assuntos
Antipsicóticos/análise , Excipientes Farmacêuticos/química , Estabilidade de MedicamentosRESUMO
Six protocols for extraction of proteins from sunflower (Helianthus annuus L.) leaves were evaluated for their abilities in both removing interferents and attaining the best resolution in two-dimensional gel electrophoresis. "Classical" phenol extraction followed by precipitation with ammonium acetate in methanol displayed the most efficient protocol, which allowed the detection of 244 protein spots with ca. 485mug of protein in gel electrophoresis. Tandem mass spectrometry was performed to identify proteins in 61 spots, and cross species identification was used for this task. Proteins from twenty two spots were identified, and 12 of these proteins are up to now not included into the ExPASy sunflower protein databank.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Helianthus/química , Folhas de Planta/química , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Bases de Dados de Proteínas , Eletroforese em Gel de Poliacrilamida , Proteínas de Plantas/análise , Proteínas de Plantas/química , Espectrometria de Massas em TandemRESUMO
abstract Valsartan was submitted to forced degradation under acid hydrolysis condition as prescribed by the ICH. Degraded sample aliquots were separated via HPLC using a Hypersil ODS (C18) column (250 x 4.6 mm i.d., 5 µm). Either photodiode array (PDA) detection or mass spectrometry (MS) full scan monitoring of HPLC runs were used. HPLC-PDA failed to indicate Valsartan degradation under forced acid degradation, showing an insignificant peak area variation and that Valsartan apparently remained pure. HPLC-MS using electrospray ionization (ESI) and total ionic current (TIC) monitoring did not reveal any peak variation either, but inspection of the ESI mass spectra showed the appearance of m/z 306 and m/z 352 ions for the same retention time as that of Valsartan (m/z 436). These ions were identified as being protonated molecules of two co-eluting degradation products formed by hydrolysis. These assignments were confirmed by ESI-MS/MS with direct infusion of the degraded samples. The results showed that the use of selective HPLC-MS is essential for monitoring Valsartan degradation. Efficient HPLC separation coupled to selective and structural diagnostic MS monitoring seems therefore mandatory for comprehensive drug degradation studies, particularly for new drugs and formulations, and for method development.
resumo Valsartana (VAL) foi submetida à degradação forçada em meio ácido conforme procedimento descrito no ICH. Os produtos de degradação (PDs) foram monitorados ao longo do tempo de degradação pela técnica de Cromatografia Líquida (LC) utilizando uma coluna Hypersil ODS (C18) (250 x 4,6 mm d.i., 5 µm). A detecção foi feita com dois detectores: espectrofotométrico (PDA) e espectrometria de massas (MS) por corrente iônica total. Ambas as técnicas falharam na identificação dos PDs obtidos ao longo do monitoramento, mostrando insignificantes variações na área do pico e permanecendo com pureza de pico ao longo de toda a eluição. Somente depois da avaliação por íon extraído (XIC), foi possível observar o aumento do íon m/z 306 e m/z 352 exatamente no mesmo tempo de retenção do íon molecular (m/z 436). Estes resultados mostram um caso simples e didático em que somente o uso de um método seletivo de LC-MS pode ser utilizado para monitorar produtos de degradação. Neste trabalho, é apresentado um caso real em que a separação por LC deve ser acoplada a métodos seletivos obtidos por MS, especialmente no estudo de PDs para novos fármacos, formulações e no desenvolvimento de métodos.
Assuntos
Espectrometria de Massas/classificação , Valsartana/farmacocinética , Metabolismo , Cromatografia Líquida de Alta Pressão , Teste de Esforço , HidróliseRESUMO
Valsartan (VAL) is a highly selective blocker of the angiotensin II receptor that has been widely used in the treatment of hypertension. Active pharmaceutical ingredient compatibility with excipients (crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose and titanium dioxide) is usually evaluated in solid pharmaceutical development. Compatibility and stability can be evaluated by liquid chromatography. Studies were performed using binary mixtures of 1:1 (w/w) VAL/excipient; samples were stored under accelerated stability test conditions (40 ºC at 75% relative humidity). The results indicate that VAL is incompatible with crospovidone and hypromellose, which reduced the VAL content and gave rise to new peaks in the chromatogram due to degradation products.
Valsartana (VAL) é um bloqueador altamente seletivo do receptor da angiotensina II, que tem sido amplamente utilizado para o tratamento da hipertensão. Testes de compatibilidade com excipientes usualmente empregados em formulações sólidas são utilizados no desenvolvimento de formulações sólidas. Neste trabalho, realizaram-se testes utilizando misturas binárias na proporção 1:1 (m/m) de VAL/excipiente e as amostras foram armazenadas em condições de estabilidade acelerada (40 ºC em 75% de umidade relativa). Os resultados obtidos indicam a incompatibilidade de VAL com crospovidona e hipromelose, através da redução do teor de VAL e a presença de novos picos no cromatograma provenientes de produtos de degradação.
Assuntos
Química Farmacêutica/classificação , Valsartana/farmacologia , Varredura Diferencial de Calorimetria , Cromatografia Líquida/métodos , Estabilidade de MedicamentosRESUMO
This work describes the optimization of a cloud point extraction (CPE) method for casein proteins from cow milk samples. To promote phase separation, polyoxyethylene(8) isooctylphenyl ether (Triton X-114) and sodium chloride (NaCl) were used as nonionic surfactant and electrolyte, respectively. Using multivariate studies, four major CPE variables were evaluated: Triton X-114 concentration, sample volume, NaCl concentration, and pH. The results show that surfactant concentration and sample volume were the main variable affecting the CPE process, with the following optimized parameters: 1% (w/v) Triton X-114 concentration, 50 microL of sample volume, 6% (w/v) NaCl concentration and extractions carried out at pH 7.0. At these conditions, 923+/-66 and 67+/-2 microg mL(-1) of total protein were found in the surfactant-rich and surfactant-poor phases, respectively. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was then used to evaluate those target proteins (alpha(s1)-casein, alpha(s2)-casein and beta-casein) separation as well as to check the efficiency of the extraction procedure, making a fingerprint of those target proteins possible.
Assuntos
Caseínas/análise , Leite/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Octoxinol , Polietilenoglicóis/química , Cloreto de Sódio/químicaRESUMO
This review describes recent tendencies for metal-binding and metalloprotein analysis, emphasizing metal quantification in proteins through X-ray, atomic absorption, mass spectrometric techniques, and others. Hyphenated techniques such as capillary electrophoresis-synchrotron radiation X-ray fluorescence (CE-SRXRF), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF-MS), etc. are also presented. As protein separation techniques electrophoresis (mainly sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) are indicated, due to their inherent sensitivity, resolution and/or easy implementation. Latest challenges in metallomics are also commented.